The histidine kinase CpHK2 has impact on spore germination, oxidative stress and fungicide resistance, and virulence of the ergot fungus Claviceps purpurea.

PubMed

Nathues, Eva; Jörgens, Cordula; Lorenz, Nicole; Tudzynski, Paul

2007-09-01

SUMMARY Histidine kinases are important mediators for adaptation of bacteria and plants to environmental signals. Genome analyses of filamentous fungi have revealed the presence of a high number of potential hybrid histidine kinase (HK)-encoding genes; the role of most of these potential sensors is so far unclear, though some members of the class III histidine kinases were shown to be involved in osmostress responses. Here we present a functional analysis of cphk2, a histidine kinase-encoding gene in the biotrophic grass pathogen Claviceps purpurea. The putative product of cphk2 (CpHK2) was shown to group within family X of fungal HKs and it had high homology to the oxidative stress sensors SpMAK2/3 of Schizosaccharomyces pombe. Analysis of a cphk2 deletion mutant indicated that this histidine kinase is involved in spore germination, sensitivity to oxidative stress and fungicide resistance. In addition, virulence of the Dcphk2 mutant on rye was significantly reduced compared with the wild-type strain, even if the conidial titre was adjusted to the lower germination rate. This is the first report of a role for a class X histidine kinase in a filamentous fungus.

Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens

NASA Astrophysics Data System (ADS)

Velikova, Nadya; Fulle, Simone; Manso, Ana Sousa; Mechkarska, Milena; Finn, Paul; Conlon, J. Michael; Oggioni, Marco Rinaldo; Wells, Jerry M.; Marina, Alberto

2016-05-01

Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials.

Characterization of the RcsC sensor kinase from Erwinia amylovora and other Enterobacteria.

PubMed

Wang, Dongping; Korban, Schuyler S; Pusey, P Lawrence; Zhao, Youfu

2011-06-01

RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain, and a receiver domain. We have previously demonstrated that, although the Erwinia amylovora rcsC mutant produces more amylovoran than the wild-type (WT) strain in vitro, the mutant remains nonpathogenic on both immature pear fruit and apple plants. In this study, we have comparatively characterized the Erwinia RcsC and its homologs from various enterobacteria. Results demonstrate that expression of the Erwinia rcsC gene suppresses amylovoran production in various amylovoran overproducing WT and mutant strains, thus suggesting the presence of a net phosphatase activity of Erwinia RcsC. Findings have also demonstrated that rcsC homologs from other enterobacteria could not rescue amylovoran production of the Erwinia rcsC mutant in vitro. However, virulence of the Erwinia rcsC mutant is partially restored by rcsC homologs from Pantoea stewartii, Yersinia pestis, and Salmonella enterica but not from Escherichia coli on apple shoots. Domain-swapping experiments have indicated that replacement of the E. coli RcsC sensor domain by those of Erwinia and Yersinia spp. partially restores virulence of the Erwinia rcsC mutant, whereas chimeric constructs containing the sensor domain of E. coli RcsC could not rescue virulence of the Erwinia rcsC mutant on apple. Interestingly, only chimeric constructs containing the histidine kinase and receiver domains of Erwinia RcsC are fully capable of rescuing amylovoran production. These results suggest that the sensor domain of RcsC may be important in regulating bacterial virulence, whereas the activity of the histidine kinase and receiver domains of Erwinia RcsC may be essential for amylovoran production in vitro.

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.

PubMed

Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I; Zhou, Xiaohui

2016-02-09

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

PubMed Central

Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I.; Zhou, Xiaohui

2016-01-01

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics. PMID:26831117

Intra- and Interprotein Phosphorylation between Two-hybrid Histidine Kinases Controls Myxococcus xanthus Developmental Progression*

PubMed Central

Schramm, Andreas; Lee, Bongsoo; Higgs, Penelope I.

2012-01-01

Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple “two-component” systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program. PMID:22661709

The accommodation index measures the perturbation associated with insertions and deletions in coiled‐coils: Application to understand signaling in histidine kinases

PubMed Central

Schmidt, Nathan W.; Grigoryan, Gevorg

2017-01-01

Abstract Coiled‐coils are essential components of many protein complexes. First discovered in structural proteins such as keratins, they have since been found to figure largely in the assembly and dynamics required for diverse functions, including membrane fusion, signal transduction and motors. Coiled‐coils have a characteristic repeating seven‐residue geometric and sequence motif, which is sometimes interrupted by the insertion of one or more residues. Such insertions are often highly conserved and critical to interdomain communication in signaling proteins such as bacterial histidine kinases. Here we develop the “accommodation index” as a parameter that allows automatic detection and classification of insertions based on the three dimensional structure of a protein. This method allows precise identification of the type of insertion and the “accommodation length” over which the insertion is structurally accommodated. A simple theory is presented that predicts the structural perturbations of 1, 3, 4 residue insertions as a function of the length over which the insertion is accommodated. Analysis of experimental structures is in good agreement with theory, and shows that short accommodation lengths give rise to greater perturbation of helix packing angles, changes in local helical phase, and increased structural asymmetry relative to long accommodation lengths. Cytoplasmic domains of histidine kinases in different signaling states display large changes in their accommodation lengths, which can now be seen to underlie diverse structural transitions including symmetry/asymmetry and local variations in helical phase that accompany signal transduction. PMID:27977891

The molecular basis of ethylene signalling in Arabidopsis

NASA Technical Reports Server (NTRS)

Woeste, K.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

1998-01-01

The simple gas ethylene profoundly influences plants at nearly every stage of growth and development. In the past ten years, the use of a genetic approach, based on the triple response phenotype, has been a powerful tool for investigating the molecular events that underlie these effects. Several fundamental elements of the pathway have been described: a receptor with homology to bacterial two-component histidine kinases (ETR1), elements of a MAP kinase cascade (CTR1) and a putative transcription factor (EIN3). Taken together, these elements can be assembled into a simple, linear model for ethylene signalling that accounts for most of the well-characterized ethylene mediated responses.

Changes at the KinA PAS-A Dimerization Interface Influence Histidine Kinase Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, James; Tomchick, Diana R.; Brautigam, Chad A.

2008-11-12

The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatorymore » role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using {beta}-sheet/{beta}-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.« less

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

PubMed Central

Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A.; Briggs, Winslow R.; Goldbaum, Fernando A.; Paris, Gastón

2015-01-01

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms. PMID:25993430

Structure-based discovery of inhibitors of the YycG histidine kinase: New chemical leads to combat Staphylococcus epidermidis infections

PubMed Central

Qin, Zhiqiang; Zhang, Jian; Xu, Bin; Chen, Lili; Wu, Yang; Yang, Xiaomei; Shen, Xu; Molin, Soeren; Danchin, Antoine; Jiang, Hualiang; Qu, Di

2006-01-01

Background Coagulase-negative Staphylococcus epidermidis has become a major frequent cause of infections in relation to the use of implanted medical devices. The pathogenicity of S. epidermidis has been attributed to its capacity to form biofilms on surfaces of medical devices, which greatly increases its resistance to many conventional antibiotics and often results in chronic infection. It has an urgent need to design novel antibiotics against staphylococci infections, especially those can kill cells embedded in biofilm. Results In this report, a series of novel inhibitors of the histidine kinase (HK) YycG protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular lead-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds displayed significant activity in inhibiting S. epidermidis growth. Furthermore, five of them displayed bactericidal effects on both planktonic and biofilm cells of S. epidermidis. Except for one, the compounds were found to bind to the YycG protein and to inhibit its auto-phosphorylation in vitro, indicating that they are potential inhibitors of the YycG/YycF two-component system (TCS), which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study. Conclusion These novel inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious than traditional screening technology. PMID:17094812

A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis.

PubMed

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-10-03

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis*

PubMed Central

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-01-01

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. PMID:25118291

How to Switch Off a Histidine Kinase: Crystal Structure of Geobacillus Stearothermophilus KinB with the Inhibitor Sda

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bick, M.; Lamour, V; Rajashankar, K

2009-01-01

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-Angstroms-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to whichmore » it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.« less

A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

PubMed Central

Galperin, Michael Y

2005-01-01

Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes, are found among the poorly studied beta-, delta- and epsilon-proteobacteria. Among all bacterial phyla, only cyanobacteria appear to be true introverts, probably due to their capacity to conduct oxygenic photosynthesis, using a complex system of intracellular membranes. The census data, available at , can be used to get an insight into metabolic and behavioral propensities of each given organism and improve prediction of the organism's properties based solely on its genome sequence. PMID:15955239

A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts.

PubMed

Galperin, Michael Y

2005-06-14

Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. This paper presents results of a comprehensive census of signal transduction proteins--histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases--encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes, are found among the poorly studied beta-, delta- and epsilon-proteobacteria. Among all bacterial phyla, only cyanobacteria appear to be true introverts, probably due to their capacity to conduct oxygenic photosynthesis, using a complex system of intracellular membranes. The census data, available at http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html, can be used to get an insight into metabolic and behavioral propensities of each given organism and improve prediction of the organism's properties based solely on its genome sequence.

The ethylene signal transduction pathway in Arabidopsis

NASA Technical Reports Server (NTRS)

Kieber, J. J.; Evans, M. L. (Principal Investigator)

1997-01-01

The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.

Aspects of eukaryotic-like signaling in Gram-positive cocci: a focus on virulence

PubMed Central

Burnside, Kellie; Rajagopal, Lakshmi

2011-01-01

Living organisms adapt to the dynamic external environment for their survival. Environmental adaptation in prokaryotes is thought to be primarily accomplished by signaling events mediated by two-component systems, consisting of histidine kinases and response regulators. However, eukaryotic-like serine/threonine kinases (STKs) have recently been described to regulate growth, antibiotic resistance and virulence of pathogenic bacteria. This article summarizes the role of STKs and their cognate phosphatases (STPs) in Gram-positive cocci that cause invasive infections in humans. Given that a large number of inhibitors to eukaryotic STKs are approved for use in humans, understanding how serine/threonine phosphorylation regulates virulence and antibiotic resistance will be beneficial for the development of novel therapeutic strategies against bacterial infections. PMID:21797690

Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor

PubMed Central

Bonomi, Hernán R.; Posadas, Diana M.; Paris, Gastón; Carrica, Mariela del Carmen; Frederickson, Marcus; Pietrasanta, Lía Isabel; Bogomolni, Roberto A.; Zorreguieta, Angeles; Goldbaum, Fernando A.

2012-01-01

Rhizobium leguminosarum is a soil bacterium that infects root hairs and induces the formation of nitrogen-fixing nodules on leguminous plants. Light, oxygen, and voltage (LOV)-domain proteins are blue-light receptors found in higher plants and many algae, fungi, and bacteria. The genome of R. leguminosarum bv. viciae 3841, a pea-nodulating endosymbiont, encodes a sensor histidine kinase containing a LOV domain at the N-terminal end (R-LOV-HK). R-LOV-HK has a typical LOV domain absorption spectrum with broad bands in the blue and UV-A regions and shows a truncated photocycle. Here we show that the R-LOV-HK protein regulates attachment to an abiotic surface and production of flagellar proteins and exopolysaccharide in response to light. Also, illumination of bacterial cultures before inoculation of pea roots increases the number of nodules per plant and the number of intranodular bacteroids. The effects of light on nodulation are dependent on a functional lov gene. The results presented in this work suggest that light, sensed by R-LOV-HK, is an important environmental factor that controls adaptive responses and the symbiotic efficiency of R. leguminosarum. PMID:22773814

The identification of four histidine kinases that influence sporulation in Clostridium thermocellum.

PubMed

Mearls, Elizabeth B; Lynd, Lee R

2014-08-01

In this study, we sought to identify genes involved in the onset of spore formation in Clostridium thermocellum via targeted gene deletions, gene over-expression, and transcriptional analysis. We determined that three putative histidine kinases, clo1313_0286, clo1313_2735 and clo1313_1942 were positive regulators of sporulation, while a fourth kinase, clo1313_1973, acted as a negative regulator. Unlike Bacillus or other Clostridium species, the deletion of a single positively regulating kinase was sufficient to abolish sporulation in this organism. Sporulation could be restored in these asporogenous strains via overexpression of any one of the positive regulators, indicating a high level of redundancy between these kinases. In addition to having a sporulation defect, deletion of clo1313_2735 produced L-forms. Thus, this kinase may play an additional role in repressing L-form formation. This work suggests that C. thermocellum enters non-growth states based on the sensory input from multiple histidine kinases. The ability to control the development of non-growth states at the genetic level has the potential to inform strategies for improved strain development, as well as provide valuable insight into C. thermocellum biology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

NASA Astrophysics Data System (ADS)

Gourley, Christopher R.

The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

PubMed Central

Biteen, Julie S.; Moerner, W.E.

2010-01-01

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells. PMID:20300204

Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B.

PubMed

Rao, Minxi; Smith, Brian C; Marletta, Michael A

2015-05-05

Nitric oxide (NO) plays an important signaling role in all domains of life. Many bacteria contain a heme-nitric oxide/oxygen binding (H-NOX) protein that selectively binds NO. These H-NOX proteins often act as sensors that regulate histidine kinase (HK) activity, forming part of a bacterial two-component signaling system that also involves one or more response regulators. In several organisms, NO binding to the H-NOX protein governs bacterial biofilm formation; however, the source of NO exposure for these bacteria is unknown. In mammals, NO is generated by the enzyme nitric oxide synthase (NOS) and signals through binding the H-NOX domain of soluble guanylate cyclase. Recently, several bacterial NOS proteins have also been reported, but the corresponding bacteria do not also encode an H-NOX protein. Here, we report the first characterization of a bacterium that encodes both a NOS and H-NOX, thus resembling the mammalian system capable of both synthesizing and sensing NO. We characterized the NO signaling pathway of the marine alphaproteobacterium Silicibacter sp. strain TrichCH4B, determining that the NOS is activated by an algal symbiont, Trichodesmium erythraeum. NO signaling through a histidine kinase-response regulator two-component signaling pathway results in increased concentrations of cyclic diguanosine monophosphate, a key bacterial second messenger molecule that controls cellular adhesion and biofilm formation. Silicibacter sp. TrichCH4B biofilm formation, activated by T. erythraeum, may be an important mechanism for symbiosis between the two organisms, revealing that NO plays a previously unknown key role in bacterial communication and symbiosis. Bacterial nitric oxide (NO) signaling via heme-nitric oxide/oxygen binding (H-NOX) proteins regulates biofilm formation, playing an important role in protecting bacteria from oxidative stress and other environmental stresses. Biofilms are also an important part of symbiosis, allowing the organism to remain in a nutrient-rich environment. In this study, we show that in Silicibacter sp. strain TrichCH4B, NO mediates symbiosis with the alga Trichodesmium erythraeum, a major marine diazotroph. In addition, Silicibacter sp. TrichCH4B is the first characterized bacteria to harbor both the NOS and H-NOX proteins, making it uniquely capable of both synthesizing and sensing NO, analogous to mammalian NO signaling. Our study expands current understanding of the role of NO in bacterial signaling, providing a novel role for NO in bacterial communication and symbiosis. Copyright © 2015 Rao et al.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

PubMed

Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

2016-03-27

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling

PubMed Central

Podgornaia, Anna I.; Casino, Patricia; Marina, Alberto; Laub, Michael T.

2013-01-01

Summary Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, E. coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes, along with a systematic mutagenesis study, reveals how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions, protein evolution, and the design of novel protein interfaces. PMID:23954504

Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System

PubMed Central

Liu, Baoquan; Wang, Jianfeng; Xiong, Wen; Zhao, Pengchao; Fan, Shengdi

2013-01-01

The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system. PMID:24303011

The protein histidine phosphatase LHPP is a tumour suppressor.

PubMed

Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

2018-03-29

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

A Rhizobium radiobacter Histidine Kinase Can Employ Both Boolean AND and OR Logic Gates to Initiate Pathogenesis.

PubMed

Fang, Fang; Lin, Yi-Han; Pierce, B Daniel; Lynn, David G

2015-10-12

The molecular logic gates that regulate gene circuits are necessarily intricate and highly regulated, particularly in the critical commitments necessary for pathogenesis. We now report simple AND and OR logic gates to be accessible within a single protein receptor. Pathogenesis by the bacterium Rhizobium radiobacter is mediated by a single histidine kinase, VirA, which processes multiple small molecule host signals (phenol and sugar). Mutagenesis analyses converged on a single signal integration node, and finer functional analyses revealed that a single residue could switch VirA from a functional AND logic gate to an OR gate where each of two signals activate independently. Host range preferences among natural strains of R. radiobacter correlate with these gate logic strategies. Although the precise mechanism for the signal integration node requires further analyses, long-range signal transmission through this histidine kinase can now be exploited for synthetic signaling circuits. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Z.; Hendrickson, W

2010-01-01

Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of themore » most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.« less

Domain Shuffling in a Sensor Protein Contributed to the Evolution of Insect Pathogenicity in Plant-Beneficial Pseudomonas protegens

PubMed Central

Kupferschmied, Peter; Péchy-Tarr, Maria; Imperiali, Nicola; Maurhofer, Monika; Keel, Christoph

2014-01-01

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity. PMID:24586167

Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation

DOE PAGES

Rivera-Cancel, Giomar; Ko, Wen-huang; Tomchick, Diana R.; ...

2014-12-02

Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. In this paper, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light–oxygen–voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain.more » The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. Finally, we suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.« less

HBCU Equipment for AFOSR Project 13RSL012: The Mechanism by which ADP Regulates the Structure and Function of the Protein KaiC

DTIC Science & Technology

2015-05-18

4 , as well as increases the risk of obesity 5-7 , diabetes 8, 9 , heart disease 10 , and cancer 11, 12 . Our lab studies the circadian clock of a...2013) Two Antagonistic Clock-Regulated Histidine Kinases Time the Activation of Circadian Gene Expression. Mol. Cell 50, 288-294. 10.1016/j.molcel...Circadian Clock-associated Histidine Kinase SasA. J. Mol. Biol. 342, 9-17. 10.1016/j.jmb.2004.07.010. 19. Smith R. M., Williams S. B. (2006) Circadian

Backbone chemical shift assignments for the sensor domain of the Burkholderia pseudomallei histidine kinase RisS: "missing" resonances at the dimer interface.

PubMed

Buchko, Garry W; Edwards, Thomas E; Hewitt, Stephen N; Phan, Isabelle Q H; Van Voorhis, Wesley C; Miller, Samuel I; Myler, Peter J

2015-10-01

Using a deuterated sample, all the observable backbone (1)H(N), (15)N, (13)C(a), and (13)C' chemical shifts for the dimeric, periplasmic sensor domain of the Burkholderia pseudomallei histidine kinase RisS were assigned. Approximately one-fifth of the amide resonances are "missing" in the (1)H-(15)N HSQC spectrum and map primarily onto α-helices at the dimer interface observed in a crystal structure suggesting this region either undergoes intermediate timescale motion (μs-ms) and/or is heterogeneous.

Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans.

PubMed

Devi, Seram Nganbiton; Kiehler, Brittany; Haggett, Lindsey; Fujita, Masaya

2015-08-01

Entry into sporulation in Bacillus subtilis is governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur in trans. To test this hypothesis, we generated a series of B. subtilis strains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in a trans fashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressed in vivo. Taken together, these in vitro and in vivo results reinforce the evidence that KinA autophosphorylation is able to occur in a trans fashion. Autophosphorylation of histidine kinases is known to occur by either the cis (one subunit of kinase phosphorylating itself within the multimer) or the trans (one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided direct in vivo and in vitro evidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur in trans within the homotetramer complex. While the physiological and mechanistic significance of the trans autophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of the RcsC sensor kinase from Erwinia amylovora and other enterobacteria

USDA-ARS?s Scientific Manuscript database

RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain and a receiver domain. We have previously demonstrated that, while the Erwinia amylovora rcsC mutant produces more amylovoran than the wild type strain in vitro, the mutant remains avirulent on both immature pea...

A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

PubMed Central

2018-01-01

ABSTRACT The alphaproteobacterial general stress response is governed by a conserved partner-switching mechanism that is triggered by phosphorylation of the response regulator PhyR. In the model organism Caulobacter crescentus, PhyR was proposed to be phosphorylated by the histidine kinase PhyK, but biochemical evidence in support of such a role of PhyK is missing. Here, we identify a single-domain response regulator, MrrA, that is essential for general stress response activation in C. crescentus. We demonstrate that PhyK does not function as a kinase but accepts phosphoryl groups from MrrA and passes them on to PhyR, adopting the role of a histidine phosphotransferase. MrrA is phosphorylated by at least six histidine kinases that likely serve as stress sensors. MrrA also transfers phosphate to LovK, a histidine kinase involved in C. crescentus holdfast production and attachment, which also negatively regulates the general stress response. We show that LovK together with the response regulator LovR acts as a phosphate sink to redirect phosphate flux away from the PhyKR branch. In agreement with the biochemical data, an mrrA mutant is unable to activate the general stress response and shows a hyperattachment phenotype, which is linked to decreased expression of the major holdfast inhibitory protein HfiA. We propose that MrrA serves as a central phosphorylation hub that coordinates the general stress response with C. crescentus development and other adaptive behaviors. The characteristic bow-tie architecture of this phosphorylation network with MrrA as the central knot may expedite the evolvability and species-specific niche adaptation of this group of bacteria. PMID:29789370

Crystal Structures of Apparent Saccharide Sensors from Histidine Kinase Receptors Prevalent in a Human Gut Symbiont

PubMed Central

Zhang, Zhen; Liu, Qun; Hendrickson, Wayne A.

2014-01-01

The adult human gut presents a complicated ecosystem where host-bacterium symbiosis plays an important role. Bacteroides thetaiotaomicron is a predominant member of the gut microflora, providing the human digestive tract with a large number of glycolytic enzymes. Expression of many of these enzymes appears to be controlled by histidine kinase receptors that are fused into unusual hybrid two-component systems that share homologous periplasmic sensor domains. These sensor domains belong to the third most populated (HK3) family based on a previous bioinformatics analysis of predicted histidine kinase sensors. Here, we present crystal structures of two sensor domains representative of the HK3 family. Each sensor is folded into three domains: two seven-bladed β-propeller domains and one β-sandwich domain. Both sensors form dimers in crystals and one sensor appears to be physiologically relevant. The folding characteristics in the individual domains, the domain organization, and the oligomeric architecture are all unique to the HK3 sensors. The sequence analysis of the HK3 sensors indicates that these sensors are shared among other signaling molecules, implying a combinatorial molecular evolution. PMID:24995510

Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

PubMed

Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

2016-01-01

Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

Prokaryotic 2-component systems and the OmpR/PhoB superfamily.

PubMed

Nguyen, Minh-Phuong; Yoon, Joo-Mi; Cho, Man-Ho; Lee, Sang-Won

2015-11-01

In bacteria, 2-component regulatory systems (TCSs) are the critical information-processing pathways that link stimuli to specific adaptive responses. Signals perceived by membrane sensors, which are generally histidine kinases, are transmitted by response regulators (RRs) to allow cells to cope rapidly and effectively with environmental challenges. Over the past few decades, genes encoding components of TCSs and their responsive proteins have been identified, crystal structures have been described, and signaling mechanisms have been elucidated. Here, we review recent findings and interesting breakthroughs in bacterial TCS research. Furthermore, we discuss structural features, mechanisms of activation and regulation, and cross-regulation of RRs, with a focus on the largest RR family, OmpR/PhoB, to provide a comprehensive overview of these critically important signaling molecules.

Light Regulation of Swarming Motility in Pseudomonas syringae Integrates Signaling Pathways Mediated by a Bacteriophytochrome and a LOV Protein

PubMed Central

Wu, Liang; McGrane, Regina S.; Beattie, Gwyn A.

2013-01-01

ABSTRACT The biological and regulatory roles of photosensory proteins are poorly understood for nonphotosynthetic bacteria. The foliar bacterial pathogen Pseudomonas syringae has three photosensory protein-encoding genes that are predicted to encode the blue-light-sensing LOV (light, oxygen, or voltage) histidine kinase (LOV-HK) and two red/far-red-light-sensing bacteriophytochromes, BphP1 and BphP2. We provide evidence that LOV-HK and BphP1 form an integrated network that regulates swarming motility in response to multiple light wavelengths. The swarming motility of P. syringae B728a deletion mutants indicated that LOV-HK positively regulates swarming motility in response to blue light and BphP1 negatively regulates swarming motility in response to red and far-red light. BphP2 does not detectably regulate swarming motility. The histidine kinase activity of each LOV-HK and BphP1 is required for this regulation based on the loss of complementation upon mutation of residues key to their kinase activity. Surprisingly, mutants lacking both lov and bphP1 were similar in motility to a bphP1 single mutant in blue light, indicating that the loss of bphP1 is epistatic to the loss of lov and also that BphP1 unexpectedly responds to blue light. Moreover, whereas expression of bphP1 did not alter motility under blue light in a bphP1 mutant, it reduced motility in a mutant lacking lov and bphP1, demonstrating that LOV-HK positively regulates motility by suppressing negative regulation by BphP1. These results are the first to show cross talk between the LOV protein and phytochrome signaling pathways in bacteria, and the similarity of this regulatory network to that of photoreceptors in plants suggests a possible common ancestry. PMID:23760465

Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus.

PubMed

Shank, Elizabeth A; Klepac-Ceraj, Vanja; Collado-Torres, Leonardo; Powers, Gordon E; Losick, Richard; Kolter, Roberto

2011-11-29

Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis--the soil--were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain's relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling

PubMed Central

Schaap, Pauline; Barrantes, Israel; Minx, Pat; Sasaki, Narie; Anderson, Roger W.; Bénard, Marianne; Biggar, Kyle K.; Buchler, Nicolas E.; Bundschuh, Ralf; Chen, Xiao; Fronick, Catrina; Fulton, Lucinda; Golderer, Georg; Jahn, Niels; Knoop, Volker; Landweber, Laura F.; Maric, Chrystelle; Miller, Dennis; Noegel, Angelika A.; Peace, Rob; Pierron, Gérard; Sasaki, Taeko; Schallenberg-Rüdinger, Mareike; Schleicher, Michael; Singh, Reema; Spaller, Thomas; Storey, Kenneth B.; Suzuki, Takamasa; Tomlinson, Chad; Tyson, John J.; Warren, Wesley C.; Werner, Ernst R.; Werner-Felmayer, Gabriele; Wilson, Richard K.; Winckler, Thomas; Gott, Jonatha M.; Glöckner, Gernot; Marwan, Wolfgang

2016-01-01

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer. PMID:26615215

A Bacillus subtilis Sensor Kinase Involved in Triggering Biofilm Formation on the Roots of Tomato Plants

PubMed Central

Chen, Yun; Cao, Shugeng; Chai, Yunrong; Clardy, Jon; Kolter, Roberto; Guo, Jian-hua; Losick, Richard

2012-01-01

SUMMARY The soil bacterium Bacillus subtilis is widely used in agriculture as a biocontrol agent able to protect plants from a variety of pathogens. Protection is thought to involve the formation of bacterial communities - biofilms - on the roots of the plants. Here we used confocal microscopy to visualize biofilms on the surface of the roots of tomato seedlings and demonstrated that biofilm formation requires genes governing the production of the extracellular matrix that holds cells together. We further show that biofilm formation was dependent on the sensor histidine kinase KinD and in particular on an extracellular CACHE domain implicated in small molecule sensing. Finally, we report that exudates of tomato roots strongly stimulated biofilm formation ex planta and that an abundant small molecule in the exudates, l-malic acid, was able to stimulate biofilm formation at high concentrations in a manner that depended on the KinD CACHE domain. We propose that small signaling molecules released by the roots of tomato plants are directly or indirectly recognized by KinD, triggering biofilm formation. PMID:22716461

Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway

PubMed Central

Espinosa, Leon; Baronian, Grégory; Molle, Virginie; Mauriello, Emilia M. F.; Brochier-Armanet, Céline; Mignot, Tâm

2015-01-01

Understanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A- and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways. PMID:26291327

Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II.

PubMed

Lu, Huasong; Yu, Dan; Hansen, Anders S; Ganguly, Sourav; Liu, Rongdiao; Heckert, Alec; Darzacq, Xavier; Zhou, Qiang

2018-06-01

Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing 1-3 . The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation 1,4,5 . The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A 6 -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH 1-3 . By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis

PubMed Central

de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Hölscher, Theresa; Kuipers, Oscar P.

2015-01-01

ABSTRACT Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. PMID:26152584

Phosphate Sink Containing Two-Component Signaling Systems as Tunable Threshold Devices

PubMed Central

Amin, Munia; Kothamachu, Varun B.; Feliu, Elisenda; Scharf, Birgit E.; Porter, Steven L.; Soyer, Orkun S.

2014-01-01

Synthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications. PMID:25357192

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

PubMed

Grau, Roberto R; de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Donato, Verónica; Hölscher, Theresa; Boland, Wilhelm; Kuipers, Oscar P; Kovács, Ákos T

2015-07-07

Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective decision to move over or stay attached to a surface controlled? Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question. Remarkably, we discover that sessile biofilm formation and social sliding motility share the same structural components and the Spo0A regulatory network via sensor kinases, KinB and KinC. Potassium, an inhibitor of KinC-dependent biofilm formation, triggers sliding via a potassium-perceiving cytosolic domain of KinB that resembles the selectivity filter of potassium channels. The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness. Copyright © 2015 Grau et al.

Structural and Biochemical Studies of TIGAR (TP53-induced Glycolysis and Apoptosis Regulator)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, H.; Jogl, G

2009-01-01

Activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. TIGAR is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. Here we describe structural and biochemical studies of TIGAR from Danio rerio. The overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalytic histidine residue and a second phosphate molecule in a position not observed in other phosphatases. The recombinant human and zebra fish enzymes hydrolyze fructose-2,6-bisphosphate as well as fructose-1,6-bisphosphate but notmore » fructose 6-phosphate in vitro. The TIGAR active site is open and positively charged, consistent with its enzymatic function as bisphosphatase. The closest related structures are the bacterial broad specificity phosphatase PhoE and the fructose-2,6-bisphosphatase domain of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The structural comparison shows that TIGAR combines an accessible active site as observed in PhoE with a charged substrate-binding pocket as seen in the fructose-2,6-bisphosphatase domain of the bifunctional enzyme.« less

Global regulation by the seven-component Pi signaling system.

PubMed

Hsieh, Yi-Ju; Wanner, Barry L

2010-04-01

This review concerns how Escherichia coli detects environmental inorganic orthophosphate (P(i)) to regulate genes of the phosphate (Pho) regulon by the PhoR/PhoB two-component system (TCS). P(i) control by the PhoR/PhoB TCS is a paradigm of a bacterial signal transduction pathway in which occupancy of a cell surface receptor(s) controls gene expression in the cytoplasm. The P(i) signaling pathway requires seven proteins, all of which probably interact in a membrane-associated signaling complex. Our latest studies show that P(i) signaling involves three distinct processes, which appear to correspond to different states of the sensory histidine kinase PhoR: an inhibition state, an activation state, and a deactivation state. We describe a revised model for P(i) signal transduction of the E. coli Pho regulon. Copyright 2010 Elsevier Ltd. All rights reserved.

The two-component signal system in rice (Oryza sativa L.): a genome-wide study of cytokinin signal perception and transduction.

PubMed

Du, Liming; Jiao, Fangchan; Chu, Jun; Jin, Gulei; Chen, Ming; Wu, Ping

2007-06-01

In this report we define the genes of two-component regulatory systems in rice through a comprehensive computational analysis of rice (Oryza sativa L.) genome sequence databases. Thirty-seven genes were identified, including 5 HKs (cytokinin-response histidine protein kinase) (OsHK1-4, OsHKL1), 5 HPs (histidine phosphotransfer proteins) (OsHP1-5), 15 type-A RRs (response regulators) (OsRR1-15), 7 type B RR genes (OsRR16-22), and 5 predicted pseudo-response regulators (OsPRR1-5). Protein motif organization, gene structure, phylogenetic analysis, chromosomal location, and comparative analysis between rice, maize, and Arabidopsis are described. Full-length cDNA clones of each gene were isolated from rice. Heterologous expression of each of the OsHKs in yeast mutants conferred histidine kinase function in a cytokinin-dependent manner. Nonconserved regions of individual cDNAs were used as probes in expression profiling experiments. This work provides a foundation for future functional dissection of the rice cytokinin two-component signaling pathway.

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

PubMed Central

Pickl, Andreas; Johnsen, Ulrike

2012-01-01

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidine-rich peptides at acidic pH.

PubMed

Mason, A James; Gasnier, Claire; Kichler, Antoine; Prévost, Gilles; Aunis, Dominique; Metz-Boutigue, Marie-Hélène; Bechinger, Burkhard

2006-10-01

The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state (2)H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.

A fungicide-responsive kinase as a tool for synthetic cell fate regulation.

PubMed

Furukawa, Kentaro; Hohmann, Stefan

2015-08-18

Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic 'suicide attack' system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs

PubMed Central

Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; Van Voorhis, Wesley C; Hol, Wim G J

2010-01-01

Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active site rearrangement upon histidine binding, but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme’s first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human histidyl-tRNA synthetases. The essentiality of the single histidyl-tRNA synthetase gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference. PMID:20132829

Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

PubMed Central

Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

2016-01-01

KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001 PMID:27542194

Functional Dissection of the CroRS Two-Component System Required for Resistance to Cell Wall Stressors in Enterococcus faecalis.

PubMed

Kellogg, Stephanie L; Kristich, Christopher J

2016-04-01

Bacteria use two-component signal transduction systems (TCSs) to sense and respond to environmental changes via a conserved phosphorelay between a sensor histidine kinase and its cognate response regulator. The opportunistic pathogen Enterococcus faecalis utilizes a TCS comprised of the histidine kinase CroS and the response regulator CroR to mediate resistance to cell wall stresses such as cephalosporin antibiotics, but the molecular details by which CroRS promotes cephalosporin resistance have not been elucidated. Here, we analyzed mutants of E. faecalis carrying substitutions in CroR and CroS to demonstrate that phosphorylated CroR drives resistance to cephalosporins, and that CroS exhibits kinase and phosphatase activities to control the level of CroR phosphorylation in vivo. Deletion of croS in various lineages of E. faecalis revealed a CroS-independent mechanism for CroR phosphorylation and led to the identification of a noncognate histidine kinase capable of influencing CroR (encoded by OG1RF_12162; here called cisS). Further analysis of this TCS network revealed that both systems respond to cell wall stress. TCSs allow bacteria to sense and respond to many different environmental conditions. The opportunistic pathogen Enterococcus faecalis utilizes the CroRS TCS to mediate resistance to cell wall stresses, including clinically relevant antibiotics such as cephalosporins and glycopeptides. In this study, we use genetic and biochemical means to investigate the relationship between CroRS signaling and cephalosporin resistance in E. faecalis cells. Through this, we uncovered a signaling network formed between the CroRS TCS and a previously uncharacterized TCS that also responds to cell wall stress. This study provides mechanistic insights into CroRS signaling and cephalosporin resistance in E. faecalis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

PHYTOCHOME STRUCTURE AND SIGNALING MECHANISMS

PubMed Central

Rockwell, Nathan C.; Su, Yi-Shin; Lagarias, J. Clark

2009-01-01

Phytochromes are a widespread family of red/far-red responsive photoreceptors first discovered in plants, where they constitute one of the three main classes of photomorphogenesis regulators. All phytochromes utilize covalently attached bilin chromophores that enable photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) forms. Phytochromes are thus photoswitchable photosensors; canonical phytochromes have a conserved N-terminal photosensory core and a C-terminal regulatory region which typically includes a histidine-kinase-related domain. The discovery of new bacterial and cyanobacterial members of the phytochrome family within the last decade has greatly aided biochemical and structural characterization of this family, with the first crystal structure of a bacteriophytochrome photosensory core appearing in 2005. This structure and other recent biochemical studies have provided exciting new insights into the structure of phytochrome, the photoconversion process that is central to light sensing, and the mechanism of signal transfer by this important family of photoreceptors. PMID:16669784

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae

PubMed Central

Band, Victor I.; Crispell, Emily K.; Napier, Brooke A.; Herrera, Carmen M.; Tharp, Greg K.; Vavikolanu, Kranthi; Pohl, Jan; Read, Timothy D.; Bosinger, Steven E.; Trent, M. Stephen; Burd, Eileen M.; Weiss, David S.

2016-01-01

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics. PMID:27572838

On the origin of fluorescence in bacteriophytochrome infrared fluorescent proteins

PubMed Central

Samma, Alex A.; Johnson, Chelsea K.; Song, Shuang; Alvarez, Samuel

2010-01-01

Tsien (Science, 2009, 324, 804-807) has recently reported the creation of the first infrared fluorescent protein (IFP). It was engineered from bacterial phytochrome by removing the PHY and histidine kinase-related domains, by optimizing the protein to prevent dimerization and by limiting the biliverdins conformational freedom, especially around its D ring. We have used database analyses and molecular dynamics simulations with freely rotating chromophoric dihedrals in order to model the dihedral freedom available to the biliverdin D ring in the excited state; to show that the tetrapyrrole ligands in phytochromes are flexible and can adopt many conformations, however their conformational space is limited/defined by the chemospatial characteristics of the protein cavity. Our simulations confirm that the reduced accessibility to conformations geared to an excited state proton transfer may be responsible for the fluorescence in IFP, just as has been suggested by Kennis (PNAS, 2010, 107, 9170-9175) for fluorescent bacteriophytochrome from Rhodopseudomonas palustris. PMID:21047084

Convergence of PASTA kinase and two-component signaling in response to cell wall stress in Enterococcus faecalis.

PubMed

Kellogg, Stephanie L; Kristich, Christopher J

2018-04-09

Two common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryotic-like Ser/Thr kinases and phosphatases (eSTK/Ps). Enterococcus faecalis is a Gram-positive bacterium and serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics, leading to modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP), which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance towards cell wall-targeting antibiotics, we hypothesized these signaling systems are intertwined. To test this hypothesis, we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition, we analyzed the phosphorylation state of CroS which revealed IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through phosphorylation of CroS to promote antimicrobial resistance in E. faecalis Importance Two-component signaling systems (TCSs) and eukaryotic-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogen Enterococcus faecalis relies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK, revealing convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance in E. faecalis This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance in E. faecalis . Copyright © 2018 American Society for Microbiology.

Reductive evolution and the loss of PDC/PAS domains from the genus Staphylococcus

PubMed Central

2013-01-01

Background The Per-Arnt-Sim (PAS) domain represents a ubiquitous structural fold that is involved in bacterial sensing and adaptation systems, including several virulence related functions. Although PAS domains and the subclass of PhoQ-DcuS-CitA (PDC) domains have a common structure, there is limited amino acid sequence similarity. To gain greater insight into the evolution of PDC/PAS domains present in the bacterial kingdom and staphylococci in specific, the PDC/PAS domains from the genomic sequences of 48 bacteria, representing 5 phyla, were identified using the sensitive search method based on HMM-to-HMM comparisons (HHblits). Results A total of 1,007 PAS domains and 686 PDC domains distributed over 1,174 proteins were identified. For 28 Gram-positive bacteria, the distribution, organization, and molecular evolution of PDC/PAS domains were analyzed in greater detail, with a special emphasis on the genus Staphylococcus. Compared to other bacteria the staphylococci have relatively fewer proteins (6–9) containing PDC/PAS domains. As a general rule, the staphylococcal genomes examined in this study contain a core group of seven PDC/PAS domain-containing proteins consisting of WalK, SrrB, PhoR, ArlS, HssS, NreB, and GdpP. The exceptions to this rule are: 1) S. saprophyticus lacks the core NreB protein; 2) S. carnosus has two additional PAS domain containing proteins; 3) S. epidermidis, S. aureus, and S. pseudintermedius have an additional protein with two PDC domains that is predicted to code for a sensor histidine kinase; 4) S. lugdunensis has an additional PDC containing protein predicted to be a sensor histidine kinase. Conclusions This comprehensive analysis demonstrates that variation in PDC/PAS domains among bacteria has limited correlations to the genome size or pathogenicity; however, our analysis established that bacteria having a motile phase in their life cycle have significantly more PDC/PAS-containing proteins. In addition, our analysis revealed a tremendous amount of variation in the number of PDC/PAS-containing proteins within genera. This variation extended to the Staphylococcus genus, which had between 6 and 9 PDC/PAS proteins and some of these appear to be previously undescribed signaling proteins. This latter point is important because most staphylococcal proteins that contain PDC/PAS domains regulate virulence factor synthesis or antibiotic resistance. PMID:23902280

Reductive evolution and the loss of PDC/PAS domains from the genus Staphylococcus.

PubMed

Shah, Neethu; Gaupp, Rosmarie; Moriyama, Hideaki; Eskridge, Kent M; Moriyama, Etsuko N; Somerville, Greg A

2013-07-31

The Per-Arnt-Sim (PAS) domain represents a ubiquitous structural fold that is involved in bacterial sensing and adaptation systems, including several virulence related functions. Although PAS domains and the subclass of PhoQ-DcuS-CitA (PDC) domains have a common structure, there is limited amino acid sequence similarity. To gain greater insight into the evolution of PDC/PAS domains present in the bacterial kingdom and staphylococci in specific, the PDC/PAS domains from the genomic sequences of 48 bacteria, representing 5 phyla, were identified using the sensitive search method based on HMM-to-HMM comparisons (HHblits). A total of 1,007 PAS domains and 686 PDC domains distributed over 1,174 proteins were identified. For 28 Gram-positive bacteria, the distribution, organization, and molecular evolution of PDC/PAS domains were analyzed in greater detail, with a special emphasis on the genus Staphylococcus. Compared to other bacteria the staphylococci have relatively fewer proteins (6-9) containing PDC/PAS domains. As a general rule, the staphylococcal genomes examined in this study contain a core group of seven PDC/PAS domain-containing proteins consisting of WalK, SrrB, PhoR, ArlS, HssS, NreB, and GdpP. The exceptions to this rule are: 1) S. saprophyticus lacks the core NreB protein; 2) S. carnosus has two additional PAS domain containing proteins; 3) S. epidermidis, S. aureus, and S. pseudintermedius have an additional protein with two PDC domains that is predicted to code for a sensor histidine kinase; 4) S. lugdunensis has an additional PDC containing protein predicted to be a sensor histidine kinase. This comprehensive analysis demonstrates that variation in PDC/PAS domains among bacteria has limited correlations to the genome size or pathogenicity; however, our analysis established that bacteria having a motile phase in their life cycle have significantly more PDC/PAS-containing proteins. In addition, our analysis revealed a tremendous amount of variation in the number of PDC/PAS-containing proteins within genera. This variation extended to the Staphylococcus genus, which had between 6 and 9 PDC/PAS proteins and some of these appear to be previously undescribed signaling proteins. This latter point is important because most staphylococcal proteins that contain PDC/PAS domains regulate virulence factor synthesis or antibiotic resistance.

Two genes with similarity to bacterial response regulators are rapidly and specifically induced by cytokinin in Arabidopsis

NASA Technical Reports Server (NTRS)

Brandstatter, I.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

1998-01-01

Cytokinins are central regulators of plant growth and development, but little is known about their mode of action. By using differential display, we identified a gene, IBC6 (for induced by cytokinin), from etiolated Arabidopsis seedlings, that is induced rapidly by cytokinin. The steady state level of IBC6 mRNA was elevated within 10 min by the exogenous application of cytokinin, and this induction did not require de novo protein synthesis. IBC6 was not induced by other plant hormones or by light. A second Arabidopsis gene with a sequence highly similar to IBC6 was identified. This IBC7 gene also was induced by cytokinin, although with somewhat slower kinetics and to a lesser extent. The pattern of expression of the two genes was similar, with higher expression in leaves, rachises, and flowers and lower transcript levels in roots and siliques. Sequence analysis revealed that IBC6 and IBC7 are similar to the receiver domain of bacterial two-component response regulators. This homology, coupled with previously published work on the CKI1 histidine kinase homolog, suggests that these proteins may play a role in early cytokinin signaling.

A Novel Sensor Kinase Is Required for Bordetella bronchiseptica To Colonize the Lower Respiratory Tract▿

PubMed Central

Kaut, Callan S.; Duncan, Mark D.; Kim, Ji Yei; Maclaren, Joshua J.; Cochran, Keith T.; Julio, Steven M.

2011-01-01

Bacterial virulence is influenced by the activity of two-component regulator systems (TCSs), which consist of membrane-bound sensor kinases that allow bacteria to sense the external environment and cytoplasmic, DNA-binding response regulator proteins that control appropriate gene expression. Respiratory pathogens of the Bordetella genus require the well-studied TCS BvgAS to control the expression of many genes required for colonization of the mammalian respiratory tract. Here we describe the identification of a novel gene in Bordetella bronchiseptica, plrS, the product of which shares sequence homology to several NtrY-family sensor kinases and is required for B. bronchiseptica to colonize and persist in the lower, but not upper, respiratory tract in rats and mice. The plrS gene is located immediately 5′ to and presumably cotranscribed with a gene encoding a putative response regulator, supporting the idea that PlrS and the product of the downstream gene may compose a TCS. Consistent with this hypothesis, the PlrS-dependent colonization phenotype requires a conserved histidine that serves as the site of autophosphorylation in other sensor kinases, and in strains lacking plrS, the production and/or cellular localization of several immune-recognized proteins is altered in comparison to that in the wild-type strain. Because plrS is required for colonization and persistence only in the lower respiratory tract, a site where innate and adaptive immune mechanisms actively target infectious agents, we hypothesize that its role may be to allow Bordetella to resist the host immune response. PMID:21606184

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moore, J.; Hendrickson, W

Histidine kinase receptors respond to diverse signals and mediate signal transduction across the plasma membrane in all prokaryotes and certain eukaryotes. Each receptor is part of a two-component system that regulates a particular cellular process. Organisms that use trimethylamine-N-oxide (TMAO) as a terminal electron acceptor typically control their anaerobic respiration through the TMAO reductase (Tor) pathway, which the TorS histidine kinase activates when sensing TMAO in the environment. We have determined crystal structures for the periplasmic sensor domains of TorS receptors from Escherichia coli and Vibrio parahaemolyticus. TorS sensor domains have a novel fold consisting of a membrane-proximal right-handed four-helicalmore » bundle and a membrane-distal left-handed four-helical bundle, but conformational dispositions differ significantly in the two structures. Isolated TorS sensor domains dimerize in solution; and from comparisons with dimeric NarX and Tar sensors, we postulate that signaling through TorS dimers involves a piston-type displacement between helices.« less

Phosphate Flow between Hybrid Histidine Kinases CheA3 and CheS3 Controls Rhodospirillum centenum Cyst Formation

PubMed Central

He, Kuang; Marden, Jeremiah N.; Quardokus, Ellen M.; Bauer, Carl E.

2013-01-01

Genomic and genetic analyses have demonstrated that many species contain multiple chemotaxis-like signal transduction cascades that likely control processes other than chemotaxis. The Che3 signal transduction cascade from Rhodospirillum centenum is one such example that regulates development of dormant cysts. This Che-like cascade contains two hybrid response regulator-histidine kinases, CheA3 and CheS3, and a single-domain response regulator CheY3. We demonstrate that cheS3 is epistatic to cheA3 and that only CheS3∼P can phosphorylate CheY3. We further show that CheA3 derepresses cyst formation by phosphorylating a CheS3 receiver domain. These results demonstrate that the flow of phosphate as defined by the paradigm E. coli chemotaxis cascade does not necessarily hold true for non-chemotactic Che-like signal transduction cascades. PMID:24367276

Light influences cytokinin biosynthesis and sensing in Nostoc (cyanobacteria).

PubMed

Frébortová, Jitka; Plíhal, Ondřej; Florová, Vendula; Kokáš, Filip; Kubiasová, Karolina; Greplová, Marta; Šimura, Jan; Novák, Ondřej; Frébort, Ivo

2017-06-01

Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation. © 2017 Phycological Society of America.

Arabidopsis Response Regulator1 and Arabidopsis Histidine Phosphotransfer Protein2 (AHP2), AHP3, and AHP5 Function in Cold Signaling1[W][OA

PubMed Central

Jeon, Jin; Kim, Jungmook

2013-01-01

The Arabidopsis (Arabidopsis thaliana) two-component signaling system, which is composed of sensor histidine kinases, histidine phosphotransfer proteins, and response regulators, mediates the cytokinin response and various other plant responses. We have previously shown that ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and cold-inducible type A ARABIDOPSIS RESPONSE REGULATORS (ARRs) play roles in cold signaling. However, the roles of type B ARRs and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHPs) have not been investigated in cold signaling. Here, we show that ARR1 and AHP2, AHP3, and AHP5 play positive roles in the cold-inducible expression of type A ARRs. arr1 mutants showed greatly reduced cold-responsive expression of type A ARRs compared with the wild type, whereas ARR1-overexpressing Arabidopsis exhibited the hypersensitive cold response of type A ARRs as well as enhanced freezing tolerance with cytokinin, suggesting that ARR1 functions as a positive factor of cold signaling. Transgenic Arabidopsis expressing ARR1ΔDDK:GR lacking the amino-terminal receiver domain showed wild-type expression levels of type A ARRs in response to cold, indicating that the signal receiver domain of ARR1 might be important for cold-responsive expression of type A ARRs. ahp2 ahp3 ahp5 triple mutations greatly reduced type A ARR expression in response to cold, whereas the single or double ahp mutants displayed wild-type levels of ARR expression, suggesting that AHP2, AHP3, and AHP5 are redundantly involved in cold signaling. Taken together, these results suggest that ARR1 mediates cold signal via AHP2, AHP3, or AHP5 from AHK2 and AHK3 to express type A ARRs. We further identified a cold transcriptome affected by ahk2 ahk3 mutations by microarray analysis, revealing a new cold-responsive gene network regulated downstream of AHK2 and AHK3. PMID:23124324

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Iain J.; Watkins, Russell F.; Samuelson, John

Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit adversity of environments.more » Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptorserine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.« less

Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities.

PubMed

Zgadzaj, Rafal; Garrido-Oter, Ruben; Jensen, Dorthe Bodker; Koprivova, Anna; Schulze-Lefert, Paul; Radutoiu, Simona

2016-12-06

Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance.

Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

PubMed Central

Zgadzaj, Rafal; Garrido-Oter, Ruben; Jensen, Dorthe Bodker; Koprivova, Anna; Schulze-Lefert, Paul; Radutoiu, Simona

2016-01-01

Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance. PMID:27864511

General Aspects of Two-Component Regulatory Circuits in Bacteria: Domains, Signals and Roles.

PubMed

Padilla-Vaca, Felipe; Mondragón-Jaimes, Verónica; Franco, Bernardo

2017-01-01

All living organisms are subject to changing environments, which must be sensed in order to respond swiftly and efficiently. Two-component systems (TCS) are signal transduction regulatory circuits based typically on a membrane bound sensor kinase and a cytoplasmic response regulator, that is activated through a histidine to aspartate phosphorelay reactions. Activated response regulator acts usually as a transcription factor. The best known examples were identified in bacteria, but they are also found in fungi, algae and plants. Thus far, they are not found in mammals. Regulatory circuits coupled to two-component systems exhibit a myriad of responses to environmental stimuli such as: redox potential, pH, specific metabolites, pressure, light and more recently to specific antimicrobial peptides that activate a sensor kinase responsible for expressing virulence factors through the active response regulator. In this review we explore general aspects on two-component systems that ultimately can play a role on virulence regulation, also the intriguing domain properties of the sensor kinases that can be a potential target for antimicrobial compounds. Only a handful of sensor kinases are extensively characterized, the vast majority belong to what we call 'the dark matter of bacterial signal transduction' since no known signal, structure and biochemical properties are available. Regulatory circuits from vertebrate pathogenic organisms can explain virulence in terms of either response to environmental factors or specific niche occupancy. Hopefully, knowledge on these signal transduction systems can lead to identify novel molecules that target two-component systems, since the increase of drug resistant microorganisms is worrisome. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mechanisms of High Temperature Resistance of Synechocystis sp. PCC 6803: An Impact of Histidine Kinase 34.

PubMed

Červený, Jan; Sinetova, Maria A; Zavřel, Tomáš; Los, Dmitry A

2015-03-02

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying responses and acclimation to different abiotic stresses. Changes in transcriptome, proteome, lipidome, and photosynthesis in response to short term heat stress are well studied in this organism, and histidine kinase 34 (Hik34) is shown to play an important role in mediating such response. Corresponding data on long term responses, however, are fragmentary and vary depending on parameters of experiments and methods of data collection, and thus are hard to compare. In order to elucidate how the early stress responses help cells to sustain long-term heat stress, as well as the role of Hik34 in prolonged acclimation, we examined the resistance to long-term heat stress of wild-type and ΔHik34 mutant of Synechocystis. In this work, we were able to precisely control the long term experimental conditions by cultivating Synechocystis in automated photobioreactors, measuring selected physiological parameters within a time range of minutes. In addition, morphological and ultrastructural changes in cells were analyzed and western blotting of individual proteins was used to study the heat stress-affected protein expression. We have shown that the majority of wild type cell population was able to recover after 24 h of cultivation at 44 °C. In contrast, while ΔHik34 mutant cells were resistant to heat stress within its first hours, they could not recover after 24 h long high temperature treatment. We demonstrated that the early induction of HspA expression and maintenance of high amount of other HSPs throughout the heat incubation is critical for successful adaptation to long-term stress. In addition, it appears that histidine kinase Hik34 is an essential component for the long term high temperature resistance.

Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

PubMed

Chourashi, Rhishita; Das, Suman; Dhar, Debarpan; Okamoto, Keinosuke; Mukhopadhyay, Asish K; Chatterjee, Nabendu Sekhar

2018-05-01

Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

Membrane fluidity controls redox-regulated cold stress responses in cyanobacteria.

PubMed

Maksimov, Eugene G; Mironov, Kirill S; Trofimova, Marina S; Nechaeva, Natalya L; Todorenko, Daria A; Klementiev, Konstantin E; Tsoraev, Georgy V; Tyutyaev, Eugene V; Zorina, Anna A; Feduraev, Pavel V; Allakhverdiev, Suleyman I; Paschenko, Vladimir Z; Los, Dmitry A

2017-09-01

Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.

The Evolution of Two-Component Signal Transduction Systems

PubMed Central

Capra, Emily J.; Laub, Michael T.

2014-01-01

To exist in a wide range of environmental niches, bacteria must sense and respond to a myriad of external signals. A primary means by which this occurs is through two-component signal transduction pathways, typically comprised of a histidine kinase that receives the input stimuli and a response regulator that effects an appropriate change in cellular physiology. Histidine kinases and response regulators have an intrinsic modularity that separates signal input, phosphotransfer, and output response; this modularity has allowed bacteria to dramatically expand and diversify their signaling capabilities. Recent work has begun to reveal the molecular basis by which two-component proteins evolve. How and why do orthologous signaling proteins diverge? How do cells gain new pathways and recognize new signals? What changes are needed to insulate a new pathway from existing pathways? What constraints are there on gene duplication and lateral gene transfer? Here, we review progress made in answering these questions, highlighting how the integration of genome sequence data with experimental studies is providing major new insights. PMID:22746333

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacques, David A.; Streamer, Margaret; Rowland, Susan L.

2009-09-02

The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomericmore » or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.« less

Signal Sensing and Transduction by Histidine Kinases as Unveiled through Studies on a Temperature Sensor.

PubMed

Abriata, Luciano A; Albanesi, Daniela; Dal Peraro, Matteo; de Mendoza, Diego

2017-06-20

Histidine kinases (HK) are the sensory proteins of two-component systems, responsible for a large fraction of bacterial responses to stimuli and environmental changes. Prototypical HKs are membrane-bound proteins that phosphorylate cognate response regulator proteins in the cytoplasm upon signal detection in the membrane or periplasm. HKs stand as potential drug targets but also constitute fascinating systems for studying proteins at work, specifically regarding the chemistry and mechanics of signal detection, transduction through the membrane, and regulation of catalytic outputs. In this Account, we focus on Bacillus subtilis DesK, a membrane-bound HK part of a two-component system that maintains appropriate membrane fluidity at low growth temperatures. Unlike most HKs, DesK has no extracytoplasmic signal-sensing domains; instead, sensing is carried out by 10 transmembrane helices (coming from two protomers) arranged in an unknown structure. The fifth transmembrane helix from each protomer connects, without any of the intermediate domains found in other HKs, into the dimerization and histidine phosphotransfer (DHp) domain located in the cytoplasm, which is followed by the ATP-binding domains (ABD). Throughout the years, genetic, biochemical, structural, and computational studies on wild-type, mutant, and truncated versions of DesK allowed us to dissect several aspects of DesK's functioning, pushing forward a more general understanding of its own structure/function relationships as well as those of other HKs. We have shown that the sensing mechanism is rooted in temperature-dependent membrane properties, most likely a combination of thickness, fluidity, and water permeability, and we have proposed possible mechanisms by which DesK senses these properties and transduces the signals. X-ray structures and computational models have revealed structural features of TM and cytoplasmic regions in DesK's kinase- and phosphatase-competent states. Biochemical and genetic experiments and molecular simulations further showed that reversible formation of a two-helix coiled coil in the fifth TM segment and the N-terminus of the cytoplasmic domain is essential for the sensing and signal transduction mechanisms. Together with other structural and functional works, the emerging picture suggests that diverse HKs possess distinct sensing and transduction mechanisms but share as rather general features (i) a symmetric phosphatase state and an asymmetric kinase state and (ii) similar functional outputs on the conserved DHp and ABD domains, achieved through different mechanisms that depend on the nature of the initial signal. We here advance (iii) an important role for TM prolines in transducing the initial signals to the cytoplasmic coiled coils, based on simulations of DesK's TM helices and our previous work on a related HK, PhoQ. Lastly, evidence for DesK, PhoQ, BvgS, and DctB HKs shows that (iv) overall catalytic output is tuned by a delicate balance between hydration potentials, coiled coil stability, and exposure of hydrophobic surface patches at their cytoplasmic coiled coils and at the N-terminal and C-terminal sides of their TM helices. This balance is so delicate that small perturbations, either physiological signals or induced by mutations, lead to large remodeling of the underlying conformational landscape achieving clear-cut changes in catalytic output, mirroring the required response speed of these systems for proper biological function.

Tissue specific and abiotic stress regulated transcription of histidine kinases in plants is also influenced by diurnal rhythm

PubMed Central

Singh, Anupama; Kushwaha, Hemant R.; Soni, Praveen; Gupta, Himanshu; Singla-Pareek, Sneh L.; Pareek, Ashwani

2015-01-01

Two-component system (TCS) is one of the key signal sensing machinery which enables species to sense environmental stimuli. It essentially comprises of three major components, sensory histidine kinase proteins (HKs), histidine phosphotransfer proteins (Hpts), and response regulator proteins (RRs). The members of the TCS family have already been identified in Arabidopsis and rice but the knowledge about their functional indulgence during various abiotic stress conditions remains meager. Current study is an attempt to carry out comprehensive analysis of the expression of TCS members in response to various abiotic stress conditions and in various plant tissues in Arabidopsis and rice using MPSS and publicly available microarray data. The analysis suggests that despite having almost similar number of genes, rice expresses higher number of TCS members during various abiotic stress conditions than Arabidopsis. We found that the TCS machinery is regulated by not only various abiotic stresses, but also by the tissue specificity. Analysis of expression of some representative members of TCS gene family showed their regulation by the diurnal cycle in rice seedlings, thus bringing-in another level of their transcriptional control. Thus, we report a highly complex and tight regulatory network of TCS members, as influenced by the tissue, abiotic stress signal, and diurnal rhythm. The insights on the comparative expression analysis presented in this study may provide crucial leads toward dissection of diverse role(s) of the various TCS family members in Arabidopsis and rice. PMID:26442025

Fungal histidine phosphotransferase plays a crucial role in photomorphogenesis and pathogenesis in Magnaporthe oryzae

NASA Astrophysics Data System (ADS)

Mohanan, Varsha C.; Chandarana, Pinal M.; Chattoo, Bharat. B.; Patkar, Rajesh N.; Manjrekar, Johannes

2017-05-01

Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

The signaling helix: a common functional theme in diverse signaling proteins

PubMed Central

Anantharaman, Vivek; Balaji, S; Aravind, L

2006-01-01

Background The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. Results Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S)-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix. Conclusion Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream signaling domains. However, upon occurrence of specific conformational changes due to binding of ligand or other sensory inputs in a linked upstream domain it transmits the signal to the downstream domain. Thus, the S-helix represents one of the most prevalent functional themes involved in the flow of signals between modules in diverse prokaryote-type multi-domain signaling proteins. Reviewers This article was reviewed by Frank Eisenhaber, Arcady Mushegian and Sandor Pongor. PMID:16953892

The two parallel photocycles of the Chlamydomonas sensory photoreceptor histidine kinase rhodopsin 1.

PubMed

Luck, Meike; Hegemann, Peter

2017-10-01

Histidine kinase rhodopsins (HKRs) belong to a class of unexplored sensory photoreceptors that share a similar modular architecture. The light sensing rhodopsin domain is covalently linked to signal-transducing modules and in some cases to a C-terminal guanylyl-cyclase effector. In spite of their wide distribution in unicellular organisms, very little is known about their physiological role and mechanistic functioning. We investigated the photochemical properties of the recombinant rhodopsin-fragment of Cr-HKR1 originating from Chlamydomonas reinhardtii. Our spectroscopic studies revealed an unusual thermal stability of the photoproducts with the deprotonated retinal Schiff base (RSB). Upon UV-irradiation these Rh-UV states with maximal absorbance in the UVA-region (Rh-UV) photochemically convert to stable blue light absorbing rhodopsin (Rh-Bl) with protonated chromophore. The heterogeneity of the sample is based on two parallel photocycles with the chromophore in C 15 =N-syn- or -anti-configuration. This report represents an attempt to decipher the underlying reaction schemes and interconversions of the two coexisting photocycles. Copyright © 2017 Elsevier GmbH. All rights reserved.

Complete Genome Sequence of the MRSA Isolate HC1335 from ST239 Lineage Displaying a Truncated AgrC Histidine Kinase Receptor

PubMed Central

Botelho, Ana M. N.; Costa, Maiana O. C.; Beltrame, Cristiana O.; Ferreira, Fabienne A.; Lima, Nicholas C. B.; Costa, Bruno S. S.; de Morais, Guilherme L.; Souza, Rangel C.; Almeida, Luiz G. P.; Vasconcelos, Ana T. R.; Nicolás, Marisa F.; Figueiredo, Agnes M. S.

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is still one of the most important hospital pathogen globally. The multiresistant isolates of the ST239-SCCmecIII lineage are spread over large geographic regions, colonizing and infecting hospital patients in virtually all continents. The balance between fitness (adaptability) and virulence potential is likely to represent an important issue in the clonal shift dynamics leading the success of some specific MRSA clones over another. The accessory gene regulator (agr) is the master quorum sensing system of staphylococci playing a role in the global regulation of key virulence factors. Consequently, agr inactivation in S. aureus may represent a significant mechanism of genetic variability in the adaptation of this healthcare-associated pathogen. We report here the complete genome sequence of the methicillin-resistant S. aureus, isolate HC1335, a variant of the ST239 lineage, which presents a natural insertion of an IS256 transposase element in the agrC gene encoding AgrC histidine kinase receptor. PMID:27635055

The Staphylococcus aureus Two-Component System AgrAC Displays Four Distinct Genomic Arrangements That Delineate Genomic Virulence Factor Signatures

PubMed Central

Choudhary, Kumari S.; Mih, Nathan; Monk, Jonathan; Kavvas, Erol; Yurkovich, James T.; Sakoulas, George; Palsson, Bernhard O.

2018-01-01

Two-component systems (TCSs) consist of a histidine kinase and a response regulator. Here, we evaluated the conservation of the AgrAC TCS among 149 completely sequenced Staphylococcus aureus strains. It is composed of four genes: agrBDCA. We found that: (i) AgrAC system (agr) was found in all but one of the 149 strains, (ii) the agr positive strains were further classified into four agr types based on AgrD protein sequences, (iii) the four agr types not only specified the chromosomal arrangement of the agr genes but also the sequence divergence of AgrC histidine kinase protein, which confers signal specificity, (iv) the sequence divergence was reflected in distinct structural properties especially in the transmembrane region and second extracellular binding domain, and (v) there was a strong correlation between the agr type and the virulence genomic profile of the organism. Taken together, these results demonstrate that bioinformatic analysis of the agr locus leads to a classification system that correlates with the presence of virulence factors and protein structural properties. PMID:29887846

Hybrid histidine kinases in pathogenic fungi.

PubMed

Defosse, Tatiana A; Sharma, Anupam; Mondal, Alok K; Dugé de Bernonville, Thomas; Latgé, Jean-Paul; Calderone, Richard; Giglioli-Guivarc'h, Nathalie; Courdavault, Vincent; Clastre, Marc; Papon, Nicolas

2015-03-01

Histidine kinases (HK) sense and transduce via phosphorylation events many intra- and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N-terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape-function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs. © 2015 John Wiley & Sons Ltd.

Potassium sensing histidine kinase in Bacillus subtilis.

PubMed

López, Daniel; Gontang, Erin A; Kolter, Roberto

2010-01-01

The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

PubMed

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

DOE PAGES

Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian; ...

2017-07-24

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

Isocitrate Lyase from Flax 1

PubMed Central

Khan, Fazal R.; McFadden, Bruce A.

1982-01-01

The cleavage of Ds-isocitrate catalyzed by isocitrate lyase from Linum usitatissimum results in the ordered release of succinate and glyoxylate. The glyoxylate analog 3-bromopyruvate irreversibly inactivates the flax enzyme in a process exhibiting saturation kinetics and protection by glyoxylate or isocitrate or the competitive inhibitor l-tartrate. Succinate provides considerably less protection. Results with 3-bromopyruvate suggest that this reagent modifies plant and prokaryotic isocitrate lyases differently. Treatment of the tetrameric 264,000-dalton flax enzyme with carboxypeptidase A results in a release of one histidine/subunit which is concordant with loss of activity. The only N-terminal residue is methionine. Treatment of flax enzyme with diethylpyrocarbonate at pH 6.5 selectively modifies two histidines per 67,000-dalton subunit. The reaction of one histidine residue is abolished by the binding of l-tartrate and the modification of one is coincident with inactivation. The carboxy-terminal and active-site modifications establish that one histidine residue/monomer is essential in the flax enzyme and considerably extend information heretofore available only for fungal and bacterial isocitrate lyase. PMID:16662648

Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis.

PubMed

Washington, Tracy A; Smith, Janet L; Grossman, Alan D

2017-10-01

DnaA is the widely conserved bacterial AAA+ ATPase that functions as both the replication initiator and a transcription factor. In many organisms, DnaA controls expression of its own gene and likely several others during growth and in response to replication stress. To evaluate the effects of DnaA on gene expression, separate from its role in replication initiation, we analyzed changes in mRNA levels in Bacillus subtilis cells with and without dnaA, using engineered strains in which dnaA is not essential. We found that dnaA was required for many of the changes in gene expression in response to replication stress. We also found that dnaA indirectly affected expression of several regulons during growth, including those controlled by the transcription factors Spo0A, AbrB, PhoP, SinR, RemA, Rok and YvrH. These effects were largely mediated by the effects of DnaA on expression of sda. DnaA activates transcription of sda, and Sda inhibits histidine protein kinases required for activation of the transcription factor Spo0A. We also found that loss of dnaA caused a decrease in the development of genetic competence. Together, our results indicate that DnaA plays an important role in modulating cell physiology, separate from its role in replication initiation. © 2017 John Wiley & Sons Ltd.

Alanine rich peptide from Populus trichocarpa inhibit growth of Staphylococcus aureus via targetting its extracellular domain of Sensor Histidine Kinase YycGex protein.

PubMed

Al Akeel, Raid; Mateen, Ayesha; Syed, Rabbani; Alqahtani, Mohammed S; Alqahtani, Ali S

2018-05-22

Due to growing concern towards microbial resistance, ongoing search for developing novel bioactive compounds such as peptides is on rise. The aim of this study was to evaluate antimicrobial effect of Populus trichocarpa extract, chemically identify the active peptide fraction and finds its target in Staphylococcus aureus. In this study the active fraction of P. trichocarpa crude extract was purified and characterized using MS/MS. This peptide PT13 antimicrobial activity was confirmed by in-vitro agar based disk diffusion and in-vivo infection model of G. mellonella. The proteomic expression analysis of S. aureus under influence of PT13 was studied using LTQ-Orbitrap-MS in-solution digestion and identity of target protein was acquired with their quantified expression using label-free approach of Progenesis QI software. Docking study was performed with peptide PT13 and its target YycG protein using CABS-dock. The active fraction PT13 sequence was identified as KVPVAAAAAAAAAVVASSMVVAAAK, with 25 amino acid including 13 alanine having M/Z 2194.2469. PT13 was uniformly inhibited growth S. aureus SA91 and MIC was determined 16 μg/mL for SA91 S. aureus strain. Sensor histidine kinase (YycG) was most significant target found differentially expressed under influence of PT13. G. mellonella larvae were killed rapidly due to S aureus infection, whereas death in protected group was insignificant in compare to control. The docking models showed ten docking models with RMSD value 1.89 for cluster 1 and RMSD value 3.95 for cluster 2 which is predicted to be high quality model. Alanine rich peptide could be useful in constructing as antimicrobial peptide for targeting extracellular Domain of Sensor Histidine Kinase YycG from S. aureus used in the study. Copyright © 2018 Elsevier Ltd. All rights reserved.

ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Celikel, Reha; Veldore, Vidya Harini; Mathews, Irimpan

The histidine WalK (YycG) plays a crucial role in coordinating murein synthesis with cell division and the crystal structure of its ATP binding domain has been determined. Interestingly the bound ATP was not hydrolyzed during crystallization and remains intact in the crystal lattice. In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferringmore » the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg{sup 2+} ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3′ of the sugar ring and O2B of the β-phosphate, implying an internal hydrogen bond. The stability of the WalK–ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.« less

Final Technical Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michael Laub

2008-12-29

Our team of investigators from MIT (Michael Laub) and Stanford (Harley McAdams and Lucy Shapiro) conducted a multi-faceted, systematic experimental analysis of the 106 Caulobacter two-component signal transduction system proteins (62 histidine kinases and 44 response regulators) to understand how they coordinate cell cycle progression, metabolism, and response to environmental changes. These two-component signaling proteins were characterized at the genetic, biochemical, and genomic levels. The results generated by our laboratories have provided numerous insights into how Caulobacter cells sense and respond to a myriad of signals. As nearly all bacteria use two-component signaling for cell regulation, the results from thismore » project help to deepen our general understanding of bacterial signal transduction. The tools and approaches developed can be applied to other bacteria. In particular, work from the Laub laboratory now enables the systematic, rational rewiring of two-component signaling proteins, a major advance that stands to impact synthetic biology and the development of biosensors and other designer molecular circuits. Results are summarized from our work. Each section lists publications and publicly-available resources which result from the work described.« less

Transcription analysis of pilS and xpsEL genes from Xylella fastidiosa.

PubMed

Coltri, Patricia P; Rosato, Yoko B

2005-04-01

Xylella fastidiosa is a xylem-limited phytopathogen responsible for diseases in several plants such as citrus and coffee. Analysis of the bacterial genome revealed some putative pathogenicity-related genes that could help to elucidate the molecular mechanisms of plant-pathogen interactions. In the present work, the transcription of three genes of the bacterium, grown in defined and rich media and also in media containing host plant extracts (sweet orange, 'ponkan' and coffee) was analyzed by RT-PCR. The pilS gene, which encodes a sensor histidine kinase responsible for the biosynthesis of fimbriae, was transcribed when the bacterium was grown in more complex media such as PW and in medium containing plant extracts. The xps genes (xpsL and xpsE) which are related to the type II secretion system were also detected when the bacterium was grown in rich media and media with 'ponkan' and coffee extracts. It was thus observed that pilS and xpsEL genes of X. fastidiosa can be modulated by environmental factors and their expression is dependent on the nutritional status of the growth medium.

Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

PubMed Central

Xie, Jianling; Mikolajek, Halina; Pigott, Craig R.; Hooper, Kelly J.; Mellows, Toby; Moore, Claire E.; Mohammed, Hafeez; Werner, Jörn M.; Thomas, Gareth J.

2015-01-01

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis. PMID:25776553

The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

PubMed Central

Pini, Francesco; Frage, Benjamin; Ferri, Lorenzo; De Nisco, Nicole J.; Mohapatra, Saswat S.; Taddei, Lucilla; Fioravanti, Antonella; Dewitte, Frederique; Galardini, Marco; Brilli, Matteo; Villeret, Vincent; Bazzicalupo, Marco; Mengoni, Alessio; Walker, Graham C.; Becker, Anke; Biondi, Emanuele G.

2013-01-01

SUMMARY Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis. PMID:23909720

Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation1[OPEN

PubMed Central

Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song

2015-01-01

Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. PMID:25941315

Characterization of the Effect of the Histidine Kinase CovS on Response Regulator Phosphorylation in Group A Streptococcus

PubMed Central

Horstmann, Nicola; Sahasrabhojane, Pranoti; Saldaña, Miguel; Ajami, Nadim J.; Flores, Anthony R.; Sumby, Paul; Liu, Chang-Gong; Yao, Hui; Su, Xiaoping; Thompson, Erika

2015-01-01

Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections. PMID:25561708

Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana.

PubMed

Dortay, Hakan; Mehnert, Nijuscha; Bürkle, Lukas; Schmülling, Thomas; Heyl, Alexander

2006-10-01

The signal of the plant hormone cytokinin is perceived by membrane-located sensor histidine kinases and transduced by other members of the plant two-component system. In Arabidopsis thaliana, 28 two-component system proteins (phosphotransmitters and response regulators) act downstream of three receptors, transmitting the signal from the membrane to the nucleus and modulating the cellular response. Although the principal signaling mechanism has been elucidated, redundancy in the system has made it difficult to understand which of the many components interact to control the downstream biological processes. Here, we present a large-scale interaction study comprising most members of the Arabidopsis cytokinin signaling pathway. Using the yeast two-hybrid system, we detected 42 new interactions, of which more than 90% were confirmed by in vitro coaffinity purification. There are distinct patterns of interaction between protein families, but only a few interactions between proteins of the same family. An interaction map of this signaling pathway shows the Arabidopsis histidine phosphotransfer proteins as hubs, which interact with members from all other protein families, mostly in a redundant fashion. Domain-mapping experiments revealed the interaction domains of the proteins of this pathway. Analyses of Arabidopsis histidine phosphotransfer protein 5 mutant proteins showed that the presence of the canonical phospho-accepting histidine residue is not required for the interactions. Interaction of A-type response regulators with Arabidopsis histidine phosphotransfer proteins but not with B-type response regulators suggests that their known activity in feedback regulation may be realized by interfering at the level of Arabidopsis histidine phosphotransfer protein-mediated signaling. This study contributes to our understanding of the protein interactions of the cytokinin-signaling system and provides a framework for further functional studies in planta.

Structure-based analysis of catalysis and substrate definition in the HIT protein family.

PubMed

Lima, C D; Klein, M G; Hendrickson, W A

1997-10-10

The histidine triad (HIT) protein family is among the most ubiquitous and highly conserved in nature, but a biological activity has not yet been identified for any member of the HIT family. Fragile histidine triad protein (FHIT) and protein kinase C interacting protein (PKCI) were used in a structure-based approach to elucidate characteristics of in vivo ligands and reactions. Crystallographic structures of apo, substrate analog, pentacovalent transition-state analog, and product states of both enzymes reveal a catalytic mechanism and define substrate characteristics required for catalysis, thus unifying the HIT family as nucleotidyl hydrolases, transferases, or both. The approach described here may be useful in identifying structure-function relations between protein families identified through genomics.

The ethylene response pathway in Arabidopsis

NASA Technical Reports Server (NTRS)

Kieber, J. J.; Evans, M. L. (Principal Investigator)

1997-01-01

The simple gas ethylene influences a diverse array of plant growth and developmental processes including germination, senescence, cell elongation, and fruit ripening. This review focuses on recent molecular genetic studies, principally in Arabidopsis, in which components of the ethylene response pathway have been identified. The isolation and characterization of two of these genes has revealed that ethylene sensing involves a protein kinase cascade. One of these genes encodes a protein with similarity to the ubiquitous Raf family of Ser/Thr protein kinases. A second gene shows similarity to the prokaryotic two-component histidine kinases and most likely encodes an ethylene receptor. Additional elements involved in ethylene signaling have only been identified genetically. The characterization of these genes and mutants will be discussed.

Changes in urinary amino acids excretion in relationship with muscle activity markers over a professional cycling stage race: in search of fatigue markers.

PubMed

Corsetti, Roberto; Barassi, Alessandra; Perego, Silvia; Sansoni, Veronica; Rossi, Alessandra; Damele, Clara Anna Linda; Melzi D'Eril, Gianlodovico; Banfi, Giuseppe; Lombardi, Giovanni

2016-01-01

The aim of this study was to identify the relationship between metabolic effort, muscular damage/activity indices, and urinary amino acids profile over the course of a strenuous prolonged endurance activity, as a cycling stage race is, in order to identify possible fatigue markers. Nine professional cyclists belonging to a single team, competing in the Giro d'Italia cycling stage race, were anthropometrically characterized and sampled for blood and urine the day before the race started, and on days 12 and 23 of the race. Diet was kept the same over the race, and power output and energy expenditure were recorded. Sera were assayed for muscle markers (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase activities, and blood urea nitrogen), and creatinine, all corrected for plasma volume changes. Urines were profiled for amino acid concentrations, normalized on creatinine excretion. Renal function, in terms of glomerular filtration rate, was monitored by MDRD equation corrected on body surface area. Creatine kinase activity and blood urea were increased during the race as did serum creatinine while kidney function remained stable. Among the amino acids, taurine, glycine, cysteine, leucine, carnosine, 1-methyl histidine, and 3-methyl histidine showed a net decreased, while homocysteine was increased. Taurine and the dipeptide carnosine (β-alanyl-L-histidine) were significantly correlated with the muscle activity markers and the indices of effort. In conclusion, the metabolic profile is modified strikingly due to the effort. Urinary taurine and carnosine seem useful tools to evaluate the muscle damage and possibly the fatigue status on a long-term basis.

Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins.

PubMed

Noda, Masafumi; Miyauchi, Rumi; Danshiitsoodol, Narandalai; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

2018-04-01

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-β and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG , are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC , encoding brevicins 174A-β and 174A-γ, respectively, for positive regulation. IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic bacteria without side effects on the human body. The bacteriocin that is produced by a Citrus iyo -derived Lactobacillus brevis strain inhibits the growth of pathogenic bacteria such as Listeria monocytogenes , Staphylococcus aureus , and Streptococcus mutans In general, lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth, which is called a quorum-sensing system. The system consists of a membrane-bound histidine protein kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response. The present study demonstrates that the expression of the genes encoding bacteriocin biosynthesis and the self-resistance determinant is positively controlled by two transcriptional regulatory proteins. Copyright © 2018 American Society for Microbiology.

Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli.

PubMed

Sporeno, E; Barbato, G; Graziani, R; Pucci, P; Nitti, G; Paonessa, G

1994-05-01

Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells. After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector. Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column. We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay. Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry. Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.

The effect of histidine ammonia-lyase on some murine tumours.

PubMed

Jack, G W; Wiblin, C N; McMahon, P C

1983-01-01

The histidine ammonia-lyase from bacterial strain CAMR 5315 was partially purified to assess its effect on the growth of murine tumours. This strain was selected as the source after an extensive screening programme for histidine ammonia-lyases. The enzyme was partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Sephadex G-150. The enzyme reduced circulating L-histidine levels in Wistar rats and in mice persisted with a half-life of 6-7 h. Neither LDH virus nor chemical modification with ethylacetimidate increased the half-life as observed with L-asparaginase and L-glutaminase. The enzyme was tested in mice against Ehrlich carcinoma, L5178Y lymphoblastic leukaemia, Mc/S sarcoma, B16 melanoma, P8157 mastocytoma, P1798 lymphosarcoma and the Gardner 6C3HED lymphosarcoma. The only tumours to show sensitivity to the enzyme were the Mc/S sarcoma against which a 65% increase in life span was observed at the highest enzyme dose, 1000 U/kg on alternate days over 14 days and the Ehrlich ascites carcinoma where cures were obtained at 250 U/kg on alternate days over 14 days, but only at inocula levels of 10(5) and 10(3) cells/animal respectively.

Complex two-component signaling regulates the general stress response in Alphaproteobacteria.

PubMed

Kaczmarczyk, Andreas; Hochstrasser, Ramon; Vorholt, Julia A; Francez-Charlot, Anne

2014-12-02

The general stress response (GSR) in Alphaproteobacteria was recently shown to be controlled by a partner-switching mechanism that is triggered by phosphorylation of the response regulator PhyR. Activation of PhyR ultimately results in release of the alternative extracytoplasmic function sigma factor σ(EcfG), which redirects transcription toward the GSR. Little is known about the signal transduction pathway(s) controlling PhyR phosphorylation. Here, we identified the single-domain response regulator (SDRR) SdrG and seven histidine kinases, PakA to PakG, belonging to the HWE/HisKA2 family as positive modulators of the GSR in Sphingomonas melonis Fr1. Phenotypic analyses, epistasis experiments, and in vitro phosphorylation assays indicate that Paks directly phosphorylate PhyR and SdrG, and that SdrG acts upstream of or in concert with PhyR, modulating its activity in a nonlinear pathway. Furthermore, we found that additional SDRRs negatively affect the GSR in a way that strictly requires PhyR and SdrG. Finally, analysis of GSR activation by thermal, osmotic, and oxidative stress indicates that Paks display different degrees of redundancy and that a specific kinase can sense multiple stresses, suggesting that the GSR senses a particular condition as a combination of, rather than individual, molecular cues. This study thus establishes the alphaproteobacterial GSR as a complex and interlinked network of two-component systems, in which multiple histidine kinases converge to PhyR, the phosphorylation of which is, in addition, subject to regulation by several SDRRs. Our finding that most HWE/HisKA2 kinases contribute to the GSR in S. melonis Fr1 opens the possibility that this notion might also be true for other Alphaproteobacteria.

Dissecting the role of histidine kinase and HOG1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat

PubMed Central

John, Evan; Lopez-Ruiz, Francisco; Rybak, Kasia; Mousley, Carl J.; Oliver, Richard P.

2016-01-01

The HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity. PMID:26978567

The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris.

PubMed

An, Shi-Qi; Allan, John H; McCarthy, Yvonne; Febrer, Melanie; Dow, J Maxwell; Ryan, Robert P

2014-05-01

A cell-cell signalling system mediated by the fatty acid signal DSF controls the virulence of Xanthomonas campestris pv. campestris (Xcc) to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon the sensor RpfC and regulator RpfG. Detailed analyses of the regulatory roles of different Rpf proteins have suggested the occurrence of further sensors for DSF. Here we have used a mutagenesis approach coupled with high-resolution transcriptional analysis to identify XC_2579 (RpfS) as a second sensor for DSF in Xcc. RpfS is a complex sensor kinase predicted to have multiple Per/Arnt/Sim (PAS) domains, a histidine kinase domain and a C-terminal receiver (REC) domain. Isothermal calorimetry showed that DSF bound to the isolated N-terminal PAS domain with a Kd of 1.4 μM. RpfS controlled expression of a sub-set of genes distinct from those controlled by RpfC to include genes involved in type IV secretion and chemotaxis. Mutation of XC_2579 was associated with a reduction in virulence of Xcc to Chinese Radish when assayed by leaf spraying but not by leaf inoculation, suggesting a role for RpfS-controlled factors in the epiphytic phase of the disease cycle. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Protein interaction networks at the host-microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen.

PubMed

Ramsey, J S; Chavez, J D; Johnson, R; Hosseinzadeh, S; Mahoney, J E; Mohr, J P; Robison, F; Zhong, X; Hall, D G; MacCoss, M; Bruce, J; Cilia, M

2017-02-01

The Asian citrus psyllid ( Diaphorina citri) is the insect vector responsible for the worldwide spread of ' Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host-microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host-microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

Protein interaction networks at the host–microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen

PubMed Central

Chavez, J. D.; Johnson, R.; Hosseinzadeh, S.; Mahoney, J. E.; Mohr, J. P.; Robison, F.; Zhong, X.; Hall, D. G.; MacCoss, M.; Bruce, J.; Cilia, M.

2017-01-01

The Asian citrus psyllid (Diaphorina citri) is the insect vector responsible for the worldwide spread of ‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host–microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host–microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening. PMID:28386418

Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib

PubMed Central

Mathea, Sebastian; Abdul Azeez, Kamal R.; Salah, Eidarus; Tallant, Cynthia; Wolfreys, Finn; Konietzny, Rebecca; Fischer, Roman; Lou, Hua Jane; Brennan, Paul E.; Schnapp, Gisela; Pautsch, Alexander; Kessler, Benedikt M.; Turk, Benjamin E.; Knapp, Stefan

2017-01-01

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The co-crystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions −1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs. PMID:26999302

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

PubMed Central

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network.

PubMed

Radeck, Jara; Fritz, Georg; Mascher, Thorsten

2017-02-01

The cell envelope stress response (CESR) encompasses all regulatory events that enable a cell to protect the integrity of its envelope, an essential structure of any bacterial cell. The underlying signaling network is particularly well understood in the Gram-positive model organism Bacillus subtilis. It consists of a number of two-component systems (2CS) and extracytoplasmic function σ factors that together regulate the production of both specific resistance determinants and general mechanisms to protect the envelope against antimicrobial peptides targeting the biogenesis of the cell wall. Here, we summarize the current picture of the B. subtilis CESR network, from the initial identification of the corresponding signaling devices to unraveling their interdependence and the underlying regulatory hierarchy within the network. In the course of detailed mechanistic studies, a number of novel signaling features could be described for the 2CSs involved in mediating CESR. This includes a novel class of so-called intramembrane-sensing histidine kinases (IM-HKs), which-instead of acting as stress sensors themselves-are activated via interprotein signal transfer. Some of these IM-HKs are involved in sensing the flux of antibiotic resistance transporters, a unique mechanism of responding to extracellular antibiotic challenge.

Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiaojing; Kuk, Jane; Moffat, Keith

2008-11-12

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 {angstrom} resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an 'arm' structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common andmore » may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.« less

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacques, David A.; Streamer, Margaret; Rowland, Susan L.

2009-06-01

The crystal structure of Sda, a DNA-replication/damage checkpoint inhibitor of sporulation in B. subtilis, has been solved via the MAD method. The subunit arrangement in the crystal has enabled a reappraisal of previous biophysical data, resulting in a new model for the behaviour of the protein in solution. The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB.more » The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.« less

Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

PubMed

Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

2011-11-01

Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

Histidine Kinase-Mediated Production and Autoassembly of Porphyromonas gingivalis Fimbriae▿ †

PubMed Central

Nishikawa, Kiyoshi; Duncan, Margaret J.

2010-01-01

Porphyromonas gingivalis, a Gram-negative oral anaerobe, is strongly associated with chronic adult periodontitis, and it utilizes FimA fimbriae to persistently colonize and evade host defenses in the periodontal crevice. The FimA-related gene cluster (the fim gene cluster) is positively regulated by the FimS-FimR two-component system. In this study, comparative analyses between fimbriate type strain ATCC 33277 and fimbria-deficient strain W83 revealed differences in their fimS loci, which encode FimS histidine kinase. Using a reciprocal gene exchange system, we established that FimS from W83 is malfunctional. Complementation analysis with chimeric fimS constructs revealed that W83 FimS has a defective kinase domain due to a truncated conserved G3 box motif that provides an ATP-binding pocket. The introduction of the functional fimS from 33277 restored the production, but not polymerization, of endogenous FimA subunits in W83. Further analyses with a fimA-exchanged W83 isogenic strain showed that even the fimbria-deficient W83 retains the ability to polymerize FimA from 33277, indicating the assembly of mature FimA by a primary structure-dependent mechanism. It also was shown that the substantial expression of 33277-type FimA fimbriae in the W83 derivative requires the introduction and expression of the functional 33277 fimS. These findings indicate that FimSR is the unique and universal regulatory system that activates the fim gene cluster in a fimA genotype-independent manner. PMID:20118268

The source of high signal cooperativity in bacterial chemosensory arrays

PubMed Central

Piñas, Germán E.; Frank, Vered; Vaknin, Ady; Parkinson, John S.

2016-01-01

The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties. PMID:26951681

CHLAMYDIA TRACHOMATIS TARP IS PHOSPHORYLATED BY SRC FAMILY TYROSINE KINASES

PubMed Central

Jewett, Travis J.; Dooley, Cheryl A.; Mead, David J.; Hackstadt, Ted

2008-01-01

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells. PMID:18442471

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

PubMed

Atack, John M; Srikhanta, Yogitha N; Djoko, Karrera Y; Welch, Jessica P; Hasri, Norain H M; Steichen, Christopher T; Vanden Hoven, Rachel N; Grimmond, Sean M; Othman, Dk Seti Maimonah Pg; Kappler, Ulrike; Apicella, Michael A; Jennings, Michael P; Edwards, Jennifer L; McEwan, Alastair G

2013-06-01

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity

PubMed Central

Lasker, Keren; Ahrens, Daniel G.; Eckart, Michael R.

2017-01-01

ABSTRACT Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment. PMID:28246363

Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajagopal, P.; Klevit, R.E.

1994-12-01

HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residuemore » at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.« less

A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

PubMed

Forster, Brian M; Bitar, Alan Pavinski; Marquis, Hélène

2014-01-01

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes *

PubMed Central

Wisniewska, Magdalena; Happonen, Lotta; Kahn, Fredrik; Varjosalo, Markku; Malmström, Lars; Rosenberger, George; Karlsson, Christofer; Cazzamali, Giuseppe; Pozdnyakova, Irina; Frick, Inga-Maria; Björck, Lars; Streicher, Werner; Malmström, Johan; Wikström, Mats

2014-01-01

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis. PMID:24825900

Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family

PubMed Central

Daugherty, Matthew; Vonstein, Veronika; Overbeek, Ross; Osterman, Andrei

2001-01-01

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi|1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi|1591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: Km,shikimate = 414 ± 33 μM, Km,ATP = 48 ± 4 μM, and kcat = 57 ± 2 s−1 for the predicted shikimate kinase and Km,homoserine = 188 ± 37 μM, Km,ATP = 101 ± 7 μM, and kcat = 28 ± 1 s−1 for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes. PMID:11114929

Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae

PubMed Central

Jacob, Stefan; Foster, Andrew J; Yemelin, Alexander; Thines, Eckhard

2014-01-01

The aim of this study is a functional characterization of 10 putative histidine kinases (HIKs)-encoding genes in the phytopathogenic fungus Magnaporthe oryzae. Two HIKs were found to be required for pathogenicity in the fungus. It was found that the mutant strains ΔMohik5 and ΔMohik8 show abnormal conidial morphology and furthermore ΔMohik5 is unable to form appressoria. Both HIKs MoHik5p and MoHik8p appear to be essential for pathogenicity since the mutants fail to infect rice plants. MoSln1p and MoHik1p were previously reported to be components of the HOG pathway in M. oryzae. The ΔMosln1 mutant is more susceptible to salt stress compared to ΔMohik1, whereas ΔMohik1 appears to be stronger affected by osmotic or sugar stress. In contrast to yeast, the HOG signaling cascade in phytopathogenic fungi apparently comprises more elements. Furthermore, vegetative growth of the mutants ΔMohik5 and ΔMohik9 was found to be sensitive to hypoxia-inducing NaNO2-treatment. Additionally, it was monitored that NaNO2-treatment resulted in MoHog1p phosphorylation. As a consequence we assume a first simplified model for hypoxia signaling in M. oryzae including the HOG pathway and the HIKs MoHik5p and MoHik9p. PMID:25103193

Mechanism of the pH-Induced Conformational Change in the Sensor Domain of the DraK Histidine Kinase via the E83, E105, and E107 Residues

PubMed Central

Jee, Jun-Goo; Lee, Jae Kyoung; Kim, Hyo Jeong; Park, Jin-Wan; Kim, Eun-Hee; Hwang, Eunha; Kim, Sang-Yoon; Lee, Eun-Gyeong; Kwon, Ohsuk; Cheong, Hae-Kap

2014-01-01

The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces. PMID:25203403

Analysis of periplasmic sensor domains from Anaeromyxobacter dehalogenans 2CP-C: Structure of one sensor domain from a histidine kinase and another from a chemotaxis protein

PubMed Central

Pokkuluri, P Raj; Dwulit-Smith, Jeff; Duke, Norma E; Wilton, Rosemarie; Mack, Jamey C; Bearden, Jessica; Rakowski, Ella; Babnigg, Gyorgy; Szurmant, Hendrik; Joachimiak, Andrzej; Schiffer, Marianne

2013-01-01

Anaeromyxobacter dehalogenans is a δ-proteobacterium found in diverse soils and sediments. It is of interest in bioremediation efforts due to its dechlorination and metal-reducing capabilities. To gain an understanding on A. dehalogenans' abilities to adapt to diverse environments we analyzed its signal transduction proteins. The A. dehalogenans genome codes for a large number of sensor histidine kinases (HK) and methyl-accepting chemotaxis proteins (MCP); among these 23 HK and 11 MCP proteins have a sensor domain in the periplasm. These proteins most likely contribute to adaptation to the organism's surroundings. We predicted their three-dimensional folds and determined the structures of two of the periplasmic sensor domains by X-ray diffraction. Most of the domains are predicted to have either PAS-like or helical bundle structures, with two predicted to have solute-binding protein fold, and another predicted to have a 6-phosphogluconolactonase like fold. Atomic structures of two sensor domains confirmed the respective fold predictions. The Adeh_2942 sensor (HK) was found to have a helical bundle structure, and the Adeh_3718 sensor (MCP) has a PAS-like structure. Interestingly, the Adeh_3718 sensor has an acetate moiety bound in a binding site typical for PAS-like domains. Future work is needed to determine whether Adeh_3718 is involved in acetate sensing by A. dehalogenans. PMID:23897711

Mechanism of the pH-induced conformational change in the sensor domain of the DraK Histidine kinase via the E83, E105, and E107 residues.

PubMed

Yeo, Kwon Joo; Hong, Young-Soo; Jee, Jun-Goo; Lee, Jae Kyoung; Kim, Hyo Jeong; Park, Jin-Wan; Kim, Eun-Hee; Hwang, Eunha; Kim, Sang-Yoon; Lee, Eun-Gyeong; Kwon, Ohsuk; Cheong, Hae-Kap

2014-01-01

The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.

Differential Expression of Two Novel Members of the Tomato Ethylene-Receptor Family

PubMed Central

Tieman, Denise M.; Klee, Harry J.

1999-01-01

The phytohormone ethylene regulates many aspects of plant growth, development, and environmental responses. Much of the developmental regulation of ethylene responses in tomato (Lycopersicon esculentum) occurs at the level of hormone sensitivity. In an effort to understand the regulation of ethylene responses, we isolated and characterized tomato genes with sequence similarity to the Arabidopsis ETR1 (ethylene response 1) ethylene receptor. Previously, we isolated three genes that exhibit high similarity to ETR1 and to each other. Here we report the isolation of two additional genes, LeETR4 and LeETR5, that are only 42% and 40% identical to ETR1, respectively. Although the amino acids known to be involved in ethylene binding are conserved, LeETR5 lacks the histidine within the kinase domain that is predicted to be phosphorylated. This suggests that histidine kinase activity is not necessary for an ethylene response, because mutated forms of both LeETR4 and LeETR5 confer dominant ethylene insensitivity in transgenic Arabidopsis plants. Expression analysis indicates that LeETR4 accounts for most of the putative ethylene-receptor mRNA present in reproductive tissues, but, like LeETR5, it is less abundant in vegetative tissues. Taken together, ethylene perception in tomato is potentially quite complex, with at least five structurally divergent, putative receptor family members exhibiting significant variation in expression levels throughout development. PMID:10318694

Group X hybrid histidine kinase Chk1 is dispensable for stress adaptation, host-pathogen interactions and virulence in the opportunistic yeast Candida guilliermondii.

PubMed

Navarro-Arias, María J; Dementhon, Karine; Defosse, Tatiana A; Foureau, Emilien; Courdavault, Vincent; Clastre, Marc; Le Gal, Solène; Nevez, Gilles; Le Govic, Yohann; Bouchara, Jean-Philippe; Giglioli-Guivarc'h, Nathalie; Noël, Thierry; Mora-Montes, Hector M; Papon, Nicolas

2017-09-01

Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine.

PubMed

Tomoike, Fumiaki; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2011-05-31

The salvage pathways of nucleotide biosynthesis are more diverse and are less well understood as compared with de novo pathways. Uridine-cytidine kinase (UCK) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. In this study, we have characterized a UCK homologue of Thermus thermophilus HB8 (ttCK) biochemically and structurally. Unlike other UCKs, ttCK had substrate specificity toward only cytidine and showed no inhibition by UTP, suggesting uridine does not bind to ttCK as substrate. Structural analysis revealed that the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK. Replacement of Tyr93 by histidine or glutamine endowed ttCK with phosphorylation activity toward uridine. These results suggested that a single amino acid residue, Tyr93, gives cytidine-limited specificity to ttCK. However, replacement of Tyr93 by Phe or Leu did not change the substrate specificity of ttCK. Therefore, we conclude that a residue at this position is essential for the recognition of uridine by UCK. In addition, thymidine phosphorylase from T. thermophilus HB8 was equally active with thymidine and uridine, which indicates that this protein is the sole enzyme metabolizing uridine in T. Thermophilus HB8. On the basis of these results, we discuss the pyrimidine-salvage pathway in T. thermophilus HB8.

A Pseudomonas putida double mutant deficient in butanol assimilation: a promising step for engineering a biological biofuel production platform.

PubMed

Cuenca, María Del Sol; Molina-Santiago, Carlos; Gómez-García, María R; Ramos, Juan L

2016-03-01

Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Functional Analysis of the Aspergillus nidulans Kinome

PubMed Central

De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Andrews, Peter; Ringelberg, Carol S.; Dunlap, Jay C.; Osmani, Stephen A.

2013-01-01

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs. PMID:23505451

Proposed Role for KaiC-Like ATPases as Major Signal Transduction Hubs in Archaea

PubMed Central

2017-01-01

ABSTRACT All organisms must adapt to ever-changing environmental conditions and accordingly have evolved diverse signal transduction systems. In bacteria, the most abundant networks are built around the two-component signal transduction systems that include histidine kinases and receiver domains. In contrast, eukaryotic signal transduction is dominated by serine/threonine/tyrosine protein kinases. Both of these systems are also found in archaea, but they are not as common and diversified as their bacterial and eukaryotic counterparts, suggesting the possibility that archaea have evolved other, still uncharacterized signal transduction networks. Here we propose a role for KaiC family ATPases, known to be key components of the circadian clock in cyanobacteria, in archaeal signal transduction. The KaiC family is notably expanded in most archaeal genomes, and although most of these ATPases remain poorly characterized, members of the KaiC family have been shown to control archaellum assembly and have been found to be a stable component of the gas vesicle system in Halobacteria. Computational analyses described here suggest that KaiC-like ATPases and their homologues with inactivated ATPase domains are involved in many other archaeal signal transduction pathways and comprise major hubs of complex regulatory networks. We predict numerous input and output domains that are linked to KaiC-like proteins, including putative homologues of eukaryotic DEATH domains that could function as adapters in archaeal signaling networks. We further address the relationships of the archaeal family of KaiC homologues to the bona fide KaiC of cyanobacteria and implications for the existence of a KaiC-based circadian clock apparatus in archaea. PMID:29208747

A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters.

PubMed

Goudenège, David; Travers, Marie Agnès; Lemire, Astrid; Petton, Bruno; Haffner, Philippe; Labreuche, Yannick; Tourbiez, Delphine; Mangenot, Sophie; Calteau, Alexandra; Mazel, Didier; Nicolas, Jean Louis; Jacq, Annick; Le roux, Frédérique

2015-11-01

Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France, since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high-throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence, the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains have lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species, we show here that VarS is a key factor of pathogenicity. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Thiol-Based Redox Switches and Gene Regulation

PubMed Central

2011-01-01

Abstract Cysteine is notable among the universal, proteinogenic amino acids for its facile redox chemistry. Cysteine thiolates are readily modified by reactive oxygen species (ROS), reactive electrophilic species (RES), and reactive nitrogen species (RNS). Although thiol switches are commonly triggered by disulfide bond formation, they can also be controlled by S-thiolation, S-alkylation, or modification by RNS. Thiol-based switches are common in both prokaryotic and eukaryotic organisms and activate functions that detoxify reactive species and restore thiol homeostasis while repressing functions that would be deleterious if expressed under oxidizing conditions. Here, we provide an overview of the best-understood examples of thiol-based redox switches that affect gene expression. Intra- or intermolecular disulfide bond formation serves as a direct regulatory switch for several bacterial transcription factors (OxyR, OhrR/2-Cys, Spx, YodB, CrtJ, and CprK) and indirectly regulates others (the RsrA anti-σ factor and RegB sensory histidine kinase). In eukaryotes, thiol-based switches control the yeast Yap1p transcription factor, the Nrf2/Keap1 electrophile and oxidative stress response, and the Chlamydomonas NAB1 translational repressor. Collectively, these regulators reveal a remarkable range of chemical modifications exploited by Cys residues to effect changes in gene expression. Antioxid. Redox Signal. 14, 1049—1063. PMID:20626317

The ArcB Leucine Zipper Domain Is Required for Proper ArcB Signaling

PubMed Central

Nuñez Oreza, Luis Alberto; Alvarez, Adrián F.; Arias-Olguín, Imilla I.; Torres Larios, Alfredo; Georgellis, Dimitris

2012-01-01

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70–121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling. PMID:22666479

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutscher, J.; Sauerwald, H.

1986-06-01

Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitivemore » manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following (/sup 32/P)PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.« less

Identification of an Antimicrobial Agent Effective against Methicillin-Resistant Staphylococcus aureus Persisters Using a Fluorescence-Based Screening Strategy

PubMed Central

Kim, Wooseong; Conery, Annie L.; Rajamuthiah, Rajmohan; Fuchs, Beth Burgwyn; Ausubel, Frederick M.; Mylonakis, Eleftherios

2015-01-01

Persisters are a subpopulation of normal bacterial cells that show tolerance to conventional antibiotics. Persister cells are responsible for recalcitrant chronic infections and new antibiotics effective against persisters would be a major development in the treatment of these infections. Using the reporter dye SYTOX Green that only stains cells with permeabilized membranes, we developed a fluorescence-based screening assay in a 384-well format for identifying compounds that can kill methicillin-resistant Staphylococcus aureus (MRSA) persisters. The assay proved robust and suitable for high throughput screening (Z`-factor: >0.7). In screening a library of hits from a previous screen, which identified compounds that had the ability to block killing of the nematode Caenorhabditis by MRSA, we discovered that the low molecular weight compound NH125, a bacterial histidine kinase inhibitor, kills MRSA persisters by causing cell membrane permeabilization, and that 5 μg/mL of the compound can kill all cells to the limit of detection in a 108 CFU/mL culture of MRSA persisters within 3h. Furthermore, NH125 disrupts 50% of established MRSA biofilms at 20 μg/mL and completely eradicates biofilms at 160 μg/mL. Our results suggest that the SYTOX Green screening assay is suitable for large-scale projects to identify small molecules effective against MRSA persisters and should be easily adaptable to a broad range of pathogens that form persisters. Since NH125 has strong bactericidal properties against MRSA persisters and high selectivity to bacteria, we believe NH125 is a good anti-MRSA candidate drug that should be further evaluated. PMID:26039584

A FRET-Based DNA Biosensor Tracks OmpR-Dependent Acidification of Salmonella during Macrophage Infection

PubMed Central

Chakraborty, Smarajit; Mizusaki, Hideaki; Kenney, Linda J.

2015-01-01

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed. PMID:25875623

Functional and structural properties of a novel protein and virulence factor (Protein sHIP) in Streptococcus pyogenes.

PubMed

Wisniewska, Magdalena; Happonen, Lotta; Kahn, Fredrik; Varjosalo, Markku; Malmström, Lars; Rosenberger, George; Karlsson, Christofer; Cazzamali, Giuseppe; Pozdnyakova, Irina; Frick, Inga-Maria; Björck, Lars; Streicher, Werner; Malmström, Johan; Wikström, Mats

2014-06-27

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Computational design of a Zn2+ receptor that controls bacterial gene expression

NASA Astrophysics Data System (ADS)

Dwyer, M. A.; Looger, L. L.; Hellinga, H. W.

2003-09-01

The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a -galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.

Screening of the two-component-system histidine kinases of Listeria monocytogenes EGD-e. LiaS is needed for growth under heat, acid, alkali, osmotic, ethanol and oxidative stresses.

PubMed

Pöntinen, Anna; Lindström, Miia; Skurnik, Mikael; Korkeala, Hannu

2017-08-01

To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HK deletion mutant strains under heat (42.5 °C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H 2 O 2 ) stresses. The growth of ΔliaS (Δlmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The ΔvirS (Δlmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of ΔagrC (Δlmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HK mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L. monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L. monocytogenes EGD-e. Copyright © 2017 Elsevier Ltd. All rights reserved.

Vancomycin tolerance in clinical and laboratory Streptococcus pneumoniae isolates depends on reduced enzyme activity of the major LytA autolysin or cooperation between CiaH histidine kinase and capsular polysaccharide.

PubMed

Moscoso, Miriam; Domenech, Mirian; García, Ernesto

2010-08-01

Vancomycin is frequently added to standard therapy for pneumococcal meningitis. Although vancomycin-resistant Streptococcus pneumoniae strains have not been isolated, reports on the emergence of vancomycin-tolerant pneumococci are a cause of concern. To date, the molecular basis of vancomycin tolerance in S. pneumoniae is essentially unknown. We examined two vancomycin-tolerant clinical isolates, i.e. a purported autolysin negative (LytA(-)), serotype 23F isolate (strain S3) and the serotype 14 strain 'Tupelo', which is considered a paradigm of vancomycin tolerance. S3 was characterized here as carrying a frameshift mutation in the lytA gene encoding the main pneumococcal autolysin. The vancomycin tolerance of strain S3 was abolished by transformation to the autolysin-proficient phenotype. The original Tupelo strain was discovered to be a mixture: a strain showing a vancomycin-tolerant phenotype (Tupelo_VT) and a vancomycin-nontolerant strain (Tupelo_VNT). The two strains differed only in terms of a single mutation in the ciaH gene present in the VT strain. Most interestingly, although the vancomycin tolerance of Tupelo_VT could be overcome by increasing the LytA dosage upon transformation by a multicopy plasmid or by externally adding the autolysin, we show that vancomycin tolerance in S. pneumoniae requires the simultaneous presence of a mutated CiaH histidine kinase and capsular polysaccharide. © 2010 Blackwell Publishing Ltd.

Transplantation of prokaryotic two-component signaling pathways into mammalian cells.

PubMed

Hansen, Jonathan; Mailand, Erik; Swaminathan, Krishna Kumar; Schreiber, Joerg; Angelici, Bartolomeo; Benenson, Yaakov

2014-11-04

Signaling pathway engineering is a promising route toward synthetic biological circuits. Histidine-aspartate phosphorelays are thought to have evolved in prokaryotes where they form the basis for two-component signaling. Tyrosine-serine-threonine phosphorelays, exemplified by MAP kinase cascades, are predominant in eukaryotes. Recently, a prokaryotic two-component pathway was implemented in a plant species to sense environmental trinitrotoluene. We reasoned that "transplantation" of two-component pathways into mammalian host could provide an orthogonal and diverse toolkit for a variety of signal processing tasks. Here we report that two-component pathways could be partially reconstituted in mammalian cell culture and used for programmable control of gene expression. To enable this reconstitution, coding sequences of histidine kinase (HK) and response regulator (RR) components were codon-optimized for human cells, whereas the RRs were fused with a transactivation domain. Responsive promoters were furnished by fusing DNA binding sites in front of a minimal promoter. We found that coexpression of HKs and their cognate RRs in cultured mammalian cells is necessary and sufficient to strongly induce gene expression even in the absence of pathways' chemical triggers in the medium. Both loss-of-function and constitutive mutants behaved as expected. We further used the two-component signaling pathways to implement two-input logical AND, NOR, and OR gene regulation. Thus, two-component systems can be applied in different capacities in mammalian cells and their components can be used for large-scale synthetic gene circuits.

Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian P

2015-01-02

Ergothioneine is an N-α-trimethyl-2-thiohistidine derivative that occurs in human, plant, fungal, and bacterial cells. Biosynthesis of this redox-active betaine starts with trimethylation of the α-amino group of histidine. The three consecutive methyl transfers are catalyzed by the S-adenosylmethionine-dependent methyltransferase EgtD. Three crystal structures of this enzyme in the absence and in the presence of N-α-dimethylhistidine and S-adenosylhomocysteine implicate a preorganized array of hydrophilic interactions as the determinants for substrate specificity and apparent processivity. We identified two active site mutations that change the substrate specificity of EgtD 10(7)-fold and transform the histidine-methyltransferase into a proficient tryptophan-methyltransferase. Finally, a genomic search for EgtD homologues in fungal genomes revealed tyrosine and tryptophan trimethylation activity as a frequent trait in ascomycetous and basidomycetous fungi. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.

PubMed

Dillon, Stephanie L; Williamson, Danielle M; Elferich, Johannes; Radler, David; Joshi, Rajendra; Thomas, Gary; Shinde, Ujwal

2012-10-12

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry

PubMed Central

Henriques, Sónia Troeira; Thorstholm, Louise; Huang, Yen-Hua; Getz, Jennifer A.; Daugherty, Patrick S.; Craik, David J.

2013-01-01

The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development. PMID:24260399

Simultaneous inhibition assay for human and microbial kinases via MALDI-MS/MS.

PubMed

Smith, Anne Marie E; Brennan, John D

2014-03-03

Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

PubMed Central

Canova, Marc J.; Molle, Virginie

2014-01-01

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

Bacterial serine/threonine protein kinases in host-pathogen interactions.

PubMed

Canova, Marc J; Molle, Virginie

2014-04-04

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

Comparative Analysis of Wolbachia Genomes Reveals Streamlining and Divergence of Minimalist Two-Component Systems

PubMed Central

Christensen, Steen; Serbus, Laura Renee

2015-01-01

Two-component regulatory systems are commonly used by bacteria to coordinate intracellular responses with environmental cues. These systems are composed of functional protein pairs consisting of a sensor histidine kinase and cognate response regulator. In contrast to the well-studied Caulobacter crescentus system, which carries dozens of these pairs, the streamlined bacterial endosymbiont Wolbachia pipientis encodes only two pairs: CckA/CtrA and PleC/PleD. Here, we used bioinformatic tools to compare characterized two-component system relays from C. crescentus, the related Anaplasmataceae species Anaplasma phagocytophilum and Ehrlichia chaffeensis, and 12 sequenced Wolbachia strains. We found the core protein pairs and a subset of interacting partners to be highly conserved within Wolbachia and these other Anaplasmataceae. Genes involved in two-component signaling were positioned differently within the various Wolbachia genomes, whereas the local context of each gene was conserved. Unlike Anaplasma and Ehrlichia, Wolbachia two-component genes were more consistently found clustered with metabolic genes. The domain architecture and key functional residues standard for two-component system proteins were well-conserved in Wolbachia, although residues that specify cognate pairing diverged substantially from other Anaplasmataceae. These findings indicate that Wolbachia two-component signaling pairs share considerable functional overlap with other α-proteobacterial systems, whereas their divergence suggests the potential for regulatory differences and cross-talk. PMID:25809075

Characterization of Lactobacillus salivarius CECT 5713, a strain isolated from human milk: from genotype to phenotype.

PubMed

Langa, Susana; Maldonado-Barragán, Antonio; Delgado, Susana; Martín, Rebeca; Martín, Virginia; Jiménez, Esther; Ruíz-Barba, José L; Mayo, Baltasar; Connor, Ruth I; Suárez, Juan Evaristo; Rodríguez, Juan M

2012-06-01

Lactobacillus salivarius CECT 5713, isolated from human milk, has immunomodulatory, anti-inflammatory and antiinfectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, the relationships between several genetic features of L. salivarius CECT 5713 and the corresponding phenotypes were evaluated. Although it contains a plasmid-encoded bacteriocin cluster, no bacteriocin biosynthesis was observed, possibly due to a 4-bp deletion at the beginning of the histidine kinase determinant abpK. The genome of L. salivarius CECT 5713 harbours two apparently complete prophages of 39.6 and 48 kbp. Upon induction, the 48-kbp prophage became liberated from the bacterial genome, but no DNA replication took place, which resulted in lysis of the cultures but not in phage progeny generation. The strain was sensitive to most antibiotics tested and no transmissible genes potentially involved in antibiotic resistance were detected. Finally, the genome of L. salivarius CECT 5713 contained four ORFs potentially involved in human molecular mimetism. Among them, protein 1230 was considered of particular relevance because of its similarity with dendritic cell-related proteins. Subsequently, in vitro assays revealed the ability of L. salivarius CECT 5713 to stimulate the maturation of immature dendritic cells and to inhibit the in vitro infectivity of HIV-1.

Identification of New Drug Targets in Multi-Drug Resistant Bacterial Infections

DTIC Science & Technology

2014-10-01

adenylation domain of ABBFA_003406. We used mass spectrometry to test if threonine once activated by the adenylating activity of ABBFA_003406 becomes...loaded with threonine , forming a thioester linkage. The ABBFA_003404 carrier protein is produced with a histidine-tag, used for purification, that is...increase in mass of 341 Da. Further addition of the threonine moiety results in an increase in mass of an additional 73 Da. 13 W81XWH-11-2-0218

A variant System I for cytochrome c biogenesis in archaea and some bacteria has a novel CcmE and no CcmH.

PubMed

Allen, James W A; Harvat, Edgar M; Stevens, Julie M; Ferguson, Stuart J

2006-09-04

C-type cytochromes are characterized by post-translational covalent attachment of heme to thiols that occur in a Cys-Xxx-Xxx-Cys-His motif. Three distinct biogenesis systems are known for this heme attachment. Archaea are now shown to contain a significantly modified form of cytochrome c maturation System I (the Ccm system). The most notable adaptation relative to the well-studied apparatus from proteobacteria and plants is a novel form of the heme chaperone CcmE, lacking the highly conserved histidine that covalently binds heme and is essential for function in Escherichia coli. In most archaeal CcmEs this histidine, normally found in a His-Xxx-Xxx-Xxx-Tyr motif, is replaced by a cysteine residue that occurs in a Cys-Xxx-Xxx-Xxx-Tyr motif. The CcmEs from two halobacteria contain yet another form of CcmE, having HxxxHxxxH approximately corresponding in alignment to the H/CxxxY motif. The CxxxY-type of CcmE is, surprisingly, also found in some bacterial genomes (including Desulfovibrio species). All of the modified CcmEs cluster together in a phylogenetic tree, as do other Ccm proteins from the same organisms. Significantly, CcmH is absent from all of the complete archaeal genomes we have studied, and also from most of the bacterial genomes that have CxxxY-type CcmE.

Scombroid poisoning: a review.

PubMed

Hungerford, James M

2010-08-15

Scombroid poisoning, also called histamine fish poisoning, is an allergy-like form of food poisoning that continues to be a major problem in seafood safety. The exact role of histamine in scombroid poisoning is not straightforward. Deviations from the expected dose-response have led to the advancement of various possible mechanisms of toxicity, none of them proven. Histamine action levels are used in regulation until more is known about the mechanism of scombroid poisoning. Scombroid poisoning and histamine are correlated but complicated. Victims of scombroid poisoning respond well to antihistamines, and chemical analyses of fish implicated in scombroid poisoning generally reveal elevated levels of histamine. Scombroid poisoning is unique among the seafood toxins since it results from product mishandling rather than contamination from other trophic levels. Inadequate cooling following harvest promotes bacterial histamine production, and can result in outbreaks of scombroid poisoning. Fish with high levels of free histidine, the enzyme substrate converted to histamine by bacterial histidine decarboxylase, are those most often implicated in scombroid poisoning. Laboratory methods and screening methods for detecting histamine are available in abundance, but need to be compared and validated to harmonize testing. Successful field testing, including dockside or on-board testing needed to augment HACCP efforts will have to integrate rapid and simplified detection methods with simplified and rapid sampling and extraction. Otherwise, time-consuming sample preparation reduces the impact of gains in detection speed on the overall analysis time. Published by Elsevier Ltd.

Characterization of the sensor domain of QseE histidine kinase from Escherichia coli.

PubMed

Yeo, Kwon Joo; Park, Jin-Wan; Kim, Eun-Hee; Jeon, Young Ho; Hwang, Kwang Yeon; Cheong, Hae-Kap

2016-10-01

In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. (1)H-(15)N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

PubMed

Dent, P; Chow, Y H; Wu, J; Morrison, D K; Jove, R; Sturgill, T W

1994-10-01

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, J. Richard; Dunham, Steve; Mochalkin, Igor

2009-06-25

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidinesmore » target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.« less

Signaling through protein kinases and transcriptional regulators in Candida albicans.

PubMed

Dhillon, Navneet K; Sharma, Sadhna; Khuller, G K

2003-01-01

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. Several signaling pathways that regulate morphogenesis have been identified, including various transcription factors that either activate or repress hypha-specific genes. Two well-characterized pathways include the MAP kinase cascade and cAMP-dependent protein kinase pathway that regulate the transcription factors Cph1p and Efg1p, respectively. cAMP also appears to interplay with other second messengers: Ca2+, inositol tri-phosphates in regulating yeast-hyphal transition. Other, less-characterized pathways include two component histidine kinases, cyclin-dependent kinase pathway, and condition specific pathways such as pH and embedded growth conditions. Nrg1 and Rfg1 function as transcriptional repressors of hyphal genes via recruitment of Tup1 co-repressor complex. Different upstream signals converge into a common downstream output during hyphal switch. The levels of expression of several genes have been shown to be associated with hyphal morphogenesis rather than with a specific hypha-inducing condition. Hyphal development is also linked to the expression of a range of other virulence factors. This review explains the relative contribution of multiple pathways that could be used by Candida albican cells to sense subtle differences in the growth conditions of its native host environment.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolodkin-Gal, I; Elsholz, AKW; Muth, C

2013-04-29

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showedmore » that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.« less

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

PubMed Central

Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

2013-01-01

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

PubMed Central

Schaenzer, Adam J.; Wlodarchak, Nathan; Drewry, David H.; Zuercher, William J.; Rose, Warren E.; Striker, Rob; Sauer, John-Demian

2017-01-01

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition. PMID:28821610

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

PubMed

Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

2017-10-13

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.

PubMed

Kim, Sung Hoon; Jeyakumar, M; Katzenellenbogen, John A

2007-10-31

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

The Copper Active Site of CBM33 Polysaccharide Oxygenases

PubMed Central

2013-01-01

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

An x-ray absorption study of the iron site in bacterial photosynthetic reaction centers.

PubMed Central

Bunker, G; Stern, E A; Blankenship, R E; Parson, W W

1982-01-01

Measurements were made of the extended x-ray absorption fine structure (EXAFS) of the iron site in photosynthetic reaction centers from the bacterium Rhodopseudomonas sphaeroides. Forms with two quinones, two quinones with added o-phenanthroline, and one quinone were studied. Only the two forms containing two quinones maintained their integrity and were analyzed. The spectra show directly that the added o-phenanthroline does not chelate the iron atom. Further analysis indicates that the iron is octahedrally coordinated by nitrogen and/or oxygen atoms located at various distances, with the average value of about 2.14 A. The analysis suggests that most of the ligands are nitrogens and that three of the nitrogen ligands belong to histidine rings. This interpretation accounts for several unusual features of the EXAFS spectrum. We speculate that the quinones are bound to the histidine rings in some manner. Qualitative features of the absorption edge spectra also are discussed and are related to the Fe-ligand distance. PMID:6977382

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-β Synthesis Induced by Lipopolysaccharide.

PubMed

Saleh, Danish; Najjar, Malek; Zelic, Matija; Shah, Saumil; Nogusa, Shoko; Polykratis, Apostolos; Paczosa, Michelle K; Gough, Peter J; Bertin, John; Whalen, Michael; Fitzgerald, Katherine A; Slavov, Nikolai; Pasparakis, Manolis; Balachandran, Siddharth; Kelliher, Michelle; Mecsas, Joan; Degterev, Alexei

2017-06-01

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-β. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-β production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-β synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-β production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-β production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-β is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella , and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-β during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors. Copyright © 2017 by The American Association of Immunologists, Inc.

Tyrosine-610 in the receptor kinase BAK1 does not play a major role in brassinosteroid signaling or innate immunity

USDA-ARS?s Scientific Manuscript database

The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING ...

Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa

PubMed Central

Wang, Dan; Chen, Weizhong; Huang, Shanqing; He, Yafeng; Liu, Xichun; Hu, Qingyuan; Wei, Tianbiao; Sang, Hong; Gan, Jianhua

2017-01-01

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/β-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1’ α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents. PMID:28732057

Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa.

PubMed

Wang, Dan; Chen, Weizhong; Huang, Shanqing; He, Yafeng; Liu, Xichun; Hu, Qingyuan; Wei, Tianbiao; Sang, Hong; Gan, Jianhua; Chen, Hao

2017-07-01

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/β-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1' α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia

PubMed Central

Pollak, Adam J.; Haghighi, Kobra; Kunduri, Swati; Arvanitis, Demetrios A.; Liu, Guan-Sheng; Singh, Vivek P.; Gonzalez, David J.; Sanoudou, Despina; Wiley, Sandra E.; Dixon, Jack E.; Kranias, Evangelia G.

2017-01-01

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias. PMID:28784772

Regulation of Response Regulator Autophosphorylation through Interdomain Contacts*♦

PubMed Central

Barbieri, Christopher M.; Mack, Timothy R.; Robinson, Victoria L.; Miller, Matthew T.; Stock, Ann M.

2010-01-01

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-β5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo. PMID:20702407

pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeo, Kwon Joo; Kim, Eun Hye; Hwang, Eunha

2013-02-15

Highlights: ► We described the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK histidine kinase. ► The ESD of DraK showed a reversible pH-dependent conformational change in a wide pH range. ► The E83 is an important residue for the pH-dependent conformational change. -- Abstract: Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism,more » structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5–10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.« less

2.3 Å X-ray Structure of the Heme-Bound GAF Domain of Sensory Histidine Kinase DosT of Mycobacterium tuberculosis†

PubMed Central

Podust, Larissa M.; Ioanoviciu, Alexandra; Ortiz de Montellano, Paul R.

2009-01-01

Mycobacterium tuberculosis responds to the changes in environmental conditions through a two-component signaling system that detects reduced O2 tension and NO and CO exposures via the heme-binding GAF domains of two sensory histidine kinases, DosT and DevS, and the transcriptional regulator DosR. We report the first x-ray structure of the DosT heme-bound GAF domain (GAFDosT) in both oxy and deoxy forms determined to a resolution of 2.3 Å. In GAFDosT, heme binds in an orientation orthogonal to that in the PAS domains via a highly conserved motif including invariant H147 as a proximal heme axial ligand. On the distal side, invariant Y169 is in stacking interactions with the heme with its long axis parallel and the plane of the ring orthogonal to the heme plane. In one of the two protein monomers in an asymmetric unit, O2 binds as a second axial ligand to the heme iron, and is stabilized via an H-bond to the OH-group of Y169. The structure reveals two small tunnel-connected cavities and a pore on the protein surface that suggest a potential route for O2 access to the sensing pocket. The limited conformational differences observed between differently heme iron-ligated GAFDosT monomers in the asymmetric unit may result from crystal lattice limitations since atmospheric oxygen binding likely occurs in the crystal as a result of x-ray induced Fe3+ photoreduction during diffraction data collection. Determination of the GAFDosT structure sets up a framework in which to address ligand-recognition, discrimination, and signal propagation schemes in the heme-based GAF domains of biological sensors. PMID:18980385

Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity.

PubMed

Erttmann, Saskia F; Härtlova, Anetta; Sloniecka, Marta; Raffi, Faizal A M; Hosseinzadeh, Ava; Edgren, Tomas; Rofougaran, Reza; Resch, Ulrike; Fällman, Maria; Ek, Torben; Gekara, Nelson O

2016-07-19

The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1β (IL-1β) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Arm-in-Arm Response Regulator Dimers Promote Intermolecular Signal Transduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Anna W.; Satyshur, Kenneth A.; Moreno Morales, Neydis

2016-02-01

ABSTRACT Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacteriumRamlibacter tataouinensis. RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, theR. tataouinensisbacteriophytochrome response regulatormore » (RtBRR), and a homolog, AtBRR fromAgrobacterium tumefaciens, crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRR mon) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems. IMPORTANCEBphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.« less

Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays*

PubMed Central

Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

2016-01-01

Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs. PMID:26902206

Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline-Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays.

PubMed

Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

2016-06-01

Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response

PubMed Central

Lamacchia, Marina; Dyrka, Witold; Breton, Annick; Saupe, Sven J.; Paoletti, Mathieu

2016-01-01

Recognition and response to non self is essential to development and survival of all organisms. It can occur between individuals of the same species or between different organisms. Fungi are established models for conspecific non self recognition in the form of vegetative incompatibility (VI), a genetically controlled process initiating a programmed cell death (PCD) leading to the rejection of a fusion cell between genetically different isolates of the same species. In Podospora anserina VI is controlled by members of the hnwd gene family encoding for proteins analogous to NOD Like Receptors (NLR) immune receptors in eukaryotes. It was hypothesized that the hnwd controlled VI reaction was derived from the fungal innate immune response. Here we analyze the P. anserina transcriptional responses to two bacterial species, Serratia fonticola to which P. anserina survives and S. marcescens to which P. anserina succumbs, and compare these to the transcriptional response induced under VI conditions. Transcriptional responses to both bacteria largely overlap, however the number of genes regulated and magnitude of regulation is more important when P. anserina survives. Transcriptional responses to bacteria also overlap with the VI reaction for both up or down regulated gene sets. Genes up regulated tend to be clustered in the genome, and display limited phylogenetic distribution. In all three responses we observed genes related to autophagy to be up-regulated. Autophagy contributes to the fungal survival in all three conditions. Genes encoding for secondary metabolites and histidine kinase signaling are also up regulated in all three conditions. Transcriptional responses also display differences. Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI. Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death. These differences are discussed in the frame of a multilayered response to non self in fungi. PMID:27148175

Molecular cloning of MSSP-2, a c-myc gene single-strand binding protein: characterization of binding specificity and DNA replication activity.

PubMed Central

Takai, T; Nishita, Y; Iguchi-Ariga, S M; Ariga, H

1994-01-01

We have previously reported the human cDNA encoding MSSP-1, a sequence-specific double- and single-stranded DNA binding protein [Negishi, Nishita, Saëgusa, Kakizaki, Galli, Kihara, Tamai, Miyajima, Iguchi-Ariga and Ariga (1994) Oncogene, 9, 1133-1143]. MSSP-1 binds to a DNA replication origin/transcriptional enhancer of the human c-myc gene and has turned out to be identical with Scr2, a human protein which complements the defect of cdc2 kinase in S.pombe [Kataoka and Nojima (1994) Nucleic Acid Res., 22, 2687-2693]. We have cloned the cDNA for MSSP-2, another member of the MSSP family of proteins. The MSSP-2 cDNA shares highly homologous sequences with MSSP-1 cDNA, except for the insertion of 48 bp coding 16 amino acids near the C-terminus. Like MSSP-1, MSSP-2 has RNP-1 consensus sequences. The results of the experiments using bacterially expressed MSSP-2, and its deletion mutants, as histidine fusion proteins suggested that the binding specificity of MSSP-2 to double- and single-stranded DNA is the same as that of MSSP-1, and that the RNP consensus sequences are required for the DNA binding of the protein. MSSP-2 stimulated the DNA replication of an SV40-derived plasmid containing the binding sequence for MSSP-1 or -2. MSSP-2 is hence suggested to play an important role in regulation of DNA replication. Images PMID:7838710

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli.

PubMed

Park, Young-Ha; Lee, Chang-Ro; Choe, Mangyu; Seok, Yeong-Jae

2013-12-24

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli

PubMed Central

Park, Young-Ha; Lee, Chang-Ro; Choe, Mangyu; Seok, Yeong-Jae

2013-01-01

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIAGlc (EIIAGlc) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIAGlc in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ70 in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ70. Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ70-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ70 and σS for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ70 activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd. PMID:24324139

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia.

PubMed

Packiriswamy, Nandakumar; Steury, Michael; McCabe, Ian C; Fitzgerald, Scott D; Parameswaran, Narayanan

2016-05-01

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants.

PubMed

Strachan, G; Grant, S D; Learmonth, D; Longstaff, M; Porter, A J; Harris, W J

1998-09-15

Single-chain antibody fragments (scAb), specific for the herbicide atrazine, have been expressed in the bacterium Escherichia coli and in transgenic tobacco plants. The scAb could be purified as a monomer (monovalent) via a hexa-histidine tail or as a dimer (divalent) by antibody affinity chromatography. In competition ELISA, the bacterial scAb showed the same specificity for atrazine and related triazine herbicides as the parental mAb cell line, but both plant and bacterial monomeric scAbs showed increased sensitivity to free atrazine. Surface plasmon resonance (BIAcore 2000) analysis confirmed that purified scAb, derived from plant or bacteria, retained similar association rates as the mAb. However, the monomeric plant and bacterial scAbs showed a lower affinity for immobilised antigen, than the equivalent dimeric scAbs or mAb. This decrease in affinity was due to a 10 fold slower dissociation rate and is likely due to loss of the avidity contribution of dimeric molecules.

IRAK4 activity controls immune responses to intracellular bacteria Listeria monocytogenes and Mycobacterium smegmatis.

PubMed

Pattabiraman, Goutham; Murphy, Michael; Agliano, Federica; Karlinsey, Keaton; Medvedev, Andrei E

2018-05-11

IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4 KI ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4 KI macrophages expressed decreased TNF-α, IL-6, IL-1β, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4 KI macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4 KI mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO. ©2018 Society for Leukocyte Biology.

Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae.

PubMed

Piñas, Germán E; Reinoso-Vizcaino, Nicolás M; Yandar Barahona, Nubia Y; Cortes, Paulo R; Duran, Rosario; Badapanda, Chandan; Rathore, Ankita; Bichara, Dario R; Cian, Melina B; Olivero, Nadia B; Perez, Daniel R; Echenique, José

2018-06-08

Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE's DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128A and wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128A mutant, the repression of spxB and sodA correlated with decreased H2O2 production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128A mutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2 production, are likely responsible for the increased survival in pneumocytes of the comET128A mutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.

Overlapping and lineage-specific roles for the type-B response regulators of monocots and dicots.

PubMed

Kim, Hyo Jung; Kieber, Joseph J; Schaller, G Eric

2012-09-01

Cytokinins are plant hormones with profound roles in growth and development. Cytokinin signaling is mediated through a 'two-component' signaling system composed of histidine kinases, histidine-containing phosphotransfer proteins, and response regulators. Phylogenetic analysis of two-component signaling elements from the monocot rice and the dicot Arabidopsis reveals lineage-specific expansions of the type-B response regulators, transcription factors that act as positive regulators for the cytokinin signal. We recently reported in Plant Physiology on a functional analysis of rice type-B response regulators. A type-B response regulator from a subfamily comprised of both monocot and dicot type-B response regulators complemented an Arabidopsis type-B response regulator mutant, but a type-B response regulator from a monocot-specific subfamily generally did not. Here, we extend this analysis to demonstrate that the promoter of an Arabidopsis cytokinin primary response gene is induced by type-B response regulators from a shared subfamily, but not by one from a lineage-specific subfamily. These results support a model in which the type-B response regulators of monocots and dicots share conserved roles in the cytokinin signaling pathway but have also diverged to take on lineage-specific roles.

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio

2013-06-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2more » is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.« less

The Salmonella enterica serovar Typhimurium QseB Response Regulator Negatively Regulates Bacterial Motility and Swine Colonization in the Absence of the QseC Sensor Kinase

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serovar Typhimurium (S. Typhimurium) responds to the catecholamine, norepinephrine by increasing bacterial growth and enhancing motility. In this study, iron with or without the siderophore, ferrioxamine E also enhanced bacterial motility. Iron-enhanced motility was growth-rate ...

Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling.

PubMed

Zhou, Xikun; Ye, Yan; Sun, Yuyang; Li, Xuefeng; Wang, Wenxue; Privratsky, Breanna; Tan, Shirui; Zhou, Zongguang; Huang, Canhua; Wei, Yu-Quan; Birnbaumer, Lutz; Singh, Brij B; Wu, Min

2015-08-01

Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca(2+) homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1(-/-) mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca(2+) entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca(2+) entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca(2+) entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

PubMed

Molenaar, D; Bosscher, J S; ten Brink, B; Driessen, A J; Konings, W N

1993-05-01

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.

High energy phosphate transfer by NDPK B/Gbetagammacomplexes--an alternative signaling pathway involved in the regulation of basal cAMP production.

PubMed

Hippe, Hans-Joerg; Wieland, Thomas

2006-08-01

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein alpha -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the beta-subunit of G proteins (Gbeta) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers in which NDPK B acts as a histidine kinase phosphorylating Gbeta at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein alpha-subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

PubMed

Bueno, Clara; Lemke, Caitlin D; Criado, Gabriel; Baroja, Miren L; Ferguson, Stephen S G; Rahman, A K M Nur-Ur; Tsoukas, Constantine D; McCormick, John K; Madrenas, Joaquin

2006-07-01

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Effect of dietary electrolytes and histidine on histidine metabolism and acid-base balance in rainbow trout (Salmo gairdneri)

USGS Publications Warehouse

Chiu, Y.N.; Austic, R.E.; Rumsey, G.L.

1984-01-01

1. Rainbow trout fingerlings were fed diets containing 1.2, 1.8 and 2.6% histidine and two mixtures of Na, K and Cl (Na + K - Cl = 0 or -200 meq/kgdiet) in a factorial design.2. Growth and efficiency of feed conversion were not affected by histidine in the diet when it contained the −200 meq/kg electrolyte mixture, but with the 0 meq/kg level, 2.6% histidine depressed both measures of response.3. Histidine increased plasma and muscle histidine levels, increased hepatic histidase activity, but did not affect hepatic histidine-pyruvate aminotransferase activity.4. Muscle-free histidine concentrations were markedly higher and lysine concentrations were lower in trout receiving 0 meq/kg than those receiving the −200 meq/kg electrolyte mixture.5. The electrolyte balance of the diet has a marked effect on the metabolism of histidine in trout.

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway.

PubMed

Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Isono, Toshihito; Nakamura, Yuki; Saitoh, Issei; Hayasaki, Haruaki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

2018-01-01

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine-threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

PubMed Central

Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

2014-01-01

SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

PubMed Central

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family

PubMed Central

Lesne, Elodie; Dupré, Elian; Lensink, Marc F.; Locht, Camille

2018-01-01

ABSTRACT Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical “X” linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family. PMID:29487240

The yeasts phosphorelay systems: a comparative view.

PubMed

Salas-Delgado, Griselda; Ongay-Larios, Laura; Kawasaki-Watanabe, Laura; López-Villaseñor, Imelda; Coria, Roberto

2017-06-01

Cells contain signal transduction pathways that mediate communication between the extracellular environment and the cell interior. These pathways control transcriptional programs and posttranscriptional processes that modify cell metabolism in order to maintain homeostasis. One type of these signal transduction systems are the so-called Two Component Systems (TCS), which conduct the transfer of phosphate groups between specific and conserved histidine and aspartate residues present in at least two proteins; the first protein is a sensor kinase which autophosphorylates a histidine residue in response to a stimulus, this phosphate is then transferred to an aspartic residue located in a response regulator protein. There are classical and hybrid TCS, whose difference consists in the number of proteins and functional domains involved in the phosphorelay. The TCS are widespread in bacteria where the sensor and its response regulator are mostly specific for a given stimulus. In eukaryotic organisms such as fungi, slime molds, and plants, TCS are present as hybrid multistep phosphorelays, with a variety of arrangements (Stock et al. in Annu Rev Biochem 69:183-215, 2000; Wuichet et al. in Curr Opin Microbiol 292:1039-1050, 2010). In these multistep phosphorelay systems, several phosphotransfer events take place between different histidine and aspartate residues localized in specific domains present in more than two proteins (Thomason and Kay, in J Cell Sci 113:3141-3150, 2000; Robinson et al. in Nat Struct Biol 7:626-633, 2000). This review presents a brief and succinct description of the Two-component systems of model yeasts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Cryptococcus neoformans and Kluyveromyces lactis. We have focused on the comparison of domain organization and functions of each component present in these phosphorelay systems.

Signal transduction and phosphoryl transfer by a FixL hybrid kinase with low oxygen affinity: importance of the vicinal PAS domain and receiver aspartate.

PubMed

Sousa, Eduardo H S; Tuckerman, Jason R; Gondim, Ana C S; Gonzalez, Gonzalo; Gilles-Gonzalez, Marie-Alda

2013-01-22

FixL is a prototype for heme-based sensors, multidomain proteins that typically couple a histidine protein kinase activity to a heme-binding domain for sensing of diatomic gases such as oxygen, carbon monoxide, and nitric oxide. Despite the relatively well-developed understanding of FixL, the importance of some of its domains has been unclear. To explore the impact of domain-domain interactions on oxygen sensing and signal transduction, we characterized and investigated Rhizobium etli hybrid sensor ReFixL. In ReFixL, the core heme-containing PAS domain and kinase region is preceded by an N-terminal PAS domain of unknown function and followed by a C-terminal receiver domain. The latter resembles a target substrate domain that usually occurs independently of the kinase and contains a phosphorylatable aspartate residue. We isolated the full-length ReFixL as a soluble holoprotein with a single heme b cofactor. Despite a low affinity for oxygen (K(d) for O₂ of 738 μM), the kinase activity was completely switched off by O₂ at concentrations well below the K(d). A deletion of the first PAS domain strongly increased the oxygen affinity but essentially prohibited autophosphorylation, although the truncated protein was competent to accept phosphoryl groups in trans. These studies provide new insights into histidyl-aspartyl phosphoryl transfers in two-component systems and suggest that the control of ligand affinity and signal transduction by PAS domains can be direct or indirect.

High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer.

PubMed

Bhoir, Siddhant; Shaik, Althaf; Thiruvenkatam, Vijay; Kirubakaran, Sivapriya

2018-03-19

Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein-drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

Transduction-like gene transfer in the methanogen Methanococcus voltae

NASA Technical Reports Server (NTRS)

Bertani, G.

1999-01-01

Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks.

PubMed

Jers, Carsten; Soufi, Boumediene; Grangeasse, Christophe; Deutscher, Josef; Mijakovic, Ivan

2008-08-01

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

PubMed

Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

2014-01-01

Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Structure and synthesis of histopine, a histidine derivative produced by crown gall tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bates, H.A.; Kaushal, A.; Deng, P.N.

1984-07-03

Histopine, an unusual amino acid derivative of histidine isolated from crown gall tumors of sunflowers (Helianthus annus) inoculated with Agrobacterium tumefaciens strain B/sub 6/, was previously assigned the gross structure N-(1-carboxyethyl)histidine. A diastereomeric mixture containing histopine was readily prepared by reductive alkylation of (S)-histidine with pyruvic acid and sodium cyanoborohydride. The individual diastereomers were prepared by reaction of (S)-histidine with (R)- and (S)-2-bromopropionic acid. (R)-N-(1-Carboxyethyl)-(S)-histidine supports the growth of A. tumefaciens whereas (S)-N-(1-carboxyethyl)-(S)-histidine is inactive.

Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana.

PubMed

Mottram, J C; McCready, B P; Brown, K G; Grant, K M

1996-11-01

The generation of homozygous null mutants for the crk1 Cdc2-Related Kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a bie-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA-content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L mexicana transfected with a Trypanosoma brucel homologue, tbcrk1, was shown to be viable in an immcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Nl(2+)-nitriloacetic acid (NTA) agarose.

21 CFR 862.1375 - Histidine test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... free histidine (an amino acid) in plasma and urine. Histidine measurements are used in the diagnosis and treatment of hereditary histidinemia characterized by excess histidine in the blood and urine...

21 CFR 862.1375 - Histidine test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... free histidine (an amino acid) in plasma and urine. Histidine measurements are used in the diagnosis and treatment of hereditary histidinemia characterized by excess histidine in the blood and urine...

Sensor kinase KinB and its pathway-associated key factors sense the signal of nutrition starvation in sporulation of Bacillus subtilis.

PubMed

Liu, Weipeng; He, Zeying; Gao, Feng; Yan, Jinyuan; Huang, Xiaowei

2018-01-03

Bacillus subtilis responds to environmental stress cues and develops endospores for survival. In the process of endospore formation, sporulation initiation is a vital stage and this stage is governed by autophosphorylation of the sensor histidine kinases. The second major sensor kinase KinB perceives the intracellular changes of GTP and ATP during sporulation. However, determination of the environmental signals as well as its related signaling pathway of KinB requires further elucidation. Our current study found that, contrary to the sporulation failure induced by ΔkinA in the nutrient-rich 2× SG medium, the sensor kinase KinB sensed the environmental cues in the nutrient-poor MM medium. Two other membrane proteins, KapB and KbaA, also responded similarly to the same external signal as KinB. Both KapB and KbaA acted upstream of KinB, but they exerted their regulation upon KinB independently. Furthermore, we demonstrated that both the SH3 domain and the α-helix structure in KapB are required for sensing or transducing the signal of sporulation initiation. Collectively, our work here supplied the direct evidences that KinB and its pathway sense the external signal of nutrient starvation in MM medium, and further analyzes the interrelationship among KinB, KbaA, and KapB. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Physical factors affecting the mutagenicity of fly ash from a coal-fired power plant.

PubMed

Fisher, G L; Chrisp, C E; Raabe, O G

1979-05-25

The two finest, most respirable coal fly ash fractions collected from the smokestack of a power plant were more mutagenic than two coarser fractions. Mutagenicity was evaluated in the histidine-requiring bacterial strains TA 1538, TA 98, and TA 100 of Salmonella typhimurium. Ash samples collected from the hoppers of an electrostatic precipitator in the plant were not mutagenic. The mutagens in coal fly ash were resistant to x-ray or ultraviolet irradiation, possibly as a result of stabilization by fly ash surfaces. All mutagenic activity is lost with heating to 350 degrees C.

The phosphoenolpyruvate:sugar phosphotransferase system is involved in sensitivity to the glucosylated bacteriocin sublancin.

PubMed

Garcia De Gonzalo, C V; Denham, E L; Mars, R A T; Stülke, J; van der Donk, W A; van Dijl, J M

2015-11-01

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Spectral Studies of Iron Coordination in Hemeprotein Complexes

PubMed Central

Brill, Arthur S.; Sandberg, Howard E.

1968-01-01

In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mμ, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mμ, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mμ is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented. PMID:5699802

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

Prokaryotic ancestry of eukaryotic protein networks mediating innate immunity and apoptosis.

PubMed

Dunin-Horkawicz, Stanislaw; Kopec, Klaus O; Lupas, Andrei N

2014-04-03

Protein domains characteristic of eukaryotic innate immunity and apoptosis have many prokaryotic counterparts of unknown function. By reconstructing interactomes computationally, we found that bacterial proteins containing these domains are part of a network that also includes other domains not hitherto associated with immunity. This network is connected to the network of prokaryotic signal transduction proteins, such as histidine kinases and chemoreceptors. The network varies considerably in domain composition and degree of paralogy, even between strains of the same species, and its repetitive domains are often amplified recently, with individual repeats sharing up to 100% sequence identity. Both phenomena are evidence of considerable evolutionary pressure and thus compatible with a role in the "arms race" between host and pathogen. In order to investigate the relationship of this network to its eukaryotic counterparts, we performed a cluster analysis of organisms based on a census of its constituent domains across all fully sequenced genomes. We obtained a large central cluster of mainly unicellular organisms, from which multicellular organisms radiate out in two main directions. One is taken by multicellular bacteria, primarily cyanobacteria and actinomycetes, and plants form an extension of this direction, connected via the basal, unicellular cyanobacteria. The second main direction is taken by animals and fungi, which form separate branches with a common root in the α-proteobacteria of the central cluster. This analysis supports the notion that the innate immunity networks of eukaryotes originated from their endosymbionts and that increases in the complexity of these networks accompanied the emergence of multicellularity. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

NMR Study Reveals the Receiver Domain of Arabidopsis ETHYLENE RESPONSE1 Ethylene Receptor as an Atypical Type Response Regulator.

PubMed

Hung, Yi-Lin; Jiang, Ingjye; Lee, Yi-Zong; Wen, Chi-Kuang; Sue, Shih-Che

2016-01-01

The gaseous plant hormone ethylene, recognized by plant ethylene receptors, plays a pivotal role in various aspects of plant growth and development. ETHYLENE RESPONSE1 (ETR1) is an ethylene receptor isolated from Arabidopsis and has a structure characteristic of prokaryotic two-component histidine kinase (HK) and receiver domain (RD), where the RD structurally resembles bacteria response regulators (RRs). The ETR1 HK domain has autophosphorylation activity, and little is known if the HK can transfer the phosphoryl group to the RD for receptor signaling. Unveiling the correlation of the receptor structure and phosphorylation status would advance the studies towards the underlying mechanisms of ETR1 receptor signaling. In this study, using the nuclear magnetic resonance technique, our data suggested that the ETR1-RD is monomeric in solution and the rigid structure of the RD prevents the conserved aspartate residue phosphorylation. Comparing the backbone dynamics with other RRs, we propose that backbone flexibility is critical to the RR phosphorylation. Besides the limited flexibility, ETR1-RD has a unique γ loop conformation of opposite orientation, which makes ETR1-RD unfavorable for phosphorylation. These two features explain why ETR1-RD cannot be phosphorylated and is classified as an atypical type RR. As a control, phosphorylation of the ETR1-RD was also impaired when the sequence was swapped to the fragment of the bacterial typical type RR, CheY. Here, we suggest a molecule insight that the ETR1-RD already exists as an active formation and executes its function through binding with the downstream factors without phosphorylation.

Apo-bacteriophytochromes modulate bacterial photosynthesis in response to low light.

PubMed

Fixen, Kathryn R; Baker, Anna W; Stojkovic, Emina A; Beatty, J Thomas; Harwood, Caroline S

2014-01-14

Bacteriophytochromes (BphPs) are light-sensing regulatory proteins encoded by photosynthetic and nonphotosynthetic bacteria. This protein class has been characterized structurally, but its biological activities remain relatively unexplored. Two BphPs in the anoxygenic photosynthetic bacterium Rhodopseudomonas palustris, designated regulatory proteins RpBphP2 and RpBphP3, are configured as light-regulated histidine kinases, which initiate a signal transduction system that controls expression of genes for the low light harvesting 4 (LH4) antenna complex. In vitro, RpBphP2 and RpBphP3 respond to light quality by reversible photoconversion, a property that requires the light-absorbing chromophore biliverdin. In vivo, RpBphP2 and RpBphP3 are both required for the expression of the LH4 antenna complex under anaerobic conditions, but biliverdin requires oxygen for its synthesis by heme oxygenase. On further investigation, we found that the apo-bacteriophytochrome forms of RpBphP2 and RpBphP3 are necessary and sufficient to control LH4 expression in response to light intensity in conjunction with other signal transduction proteins. One possibility is that the system senses a reduced quinone pool generated when light energy is absorbed by bacteriochlorophyll. The biliverdin-bound forms of the BphPs have the additional property of being able to fine-tune LH4 expression in response to light quality. These observations support the concept that some bacteriophytochromes can function with or without a chromophore and may be involved in regulating physiological processes not directly related to light sensing.

Pharmacokinetics of stable isotopically labeled L-histidine in humans and the assessment of in vivo histidine ammonia lyase activities.

PubMed

Furuta, T; Okamiya, K; Shibasaki, H; Kasuya, Y

1996-01-01

The pharmacokinetics of L-histidine in humans has been investigated to evaluate the in vivo histidine ammonia lyase system for the conversion of L-histidine to urocanic acid. Two healthy volunteers (subjects A and B) received a single 100-mg oral dose of L-[3,3-2H2,1',3'-15N2]histidine. Blood and urine samples were obtained over 24 hr after the administration and analyzed by stable isotope dilution ms. Labeled L-histidine was rapidly absorbed, and a maximum plasma concentration of L-histidine was observed at 30 min (1057.6 ng/ml) in subject A and at 60 min (1635.6 ng/ml) in subject B after oral administration. Pharmacokinetic parameters were calculated based on a two-compartment model. Labeled L-histidine in subject A (t1/2 = 1.0 hr) was eliminated approximately twice faster than that in subject B (t1/2 = 1.9 hr). Total body clearances were 70.0 liters/hr in subject A and 30.0 liters/hr in subject B. The low ratios of the renal clearance to the total body clearance (1.04% for subject A and 0.43% for subject B) indicated that most of L-histidine was eliminated via the nonrenal processes. L-Histidine was rapidly metabolized to urocanic acid. Maximum plasma concentrations of urocanic acid were 59.61 ng/ml at 30 min for subject A and 46.10 ng/ml at 60 min for subject B. The slope of the plot of urinary excretion rate of urocanic acid vs. the plasma concentration of unchanged L-histidine was demonstrated to reflect the metabolic clearance of L-histidine to urocanic acid. The method of evaluating the in vivo human histidine ammonia lyase activities discussed in this study offers a significant value with regard to the biochemical and clinical elucidations of the heterogeneity of histidinemia.

Interplay between the Localization and Kinetics of Phosphorylation in Flagellar Pole Development of the Bacterium Caulobacter crescentus

PubMed Central

Tropini, Carolina; Huang, Kerwyn Casey

2012-01-01

Bacterial cells maintain sophisticated levels of intracellular organization that allow for signal amplification, response to stimuli, cell division, and many other critical processes. The mechanisms underlying localization and their contribution to fitness have been difficult to uncover, due to the often challenging task of creating mutants with systematically perturbed localization but normal enzymatic activity, and the lack of quantitative models through which to interpret subtle phenotypic changes. Focusing on the model bacterium Caulobacter crescentus, which generates two different types of daughter cells from an underlying asymmetric distribution of protein phosphorylation, we use mathematical modeling to investigate the contribution of the localization of histidine kinases to the establishment of cellular asymmetry and subsequent developmental outcomes. We use existing mutant phenotypes and fluorescence data to parameterize a reaction-diffusion model of the kinases PleC and DivJ and their cognate response regulator DivK. We then present a systematic computational analysis of the effects of changes in protein localization and abundance to determine whether PleC localization is required for correct developmental timing in Caulobacter. Our model predicts the developmental phenotypes of several localization mutants, and suggests that a novel strain with co-localization of PleC and DivJ could provide quantitative insight into the signaling threshold required for flagellar pole development. Our analysis indicates that normal development can be maintained through a wide range of localization phenotypes, and that developmental defects due to changes in PleC localization can be rescued by increased PleC expression. We also show that the system is remarkably robust to perturbation of the kinetic parameters, and while the localization of either PleC or DivJ is required for asymmetric development, the delocalization of one of these two components does not prevent flagellar pole development. We further find that allosteric regulation of PleC observed in vitro does not affect the predicted in vivo developmental phenotypes. Taken together, our model suggests that cells can tolerate perturbations to localization phenotypes, whose evolutionary origins may be connected with reducing protein expression or with decoupling pre- and post-division phenotypes. PMID:22876167

Protein Kinase LegK2 Is a Type IV Secretion System Effector Involved in Endoplasmic Reticulum Recruitment and Intracellular Replication of Legionella pneumophila ▿

PubMed Central

Hervet, Eva; Charpentier, Xavier; Vianney, Anne; Lazzaroni, Jean-Claude; Gilbert, Christophe; Atlan, Danièle; Doublet, Patricia

2011-01-01

Legionella pneumophila is the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses. PMID:21321072

Mutagenic activity of overnight urine from healthy non-smoking subjects.

PubMed

Pavanello, Sofia; Lupi, Silvia; Pulliero, Alessandra; Gregorio, Pasquale; Saia, Bruno Onofrio; Clonfero, Erminio

2007-03-01

Urinary mutagenicity was evaluated in relation to environmental mutagen exposure (i.e., diet, indoor/outdoor activities, residential area etc.) on the day prior to sample collection, and also considering factors that contribute to the variability of Salmonella mutagenicity assay results. Overnight urine samples from 283 healthy non-smoking residents of northeast Italy (46% males, 20-62 years) were analyzed for mutagenicity on sensitive Salmonella typhimurium strain YG1024 with S9 mix employing the preincubation version of the plate incorporation assay (i.e., the Salmonella reverse mutation test). Urinary mutagenicity varied between 0.02 and 9.84 rev/ equiv. ml, and 7% of samples were positive (i.e., sample elicited a two-fold increase in revertants). There was an evident increase in mutagenicity in subjects with increased intake of mutagen-rich meals (n = 80) (P < 0.01 and positive urine 13% vs. 5%, P = 0.025). Indoor-exposed subjects (n = 65) also showed a higher percentage of positive urine (14% vs. 5%, P = 0.015). In particular, those subjects exposed to cooking fumes the previous evening (n = 28) revealed higher urinary mutagenicity (P = 0.035, positive urine 25% vs. 5%, P < 0.001) than non-indoor exposed. The sources of variability of the mutagenicity assay, mainly the histidine content of the urine concentrate (z = 4.06, P < 0.0001), and to a lesser extent bacterial inoculum size (z = 2.33, P = 0.019), also significantly influenced urinary mutagenicity values. In a linear multiple regression analysis, their effects were still significant (i.e., histidine content P = 0.026 and inoculum size P = 0.021), but the effects of diet, indoor exposure, and other environmental exposures (i.e., traffic and heating system exhausts, residential area) were not. It is concluded that the previous day's exposure to mutagen-rich meals and cooking fumes may influence the presence of mutagenic activity in the overnight urine of non-smoking subjects. This mutagenic activity, which remains in contact with bladder mucosa for several hours, could be considered risk factors for colorectal adenoma and possibly other cancers (i.e., bladder) in non-smokers. Accurate control of histidine content and bacterial inoculum size is strongly recommended when investigating the mutagenic activity of urine from non-smokers. (c) 2007 Wiley-Liss, Inc.

Histamine fish poisoning revisited.

PubMed

Lehane, L; Olley, J

2000-06-30

Histamine (or scombroid) fish poisoning (HFP) is reviewed in a risk-assessment framework in an attempt to arrive at an informed characterisation of risk. Histamine is the main toxin involved in HFP, but the disease is not uncomplicated histamine poisoning. Although it is generally associated with high levels of histamine (> or =50 mg/100 g) in bacterially contaminated fish of particular species, the pathogenesis of HFP has not been clearly elucidated. Various hypotheses have been put forward to explain why histamine consumed in spoiled fish is more toxic than pure histamine taken orally, but none has proved totally satisfactory. Urocanic acid, like histamine, an imidazole compound derived from histidine in spoiling fish, may be the "missing factor" in HFP. cis-Urocanic acid has recently been recognised as a mast cell degranulator, and endogenous histamine from mast cell degranulation may augment the exogenous histamine consumed in spoiled fish. HFP is a mild disease, but is important in relation to food safety and international trade. Consumers are becoming more demanding, and litigation following food poisoning incidents is becoming more common. Producers, distributors and restaurants are increasingly held liable for the quality of the products they handle and sell. Many countries have set guidelines for maximum permitted levels of histamine in fish. However, histamine concentrations within a spoiled fish are extremely variable, as is the threshold toxic dose. Until the identity, levels and potency of possible potentiators and/or mast-cell-degranulating factors are elucidated, it is difficult to establish regulatory limits for histamine in foods on the basis of potential health hazard. Histidine decarboxylating bacteria produce histamine from free histidine in spoiling fish. Although some are present in the normal microbial flora of live fish, most seem to be derived from post-catching contamination on board fishing vessels, at the processing plant or in the distribution system, or in restaurants or homes. The key to keeping bacterial numbers and histamine levels low is the rapid cooling of fish after catching and the maintenance of adequate refrigeration during handling and storage. Despite the huge expansion in trade in recent years, great progress has been made in ensuring the quality and safety of fish products. This is largely the result of the introduction of international standards of food hygiene and the application of risk analysis and hazard analysis and critical control point (HACCP) principles.

Role of the Putative Osmosensor Arabidopsis Histidine Kinase1 in Dehydration Avoidance and Low-Water-Potential Response1[W][OA

PubMed Central

Kumar, M. Nagaraj; Jane, Wann-Neng; Verslues, Paul E.

2013-01-01

The molecular basis of plant osmosensing remains unknown. Arabidopsis (Arabidopsis thaliana) Histidine Kinase1 (AHK1) can complement the osmosensitivity of yeast (Saccharomyces cerevisiae) osmosensor mutants lacking Synthetic Lethal of N-end rule1 and SH3-containing Osmosensor and has been proposed to act as a plant osmosensor. We found that ahk1 mutants in either the Arabidopsis Nossen-0 or Columbia-0 background had increased stomatal density and stomatal index consistent with greater transpirational water loss. However, the growth of ahk1 mutants was not more sensitive to controlled moderate low water potential (ψw) or to salt stress. Also, ahk1 mutants had increased, rather than reduced, solute accumulation across a range of low ψw severities. ahk1 mutants had reduced low ψw induction of Δ1-Pyrroline-5-Carboxylate Synthetase1 (P5CS1) and 9-cis-Epoxycarotenoid Dioxygenase3, which encode rate-limiting enzymes in proline and abscisic acid (ABA) synthesis, respectively. However, neither Pro nor ABA accumulation was reduced in ahk1 mutants at low ψw. P5CS1 protein level was not reduced in ahk1 mutants. This indicated that proline accumulation was regulated in part by posttranscriptional control of P5CS1 that was not affected by AHK1. Expression of AHK1 itself was reduced by low ψw, in contrast to previous reports. These results define a role of AHK1 in controlling stomatal density and the transcription of stress-responsive genes. These phenotypes may be mediated in part by reduced ABA sensitivity. More rapid transpiration and water depletion can also explain the previously reported sensitivity of ahk1 to uncontrolled soil drying. The unimpaired growth, ABA, proline, and solute accumulation of ahk1 mutants at low ψw suggest that AHK1 may not be the main plant osmosensor required for low ψw tolerance. PMID:23184230

A Retentive Memory of Tetrachloroethene Respiration in Sulfurospirillum halorespirans - involved Proteins and a possible link to Acetylation of a Two-Component Regulatory System.

PubMed

Türkowsky, Dominique; Esken, Jens; Goris, Tobias; Schubert, Torsten; Diekert, Gabriele; Jehmlich, Nico; von Bergen, Martin

2018-06-15

Organohalide respiration (OHR), comprising the reductive dehalogenation of halogenated organic compounds, is subject to a unique memory effect and long-term transcriptional downregulation of the involved genes in Sulfurospirillum multivorans. Gene expression ceases slowly over approximately 100 generations in the absence of tetrachloroethene (PCE). However, the molecular mechanisms of this regulation process are not understood. We show here that Sulfurospirillum halorespirans undergoes the same type of regulation when cultivated without chlorinated ethenes for a long period of time. In addition, we compared the proteomes of S. halorespirans cells cultivated in the presence of PCE with those of cells long- and short-term cultivated with nitrate as the sole electron acceptor. Important OHR-related proteins previously unidentified in S. multivorans include a histidine kinase, a putative quinol dehydrogenase membrane protein, and a PCE-induced porin. Since for some regulatory proteins a posttranslational regulation of activity by lysine acetylations is known, we also analyzed the acetylome of S. halorespirans, revealing that 32% of the proteome was acetylated in at least one condition. The data indicate that the response regulator and the histidine kinase of a two-component system most probably involved in induction of PCE respiration are highly acetylated during short-term cultivation with nitrate in the absence of PCE. The so far unique long-term downregulation of organohalide respiration is now identified in a second species suggesting a broader distribution of this regulatory phenomenon. An improved protein extraction method allowed the identification of proteins most probably involved in transcriptional regulation of OHR in Sulfurospirillum spp. Our data indicate that acetylations of regulatory proteins are involved in this extreme, sustained standby-mode of metabolic enzymes in the absence of a substrate. This first published acetylome of Epsilonproteobacteria might help to study other ecologically or medically important species of this clade. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

PubMed

Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

2014-08-01

Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae.

PubMed

Watanabe, Daisuke; Kikushima, Rie; Aitoku, Miho; Nishimura, Akira; Ohtsu, Iwao; Nasuno, Ryo; Takagi, Hiroshi

2014-07-07

The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1 , which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.

Gold Nanoparticles as a Direct and Rapid Sensor for Sensitive Analytical Detection of Biogenic Amines

NASA Astrophysics Data System (ADS)

El-Nour, K. M. A.; Salam, E. T. A.; Soliman, H. M.; Orabi, A. S.

2017-03-01

A new optical sensor was developed for rapid screening with high sensitivity for the existence of biogenic amines (BAs) in poultry meat samples. Gold nanoparticles (GNPs) with particle size 11-19 nm function as a fast and sensitive biosensor for detection of histamine resulting from bacterial decarboxylation of histidine as a spoilage marker for stored poultry meat. Upon reaction with histamine, the red color of the GNPs converted into deep blue. The appearance of blue color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. This biosensor enables a semi-quantitative detection of analyte in real samples by eye-vision. Quality evaluation is carried out by measuring histamine and histidine using different analytical techniques such as UV-vis, FTIR, and fluorescence spectroscopy as well as TEM. A rapid quantitative readout of samples by UV-vis and fluorescence methods with standard instrumentation were proposed in a short time unlike chromatographic and electrophoretic methods. Sensitivity and limit of detection (LOD) of 6.59 × 10-4 and 0.6 μM, respectively, are determined for histamine as a spoilage marker with a correlation coefficient ( R 2) of 0.993.

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-05-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.

Murein Lytic Enzyme TgaA of Bifidobacterium bifidum MIMBb75 Modulates Dendritic Cell Maturation through Its Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase (CHAP) Amidase Domain

PubMed Central

Zanoni, Ivan; Balzaretti, Silvia; Miriani, Matteo; Taverniti, Valentina; De Noni, Ivano; Presti, Ilaria; Stuknyte, Milda; Scarafoni, Alessio; Arioli, Stefania; Iametti, Stefania; Bonomi, Francesco; Mora, Diego; Karp, Matti; Granucci, Francesca

2014-01-01

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system. PMID:24814791

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

PubMed Central

Hoffmeyer, J.; Neuhard, J.

1971-01-01

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate. PMID:4928005

The multiple roles of histidine in protein interactions

PubMed Central

2013-01-01

Background Among the 20 natural amino acids histidine is the most active and versatile member that plays the multiple roles in protein interactions, often the key residue in enzyme catalytic reactions. A theoretical and comprehensive study on the structural features and interaction properties of histidine is certainly helpful. Results Four interaction types of histidine are quantitatively calculated, including: (1) Cation-π interactions, in which the histidine acts as the aromatic π-motif in neutral form (His), or plays the cation role in protonated form (His+); (2) π-π stacking interactions between histidine and other aromatic amino acids; (3) Hydrogen-π interactions between histidine and other aromatic amino acids; (4) Coordinate interactions between histidine and metallic cations. The energies of π-π stacking interactions and hydrogen-π interactions are calculated using CCSD/6-31+G(d,p). The energies of cation-π interactions and coordinate interactions are calculated using B3LYP/6-31+G(d,p) method and adjusted by empirical method for dispersion energy. Conclusions The coordinate interactions between histidine and metallic cations are the strongest one acting in broad range, followed by the cation-π, hydrogen-π, and π-π stacking interactions. When the histidine is in neutral form, the cation-π interactions are attractive; when it is protonated (His+), the interactions turn to repulsive. The two protonation forms (and pKa values) of histidine are reversibly switched by the attractive and repulsive cation-π interactions. In proteins the π-π stacking interaction between neutral histidine and aromatic amino acids (Phe, Tyr, Trp) are in the range from -3.0 to -4.0 kcal/mol, significantly larger than the van der Waals energies. PMID:23452343

In Silico Analysis of Putrefaction Pathways in Bacteria and Its Implication in Colorectal Cancer

PubMed Central

Kaur, Harrisham; Das, Chandrani; Mande, Sharmila S.

2017-01-01

Fermentation of undigested proteins in human gastrointestinal tract (gut) by the resident microbiota, a process called bacterial putrefaction, can sometimes disrupt the gut homeostasis. In this process, essential amino acids (e.g., histidine, tryptophan, etc.) that are required by the host may be utilized by the gut microbes. In addition, some of the products of putrefaction, like ammonia, putrescine, cresol, indole, phenol, etc., have been implicated in the disease pathogenesis of colorectal cancer (CRC). We have investigated bacterial putrefaction pathways that are known to be associated with such metabolites. Results of the comprehensive in silico analysis of the selected putrefaction pathways across bacterial genomes revealed presence of these pathways in limited bacterial groups. Majority of these bacteria are commonly found in human gut. These include Bacillus, Clostridium, Enterobacter, Escherichia, Fusobacterium, Salmonella, etc. Interestingly, while pathogens utilize almost all the analyzed pathways, commensals prefer putrescine and H2S production pathways for metabolizing the undigested proteins. Further, comparison of the putrefaction pathways in the gut microbiomes of healthy, carcinoma and adenoma datasets indicate higher abundances of putrefying bacteria in the carcinoma stage of CRC. The insights obtained from the present study indicate utilization of possible microbiome-based therapies to minimize the adverse effects of gut microbiome in enteric diseases. PMID:29163445

Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus

PubMed Central

Dietl, Anna-Maria; Amich, Jorge; Leal, Sixto; Beckmann, Nicola; Binder, Ulrike; Beilhack, Andreas; Pearlman, Eric; Haas, Hubertus

2016-01-01

Abstract Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches. PMID:26854126

Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus.

PubMed

Dietl, Anna-Maria; Amich, Jorge; Leal, Sixto; Beckmann, Nicola; Binder, Ulrike; Beilhack, Andreas; Pearlman, Eric; Haas, Hubertus

2016-05-18

Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches.

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates.

PubMed

Molle, Virginie; Leiba, Jade; Zanella-Cléon, Isabelle; Becchi, Michel; Kremer, Laurent

2010-11-01

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle

2012-11-01

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitormore » design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k{sub cat}. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.« less

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

PubMed Central

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J.

2012-01-01

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens. PMID:22646091

Crystal structures of Trypanosoma cruzi UDP-galactopyranose mutase implicate flexibility of the histidine loop in enzyme activation.

PubMed

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J

2012-06-19

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k(cat). Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

PubMed Central

Devi, Savita; Ansari, Suhail A.; Tenguria, Shivendra; Kumar, Naveen; Ahmed, Niyaz

2016-01-01

Helicobacter pylori portrays a classical paradigm of persistent bacterial infections. A well balanced homeostasis of bacterial effector functions and host responses is purported to be the key in achieving long term colonization in specific hosts. H. pylori nucleases have been shown to assist in natural transformation, but their role in virulence and colonization remains elusive. Therefore, it is imperative to understand the involvement of these nucleases in the pathogenesis of H. pylori. Here, we report the multifaceted role of a TNFR-1 interacting endonuclease A (TieA) from H. pylori. tieA expression is differentially regulated in response to environmental stress and post adherence to gastric epithelial cells. Studies with isogenic knockouts of tieA revealed it to be a secretory protein which translocates into the host gastric epithelial cells independent of a type IV secretion system, gets phosphorylated by DNA-PK kinase and auto-phosphorylates as serine kinase. Furthermore, TieA binds to and cleaves DNA in a non-specific manner and promotes Fas mediated apoptosis in AGS cells. Additionally, TieA induced pro-inflammatory cytokine secretion via activation of transcription factor AP-1 and signaled through MAP kinase pathway. Collectively, TieA with its multipronged and moonlighting functions could facilitate H. pylori in maintaining a balance of bacterial adaptation, and elimination by the host responses. PMID:27550181

21 CFR 862.1375 - Histidine test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... free histidine (an amino acid) in plasma and urine. Histidine measurements are used in the diagnosis... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Histidine test system. 862.1375 Section 862.1375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

21 CFR 862.1375 - Histidine test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... free histidine (an amino acid) in plasma and urine. Histidine measurements are used in the diagnosis... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Histidine test system. 862.1375 Section 862.1375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED...

A phenylalanine rotameric switch for signal-state control in bacterial chemoreceptors

NASA Astrophysics Data System (ADS)

Ortega, Davi R.; Yang, Chen; Ames, Peter; Baudry, Jerome; Parkinson, John S.; Zhulin, Igor B.

2013-12-01

Bacterial chemoreceptors are widely used as a model system for elucidating the molecular mechanisms of transmembrane signalling and have provided a detailed understanding of how ligand binding by the receptor modulates the activity of its associated kinase CheA. However, the mechanisms by which conformational signals move between signalling elements within a receptor dimer and how they control kinase activity remain unknown. Here, using long molecular dynamics simulations, we show that the kinase-activating cytoplasmic tip of the chemoreceptor fluctuates between two stable conformations in a signal-dependent manner. A highly conserved residue, Phe396, appears to serve as the conformational switch, because flipping of the stacked aromatic rings of an interacting F396-F396‧ pair in the receptor homodimer takes place concomitantly with the signal-related conformational changes. We suggest that interacting aromatic residues, which are common stabilizers of protein tertiary structure, might serve as rotameric molecular switches in other biological processes as well.

Structural studies of ROK fructokinase YdhR from Bacillus subtilis : insights into substrate binding and fructose specificity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nocek, B.; Stein, A.; Jedrzejczak, R.

2011-02-18

The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the 'ROK' (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo andmore » in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar {alpha}/{beta} domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.« less

Effects of Histidine Supplementation on Global Serum and Urine 1H NMR-based Metabolomics and Serum Amino Acid Profiles in Obese Women from a Randomized Controlled Study.

PubMed

Du, Shanshan; Sun, Shuhong; Liu, Liyan; Zhang, Qiao; Guo, Fuchuan; Li, Chunlong; Feng, Rennan; Sun, Changhao

2017-06-02

The aim of current study was to investigate the metabolic changes associated with histidine supplementation in serum and urine metabolic signatures and serum amino acid (AA) profiles. Serum and urine 1 H NMR-based metabolomics and serum AA profiles were employed in 32 and 37 obese women with metabolic syndrome (MetS) intervened with placebo or histidine for 12 weeks. Multivariable statistical analysis were conducted to define characteristic metabolites. In serum 1 H NMR metabolic profiles, increases in histidine, glutamine, aspartate, glycine, choline, and trimethylamine-N-oxide (TMAO) were observed; meanwhile, decreases in cholesterol, triglycerides, fatty acids and unsaturated lipids, acetone, and α/β-glucose were exhibited after histidine supplement. In urine 1 H NMR metabolic profiles, citrate, creatinine/creatine, methylguanidine, and betaine + TMAO were higher, while hippurate was lower in histidine supplement group. In serum AA profiles, 10 AAs changed after histidine supplementation, including increased histidine, glycine, alanine, lysine, asparagine, and tyrosine and decreased leucine, isoleucine, ornithine, and citrulline. The study showed a systemic metabolic response in serum and urine metabolomics and AA profiles to histidine supplementation, showing significantly changed metabolism in AAs, lipid, and glucose in obese women with MetS.

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism.

PubMed

Campion, Katherine L; McCormick, Wanda D; Warwicker, Jim; Khayat, Mohd Ezuan Bin; Atkinson-Dell, Rebecca; Steward, Martin C; Delbridge, Leigh W; Mun, Hee-Chang; Conigrave, Arthur D; Ward, Donald T

2015-09-01

The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pHo) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pHo can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pHo from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca(2+)i) mobilization, whereas raising pHo to 7.6 potentiated responsiveness to extracellular calcium (Ca(2+)o). Similar pHo effects were observed for Ca(2+)o-induced extracellular signal-regulated kinase phosphorylation and actin polymerization and for L-Phe-induced Ca(2+)i mobilization. Intracellular pH was unaffected by acute 0.4-unit pHo changes, and the presence of physiologic albumin concentrations failed to attenuate the pHo-mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pHo sensitivity. Finally, pathophysiologic pHo elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pHo changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo. Copyright © 2015 by the American Society of Nephrology.

Regioselective copper-catalyzed N(1)-(hetero)arylation of protected histidine.

PubMed

Sharma, Krishna K; Mandloi, Meenakshi; Jain, Rahul

2016-09-26

We report regioselective N(1)-arylation of protected histidine using copper(i) iodide as a catalyst, trans-N,N'-dimethylcyclohexane-1,2-diamine as a ligand and readily available aryl iodides as coupling partners under microwave irradiation at 130 °C for 40 min. The reaction provides rapid access to electron-donating, electron-withdrawing and bulky group substituted N-arylated histidines in high yields, including previously inaccessible N-heteroaryl histidines. These N(1)-(hetero)aryl histidines are promising building blocks in peptide-based drug design and discovery.

Characterization of cytokinin signaling and homeostasis gene families in two hardwood tree species: Populus trichocarpa and Prunus persica.

PubMed

Immanen, Juha; Nieminen, Kaisa; Duchens Silva, Héctor; Rodríguez Rojas, Fernanda; Meisel, Lee A; Silva, Herman; Albert, Victor A; Hvidsten, Torgeir R; Helariutta, Ykä

2013-12-16

Through the diversity of cytokinin regulated processes, this phytohormone has a profound impact on plant growth and development. Cytokinin signaling is involved in the control of apical and lateral meristem activity, branching pattern of the shoot, and leaf senescence. These processes influence several traits, including the stem diameter, shoot architecture, and perennial life cycle, which define the development of woody plants. To facilitate research about the role of cytokinin in regulation of woody plant development, we have identified genes associated with cytokinin signaling and homeostasis pathways from two hardwood tree species. Taking advantage of the sequenced black cottonwood (Populus trichocarpa) and peach (Prunus persica) genomes, we have compiled a comprehensive list of genes involved in these pathways. We identified genes belonging to the six families of cytokinin oxidases (CKXs), isopentenyl transferases (IPTs), LONELY GUY genes (LOGs), two-component receptors, histidine containing phosphotransmitters (HPts), and response regulators (RRs). All together 85 Populus and 45 Prunus genes were identified, and compared to their Arabidopsis orthologs through phylogenetic analyses. In general, when compared to Arabidopsis, differences in gene family structure were often seen in only one of the two tree species. However, one class of genes associated with cytokinin signal transduction, the CKI1-like family of two-component histidine kinases, was larger in both Populus and Prunus than in Arabidopsis.

Characterization of cytokinin signaling and homeostasis gene families in two hardwood tree species: Populus trichocarpa and Prunus persica

PubMed Central

2013-01-01

Background Through the diversity of cytokinin regulated processes, this phytohormone has a profound impact on plant growth and development. Cytokinin signaling is involved in the control of apical and lateral meristem activity, branching pattern of the shoot, and leaf senescence. These processes influence several traits, including the stem diameter, shoot architecture, and perennial life cycle, which define the development of woody plants. To facilitate research about the role of cytokinin in regulation of woody plant development, we have identified genes associated with cytokinin signaling and homeostasis pathways from two hardwood tree species. Results Taking advantage of the sequenced black cottonwood (Populus trichocarpa) and peach (Prunus persica) genomes, we have compiled a comprehensive list of genes involved in these pathways. We identified genes belonging to the six families of cytokinin oxidases (CKXs), isopentenyl transferases (IPTs), LONELY GUY genes (LOGs), two-component receptors, histidine containing phosphotransmitters (HPts), and response regulators (RRs). All together 85 Populus and 45 Prunus genes were identified, and compared to their Arabidopsis orthologs through phylogenetic analyses. Conclusions In general, when compared to Arabidopsis, differences in gene family structure were often seen in only one of the two tree species. However, one class of genes associated with cytokinin signal transduction, the CKI1-like family of two-component histidine kinases, was larger in both Populus and Prunus than in Arabidopsis. PMID:24341635

Histidine augments the suppression of hepatic glucose production by central insulin action.

PubMed

Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-Ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

2013-07-01

Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.

Histidine Augments the Suppression of Hepatic Glucose Production by Central Insulin Action

PubMed Central

Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

2013-01-01

Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes. PMID:23474485

Chiral recognition of proteins having L-histidine residues on the surface with lanthanide ion complex incorporated-molecularly imprinted fluorescent nanoparticles.

PubMed

Uzun, Lokman; Uzek, Recep; Senel, Serap; Say, Ridvan; Denizli, Adil

2013-08-01

In this study, lanthanide ion complex incorporated molecularly imprinted fluorescent nanoparticles were synthesized. A combination of three novel approaches was applied for the purpose. First, lanthanide ions [Terbium(III)] were complexed with N-methacryloyl-L-histidine (MAH), polymerizable derivative of L-histidine amino acid, in order to incorporate the complex directly into the polymeric backbone. At the second stage, L-histidine molecules imprinted nanoparticles were utilized instead of whole protein imprinting in order to avoid whole drawbacks such as fragility, complexity, denaturation tendency, and conformation dependency. At the third stage following the first two steps mentioned above, imprinted L-histidine was coordinated with cupric ions [Cu(II)] to conduct the study under mild conditions. Then, molecularly imprinted fluorescent nanoparticles synthesized were used for L-histidine adsorption from aqueous solution to optimize conditions for adsorption and fluorimetric detection. Finally, usability of nanoparticles was investigated for chiral biorecognition using stereoisomer, D-histidine, racemic mixture, D,L-histidine, proteins with surface L-histidine residue, lysozyme, cytochrome C, or without ribonuclease A. The results revealed that the proposed polymerization strategy could make significant contribution to the solution of chronic problems of fluorescent component introduction into polymers. Additionally, the fluorescent nanoparticles reported here could be used for selective separation and fluorescent monitoring purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

Biological functions of histidine-dipeptides and metabolic syndrome.

PubMed

Song, Byeng Chun; Joo, Nam-Seok; Aldini, Giancarlo; Yeum, Kyung-Jin

2014-02-01

The rapid increase in the prevalence of metabolic syndrome, which is associated with a state of elevated systemic oxidative stress and inflammation, is expected to cause future increases in the prevalence of diabetes and cardiovascular diseases. Oxidation of polyunsaturated fatty acids and sugars produces reactive carbonyl species, which, due to their electrophilic nature, react with the nucleophilic sites of certain amino acids. This leads to formation of protein adducts such as advanced glycoxidation/lipoxidation end products (AGEs/ALEs), resulting in cellular dysfunction. Therefore, an effective reactive carbonyl species and AGEs/ALEs sequestering agent may be able to prevent such cellular dysfunction. There is accumulating evidence that histidine containing dipeptides such as carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-methyl-L-histidine) detoxify cytotoxic reactive carbonyls by forming unreactive adducts and are able to reverse glycated protein. In this review, 1) reaction mechanism of oxidative stress and certain chronic diseases, 2) interrelation between oxidative stress and inflammation, 3) effective reactive carbonyl species and AGEs/ALEs sequestering actions of histidine-dipeptides and their metabolism, 4) effects of carnosinase encoding gene on the effectiveness of histidine-dipeptides, and 5) protective effects of histidine-dipeptides against progression of metabolic syndrome are discussed. Overall, this review highlights the potential beneficial effects of histidine-dipeptides against metabolic syndrome. Randomized controlled human studies may provide essential information regarding whether histidine-dipeptides attenuate metabolic syndrome in humans.

Arginine in the salt-induced peptide formation reaction: enantioselectivity facilitated by glycine, L- and D-histidine.

PubMed

Li, Feng; Fitz, Daniel; Fraser, Donald G; Rode, Bernd M

2010-07-01

The salt-induced peptide formation reaction has been proposed as a conceivable preliminary to the prebiotic evolution of peptides. In the present paper, the behaviour of arginine is reported for this reaction together with a discussion of the catalytic effects of glycine, and L- and D-histidine. Importantly, the behaviour of the two histidine enantiomers is different. Both histidine enantiomers perform better than glycine in enhancing the yields of arginine dipeptide with L-histidine being more effective than D-histidine. Yields in the presence of histidine are up to 70 times greater than for arginine solutions alone. This compares with 4.2 times higher in the presence of glycine. This difference is most pronounced in the most concentrated (containing 80 mM arginine) reaction solution where arginine has the lowest reactivity. A distinct preference for dimerisation of L-arginine also appears in the 80 mM cases for catalyses of other amino acids. This phenomenon is different from the behaviour of aliphatic amino acids, which display obvious inherent enantioselectivity for the L-stereomers in the SIPF reaction on their own rather than when catalysed by glycine or histidine.

Protein-induced geometric constraints and charge transfer in bacteriochlorophyll-histidine complexes in LH2.

PubMed

Wawrzyniak, Piotr K; Alia, A; Schaap, Roland G; Heemskerk, Mattijs M; de Groot, Huub J M; Buda, Francesco

2008-12-14

Bacteriochlorophyll-histidine complexes are ubiquitous in nature and are essential structural motifs supporting the conversion of solar energy into chemically useful compounds in a wide range of photosynthesis processes. A systematic density functional theory study of the NMR chemical shifts for histidine and for bacteriochlorophyll-a-histidine complexes in the light-harvesting complex II (LH2) is performed using the BLYP functional in combination with the 6-311++G(d,p) basis set. The computed chemical shift patterns are consistent with available experimental data for positive and neutral(tau) (N(tau) protonated) crystalline histidines. The results for the bacteriochlorophyll-a-histidine complexes in LH2 provide evidence that the protein environment is stabilizing the histidine close to the Mg ion, thereby inducing a large charge transfer of approximately 0.5 electronic equivalent. Due to this protein-induced geometric constraint, the Mg-coordinated histidine in LH2 appears to be in a frustrated state very different from the formal neutral(pi) (N(pi) protonated) form. This finding could be important for the understanding of basic functional mechanisms involved in tuning the electronic properties and exciton coupling in LH2.

L-histidine inhibits biofilm formation and FLO11-associated phenotypes in Saccharomyces cerevisiae flor yeasts.

PubMed

Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

2014-01-01

Flor yeasts of Saccharomyces cerevisiae have an innate diversity of Flo11p which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling Flo11p alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce Flo11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to Flo11p expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air-liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the Flo11p gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts [corrected].

L-Histidine Inhibits Biofilm Formation and FLO11-Associated Phenotypes in Saccharomyces cerevisiae Flor Yeasts

PubMed Central

Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

2014-01-01

Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to FLO11 expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air–liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the FLO11 gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts. PMID:25369456

A genome-wide study of two-component signal transduction systems in eight newly sequenced mutans streptococci strains

PubMed Central

2012-01-01

Background Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens. Results HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments. Conclusions Differences in the TCS repertoires of mutans streptococci strains, especially those of S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains. PMID:22475007

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

PubMed

Pragman, Alexa A; Yarwood, Jeremy M; Tripp, Timothy J; Schlievert, Patrick M

2004-04-01

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

Transcriptomic analysis of the response of Pseudomonas fluorescens to epigallocatechin gallate by RNA-seq

PubMed Central

Liu, Xiaoxiang; Shen, Bimiao; Du, Peng; Wang, Nan; Wang, Jiaxue; Li, Jianrong

2017-01-01

Epigallocatechin gallate (EGCG) is a main constituent of green tea polyphenols that are widely used as food preservatives and are considered to be safe for consumption. However, the underlying antimicrobial mechanism of EGCG and the bacterial response to EGCG are not clearly understood. In the present study, a genome-wide transcriptional analysis of a typical spoilage bacterium, Pseudomonas fluorescens that responded to EGCG was performed using RNA-seq technology. A total of 26,365,414 and 23,287,092 clean reads were generated from P. fluorescens treated with or without 1 mM EGCG and the clean reads were aligned to the reference genome. Differential expression analysis revealed 291 upregulated genes and 134 downregulated genes and the differentially expressed genes (DEGs) were verified using RT-qPCR. Most of the DGEs involved in iron uptake, antioxidation, DNA repair, efflux system, cell envelope and cell-surface component synthesis were significantly upregulated by EGCG treatment, while most genes associated with energy production were downregulated. These transcriptomic changes are likely to be adaptive responses of P. fluorescens to iron limitation and oxidative stress, as well as DNA and envelope damage caused by EGCG. The expression of specific genes encoding the extra-cytoplasmic function sigma factor (PvdS, RpoE and AlgU) and the two-component sensor histidine kinase (BaeS and RpfG) were markedly changed by EGCG treatment, which may play important roles in regulating the stress responses of P. fluorescens to EGCG. The present data provides important insights into the molecular action of EGCG and the possible cross-resistance mediated by EGCG on P. fluorescens, which may ultimately contribute to the optimal application of green tea polyphenols in food preservation. PMID:28545064

Shedding light on the paradox of high alkaline phosphatase utilization at high end-product concentrations

NASA Astrophysics Data System (ADS)

Baltar, F.; Lundin, D.; Palovaara, J.; Reinthaler, T.; Herndl, G. J.; Pinhassi, J.

2016-02-01

Alkaline phosphatase (APase) activity is supposed to be regulated by the concentration of its endproduct, decreasing with increasing inorganic phosphate (Pi) concentrations. Since Pi is readily available in the deep ocean, APase activity would be expected to be low. However, high APase activities at high Pi concentrations have been found in the deep Indian and Atlantic Ocean. To understand how APase activities are regulated and what mechanisms are responsible for its regulation we performed microcosm experiments with mesopelagic North Atlantic waters. Treatments consisted of enrichment with either ammonium or organic carbon, and were compared to unamended controls. We assessed changes in prokaryotic abundance, APase, leucine aminopeptidase, heterotrophic production, dark CO2 fixation and community gene expression (metatranscriptomics) between treatments and control. In the organic matter enrichments, APase increased along with all measured rates, whereas only dark CO2 fixation and APase were enhanced in the ammonium enrichment. In the organic matter enrichment, genes for heterotrophic metabolism were strongly upregulated, whereas genes for ammonia oxidation and CO2 fixation were upregulated in the ammonium treatment. In both treatments, the Pho regulon -a global regulatory mechanism involved in bacterial Pi management- was also upregulated, including genes encoding alkaline phosphatases. The activation of the Pho regulon seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. Increased C or N bioavailability thus appear to elicit a Pi deficiency inside cells and activate the Pho regulon. These results indicate possible ways (e.g. pulses of C or N or changes in elemental ratios) in which APase can be activated irrespectively of the environmental Pi concentration.

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

A chemical-genetic approach for functional analysis of plant protein kinases

PubMed Central

Salomon, Dor; Bonshtien, Arale

2009-01-01

Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N6-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases. PMID:19820342

Growth and histamine formation of Morganella morganii in determining the safety and quality of inoculated and uninoculated bluefish (Pomatomus saltatrix).

PubMed

Lorca, T A; Gingerich, T M; Pierson, M D; Flick, G J; Hackney, C R; Sumner, S S

2001-12-01

The objective of this study was to determine the effect of normal microflora and Morganella morganii on histamine formation and olfactory acceptability in raw bluefish under controlled storage conditions. Fillets inoculated with and without M. morganii were stored at 5, 10, and 15 degrees C for 7 days. Microbial isolates from surface swabs were identified and screened for histidine decarboxylase activity. Olfactory acceptance was performed by an informal sensory panel. Histamine levels were quantified using high-performance liquid chromatography and fluorescence detection. While olfactory acceptance decreased, histamine concentration and bacterial counts increased. Storage temperature had a significant effect on histamine levels, bacterial counts, and olfactory acceptance of the bluefish. Inoculation with M. morganii had a positive significant effect on histamine formation for bluefish held at 10 and 15 degrees C (P < 0.0001). The results of the study will serve in supporting U.S. Food and Drug Administration (FDA) regulations regarding guidance and hazard levels of histamine in fresh bluefish.

Constructing bioactive peptides with pH-dependent activities.

PubMed

Tu, Zhigang; Volk, Melanie; Shah, Khushali; Clerkin, Kevin; Liang, Jun F

2009-08-01

Many bioactive peptides are featured by their arginine and lysine rich contents. In this study, lysine and arginine residues in lytic peptides were selectively replaced by histidines. Although resulting histidine-containing lytic peptides had decreased activity, they did show pH-dependent cytotoxicity. The activity of the constructed histidine-containing lytic peptides increased 2-8 times as the solution pH changed from 7.4 to 5.5. More importantly, these histidine-containing peptides maintain the same cell killing mechanism as their parent peptides by causing cell lysis. Both the activity and pH-sensitivity of histidine-containing peptides are tunable by adjusting histidine substitution numbers and positions. This study has presented a general strategy to create bioactive peptides with desired pH-sensitivity to meet the needs of various applications such as cancer treatments.

Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori.

PubMed

Devi, Savita; Ansari, Suhail A; Tenguria, Shivendra; Kumar, Naveen; Ahmed, Niyaz

2016-11-02

Helicobacter pylori portrays a classical paradigm of persistent bacterial infections. A well balanced homeostasis of bacterial effector functions and host responses is purported to be the key in achieving long term colonization in specific hosts. H. pylori nucleases have been shown to assist in natural transformation, but their role in virulence and colonization remains elusive. Therefore, it is imperative to understand the involvement of these nucleases in the pathogenesis of H. pylori Here, we report the multifaceted role of a TNFR-1 interacting endonuclease A (TieA) from H. pylori. tieA expression is differentially regulated in response to environmental stress and post adherence to gastric epithelial cells. Studies with isogenic knockouts of tieA revealed it to be a secretory protein which translocates into the host gastric epithelial cells independent of a type IV secretion system, gets phosphorylated by DNA-PK kinase and auto-phosphorylates as serine kinase. Furthermore, TieA binds to and cleaves DNA in a non-specific manner and promotes Fas mediated apoptosis in AGS cells. Additionally, TieA induced pro-inflammatory cytokine secretion via activation of transcription factor AP-1 and signaled through MAP kinase pathway. Collectively, TieA with its multipronged and moonlighting functions could facilitate H. pylori in maintaining a balance of bacterial adaptation, and elimination by the host responses. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L)

PubMed Central

Waagbø, Rune; Breck, Olav; Stavrum, Anne-Kristin; Petersen, Kjell; Olsvik, Pål A.

2009-01-01

Purpose Elevated levels of dietary histidine have previously been shown to prevent or mitigate cataract formation in farmed Atlantic salmon (Salmo salar L). The aim of this study was to shed light on the mechanisms by which histidine acts. Applying microarray analysis to the lens transcriptome, we screened for differentially expressed genes in search for a model explaining cataract development in Atlantic salmon and possible markers for early cataract diagnosis. Methods Adult Atlantic salmon (1.7 kg) were fed three standard commercial salmon diets only differing in the histidine content (9, 13, and 17 g histidine/kg diet) for four months. Individual cataract scores for both eyes were assessed by slit-lamp biomicroscopy. Lens N-acetyl histidine contents were measured by high performance liquid chromatography (HPLC). Total RNA extracted from whole lenses was analyzed using the GRASP 16K salmonid microarray. The microarray data were analyzed using J-Express Pro 2.7 and validated by quantitative real-time polymerase chain reaction (qRT–PCR). Results Fish developed cataracts with different severity in response to dietary histidine levels. Lens N-acetyl histidine contents reflected the dietary histidine levels and were negatively correlated to cataract scores. Significance analysis of microarrays (SAM) revealed 248 significantly up-regulated transcripts and 266 significantly down-regulated transcripts in fish that were fed a low level of histidine compared to fish fed a higher histidine level. Among the differentially expressed transcripts were metallothionein A and B as well as transcripts involved in lipid metabolism, carbohydrate metabolism, regulation of ion homeostasis, and protein degradation. Hierarchical clustering and correspondence analysis plot confirmed differences in gene expression between the feeding groups. The differentially expressed genes could be categorized as “early” and “late” responsive according to their expression pattern relative to progression in cataract formation. Conclusions Dietary histidine regimes affected cataract formation and lens gene expression in adult Atlantic salmon. Regulated transcripts selected from the results of this genome-wide transcription analysis might be used as possible biological markers for cataract development in Atlantic salmon. PMID:19597568

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD

PubMed Central

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-01-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosyn­thesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-l-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit. PMID:24817736

The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices.

PubMed

Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, Lorena

2017-10-20

The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.

Purification and genetic characterization of gassericin E, a novel co-culture inducible bacteriocin from Lactobacillus gasseri EV1461 isolated from the vagina of a healthy woman.

PubMed

Maldonado-Barragán, Antonio; Caballero-Guerrero, Belén; Martín, Virginia; Ruiz-Barba, José Luis; Rodríguez, Juan Miguel

2016-03-12

Lactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed. We found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI). The origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.

A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death1[OPEN

PubMed Central

Elmore, James M.; Creer, Athena Y.; Feng, Baomin; Franco, Jessica Y.; He, Ping; Phinney, Brett

2017-01-01

Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae. Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses. PMID:27852951

The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response1[OPEN

PubMed Central

Brauer, Elizabeth K.; Ahsan, Nagib; Kato, Naohiro; Coluccio, Alison E.; Thelen, Jay J.

2016-01-01

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca2+ uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H+)/K+ symporter that mediates a high-affinity uptake during K+ deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K+ efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K+ loss. Taken together, our results position ILK1 as a link between plant defense pathways and K+ homeostasis. PMID:27208244

Characterization of a bacterial tyrosine kinase in Porphyromonas gingivalis involved in polymicrobial synergy.

PubMed

Wright, Christopher J; Xue, Peng; Hirano, Takanori; Liu, Chengcheng; Whitmore, Sarah E; Hackett, Murray; Lamont, Richard J

2014-06-01

Interspecies communication between Porphyromonas gingivalis and Streptococcus gordonii underlies the development of synergistic dual species communities. Contact with S. gordonii initiates signal transduction within P. gingivalis that is based on protein tyrosine (de)phosphorylation. In this study, we characterize a bacterial tyrosine (BY) kinase (designated Ptk1) of P. gingivalis and demonstrate its involvement in interspecies signaling. Ptk1 can utilize ATP for autophosphorylation and is dephosphorylated by the P. gingivalis tyrosine phosphatase, Ltp1. Community development with S. gordonii is severely abrogated in a ptk1 mutant of P. gingivalis, indicating that tyrosine kinase activity is required for maximal polymicrobial synergy. Ptk1 controls the levels of the transcriptional regulator CdhR and the fimbrial adhesin Mfa1 which mediates binding to S. gordonii. The ptk1 gene is in an operon with two genes involved in exopolysaccharide synthesis, and similar to other BY kinases, Ptk1 is necessary for exopolysaccharide production in P. gingivalis. Ptk1 can phosphorylate the capsule related proteins PGN_0224, a UDP-acetyl-mannosamine dehydrogenase, and PGN_0613, a UDP-glucose dehydrogenase, in P. gingivalis. Knockout of ptk1 in an encapsulated strain of P. gingivalis resulted in loss of capsule production. Collectively these results demonstrate that the P. gingivalis Ptk1 BY kinase regulates interspecies communication and controls heterotypic community development with S. gordonii through adjusting the levels of the Mfa1 adhesin and exopolysaccharide. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens

PubMed Central

2012-01-01

Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection. PMID:22691598

Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens.

PubMed

Cortés-Vieyra, Ricarda; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J; Juárez, Marcos Cajero; Finlay, B Brett; Baizabal-Aguirre, Víctor M

2012-06-12

Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

Pyrazole derivatives as antitumor, anti-inflammatory and antibacterial agents.

PubMed

Liu, Jia-Jia; Zhao, Meng-Yue; Zhang, Xin; Zhao, Xin; Zhu, Hai-Liang

2013-11-01

Within the past years, many researches on the synthesis, structure-activity relationships (SAR), antitumor, antiinflammatory and anti-bacterial activities of the pyrazole derivatives have been reported. Several pyrazole derivatives possess important pharmacological activities and they have been proved useful materials in drug research. Pyrazole derivatives play an important role in antitumor agents because of their good inhibitory activity against BRAF(V600E), EGFR, telomerase, ROS Receptor Tyrosine Kinase and Aurora-A kinase. In addition, pyrazole derivatives also show good antiinflammatory and anti-bacterial activities. In this review, the bioactivities of the pyrazole derivatives mentioned above will be summarized in detail. We sincerely hope that increasing knowledge of the SAR and cellular processes underlying the bioactivity of pyrazole derivatives will be beneficial to the rational design of new generation of small molecule drugs.

5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

PubMed

Kumar, Ajay; Giri, Shailendra; Kumar, Ashok

2016-12-01

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections. © 2016 John Wiley & Sons Ltd.

Stress Sensors and Signal Transducers in Cyanobacteria

PubMed Central

Los, Dmitry A.; Zorina, Anna; Sinetova, Maria; Kryazhov, Sergey; Mironov, Kirill; Zinchenko, Vladislav V.

2010-01-01

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress. PMID:22294932

Role of Heavy Meromyosin in Heat-Induced Gelation in Low Ionic Strength Solution Containing L-Histidine.

PubMed

Hayakawa, Toru; Yoshida, Yuri; Yasui, Masanori; Ito, Toshiaki; Wakamatsu, Jun-ichi; Hattori, Akihito; Nishimura, Takanori

2015-08-01

The gelation of myosin has a very important role in meat products. We have already shown that myosin in low ionic strength solution containing L-histidine forms a transparent gel after heating. To clarify the mechanism of this unique gelation, we investigated the changes in the nature of myosin subfragments during heating in solutions with low and high ionic strengths with and without L-histidine. The hydrophobicity of myosin and heavy meromyosin (HMM) in low ionic strength solution containing L-histidine was lower than in high ionic strength solution. The SH contents of myosin and HMM in low ionic strength solution containing l-histidine did not change during the heating process, whereas in high ionic strength solution they decreased slightly. The heat-induced globular masses of HMM in low ionic strength solution containing L-histidine were smaller than those in high ionic strength solution. These findings suggested that the polymerization of HMM molecules by heating was suppressed in low ionic strength solution containing L-histidine, resulting in formation of the unique gel. © 2015 Institute of Food Technologists®

Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae.

PubMed

Konte, Tilen; Terpitz, Ulrich; Plemenitaš, Ana

2016-01-01

The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like other fungi, W. ichthyophaga detects changes in environmental salinity mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with protein modeling data, our study reveals conserved and non-conserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.

Complexes of yeast adenylate kinase and nucleotides investigated by sup 1 H NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vetter, I.R.; Konrad, M.; Rosch, P.

1991-04-30

The role of one of the histidine residues present in many adenylate kinases (H36 in the porcine cytosolic enzyme) is highly disputed. The authors studied the yeast enzyme (AK{sub ye}) containing this His residue. AK{sub ye} is highly homologous to the Escherichia coli enzyme (AK{sub ec}), a protein that is already well characterized by NMR and does not contain the His residue in question. In addition, discrepancies between solution structural and X-ray crystallographic studies on the location of the nucleotide binding sites of adenylate kinases are clarified. One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to investigate AK{submore » ye} and its complex with the bisubstrate analogue P{sup 1},P{sup 5}-bis(5{prime}-adenosyl)pentaphosphate (AP{sub 5}A). From these studies, all aromatic residues of AK{sub ec} involved in the binding of ATP{center dot}Mg{sup 2+} have functional analogues in AK{sub ye}. The AMP site seems to make no contacts to aromatic side chains, neither in the AK{sub ye}{center dot}AP{sub 5}A{center dot}Mg{sup 2+} nor in the AK{sub ec}{center dot}AP{sub 5}A{center dot}Mg{sup 2+} complexes, so that it is presently not possible to localize this binding site by NMR. In combination with the recent X-ray results on the AP{sub 5}A complexes AK{sub ye} and AK{sub ec} and the GMP complex of guanylate kinase the latter one leading to the definition of the monophosphate site, the problem of the location of the nucleotide sites can be considered to be solved in a way contradicting earlier work and denying the His residue homologous to H36 in porcine adenylate kinase a direct role in substrate binding.« less

Roles of the redox-active disulfide and histidine residues forming a catalytic dyad in reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase/carboxylase.

PubMed

Kofoed, Melissa A; Wampler, David A; Pandey, Arti S; Peters, John W; Ensign, Scott A

2011-09-01

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK(a) value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK(a) value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

A novel kinase function of a nucleoside-diphosphate-kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat-shock protein 27.

PubMed

Lee, Jungnam; Roberts, JoAnn S; Atanasova, Kalina R; Chowdhury, Nityananda; Yilmaz, Özlem

2018-05-01

We have previously shown that a homologue of a conserved nucleoside-diphosphate-kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive-oxygen-species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis-Ndk, in abrogating epithelial cell death by phosphorylating heat-shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer-epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho-HSP27 during infection has never been characterised. Interestingly, using glutathione S-transferase-rNdk pull-down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis-Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis-Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis-Ndk protein constructs and an isogenic P. gingivalis-ndk-deficient-mutant strain for functional examination. P. gingivalis-infected GECs displayed significantly increased phospho-HSP27 compared with ndk-deficient-strain during 24 hr infection. Phospho-HSP27 was significantly increased by transfection of GFP-tagged-Ndk into uninfected-GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis-Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine-mediated-apoptosis during infection. Transfection of recombinant P. gingivalis-Ndk protein into GECs substantially decreased staurosporine-induced-apoptosis. Finally, ndk-deficient-mutant strain was unable to inhibit staurosporine-induced Cytochrome C release/Caspase-9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector-P. gingivalis-Ndk-and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival. © 2018 John Wiley & Sons Ltd.

The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity.

PubMed

Kiehler, Brittany; Haggett, Lindsey; Fujita, Masaya

2017-08-01

Sporulation in Bacillus subtilis is induced upon starvation. In a widely accepted model, an N-terminal "sensor" domain of the major sporulation kinase KinA recognizes a hypothetical starvation signal(s) and autophosphorylates a histidine residue to activate the master regulator Spo0A via a multicomponent phosphorelay. However, to date no confirmed signal has been found. Here, we demonstrated that PAS-A, the most N-terminal of the three PAS domains (PAS-ABC), is dispensable for the activity, contrary to a previous report. Our data indicated that the autokinase activity is dependent on the formation of a functional tetramer, which is mediated by, at least, PAS-B and PAS-C. Additionally, we ruled out the previously proposed notion that NAD + /NADH ratio controls KinA activity through the PAS-A domain by demonstrating that the cofactors show no effects on the kinase activity in vitro. In support of these data, we found that the cofactors exist in approximately 1000-fold excess of KinA in the cell and the cofactors' ratio does not change significantly during growth and sporulation, suggesting that changes in the cofactor ratio might not play a role in controlling KinA activity. These data may refute the widely-held belief that the activity of KinA is regulated in response to an unknown starvation signal(s). © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

A New Type of Metal-Binding Site in Cobalt- And Zinc-Containing Adenylate Kinases Isolated From Sulfate-Reducers D. Gigas And D. Desulfuricans ATCC 27774

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gavel, O.Y.; Bursakov, S.A.; Rocco, G.Di

2009-05-18

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterized in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorptionmore » spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the 'LID' domain. The sequence {sup 129}Cys-X{sub 5}-His-X{sub 15}-Cys-X{sub 2}-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.« less

Identification of histidine residues that act as zinc ligands in beta-lactamase II by differential tritium exchange.

PubMed Central

Baldwin, G S; Waley, S G; Abraham, E P

1979-01-01

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified. PMID:314287

The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

PubMed

Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

2014-05-20

Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for phagocytic activity. Haemophilus ducreyi, a sexually transmitted pathogen, secretes a 4,153-amino-acid (aa) protein (LspA1) that effectively inhibits FcγR-mediated phagocytic activity. In this study, we show that a 294-aa domain within this bacterial protein binds to C-terminal Src kinase (Csk) and stimulates its catalytic activity, resulting in a significant attenuation of Src kinase activity and consequent inhibition of phagocytosis. The ability to inhibit phagocytosis via Csk is not unique to H. ducreyi, because we found that the Helicobacter pylori CagA protein also inhibits phagocytosis in a Csk-dependent manner. Harnessing Csk to subvert the FcγR-mediated phagocytic pathway represents a new bacterial effector mechanism for circumventing the innate immune response. Copyright © 2014 Dodd et al.

Changes in Quaternary Structure in the Signaling Mechanisms of PAS Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ayers, Rebecca A.; Moffat, Keith

2008-12-15

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group (P1) and at resolutions (1.5--1.8 {angstrom}) higher than the resolutions of those previously obtained. Interestingly, bjFixLHmore » can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated {approx}175 deg. relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to 2 deg. relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.« less

A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain.

PubMed

Alexandre, Maxime T A; Arents, Jos C; van Grondelle, Rienk; Hellingwerf, Klaas J; Kennis, John T M

2007-03-20

Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the C-terminal part of Avena sativa phototropin 1. The N-terminal part contains two light, oxygen, or voltage (LOV) sensing domains, LOV1 and LOV2; each binds a flavin mononucleotide (FMN) chromophore (lambdamax = 447 nm, termed D447) and forms the light-sensitive domains, of which LOV2 is the principal component. Blue-light absorption produces a covalent adduct between a very conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary by several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well-characterized LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by 3 orders of magnitude in the submolar concentration range, via a base-catalyzed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cysteine. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethyl pyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)-1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contibute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)-1, leading to a net adduct decay time constant of 30 s at pH 8.

Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7.

PubMed

Gullett, Jessica M; Bible, Amber; Alexandre, Gladys

2017-07-01

Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense , Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms. IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions. Copyright © 2017 American Society for Microbiology.

Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7

PubMed Central

Gullett, Jessica M.

2017-01-01

ABSTRACT Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms. IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions. PMID:28416707

Effects of histidine and n-acetylcysteine on experimental lesions induced by doxorubicin in sciatic nerve of rats.

PubMed

Farshid, Amir Abbas; Tamaddonfard, Esmaeal; Najafi, Sima

2015-10-01

In this study, the effect of separate and combined intraperitoneal (i.p.) injections of histidine and n-acetylcysteine were investigated on experimental damage induced by doxorubicin (DOX) in sciatic nerve of rats. DOX was i.p. injected at a dose of 4 mg/kg once weekly for four weeks. Histidine and n-acetylcysteine were i.p. injected at a same dose of 20 mg/kg. Cold and mechanical allodynia were recorded using acetone spray and von Frey filaments tests, respectively. The sciatic nerve damage was evaluated by light microscopy. Plasma levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured. Histidine and especially n-acetylcysteine at a same dose of 20 mg/kg suppressed cold and mechanical allodynia, improved sciatic nerve lesions and reversed MDA and TAC levels in DOX-treated groups. Combination treatment with histidine and n-acetylcysteine showed better responses when compared with them used alone. The results of the present study showed peripheral neuroprotective effects for histidine and n-acetylcysteine. Reduction of free radical-induced toxic effects may have a role in neuroprotective properties of histidine and n-acetylcysteine.

Interaction of Pd(II) and Pt(II) Amino Acid Complexes With Dinucleotides

PubMed Central

Vicens, Margarita; Caubet, Amparo

1997-01-01

The interaction of the dinucleotides d(ApG) and d(ApA) with [Pd(aa)Cl2], where aa = L- or D-histidine or the methyl ester of L-histidine, and with [Pt(Met)Cl2], where Met = L-methionine was studied by 1H and 13C NMR and CD measurements. In the case of the L-histidine and L-histidineOMe, the reaction with d(ApG) appeared to give the bifunctional adducts Pd(L-Histidine)N1(1)N7(2) and Pd(L-HisOMe)N1(1)N7(2), but the behavior with D-histidine suggested the formation of the monofunctional adduct Pd(D-His)N7(2). The reaction of L-histidine with d(ApA) seemed to form the bimetallic adduct (L-His)PdN7(1)N7(2)Pd(L-His). The Pt(II)-L-methionine complex in both reactions with d(ApG) and d(ApA) seemed to yield mainly adducts Pt(L-Met)N7(1)N7(2) but the existence of adducts Pt(L-Met)N1(1)N7(2) cannot be ruled out. PMID:18475765

Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes.

PubMed

Diehl, Carl; Wisniewska, Magdalena; Frick, Inga-Maria; Streicher, Werner; Björck, Lars; Malmström, Johan; Wikström, Mats

2016-01-01

Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular details into the interactions involving protein sHIP.

Molecular Evolution of the Oxygen-Binding Hemerythrin Domain

PubMed Central

Alvarez-Carreño, Claudia; Becerra, Arturo; Lazcano, Antonio

2016-01-01

Background The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins) are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes. Results Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid) cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role. Conclusions Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later evolution of the oxygen-binding hemerythrin domain in both prokaryotes and eukaryotes led to a wide variety of functions, ranging from protection against oxidative damage in anaerobic and microaerophilic organisms, to oxygen supplying to particular enzymes and pathways in aerobic and facultative species. PMID:27336621

Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

PubMed

Ormazabal, Valeska; Zuñiga, Felipe A; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

2010-11-19

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

Impact of Stainless Steel Exposure on the Oxidation of Polysorbate 80 in Histidine Placebo and Active Monoclonal Antibody Formulation.

PubMed

Gopalrathnam, Ganapathy; Sharma, Anant Navanithan; Dodd, Steven Witt; Huang, Lihua

2018-01-01

Rapid oxidation of polysorbate 80 in histidine buffer was observed upon brief exposure to stainless steel. Liquid chromatography-mass spectrometry analysis indicates degradation of both polyoxyethylene sorbitan and polyoxyethylene head groups and unsaturated fatty acid chains, with further confirmation by reversed-phase high-performance liquid chromatography data. Both Fe 2+ and Fe 3+ were shown to induce polysorbate 80 oxidation. The degree of oxidation in polysorbate 20 and polysorbate 80 are comparable for the head groups and saturated fatty acid esters. However, the same phenomenon was not observed with placebo or monoclonal antibody at a threshold protein concentration, formulated in sodium citrate, in combination with histidine and sodium citrate, or with Na 2 ethylenediaminetetraacetic acid (EDTA). Further, polysorbate 80 oxidation was not observed with Lilly's antibody containing the active ingredient LY2951742, at or above a threshold concentration. Finally, no major polysorbate 80 degradation was observed in histidine buffer, with or without protein, in containers composed of glass or plastic, or when stainless steel exposure was otherwise completely absent. Finally, the 2-oxo oxidation form of histidine was not observed, but the other oxidation products and modifications of histidine were identified. LAY ABSTRACT: Rapid oxidation of polysorbate 80 in histidine buffer was observed upon brief exposure to stainless steel. The degree of oxidation in polysorbate 80 and polysorbate 20 were comparable. However, the same phenomenon was not observed with placebo when formulated in sodium citrate, in combination with histidine and sodium citrate, or with Na 2 ethylenediaminetetraacetic acid (EDTA). Polysorbate 80 oxidation was not observed with Lilly's antibody containing the active ingredient, LY2951742, at or above a threshold concentration. No major polysorbate 80 degradation in histidine buffer was observed when stainless steel contact was completely absent. © PDA, Inc. 2018.

Association of Rare Loss-Of-Function Alleles in HAL, Serum Histidine: Levels and Incident Coronary Heart Disease.

PubMed

Yu, Bing; Li, Alexander H; Muzny, Donna; Veeraraghavan, Narayanan; de Vries, Paul S; Bis, Joshua C; Musani, Solomon K; Alexander, Danny; Morrison, Alanna C; Franco, Oscar H; Uitterlinden, André; Hofman, Albert; Dehghan, Abbas; Wilson, James G; Psaty, Bruce M; Gibbs, Richard; Wei, Peng; Boerwinkle, Eric

2015-04-01

Histidine is a semiessential amino acid with antioxidant and anti-inflammatory properties. Few data are available on the associations between genetic variants, histidine levels, and incident coronary heart disease (CHD) in a population-based sample. By conducting whole exome sequencing on 1152 African Americans in the Atherosclerosis Risk in Communities (ARIC) study and focusing on loss-of-function (LoF) variants, we identified 3 novel rare LoF variants in HAL, a gene that encodes histidine ammonia-lyase in the first step of histidine catabolism. These LoF variants had large effects on blood histidine levels (β=0.26; P=1.2×10(-13)). The positive association with histidine levels was replicated by genotyping an independent sample of 718 ARIC African Americans (minor allele frequency=1%; P=1.2×10(-4)). In addition, high blood histidine levels were associated with reduced risk of developing incident CHD with an average of 21.5 years of follow-up among African Americans (hazard ratio=0.18; P=1.9×10(-4)). This finding was validated in an independent sample of European Americans from the Framingham Heart Study (FHS) Offspring Cohort. However, LoF variants in HAL were not directly significantly associated with incident CHD after meta-analyzing results from the CHARGE Consortium. Three LoF mutations in HAL were associated with increased histidine levels, which in turn were shown to be inversely related to the risk of CHD among both African Americans and European Americans. Future investigations on the association between HAL gene variation and CHD are warranted. © 2015 American Heart Association, Inc.

Relationships of Dietary Histidine and Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study.

PubMed

Li, Yan-Chuan; Li, Chun-Long; Qi, Jia-Yue; Huang, Li-Na; Shi, Dan; Du, Shan-Shan; Liu, Li-Yan; Feng, Ren-Nan; Sun, Chang-Hao

2016-07-11

Our previous studies have demonstrated that histidine supplementation significantly ameliorates inflammation and oxidative stress in obese women and high-fat diet-induced obese rats. However, the effects of dietary histidine on general population are not known. The objective of this Internet-based cross-sectional study was to evaluate the associations between dietary histidine and prevalence of overweight/obesity and abdominal obesity in northern Chinese population. A total of 2376 participants were randomly recruited and asked to finish our Internet-based dietary questionnaire for the Chinese (IDQC). Afterwards, 88 overweight/obese participants were randomly selected to explore the possible mechanism. Compared with healthy controls, dietary histidine was significantly lower in overweight (p < 0.05) and obese (p < 0.01) participants of both sexes. Dietary histidine was inversely associated with body mass index (BMI), waist circumference (WC) and blood pressure in overall population and stronger associations were observed in women and overweight/obese participants. Higher dietary histidine was associated with lower prevalence of overweight/obesity and abdominal obesity, especially in women. Further studies indicated that higher dietary histidine was associated with lower fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), 2-h postprandial glucose (2 h-PG), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), C-reactive protein (CRP), malonaldehyde (MDA) and vaspin and higher glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and adiponectin of overweight/obese individuals of both sexes. In conclusion, higher dietary histidine is inversely associated with energy intake, status of insulin resistance, inflammation and oxidative stress in overweight/obese participants and lower prevalence of overweight/obesity in northern Chinese adults.

Relationships of Dietary Histidine and Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study

PubMed Central

Li, Yan-Chuan; Li, Chun-Long; Qi, Jia-Yue; Huang, Li-Na; Shi, Dan; Du, Shan-Shan; Liu, Li-Yan; Feng, Ren-Nan; Sun, Chang-Hao

2016-01-01

Our previous studies have demonstrated that histidine supplementation significantly ameliorates inflammation and oxidative stress in obese women and high-fat diet-induced obese rats. However, the effects of dietary histidine on general population are not known. The objective of this Internet-based cross-sectional study was to evaluate the associations between dietary histidine and prevalence of overweight/obesity and abdominal obesity in northern Chinese population. A total of 2376 participants were randomly recruited and asked to finish our Internet-based dietary questionnaire for the Chinese (IDQC). Afterwards, 88 overweight/obese participants were randomly selected to explore the possible mechanism. Compared with healthy controls, dietary histidine was significantly lower in overweight (p < 0.05) and obese (p < 0.01) participants of both sexes. Dietary histidine was inversely associated with body mass index (BMI), waist circumference (WC) and blood pressure in overall population and stronger associations were observed in women and overweight/obese participants. Higher dietary histidine was associated with lower prevalence of overweight/obesity and abdominal obesity, especially in women. Further studies indicated that higher dietary histidine was associated with lower fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), 2-h postprandial glucose (2 h-PG), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), C-reactive protein (CRP), malonaldehyde (MDA) and vaspin and higher glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and adiponectin of overweight/obese individuals of both sexes. In conclusion, higher dietary histidine is inversely associated with energy intake, status of insulin resistance, inflammation and oxidative stress in overweight/obese participants and lower prevalence of overweight/obesity in northern Chinese adults. PMID:27409634

TULA-2, a novel histidine phosphatase regulates bone remodeling by modulating osteoclast function

PubMed Central

Back, Steven H.; Adapala, Naga Suresh; Barbe, Mary F.; Carpino, Nick C.; Tsygankov, Alexander Y.; Sanjay, Archana

2013-01-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The novel protein T-cell Ubiquitin Ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO Mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of Immune-receptor-Tyrosine-based-Activation-Motif (ITAM)–mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function. PMID:23149425

TULA-2, a novel histidine phosphatase, regulates bone remodeling by modulating osteoclast function.

PubMed

Back, Steven H; Adapala, Naga Suresh; Barbe, Mary F; Carpino, Nick C; Tsygankov, Alexander Y; Sanjay, Archana

2013-04-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The protein T cell ubiquitin ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members, only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation, suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of immune-receptor-tyrosine-based-activation-motif (ITAM)-mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function.

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

Mussel-inspired histidine-based transient network metal coordination hydrogels

PubMed Central

Fullenkamp, Dominic E.; He, Lihong; Barrett, Devin G.; Burghardt, Wesley R.; Messersmith, Phillip B.

2013-01-01

Transient network hydrogels cross-linked through histidine-divalent cation coordination bonds were studied by conventional rheologic methods using histidine-modified star poly(ethylene glycol) (PEG) polymers. These materials were inspired by the mussel, which is thought to use histidine-metal coordination bonds to impart self-healing properties in the mussel byssal thread. Hydrogel viscoelastic mechanical properties were studied as a function of metal, pH, concentration, and ionic strength. The equilibrium metal-binding constants were determined by dilute solution potentiometric titration of monofunctional histidine-modified methoxy-PEG and were found to be consistent with binding constants of small molecule analogs previously studied. pH-dependent speciation curves were then calculated using the equilibrium constants determined by potentiometric titration, providing insight into the pH dependence of histidine-metal ion coordination and guiding the design of metal coordination hydrogels. Gel relaxation dynamics were found to be uncorrelated with the equilibrium constants measured, but were correlated to the expected coordination bond dissociation rate constants. PMID:23441102

Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes

PubMed Central

Bhalla, Manmeet; Law, Daria; Dowd, Georgina C.

2017-01-01

ABSTRACT The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-α (PKC-α) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1α (HIF-1α) and the mTORC2 substrate PKC-α each control Listeria uptake. Genetic or pharmacological inhibition of PKC-α reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-α to be host factors exploited by Listeria to promote infection. PKC-α controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor. PMID:28461391

Effects of the location of distal histidine in the reaction of myoglobin with hydrogen peroxide.

PubMed

Matsui, T; Ozaki, S i; Liong, E; Phillips, G N; Watanabe, Y

1999-01-29

To clarify how the location of distal histidine affects the activation process of H2O2 by heme proteins, we have characterized reactions with H2O2 for the L29H/H64L and F43H/H64L mutants of sperm whale myoglobin (Mb), designed to locate the histidine farther from the heme iron. Whereas the L29H/H64L double substitution retarded the reaction with H2O2, an 11-fold rate increase versus wild-type Mb was observed for the F43H/H64L mutant. The Vmax values for 1-electron oxidations by the myoglobins correlate well with the varied reactivities with H2O2. The functions of the distal histidine as a general acid-base catalyst were examined based on the reactions with cumene hydroperoxide and cyanide, and only the histidine in F43H/H64L Mb was suggested to facilitate heterolysis of the peroxide bond. The x-ray crystal structures of the mutants confirmed that the distal histidines in F43H/H64L Mb and peroxidase are similar in distance from the heme iron, whereas the distal histidine in L29H/H64L Mb is located too far to enhance heterolysis. Our results indicate that the proper positioning of the distal histidine is essential for the activation of H2O2 by heme enzymes.

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice

PubMed Central

Eigenbrod, Sabina; Frick, Petra; Bertsch, Uwe; Mitteregger-Kretzschmar, Gerda; Mielke, Janina; Maringer, Marko; Piening, Niklas; Hepp, Alexander; Daude, Nathalie; Windl, Otto; Levin, Johannes; Giese, Armin; Sakthivelu, Vignesh; Tatzelt, Jörg

2017-01-01

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1–4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain. PMID:29220360

The mechanism of signal transduction by two-component systems.

PubMed

Casino, Patricia; Rubio, Vicente; Marina, Alberto

2010-12-01

Two-component systems, composed of a homodimeric histidine kinase (HK) and a response regulator (RR), are major signal transduction devices in bacteria. Typically the signal triggers HK autophosphorylation at one His residue, followed by phosphoryl transfer from the phospho-His to an Asp residue in the RR. Signal extinction frequently involves phospho-RR dephosphorylation by a phosphatase activity of the HK. Our understanding of these reactions and of the determinants of partner specificity among HK-RR couples has been greatly increased by recent crystal structures and biochemical experiments on HK-RR complexes. Cis-autophosphorylation (one subunit phosphorylates itself) occurs in some HKs while trans-autophosphorylation takes place in others. We review and integrate this new information, discuss the mechanism of the three reactions and propose a model for transmembrane signaling by these systems. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effect of starvation on free histidine and amino acids in white muscle of milkfish Chanos chanos.

PubMed

Shiau, C Y; Pong, Y P; Chiou, T K; Tin, Y Y

2001-03-01

Milkfish (Chanos chanos) decreased their body weight from 47 to 28 g over the 60-day period of starvation. Starvation also resulted in the reduction of muscle lipid and protein, and hepatosomatic index. The predominant free amino acid (FAA) in white muscle of milkfish was histidine, followed by taurine and glycine. In the first 25 days of starvation, no significant change in histidine was found. After 40 days of starvation, however, the histidine concentration was significantly decreased by 46%, and remained unchanged thereafter. As compared to control group fish, the 60-day-starved fish possessed only half the amount of histidine. Taurine and glycine, on the other hand, showed no significant changes throughout starvation. Taurine became the most predominant in the FAA pool after 40 days of starvation, and the concentration of 60-day-starved fish was two times higher than that of control group fish without starvation. The ratios of histidine, taurine, and glycine to total FAAs remained approximately the same although the individual contributions varied considerably to the total FAAs during starvation. The results of this study suggested that a good strategy would be to keep taurine and glycine in milkfish muscle at relatively high levels for physiological function as histidine decreased drastically for energy source under conditions of food deprivation.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

PubMed

Hoch, Eitan; Lin, Wei; Chai, Jin; Hershfinkel, Michal; Fu, Dax; Sekler, Israel

2012-05-08

Zinc and cadmium are similar metal ions, but though Zn(2+) is an essential nutrient, Cd(2+) is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn(2+) vs. Cd(2+) suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn(2+) transport, but reject Cd(2+), thus constituting the first mammalian metal transporter with a refined selectivity against Cd(2+). Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn(2+) and Cd(2+). A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn(2+) transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd(2+) by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn(2+) and Cd(2+), and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd(2+) binding.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity

PubMed Central

Hoch, Eitan; Lin, Wei; Chai, Jin; Hershfinkel, Michal; Fu, Dax; Sekler, Israel

2012-01-01

Zinc and cadmium are similar metal ions, but though Zn2+ is an essential nutrient, Cd2+ is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn2+ vs. Cd2+ suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn2+ transport, but reject Cd2+, thus constituting the first mammalian metal transporter with a refined selectivity against Cd2+. Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn2+ and Cd2+. A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn2+ transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd2+ by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn2+ and Cd2+, and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd2+ binding. PMID:22529353

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

PubMed Central

Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

2017-01-01

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin.

PubMed

Gutowska-Owsiak, D; Greenwald, L; Watson, C; Selvakumar, T A; Wang, X; Ogg, G S

2014-10-01

Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. We detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-α, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention. © 2014 British Association of Dermatologists.

X-Ray Absorption Near-Edge Structure (XANES) Spectroscopy Study of the Interaction of Silver Ions with Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli

PubMed Central

Zanzen, Ulrike; Krishna, Katla Sai; Hormes, Josef

2013-01-01

Silver ions are widely used as antibacterial agents, but the basic molecular mechanism of this effect is still poorly understood. X-ray absorption near-edge structure (XANES) spectroscopy at the Ag LIII, S K, and P K edges reveals the chemical forms of silver in Staphylococcus aureus and Escherichia coli (Ag+ treated). The Ag LIII-edge XANES spectra of the bacteria are all slightly different and very different from the spectra of silver ions (silver nitrate and silver acetate), which confirms that a reaction occurs. Death or inactivation of bacteria was observed by plate counting and light microscopy. Silver bonding to sulfhydryl groups (Ag-S) in cysteine and Ag-N or Ag-O bonding in histidine, alanine, and dl-aspartic acid was detected by using synthesized silver-amino acids. Significantly lower silver-cysteine content, coupled with higher silver-histidine content, in Gram-positive S. aureus and Listeria monocytogenes cells indicates that the peptidoglycan multilayer could be buffering the biocidal effect of silver on Gram-positive bacteria, at least in part. Bonding of silver to phosphate groups was not detected. Interaction with DNA or proteins can occur through Ag-N bonding. The formation of silver-cysteine can be confirmed for both bacterial cell types, which supports the hypothesis that enzyme-catalyzed reactions and the electron transport chain within the cell are disrupted. PMID:23934494

Kounis syndrome following canned tuna fish ingestion.

PubMed

De Gennaro, Luisa; Brunetti, Natale Daniele; Locuratolo, Nicola; Ruggiero, Massimo; Resta, Manuela; Diaferia, Giuseppe; Rana, Michele; Caldarola, Pasquale

2017-04-01

Kounis syndrome (KS) is a complex of cardiovascular symptoms and signs following either allergy or hypersensitivity and anaphylactic or anaphylactoid insults. We report the case of 57-year-old man, with hypertension and history of allergy, referred for facial rash and palpitations appeared after consumption of canned tuna fish. Suddenly, the patient collapsed: electrocardiogram showed ST-elevation in inferior leads. The patient was transferred from the spoke emergency room for coronary angio, which did not show any sign of coronary atherosclerosis. A transient coronary spasm was therefore hypothesized and the final diagnosis was KS. To the best of our knowledge, this is one of the first cases of KS following the ingestion of tuna fish. KS secondary to food allergy has also been reported, and shellfish ingestion has been considered as one of the most active KS inducer foods. Canned tuna fish too is well known as an allergy inducer. Tuna fish allergy should be considered, however, within the context of scombroid food poisoning, also called histamine fish poisoning. Fish with high levels of free histidine, the enzyme substrate converted to histamine by bacterial histidine decarboxylase, are those most often implicated in scombroid poisoning. Inflammatory mediators such as histamine constitute the pathophysiologic basis of Kounis hypersensitivity-associated acute coronary syndrome. Patients with coronary risk factors, allergic reaction after food ingestion, and suspected scombroid poisoning should be therefore carefully monitored for a prompt diagnosis of possible coronary complications.

Structural basis for the sequestration of the anti-σ(70) factor Rsd from σ(70) by the histidine-containing phosphocarrier protein HPr.

PubMed

Park, Young Ha; Um, Si Hyeon; Song, Saemee; Seok, Yeong Jae; Ha, Nam Chul

2015-10-01

Histidine-containing phosphocarrier protein (HPr) is a general component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) involved in the phosphorylation-coupled transport of numerous sugars called PTS sugars. HPr mainly exists in a dephosphorylated form in the presence of PTS sugars in the medium, while its phosphorylation increases in the absence of PTS sugars. A recent study revealed that the dephosphorylated form of HPr binds and antagonizes the function of the antisigma factor Rsd. This anti-sigma factor sequesters the housekeeping sigma factor σ(70) to facilitate switching of the sigma subunit on RNA polymerase from σ(70) to the stress-responsive sigma factor σ(S) in stationary-phase cells. In this study, the structure of the complex of Rsd and HPr was determined at 2.1 Å resolution and revealed that the binding site for HPr on the surface of Rsd partly overlaps with that for σ(70). The localization of the phosphorylation site on HPr at the binding interface for Rsd explains why phosphorylation of HPr abolishes its binding to Rsd. The mutation of crucial residues involved in the HPr-Rsd interaction significantly influenced the competition between HPr and σ(70) for binding to Rsd both in vitro and in vivo. The results provide a structural basis for the linkage of global gene regulation to nutrient availability in the external environment.

[Ionization energies and infrared spectra studies of histidine using density functional theory].

PubMed

Hu, Qiong; Wang, Guo-Ying; Liu, Gang; Ou, Jia-Ming; Wang, Rui-Li

2010-05-01

Histidines provide axial ligands to the primary electron donors in photosynthetic reaction centers (RCs) and play an important role in the protein environments of these donors. In this paper the authors present a systematic study of ionization energies and vibrational properties of histidine using hybrid density functional theory (DFT). All calculations were undertaken by using B3LYP method in combination with four basis sets: 6-31G(d), 6-31G(df, p), 6-31+G(d) and 6-311+G(2d, 2p) with the aim to investigate how the basis sets influence the calculation results. To investigate solvent effects and gain a detailed understanding of marker bands of histidine, the ionization energies of histidine and the vibrational frequencies of histidine which are unlabeled and 13C, 15N, and 2H labeled in the gas phase, CCl4, protein environment, THF and water solution, which span a wide range of dielectric constant, were also calculated. Our results showed that: (1) The main geometry parameters of histidine were impacted by basis sets and mediums, and C2-N3 and N3-C4 bond of imidazole ring of histidine side chain display the maximum bond lengths in the gas phase; (2) single point energies and frequencies calculated were decreased while ionization energies increased with the increasing level of basis sets and diffuse function applied in the same solvent; (3) with the same computational method, the higher the dielectric constant of the solvent used, the lower the ionization energy and vibrational frequency and the higher the intensity obtained. In addition, calculated ionization energy in the gas phase and marker bands of histidine as well as frequency shift upon 13C and 15N labeling at the computationally more expensive 6-311+G(2d, 2p) level are in good agreement with experimental observations available in literatures. All calculations indicated that the results calculated by using higher level basis set with diffuse function were more accurate and closer to the experimental value. In conclusion, the results provide useful information for the further studies of the functional and vibrational properties of chlorophyll-a ligated to histidine residue in photosynthetic reaction center.

Identification of gold sensing peptide by integrative proteomics and a bacterial two-component system

NASA Astrophysics Data System (ADS)

Ng, I.-Son; Yu, You-Jin; Yi, Ying-Chen; Tan, Shih-I.; Huang, Bo-Chuan; Han, Yin-Lung

2017-12-01

The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from Tepidimonas fonticaldi AT-A2, due to its outstanding performance in gold-binding and recovery. The results showed that three small proteins, including histidine biosynthesis protein (HisIE), iron donor protein (CyaY) and hypothetical protein_65aa, have a higher ability to adsorb gold ions because of the negatively charged domains or metal binding sites. On the other hand, the Salmonella PmrA/PmrB two-component system first replaces the iron (III)-binding motif using the peptide sequence from hypothetical protein_65aa, and this is then used to reveal the sensing and responsiveness to gold metal ions, which is totally different from the performance of traditional gold binding peptide (GBP) on the crystals on the surface of gold (111). We have successfully demonstrated an integrative proteomics and bacterial two-component system to explore the novel gold binding peptide. Finally, the heterologous over-expression of gold binding peptide by E. coli and the equilibrium of binding capacity for Au(III) have been conducted.

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

PubMed

Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation

PubMed Central

Khan, Mohammed A S; Kang, Jian; Steiner, Theodore S

2004-01-01

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings. EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines. The host response to flagellins from E. coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-κB) to induce transcription of pro-inflammatory cytokines. p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products. We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells. We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC. The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct. Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC. Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-κB activation. Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-κB nuclear migration. PMID:15270737

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

2016-06-01

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins

PubMed Central

Valéry, Céline; Deville-Foillard, Stéphanie; Lefebvre, Christelle; Taberner, Nuria; Legrand, Pierre; Meneau, Florian; Meriadec, Cristelle; Delvaux, Camille; Bizien, Thomas; Kasotakis, Emmanouil; Lopez-Iglesias, Carmen; Gall, Andrew; Bressanelli, Stéphane; Le Du, Marie-Hélène; Paternostre, Maïté; Artzner, Franck

2015-01-01

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended β-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials. PMID:26190377

Regulatory Mutants at the his1 Locus of Yeast

PubMed Central

Lax, Carol; Fogel, Seymour; Cramer, Carole

1979-01-01

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1–7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auoxtrophic parental alleles. The double mutants, HIS1–7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1–7 background, the mutant regulatory site (HIS1–e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine. PMID:385447

Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters Resistance to Bacterial and Fungal Pathogens.

PubMed

Harkenrider, Mitch; Sharma, Rita; De Vleesschauwer, David; Tsao, Li; Zhang, Xuting; Chern, Mawsheng; Canlas, Patrick; Zuo, Shimin; Ronald, Pamela C

2016-01-01

Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus.

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

PubMed Central

Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B

1997-01-01

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm. PMID:9405352

Identification of the PhoB Regulon and Role of PhoU in the Phosphate Starvation Response of Caulobacter crescentus.

PubMed

Lubin, Emma A; Henry, Jonathan T; Fiebig, Aretha; Crosson, Sean; Laub, Michael T

2016-01-01

An ability to sense and respond to changes in extracellular phosphate is critical for the survival of most bacteria. For Caulobacter crescentus, which typically lives in phosphate-limited environments, this process is especially crucial. Like many bacteria, Caulobacter responds to phosphate limitation through a conserved two-component signaling pathway called PhoR-PhoB, but the direct regulon of PhoB in this organism is unknown. Here we used chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) to map the global binding patterns of the phosphate-responsive transcriptional regulator PhoB under phosphate-limited and -replete conditions. Combined with genome-wide expression profiling, our work demonstrates that PhoB is induced to regulate nearly 50 genes under phosphate-starved conditions. The PhoB regulon is comprised primarily of genes known or predicted to help Caulobacter scavenge for and import inorganic phosphate, including 15 different membrane transporters. We also investigated the regulatory role of PhoU, a widely conserved protein proposed to coordinate phosphate import with expression of the PhoB regulon by directly modulating the histidine kinase PhoR. However, our studies show that it likely does not play such a role in Caulobacter, as PhoU depletion has no significant effect on PhoB-dependent gene expression. Instead, cells lacking PhoU exhibit striking accumulation of large polyphosphate granules, suggesting that PhoU participates in controlling intracellular phosphate metabolism. The transcription factor PhoB is widely conserved throughout the bacterial kingdom, where it helps organisms respond to phosphate limitation by driving the expression of a battery of genes. Most of what is known about PhoB and its target genes is derived from studies of Escherichia coli. Our work documents the PhoB regulon in Caulobacter crescentus, and comparison to the regulon in E. coli reveals significant differences, highlighting the evolutionary plasticity of transcriptional responses driven by highly conserved transcription factors. We also demonstrated that the conserved protein PhoU, which is implicated in bacterial persistence, does not regulate PhoB activity, as previously suggested. Instead, our results favor a model in which PhoU affects intracellular phosphate accumulation, possibly through the high-affinity phosphate transporter. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

PubMed

Tenconi, Paula E; Giusto, Norma M; Salvador, Gabriela A; Mateos, Melina V

2016-12-01

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/βII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and β protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, β and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Conserved p38 Mitogen-Activated Protein Kinase Pathway Regulates Drosophila Immunity Gene Expression

PubMed Central

Han, Zhiqiang Stanley; Enslen, Hervé; Hu, Xiaodi; Meng, Xiangjun; Wu, I-Huan; Barrett, Tamera; Davis, Roger J.; Ip, Y. Tony

1998-01-01

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-κB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide. PMID:9584193

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

PubMed

Athwal, Navjot Singh; Alagurajan, Jagannathan; Andreotti, Amy H; Hargrove, Mark S

2016-10-18

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

Study of the affinity between the protein kinase PKA and homoarginine-containing peptides derived from kemptide: Free energy perturbation (FEP) calculations.

PubMed

Mena-Ulecia, Karel; Gonzalez-Norambuena, Fabian; Vergara-Jaque, Ariela; Poblete, Horacio; Tiznado, William; Caballero, Julio

2018-06-15

Protein kinases (PKs) discriminate between closely related sequences that contain serine, threonine, and/or tyrosine residues. Such specificity is defined by the amino acid sequence surrounding the phosphorylatable residue, so that it is possible to identify an optimal recognition motif (ORM) for each PK. The ORM for the protein kinase A (PKA), a well-known member of the PK family, is the sequence RRX(S/T)X, where arginines at the -3 and -2 positions play a key role with respect to the primed phosphorylation site. In this work, differential affinities of PKA for the peptide substrate Kemptide (LRRASLG) and mutants that substitute the arginine residues by the unnatural peptide homoarginine were evaluated through molecular dynamics (MD) and free energy perturbation (FEP) calculations. The FEP study for the homoarginine mutants required previous elaboration of a CHARMM "arginine to homoarginine" (R2B) hybrid topology file which is available in this manuscript as Supporting Information. Mutants substituting the arginine residues by alanine, lysine, and histidine were also considered in the comparison by using the same protocol. FEP calculations allowed estimating the free energy changes from the free PKA to PKA-substrate complex (ΔΔG E→ES ) when Kemptide structure was mutated. Both ΔΔG S→ES values for homoarginine mutants were predicted with a difference below 1 kcal/mol. In addition, FEP correctly predicted that all the studied mutations decrease the catalytic efficiency of Kemptide for PKA. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.

PubMed

Burczyk, Martina; Burkhalter, Martin D; Blätte, Tamara; Matysik, Sabrina; Caron, Marc G; Barak, Lawrence S; Philipp, Melanie

2015-01-27

The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

NASA Astrophysics Data System (ADS)

Mishra, Arjun K.; Singh, Nidhi; Agnihotri, Pragati; Mishra, Shikha; Singh, Saurabh P.; Kolli, Bala K.; Chang, Kwang Poo; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Pratap, J. Venkatesh

2017-06-01

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation -based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed 45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

PubMed Central

2016-01-01

The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m93 (milm93), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling. PMID:25555130

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

PubMed

Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

PubMed Central

Lensink, Marc F.; Wintjens, René; Vagin, Alexey; Lebedev, Andrey; Crosson, Sean; Villeret, Vincent; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

2015-01-01

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions. PMID:25738876

Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes.

PubMed

Anantharaman, Vivek; Aravind, L

2003-01-01

Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-gamma-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages. Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include lecithin retinol acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.

The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

PubMed

Li, Zhiru; Dugan, Aisling S; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R

2009-09-17

The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond.

PubMed

Kulis-Horn, Robert Kasimir; Rückert, Christian; Kalinowski, Jörn; Persicke, Marcus

2017-07-18

The eighth step of L-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in L-histidine biosynthesis. Deletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete L-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases. Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two paralog groups from each other and other IMPases. The kinetic data obtained for HisN from C. glutamicum, as an example for an IMPase-like HolPases, shows remarkable differences in enzyme properties as compared to HAD- or PHP-type HolPases. The six sequence motifs and the HMMs presented in this study can be used to reliably differentiate between IMPase-like HolPases and IMPase-like proteins with no such activity, with the potential to enhance current and future genome annotations. A phylogenetic analysis reveals that IMPase-like HolPases are not only present in Actinobacteria and plant but can be found in further bacterial phyla, including, among others, Proteobacteria, Chlorobi and Planctomycetes.

Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition.

PubMed

Das, Ushati; Wang, Li Kai; Smith, Paul; Jacewicz, Agata; Shuman, Stewart

2014-01-01

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.

Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

NASA Technical Reports Server (NTRS)

White, D. H.; Erickson, J. C.

1980-01-01

The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic proto-enzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.

Phosphatase activity tunes two-component system sensor detection threshold.

PubMed

Landry, Brian P; Palanki, Rohan; Dyulgyarov, Nikola; Hartsough, Lucas A; Tabor, Jeffrey J

2018-04-12

Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larimer, Frank W; Chain, Patrick S. G.; Hauser, Loren John

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant-derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustrismore » encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.« less

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets.

PubMed

Bott, Michael; Brocker, Melanie

2012-06-01

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m(3) volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species.

Protein Kinase C Processes and Their Relation to Apoptosis in Human Breast Carcinoma Cells

DTIC Science & Technology

1997-08-01

with two members of this library indicated competitive inhibition with K4 values of < 15 uM for Cdc25 DSPases that were also noncompetitive PTP1B ...the PTP1B . AC-aa59 had antiproliferative activity against and caused a Gi block in MDA-MB-231 cells. 14. SUBJECT TERMS Apoptosis, Protein Kinase C...Recombinant PTP1B was obatained from Upstate Biotechnology(Lake Placid, NY). Bacterial growth and fusion protein production. E. co//strain BL21 (DE3

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

PubMed

Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

2017-09-01

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

PubMed

Stranava, Martin; Man, Petr; Skálová, Tereza; Kolenko, Petr; Blaha, Jan; Fojtikova, Veronika; Martínek, Václav; Dohnálek, Jan; Lengalova, Alzbeta; Rosůlek, Michal; Shimizu, Toru; Martínková, Markéta

2017-12-22

The heme-based oxygen sensor histidine kinase Af GcHK is part of a two-component signal transduction system in bacteria. O 2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His 183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH - and -CN - complexes of Af GcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN - and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length Af GcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of Af GcHK. We conclude that Af GcHK functions as an ensemble of molecules sampling at least two conformational states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

PubMed Central

Morona, Renato

2014-01-01

Abstract Significance: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Recent Advances: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Critical Issues: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Future Directions: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria. Antioxid. Redox Signal. 20, 2274–2289. PMID:24295407

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

PubMed Central

Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

1992-01-01

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

Proton affinity of the histidine-tryptophan cluster motif from the influenza A virus from ab initio molecular dynamics

NASA Astrophysics Data System (ADS)

Bankura, Arindam; Klein, Michael L.; Carnevale, Vincenzo

2013-08-01

Ab initio molecular dynamics calculations have been used to compare and contrast the deprotonation reaction of a histidine residue in aqueous solution with the situation arising in a histidine-tryptophan cluster. The latter is used as a model of the proton storage unit present in the pore of the M2 proton conducting ion channel. We compute potentials of mean force for the dissociation of a proton from the Nδ and Nɛ positions of the imidazole group to estimate the pKas. Anticipating our results, we will see that the estimated pKa for the first protonation event of the M2 channel is in good agreement with experimental estimates. Surprisingly, despite the fact that the histidine is partially desolvated in the M2 channel, the affinity for protons is similar to that of a histidine in aqueous solution. Importantly, the electrostatic environment provided by the indoles is responsible for the stabilization of the charged imidazolium.

Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor*

PubMed Central

An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W.; Kaplan, David L.; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

2016-01-01

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities. PMID:26702058

The Impact of Protein Phosphorylation on Chlamydial Physiology

PubMed Central

Claywell, Ja E.; Matschke, Lea M.; Fisher, Derek J.

2016-01-01

Chlamydia are Gram negative bacterial pathogens responsible for disease in humans and economically important domesticated animals. As obligate intracellular bacteria, they must gain entry into a host cell where they propagate within a parasitophorous organelle that serves as an interactive interface between the bacterium and the host. Nutrient acquisition, growth, and evasion of host defense mechanisms occur from this location. In addition to these cellular and bacterial dynamics, Chlamydia differentiate between two morphologically distinct forms, the elementary body and reticulate body, that are optimized for either extracellular or intracellular survival, respectively. The mechanisms regulating and mediating these diverse physiological events remain largely unknown. Reversible phosphorylation, including classical two-component signaling systems, partner switching mechanisms, and the more recently appreciated bacterial Ser/Thr/Tyr kinases and phosphatases, has gained increasing attention for its role in regulating important physiological processes in bacteria including metabolism, development, and virulence. Phosphorylation modulates these events via rapid and reversible modification of protein substrates leading to changes in enzyme activity, protein oligomerization, cell signaling, and protein localization. The characterization of several conserved chlamydial protein kinases and phosphatases along with phosphoproteome analysis suggest that Chlamydia are capable of global and growth stage-specific protein phosphorylation. This mini review will highlight the current knowledge of protein phosphorylation in Chlamydia and its potential role in chlamydial physiology and, consequently, virulence. Comparisons with other minimal genome intracellular bacterial pathogens also will be addressed with the aim of illustrating the importance of this understudied regulatory mechanism on pathogenesis and the principle questions that remain unanswered. PMID:28066729

An alteration of the gut-liver axis drives pulmonary inflammation after intoxication and burn injury in mice

PubMed Central

Chen, Michael M.; Zahs, Anita; Brown, Mary M.; Ramirez, Luis; Turner, Jerrold R.; Choudhry, Mashkoor A.

2014-01-01

Approximately half of all adult burn patients are intoxicated at the time of their injury and have worse clinical outcomes than those without prior alcohol exposure. This study tested the hypothesis that intoxication alters the gut-liver axis, leading to increased pulmonary inflammation mediated by burn-induced IL-6 in the liver. C57BL/6 mice were given 1.2 g/kg ethanol 30 min prior to a 15% total body surface area burn. To restore gut barrier function, the specific myosin light chain kinase inhibitor membrane-permeant inhibitor of kinase (PIK), which we have demonstrated to reduce bacterial translocation from the gut, was administered 30 min after injury. Limiting bacterial translocation with PIK attenuated hepatic damage as measured by a 47% reduction in serum alanine aminotransferase (P < 0.05), as well as a 33% reduction in hepatic IL-6 mRNA expression (P < 0.05), compared with intoxicated and burn-injured mice without PIK. This mitigation of hepatic damage was associated with a 49% decline in pulmonary neutrophil infiltration (P < 0.05) and decreased alveolar wall thickening compared with matched controls. These results were reproduced by prophylactic reduction of the bacterial load in the intestines with oral antibiotics before intoxication and burn injury. Overall, these data suggest that the gut-liver axis is deranged when intoxication precedes burn injury and that limiting bacterial translocation in this setting attenuates hepatic damage and pulmonary inflammation. PMID:25104501

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells*

PubMed Central

Agarwal, Vaibhav; Asmat, Tauseef M.; Luo, Shanshan; Jensch, Inga; Zipfel, Peter F.; Hammerschmidt, Sven

2010-01-01

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase. PMID:20504767

EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

PubMed

Pearson, Jaclyn S; Giogha, Cristina; Mühlen, Sabrina; Nachbur, Ueli; Pham, Chi L L; Zhang, Ying; Hildebrand, Joanne M; Oates, Clare V; Lung, Tania Wong Fok; Ingle, Danielle; Dagley, Laura F; Bankovacki, Aleksandra; Petrie, Emma J; Schroeder, Gunnar N; Crepin, Valerie F; Frankel, Gad; Masters, Seth L; Vince, James; Murphy, James M; Sunde, Margaret; Webb, Andrew I; Silke, John; Hartland, Elizabeth L

2017-01-13

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling 1-3 . RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis 4,5 . Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

PubMed

Parker, Antony R

2003-10-01

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.

The active transport of histidine and its role in ATP production in Trypanosoma cruzi.

PubMed

Barisón, M J; Damasceno, F S; Mantilla, B S; Silber, A M

2016-08-01

Trypanosoma cruzi, the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of T. cruzi, suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in T. cruzi, showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens.

PubMed

Rodríguez-Moya, Javier; Argandoña, Montserrat; Reina-Bueno, Mercedes; Nieto, Joaquín J; Iglesias-Guerra, Fernando; Jebbar, Mohamed; Vargas, Carmen

2010-10-13

Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions. A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain. This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Contributions of the Histidine Side Chain and the N-terminal α-Amino Group to the Binding Thermodynamics of Oligopeptides to Nucleic Acids as a Function of pH

PubMed Central

Ballin, Jeff D.; Prevas, James P.; Ross, Christina R.; Toth, Eric A.; Wilson, Gerald M.; Record, M. Thomas

2010-01-01

Interactions of histidine with nucleic acid phosphates and histidine pKa shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK (+NH3-His-Trp-Lys-Lys-NH2) and its lysine analog KWKK (+NH3-Lys-Trp-Lys-Lys-NH2) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, Kobs, and magnitudes of log-log salt derivatives SKobs ≡ (∂logKobs/∂log[Na+]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and α-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same Kobs and SKobs within uncertainty, and these SKobs values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pKa shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pKa shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the α-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific versus nonspecific nucleic acid binding. PMID:20108951

Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligands.

PubMed

Amorim, Lúcia F A; Gaspar, Rita; Pereira, Patrícia; Černigoj, Urh; Sousa, Fani; Queiroz, João António; Sousa, Ângela

2017-11-01

Affinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH 4 ) 2 SO 4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase.

PubMed

Hsiao, Yi Y; Van, Ru C; Hung, Shu H; Lin, Hsin H; Pan, Rong L

2004-02-15

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.

Molecular switch-modulated fluorescent copper nanoclusters for selective and sensitive detection of histidine and cysteine.

PubMed

Gu, Zefeng; Cao, Zhijuan

2018-06-07

A novel assay for histidine and cysteine has been constructed based on modulation of fluorescent copper nanoclusters (CuNCs) by molecular switches. In our previous work, a dumbbell DNA template with a poly-T (thymine) loop has been developed as an excellent template for the formation of strongly fluorescent CuNCs. Herein, for the first time, we established this biosensor for sensing two amino acids by using dumbbell DNA-templated CuNCs as the single probe. Among 20 natural amino acids, only histidine and cysteine can selectively quench fluorescence emission of CuNCs, because of the specific interaction of these compounds with copper ions. Furthermore, by using nickel ions (Ni 2+ ) and N-ethylmaleimide as the masking agents for histidine and cysteine respectively, an integrated logic gate system was designed by coupling with the fluorescent CuNCs and demonstrated selective and sensitive detection of cysteine and histidine. Under optimal conditions, cysteine can be detected in the concentration ranges of 0.01-10.0 μM with the detection limit (DL) of as low as 98 pM, while histidine can be detected in the ranges of 0.05-40.0 μM with DL of 1.6 nM. In addition, histidine and cysteine can be observed with the naked eye under a hand-held UV lamp (DL, 50 nM), which can be easily adapted to automated high-throughput screening. Finally, the strategy has been successfully utilized for biological fluids. The proposed system can be conducted in homogeneous solution, eliminating the need for organic cosolvents, separation processes of nanomaterials, or any chemical modifications. Overall, the assay provides an alternative method for simultaneous detection of cysteine and histidine by taking the advantages of high speed, no label and enzyme requirement, and good sensitivity and specificity, and will satisfy the great demand for determination of amino acids in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. Graphical abstract.

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

PubMed Central

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

2015-01-01

Summary The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. PMID:25402841

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

PubMed

Hammerstrom, Troy G; Horton, Lori B; Swick, Michelle C; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M

2015-02-01

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism. © 2014 John Wiley & Sons Ltd.

Acyltransferases in Bacteria

PubMed Central

Röttig, Annika

2013-01-01

SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue. PMID:23699259

Sinorhizobium meliloti CtrA Stability Is Regulated in a CbrA-Dependent Manner That Is Influenced by CpdR1

PubMed Central

Schallies, Karla B.; Sadowski, Craig; Meng, Julia; Chien, Peter

2015-01-01

ABSTRACT CbrA is a DivJ/PleC-like histidine kinase of DivK that is required for cell cycle progression and symbiosis in the alphaproteobacterium Sinorhizobium meliloti. Loss of cbrA results in increased levels of CtrA as well as its phosphorylation. While many of the known Caulobacter crescentus regulators of CtrA phosphorylation and proteolysis are phylogenetically conserved within S. meliloti, the latter lacks the PopA regulator that is required for CtrA degradation in C. crescentus. In order to investigate whether CtrA proteolysis occurs in S. meliloti, CtrA stability was assessed. During exponential growth, CtrA is unstable and therefore likely to be degraded in a cell cycle-regulated manner. Loss of cbrA significantly increases CtrA stability, but this phenotype is restored to that of the wild type by constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated (CpdR1D53A). Addition of CpdR1D53A fully suppresses cbrA mutant cell cycle defects, consistent with regulation of CtrA stability playing a key role in mediating proper cell cycle progression in S. meliloti. Importantly, the cbrA mutant symbiosis defect is also suppressed in the presence of CpdR1D53A. Thus, regulation of CtrA stability by CbrA and CpdR1 is associated with free-living cell cycle outcomes and symbiosis. IMPORTANCE The cell cycle is a fundamental process required for bacterial growth, reproduction, and developmental differentiation. Our objective is to understand how a two-component signal transduction network directs cell cycle events during free-living growth and host colonization. The Sinorhizobium meliloti nitrogen-fixing symbiosis with plants is associated with novel cell cycle events. This study identifies a link between the regulated stability of an essential response regulator, free-living cell cycle progression, and symbiosis. PMID:25897034

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America.

PubMed

Giampetruzzi, Annalisa; Saponari, Maria; Loconsole, Giuliana; Boscia, Donato; Savino, Vito Nicola; Almeida, Rodrigo P P; Zicca, Stefania; Landa, Blanca B; Chacón-Diaz, Carlos; Saldarelli, Pasquale

2017-07-01

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

USDA-ARS?s Scientific Manuscript database

Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation.

PubMed

Labesse, Gilles; Benkali, Khaled; Salard-Arnaud, Isabelle; Gilles, Anne-Marie; Munier-Lehmann, Hélène

2011-04-01

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(-) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

DupA: a key regulator of the amoebal MAP kinase response to Legionella pneumophila

PubMed Central

Li, Zhiru; Dugan, Aisling S.; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R.

2009-01-01

SUMMARY The dupA gene, encoding a putative tyrosine kinase/dual specificity phosphatase (Dusp), was identified in a screen for Dictyostelium discoideum mutants altered in supporting Legionella pneumophila intracellular replication. The absence of dupA resulted in hyperphosphorylation of ERK1, consistent with the loss of a phosphatase activity, as well as degradation of ERK2. ERK1 hyperphosphorylation mimicked the response of this protein after bacterial challenge of wild type amoebae. Similar to Dusps in higher eukaryotic cells, the amoebal dupA gene was induced after bacterial contact, indicating a response of Dusps that is conserved from amoebae to mammals. A large set of genes was misregulated in the dupA− mutant that largely overlaps with genes responding to L. pneumophila infection. Some of the amoebal genes appear to be involved in a response similar to innate immunity in higher eukaryotes, indicating there was misregulation of a conserved response to bacteria. PMID:19748467

Phosphorylation of spore coat proteins by a family of atypical protein kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

PubMed Central

Myers, M G; Backer, J M; Sun, X J; Shoelson, S; Hu, P; Schlessinger, J; Yoakim, M; Schaffhausen, B; White, M F

1992-01-01

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains. Images PMID:1332046

Phosphorylation of spore coat proteins by a family of atypical protein kinases

DOE PAGES

Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

2016-05-16

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

Metabolism of. cap alpha. -C/sup 14/-histidine in the intact rat. II. Radioactive excretion products in urine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, G.; Wu, P.H.L.; Heck, W.W.

1956-09-01

The normal metabolic pathways in the intact rat was investigated via the radioactive urinary excretion products following administration of a physiological dose of a radioactive compound such as ..cap alpha..-C/sup 14/-DL-histidine. The major metabolites, except one, excreted in the urine 5 hours after administration of ..cap alpha..-C/sup 14/-DL-histidine were isolated and identified. Glutamic acid and urocanic acids had simlar and low activities, whereas carboxyl-labeled imidazoacetic acid was found to be the principal metabolite with a high level of activity. It was concluded that the main end-product of the catabolism of DL-histidine is imidazoleacetic acid probably formed through imidazolepyruvic acid.

Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization

PubMed Central

Vorontsov, Egor; Gallaud, Julien; Malosse, Christian; Michel, Valérie; Cavazza, Christine; Robbe-Saule, Marie; Richaud, Pierre; Chamot-Rooke, Julia; Brochier-Armanet, Céline; De Reuse, Hilde

2015-01-01

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori. PMID:26641249

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

PubMed

Gray, K A; Dutton, P L; Daldal, F

1994-01-25

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET.

PubMed

Mons, Cécile; Ferecatu, Ioana; Riquier, Sylvie; Lescop, Ewen; Bouton, Cécile; Golinelli-Cohen, Marie-Pierre

2017-01-01

MitoNEET is the first identified Fe-S protein anchored to mammalian outer mitochondrial membranes with the vast majority of the protein polypeptide located in the cytosol, including its [2Fe-2S] cluster-binding domain. The coordination of the cluster is unusual and involves three cysteines and one histidine. MitoNEET is capable of transferring its redox-active Fe-S cluster to a bacterial apo-ferredoxin in vitro even under aerobic conditions, unlike other Fe-S transfer proteins such as ISCU. This specificity suggests its possible involvement in Fe-S repair after oxidative and/or nitrosative stress. Recently, we identified cytosolic aconitase/iron regulatory protein 1 (IRP1) as the first physiological protein acceptor of the mitoNEET Fe-S cluster in an Fe-S repair process. This chapter describes methods to study in vitro mitoNEET Fe-S cluster transfer/repair to a bacterial ferredoxin used as a model aporeceptor and in a more comprehensive manner to cytosolic aconitase/IRP1 after a nitrosative stress using in vitro, in cellulo, and in vivo methods. © 2017 Elsevier Inc. All rights reserved.

Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

PubMed

Munir, Annum; Shuman, Stewart

2016-11-28

5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Mechanism of Fine-tuning pH Sensors in Proprotein Convertases: IDENTIFICATION OF A pH-SENSING HISTIDINE PAIR IN THE PROPEPTIDE OF PROPROTEIN CONVERTASE 1/3.

PubMed

Williamson, Danielle M; Elferich, Johannes; Shinde, Ujwal

2015-09-18

The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a "gatekeeper" that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of the active site mechanism of Tyrosyl-DNA phosphodiesterase I: a member of the phospholipase D superfamily

PubMed Central

Gajewski, Stefan; Comeaux, Evan Q.; Jafari, Nauzanene; Bharatham, Nagakumar; Bashford, Donald; White, Stephen W.; van Waardenburg, Robert C.A.M.

2011-01-01

Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily. PMID:22155078

Analysis of the Active-Site Mechanism of Tyrosyl-DNA Phosphodiesterase I: A Member of the Phospholipase D Superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gajewski, Stefan; Comeaux, Evan Q.; Jafari, Nauzanene

2012-03-15

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines - one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observedmore » in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK{sub a} of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.« less

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

PubMed Central

2011-01-01

Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion. PMID:22204307

The protease ClpXP and the PAS-domain protein DivL regulate CtrA and gene transfer agent production in Rhodobacter capsulatus.

PubMed

Westbye, A B; Kater, L; Wiesmann, C; Ding, H; Yip, C K; Beatty, J T

2018-04-06

Several members of the Rhodobacterales ( Alphaproteobacteria ) produce a conserved horizontal gene transfer vector, called gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system to study GTA biology is the R. capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that a dysregulation of the CckA-ChpT-CtrA phosphorelay - either by loss of the PAS-domain protein DivL or by substituting the autophosphorylation residue of the hybrid histidine kinase CckA - decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that a loss of the ClpXP protease or the three C-terminal residues of CtrA resulted in increased CtrA levels in R. capsulatus, and identify ClpX(P) as essential for maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Rhodobacterales Importance Members of the Rhodobacterales are abundant in ocean and fresh water environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay, however several questions about GTA regulation remains. Our identification that a 'short' DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales. We found that the magnitude of RcGTA production greatly depended on DivL and CckA kinase activity, adding yet a layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles. Copyright © 2018 American Society for Microbiology.

Changes in the free amino acid composition with maturity of the noble cultivar of Vitis rotundifolia Michx. grape.

PubMed

Lamikanra, O; Kassa, A K

1999-12-01

The changes in amino acid composition that occur with maturity of the Noble cultivar of the Vitis rotundifolia Michx. (muscadine) grape were determined by HPLC. Eighteen amino acids were identified. Histidine was the most prominent amino acid followed by alanine. The concentrations of most of the major amino acids (alanine, glycine, histidine, valine, isoleucine, aspartic acid, and serine) were highest at verasion. Glutamine and threonine contents dropped sharply after fruit set, while those of arginine and proline increased gradually with maturity and ripening. Tyrosine content increased gradually with maturity and ripening following a slight drop after fruit set. In ripe grapes, seeds contained most of the amino acids in mature grapes (50%) followed by the pulp (23%), the juice (15%), and the skin (11%). Alanine, histidine, and arginine were the principal amino acids identified in the juice. Alanine, histidine, arginine, valine, glutamine, aspartic acid, proline, serine, and threonine accounted for about 90% of the amino acids in the pulp. In seeds, alanine, proline, asparagine, and histidine accounted for over 55% of the amino acids, while alanine and histidine were found to be the predominant free amino acids in the skin. The profile indicates some differences in the changes in amino acid composition with berry maturity and relative amounts of amino acids present in muscadine compared to those in nonmuscadine grape species.

The histidine permease gene (HIP1) of Saccharomyces cerevisiae.

PubMed

Tanaka, J; Fink, G R

1985-01-01

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.

Disruption of Chemoreceptor Signaling Arrays by High Levels of CheW, the Receptor-Kinase Coupling Protein

PubMed Central

Cardozo, Marcos J.; Massazza, Diego A.; Parkinson, John S.; Studdert, Claudia A.

2017-01-01

Summary During chemotactic signaling by Escherichia coli, the small cytoplasmic CheW protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signaling complexes, CheW in stoichiometric excess disrupts chemotactic behavior. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability, and elevated receptor methylation levels. The severity of these effects paralleled the receptor binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signaling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signaling. PMID:20487303

In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis

PubMed Central

Quisel, John D.; Burkholder, William F.; Grossman, Alan D.

2001-01-01

The phosphorylated form of the response regulator Spo0A (Spo0A∼P) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A∼P-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants. PMID:11673427

Activation of choline kinase drives aberrant choline metabolism in esophageal squamous cell carcinomas.

PubMed

Ma, Wang; Wang, Shuangyuan; Zhang, Tengfei; Zhang, Erik Y; Zhou, Lina; Hu, Chunxiu; Yu, Jane J; Xu, Guowang

2018-06-05

Esophageal squamous cell carcinoma (ESCC) is a major health threat worldwide. Research focused on molecular events associated with ESCC carcinogenesis for diagnosis, treatment and prevention is needed. Our goal is to discover novel biomarkers and investigate the underlying molecular mechanisms of ESCC progression by employing a global metabolomic approach. Sera from 34 ESCC patients and 32 age and sex matched healthy controls were profiled using two-dimensional liquid chromatography-mass spectrometry (2D LC-MS). We identified 120 differential metabolites in ESCC patient serums compared to healthy controls. Several amino acids, serine, arginine, lysine and histidine were significantly changed in ESCC patients. Most importantly, we found dysregulated lipid metabolism as an important characteristic in ESCC patients. Several free fat acids (FFA) and carnitines were found down-regulated in ESCC patients. Choline was significantly increased and phosphatidylcholines (PC) were significantly decreased in ESCC serum. The high expression of choline and low expression of total PC in patient serum were associated with the high expression of choline kinase (Chok) and activated Kennedy pathway in ESCC cells. Chok expression can serve as a significant biomarker for ESCC prognosis. In conclusion, metabolite profiles in the ESCC patient serum were significantly different from those in the healthy controls. Phosphatidylcholines and Chok, the key enzyme in the PC metabolism pathway, may serve as novel biomarkers for ESCC. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Germane, Katherine L.; Spiller, Benjamin W.

2011-09-20

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase.

PubMed

Liyasova, Mariya S; Schopfer, Lawrence M; Lockridge, Oksana

2013-03-25

Cresyl saligenin phosphate (CBDP) is a suspected causative agent of "aerotoxic syndrome", affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106 amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free L-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase

PubMed Central

Liyasova, Mariya S.; Schopfer, Lawrence M.; Lockridge, Oksana

2012-01-01

Cresyl saligenin phosphate (CBDP) is a suspected causative agent of “aerotoxic syndrome”, affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106 amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free L-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins. PMID:22898212

Heat-induced gelation of myosin in a low ionic strength solution containing L-histidine.

PubMed

Hayakawa, T; Yoshida, Y; Yasui, M; Ito, T; Iwasaki, T; Wakamatsu, J; Hattori, A; Nishimura, T

2012-01-01

Binding properties are important for meat products and are substantially derived from the heat-induced gelation of myosin. We have shown that myosin is solubilized in a low ionic strength solution containing L-histidine. To clarify its processing characteristics, we investigated properties and structures of heat-induced gels of myosin solubilized in a low ionic strength solution containing L-histidine. Myosin in a low ionic strength solution formed transparent gels at 40-50°C, while myosin in a high ionic strength solution formed opaque gels at 60-70°C. The gel of myosin in a low ionic strength solution with L-histidine showed a fine network consisting of thin strands and its viscosity was lower than that of myosin in a high ionic strength solution at 40-50°C. The rheological properties of heat-induced gels of myosin at low ionic strength are different from those at high ionic strength. This difference might be caused by structural changes in the rod region of myosin in a low ionic strength solution containing L-histidine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators

PubMed Central

Yan, Dalai; Cho, Ho S.; Hastings, Curtis A.; Igo, Michele M.; Lee, Seok-Yong; Pelton, Jeffrey G.; Stewart, Valley; Wemmer, David E.; Kustu, Sydney

1999-01-01

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains. PMID:10611291

Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

PubMed

Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

2014-12-01

Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

Getting to PTI of bacterial RNAs: Triggering plant innate immunity by extracellular RNAs from bacteria.

PubMed

Park, Yong-Soon; Lee, Boyoung; Ryu, Choong-Min

2016-07-02

Defense against diverse biotic and abiotic stresses requires the plant to distinguish between self and non-self signaling molecules. Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) are pivotal for triggering innate immunity in plants. Unlike in animals and humans, the precise roles of nucleic acids in plant innate immunity are unclear. We therefore investigated the effects of infiltration of total Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) RNAs into Arabidopsis plants. The pathogen population was 10-fold lower in bacterial RNAs pre-treated Arabidopsis plants than in the control. Bacterial RNAs purity was confirmed by physical (sonication) and chemical (RNase A and proteinase K digestion) methods. The perception of bacterial RNAs, especially rRNAs, positively regulated mitogen-activated protein kinase (MAPK) and induced a reactive oxygen species burst, callose deposition, salicylic acid (SA) and jasmonic acid (JA) signaling, and defense-related genes. Therefore, bacterial RNAs function as a new MAMP that activates plant innate immunity, providing a new paradigm for plant-microbe interactions.

The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain

PubMed Central

Husain, S. S.; Lowe, G.

1970-01-01

Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin. PMID:5420046

[Influence of intestinal microflora on the amino acid composition of lamb feces].

PubMed

Combe, E

1976-01-01

6 conventional and 5 germfree male lambs were fed ad libitum a UHT sterilized cow milk. Body weight and food intake were recorded. Whole feces were collected for 5 consecutive days. Growth rate reached 259 g/d for the germfree. Daily fecal excretion of dry matter and nitrogenous compounds are not found different in the two groups of animals. The influence of intestinal microflora appears on the biochemical composition of the feces. As compared to the conventional fecal proteins from germfree lambs are very high in threonine and serine and low in lysine. Moreover the difference of amino acid composition between these two groups come not only from the histidine alanine and arginine composition of bacteries; it also involves the high levels of threonine serine cystine and tyrosine of the endogenous digestive proteins.

Evidence against a bacterial endotoxin masking effect in biologic drug products by limulus amebocyte lysate detection.

PubMed

Bolden, Jay S; Claerbout, Mark E; Miner, Matthew K; Murphy, Marie A; Smith, Kelly R; Warburton, Rob E

2014-01-01

The inability to detect endotoxin using compendia methods is a potential safety concern for patients due to the lack of endotoxin removal capabilities at the fill-finish stage in typical aseptic biologic drug product manufacturing. We have successfully demonstrated endotoxin challenge study recovery methodology using mammalian cell-produced biologic drug products and drug substances in citrate, histidine, phosphate, and sodium acetate buffer formulations containing polysorbate, challenged with an endotoxin analyte, for up to 6 months of storage. Successful recovery was similarly demonstrated for a preserved, peptide-containing drug product formulation. To isolate a potential masking-or low-endotoxin recovery-source, additional studies were performed to evaluate factors including product manufacturing contact surfaces, drug product matrix with and without polysorbate, individual matrix components, protein concentration, reagent suppliers, an orthogonal test method, and storage conditions. In all cases, acceptable recoveries were observed. Bacterial endotoxin is known to be chemically stable at physiological conditions. Purified endotoxin in aqueous conditions is likely to self-aggregate or bind to surfaces. Neither the nature of, nor the storage conditions of, the studied formulation matrices were shown experimentally to render the challenge endotoxin biologically inactive. The results highlight the importance of appropriate study design in assessing the recovery of endotoxins. Bacterial endotoxin is a Gram-negative bacterial cell wall component that is harmful to humans at threshold concentrations, and it is not expected to be in aseptically-produced pharmaceutical medicines. It has been suggested that endotoxin cannot be detected over time in certain biopharmaceutical drug product formulations containing citrate, phosphate, and polysorbate components via an unknown masking mechanism. We have generated and present data here that indicate that endotoxin can be recovered in a variety of matrices, and under various experimental conditions. © PDA, Inc. 2014.

Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude-Nernst equation.

PubMed

Mauzerall, David; Hou, Jian-Min; Boichenko, Vladimir A

2002-01-01

Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1-10 mus time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (-26 A(3)) and reaction enthalpy (-0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, -1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of approximately 4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe(4)S(4))(Cys(4)) with charge change from -1 to -2. The small volume change in PS II is explained by sub-mus electron transfer from Y(Z) anion to P(680) cation, in which charge is only moved from the Y(Z) anion to the Q(A) with no charge separation or with rapid proton transfer from oxidized Y(Z) to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P(680) cation.

Factors Required for Activation of Urease as a Virulence Determinant in Cryptococcus neoformans

PubMed Central

Singh, Arpita; Panting, Robert J.; Varma, Ashok; Saijo, Tomomi; Waldron, Kevin J.; Jong, Ambrose; Ngamskulrungroj, Popchai; Chang, Yun C.; Rutherford, Julian C.; Kwon-Chung, Kyung J.

2013-01-01

ABSTRACT Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2+ into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria. PMID:23653445

Salivary Histatin 5 Is an Inhibitor of Both Host and Bacterial Enzymes Implicated in Periodontal Disease

PubMed Central

Gusman, Heloisa; Travis, James; Helmerhorst, Eva J.; Potempa, Jan; Troxler, Robert F.; Oppenheim, Frank G.

2001-01-01

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 μM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 μM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a Ki of 15 μM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity. PMID:11179305

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways.

PubMed

Liu, Dali; Yumoto, Hiromichi; Hirota, Katsuhiko; Murakami, Keiji; Takahashi, Kanako; Hirao, Kouji; Matsuo, Takashi; Ohkura, Kazuto; Nagamune, Hideaki; Miyake, Yoichiro

2008-01-01

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.

A novel initiation mechanism of death in Streptococcus pneumoniae induced by the human milk protein-lipid complex HAMLET and activated during physiological death.

PubMed

Clementi, Emily A; Marks, Laura R; Duffey, Michael E; Hakansson, Anders P

2012-08-03

To cause colonization or infection, most bacteria grow in biofilms where differentiation and death of subpopulations is critical for optimal survival of the whole population. However, little is known about initiation of bacterial death under physiological conditions. Membrane depolarization has been suggested, but never shown to be involved, due to the difficulty of performing such studies in bacteria and the paucity of information that exists regarding ion transport mechanisms in prokaryotes. In this study, we performed the first extensive investigation of ion transport and membrane depolarization in a bacterial system. We found that HAMLET, a human milk protein-lipid complex, kills Streptococcus pneumoniae (the pneumococcus) in a manner that shares features with activation of physiological death from starvation. Addition of HAMLET to pneumococci dissipated membrane polarity, but depolarization per se was not enough to trigger death. Rather, both HAMLET- and starvation-induced death of pneumococci specifically required a sodium-dependent calcium influx, as shown using calcium and sodium transport inhibitors. This mechanism was verified under low sodium conditions, and in the presence of ionomycin or monensin, which enhanced pneumococcal sensitivity to HAMLET- and starvation-induced death. Pneumococcal death was also inhibited by kinase inhibitors, and indicated the involvement of Ser/Thr kinases in these processes. The importance of this activation mechanism was made evident, as dysregulation and manipulation of physiological death was detrimental to biofilm formation, a hallmark of bacterial colonization. Overall, our findings provide novel information on the role of ion transport during bacterial death, with the potential to uncover future antimicrobial targets.

A Novel Initiation Mechanism of Death in Streptococcus pneumoniae Induced by the Human Milk Protein-Lipid Complex HAMLET and Activated during Physiological Death*

PubMed Central

Clementi, Emily A.; Marks, Laura R.; Duffey, Michael E.; Hakansson, Anders P.

2012-01-01

To cause colonization or infection, most bacteria grow in biofilms where differentiation and death of subpopulations is critical for optimal survival of the whole population. However, little is known about initiation of bacterial death under physiological conditions. Membrane depolarization has been suggested, but never shown to be involved, due to the difficulty of performing such studies in bacteria and the paucity of information that exists regarding ion transport mechanisms in prokaryotes. In this study, we performed the first extensive investigation of ion transport and membrane depolarization in a bacterial system. We found that HAMLET, a human milk protein-lipid complex, kills Streptococcus pneumoniae (the pneumococcus) in a manner that shares features with activation of physiological death from starvation. Addition of HAMLET to pneumococci dissipated membrane polarity, but depolarization per se was not enough to trigger death. Rather, both HAMLET- and starvation-induced death of pneumococci specifically required a sodium-dependent calcium influx, as shown using calcium and sodium transport inhibitors. This mechanism was verified under low sodium conditions, and in the presence of ionomycin or monensin, which enhanced pneumococcal sensitivity to HAMLET- and starvation-induced death. Pneumococcal death was also inhibited by kinase inhibitors, and indicated the involvement of Ser/Thr kinases in these processes. The importance of this activation mechanism was made evident, as dysregulation and manipulation of physiological death was detrimental to biofilm formation, a hallmark of bacterial colonization. Overall, our findings provide novel information on the role of ion transport during bacterial death, with the potential to uncover future antimicrobial targets. PMID:22700972

Evolution of bacterial-like phosphoprotein phosphatases in photosynthetic eukaryotes features ancestral mitochondrial or archaeal origin and possible lateral gene transfer.

PubMed

Uhrig, R Glen; Kerk, David; Moorhead, Greg B

2013-12-01

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.

Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

PubMed Central

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand

2012-01-01

Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology. PMID:22833816

Histamine production by Enterobacter aerogenes in sailfish and milkfish at various storage temperatures.

PubMed

Tsai, Yung-Hsiang; Chang, Shiou-Chung; Kung, Hsien-Feng; Wei, Cheng-I; Hwang, Deng-Fwu

2005-08-01

Enterobacter aerogenes was studied for its growth and ability to promote the formation of total volatile base nitrogen (TVBN) and histamine in sailfish (Istiophorus platypterus) and milkfish (Chanos chanos) stored at various temperatures from -20 to 37 degrees C. The optimal temperature for bacterial growth in both fish species was 25 degrees C, whereas the optimal temperature for histamine formation was 37 degrees C. The two fish species inoculated with E. aerogenes, when not properly stored at low temperatures such as 15 degrees C for 36 h, formed histamine at above the U.S. Food and Drug Administration hazardous guideline level of 50 mg/100 g. Milkfish was a better substrate than sailfish for histamine formation by bacterial histidine decarboxylation at elevated temperatures (> 15 degrees C). Although higher contents of TVBN were detected in the spiked sailfish than milkfish during the same storage time at temperatures above 15 degrees C, the use of the 30-mg/100 g level of TVBN as a determination index for fish quality and decomposition was not a good criterion for assessing potential histamine hazard for both fish species. Bacterial growth was controlled by cold storage of the fish at 4 degrees C or below, but histamine formation was stopped only by frozen storage. Once the frozen fish samples were thawed and stored at 25 degrees C, histamine started to accumulate rapidly and reached levels greater than the hazardous action level in 36 h.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

PubMed Central

Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

2008-01-01

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

PubMed Central

Covian, Raul

2012-01-01

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria. PMID:22886415

The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

PubMed Central

Bakó, László; Umeda, Masaaki; Tiburcio, Antonio F.; Schell, Jeff; Koncz, Csaba

2003-01-01

The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo. These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes. PMID:12900506

Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin, a Major Red Wine Polyphenol

PubMed Central

Bognar, Eszter; Sarszegi, Zsolt; Szabo, Aliz; Debreceni, Balazs; Kalman, Nikoletta; Tucsek, Zsuzsanna; Sumegi, Balazs; Gallyas, Ferenc

2013-01-01

Background Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. Methods & Findings The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. Conclusions These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease. PMID:23755222

Capture and separation of l-histidine through optimized zinc-decorated magnetic silica spheres.

PubMed

Cardoso, Vanessa F; Sebastián, Víctor; Silva, Carlos J R; Botelho, Gabriela; Lanceros-Méndez, Senentxu

2017-09-01

Zinc-decorated magnetic silica spheres were developed, optimized and tested for the capture and separation of l-histidine. The magnetic silica spheres were prepared using a simple sol-gel method and show excellent magnetic characteristics, adsorption capacity toward metal ions, and stability in aqueous solution in a wide pH range. The binding capacity of zinc-decorated magnetic silica spheres to histidine proved to be strongly influenced by the morphology, composition and concentration of metal at the surface of the magnetic silica spheres and therefore these parameters should be carefully controlled in order to maximize the performance for protein purification purposes. Optimized zinc-decorated magnetic silica spheres demonstrate a binding capacity to l-histidine of approximately 44mgg -1 at the optimum binding pH buffer. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure and reactivity of hexacoordinate hemoglobins

PubMed Central

Kakar, Smita; Hoffman, Federico G.; Storz, Jay F.; Fabian, Marian; Hargrove, Mark S.

2015-01-01

The heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called “pentacoordinate” hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called “hexacoordinate hemoglobins”, which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal. PMID:20933319

Examination of correlation between histidine and nickel absorption by Morus L., Robinia pseudoacacia L. and Populus nigra L. using HPLC-MS and ICP-MS.

PubMed

Ozen, Sukran Akkus; Yaman, Mehmet

2016-08-02

In this study, HPLC-MS and ICP-MS methods were used for the determination of histidine and nickel in Morus L., Robinia pseudoacacia L., and Populus nigra L. leaves taken from industrial areas including Gaziantep and Bursa cities. In the determination of histidine by HPLC-MS, all of the system parameters such as flow rate of mobile phase, fragmentor potential, injection volume and column temperature were optimized and found to be 0.2 mL min(-1), 70 V, 15 µL, and 20°C, respectively. Under the optimum conditions, histidine was extracted from plant sample by distilled water at 90°C for 30 min. Concentrations of histidine as mg kg(-1) were found to be between 2-9 for Morus L., 6-13 for Robinia pseudoacacia L., and 2-10 for Populus nigra L. Concentrations of nickel were in the ranges of 5-10 mg kg(-1) for Morus L., 3-10 mg kg(-1) for Robinia pseudoacacia L., and 0.6-4 mg kg(-1) for Populus nigra L. A significant linear correlation (r = 0.78) between histidine and Ni was observed for Populus nigra L., whereas insignificant linear correlation for Robinia pseudoacacia L. (r = 0.22) were seen. Limits of detection (LOD) and quantitation (LOQ) were found to be 0.025 mg Ni L(-1) and 0.075 mg Ni L(-1), respectively.

Discovery and characterization of a photo-oxidative histidine-histidine cross-link in IgG1 antibody utilizing ¹⁸O-labeling and mass spectrometry.

PubMed

Liu, Min; Zhang, Zhongqi; Cheetham, Janet; Ren, Da; Zhou, Zhaohui Sunny

2014-05-20

A novel photo-oxidative cross-linking between two histidines (His-His) has been discovered and characterized in an IgG1 antibody via the workflow of XChem-Finder, (18)O labeling and mass spectrometry (Anal. Chem. 2013, 85, 5900-5908). Its structure was elucidated by peptide mapping with multiple proteases with various specificities (e.g., trypsin, Asp-N, and GluC combined with trypsin or Asp-N) and mass spectrometry with complementary fragmentation modes (e.g., collision-induced dissociation (CID) and electron-transfer dissociation (ETD)). Our data indicated that cross-linking occurred across two identical conserved histidine residues on two separate heavy chains in the hinge region, which is highly flexible and solvent accessible. On the basis of model studies with short peptides, it has been proposed that singlet oxygen reacts with the histidyl imidazole ring to form an endoperoxide and then converted to the 2-oxo-histidine (2-oxo-His) and His+32 intermediates, the latter is subject to a nucleophilic attack by the unmodified histidine; and finally, elimination of a water molecule leads to the final adduct with a net mass increase of 14 Da. Our findings are consistent with this mechanism. Successful discovery of cross-linked His-His again demonstrates the broad applicability and utility of our XChem-Finder approach in the discovery and elucidation of protein cross-linking, particularly without a priori knowledge of the chemical nature and site of cross-linking.

Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer

NASA Technical Reports Server (NTRS)

Schweighofer, K. J.; Pohorille, A.

2000-01-01

The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2).

A highly sensitive luminescent probe based on Ru(II)-bipyridine complex for Cu2+, l-Histidine detection and cellular imaging.

PubMed

Zhang, Shi-Ting; Li, Panpan; Liao, Caiyun; Luo, Tingting; Kou, Xingming; Xiao, Dan

2018-05-02

A ruthenium(II) bipyridyl complex conjugated with functionalized Schiff base (RuA) has been synthesized and functioned as a luminescent probe. The luminescence of RuA was greatly quenched by Cu 2+ due to its molecular coordination with paramagnetic Cu 2+ . Subsequently, the addition of l-Histidine can turn on the luminescence of the RuA-Cu(II) ensemble, which can be attributed to the replacement of RuA in RuA-Cu(II) ensemble by l-Histidine. On the basis of the quenching and recovery of the luminescence of RuA, we proposed a rapid and highly sensitive on-off-on luminescent assay for sensing Cu 2+ and l-Histidine in aqueous solution. Under the optimal conditions, Cu 2+ and l-Histidine can be detected in the concentration range of 5 nM-9.0 μM and 50 nM-30 μM, respectively, and the corresponding detection limits were calculated to be 0.35 and 0.44 nM (S/N=3), separately. The proposed luminescent probe has been successfully utilized for the analysis of Cu 2+ and l-Histidine in real samples (drinking water and biological fluids). Furthermore, the probe revealed good photostability, low cytotoxicity and excellent permeability, making it a suitable candidate for cell imaging and labeling in vitro. Copyright © 2018 Elsevier B.V. All rights reserved.

Highly Efficient Photocatalytic Hydrogen Production of Flower-like Cadmium Sulfide Decorated by Histidine

PubMed Central

Wang, Qizhao; Lian, Juhong; Li, Jiajia; Wang, Rongfang; Huang, Haohao; Su, Bitao; Lei, Ziqiang

2015-01-01

Morphology-controlled synthesis of CdS can significantly enhance the efficiency of its photocatalytic hydrogen production. In this study, a novel three-dimensional (3D) flower-like CdS is synthesized via a facile template-free hydrothermal process using Cd(NO3)2•4H2O and thiourea as precursors and L-Histidine as a chelating agent. The morphology, crystal phase, and photoelectrochemical performance of the flower-like CdS and pure CdS nanocrystals are carefully investigated via various characterizations. Superior photocatalytic activity relative to that of pure CdS is observed on the flower-like CdS photocatalyst under visible light irradiation, which is nearly 13 times of pure CdS. On the basis of the results from SEM studies and our analysis, a growth mechanism of flower-like CdS is proposed by capturing the shape evolution. The imidazole ring of L-Histidine captures the Cd ions from the solution, and prevents the growth of the CdS nanoparticles. Furthermore, the photocatalytic contrast experiments illustrate that the as-synthesized flower-like CdS with L-Histidine is more stable than CdS without L-Histidine in the hydrogen generation. PMID:26337119

Highly Efficient Photocatalytic Hydrogen Production of Flower-like Cadmium Sulfide Decorated by Histidine

NASA Astrophysics Data System (ADS)

Wang, Qizhao; Lian, Juhong; Li, Jiajia; Wang, Rongfang; Huang, Haohao; Su, Bitao; Lei, Ziqiang

2015-09-01

Morphology-controlled synthesis of CdS can significantly enhance the efficiency of its photocatalytic hydrogen production. In this study, a novel three-dimensional (3D) flower-like CdS is synthesized via a facile template-free hydrothermal process using Cd(NO3)2•4H2O and thiourea as precursors and L-Histidine as a chelating agent. The morphology, crystal phase, and photoelectrochemical performance of the flower-like CdS and pure CdS nanocrystals are carefully investigated via various characterizations. Superior photocatalytic activity relative to that of pure CdS is observed on the flower-like CdS photocatalyst under visible light irradiation, which is nearly 13 times of pure CdS. On the basis of the results from SEM studies and our analysis, a growth mechanism of flower-like CdS is proposed by capturing the shape evolution. The imidazole ring of L-Histidine captures the Cd ions from the solution, and prevents the growth of the CdS nanoparticles. Furthermore, the photocatalytic contrast experiments illustrate that the as-synthesized flower-like CdS with L-Histidine is more stable than CdS without L-Histidine in the hydrogen generation.

Highly Efficient Photocatalytic Hydrogen Production of Flower-like Cadmium Sulfide Decorated by Histidine.

PubMed

Wang, Qizhao; Lian, Juhong; Li, Jiajia; Wang, Rongfang; Huang, Haohao; Su, Bitao; Lei, Ziqiang

2015-09-04

Morphology-controlled synthesis of CdS can significantly enhance the efficiency of its photocatalytic hydrogen production. In this study, a novel three-dimensional (3D) flower-like CdS is synthesized via a facile template-free hydrothermal process using Cd(NO3)2•4H2O and thiourea as precursors and L-Histidine as a chelating agent. The morphology, crystal phase, and photoelectrochemical performance of the flower-like CdS and pure CdS nanocrystals are carefully investigated via various characterizations. Superior photocatalytic activity relative to that of pure CdS is observed on the flower-like CdS photocatalyst under visible light irradiation, which is nearly 13 times of pure CdS. On the basis of the results from SEM studies and our analysis, a growth mechanism of flower-like CdS is proposed by capturing the shape evolution. The imidazole ring of L-Histidine captures the Cd ions from the solution, and prevents the growth of the CdS nanoparticles. Furthermore, the photocatalytic contrast experiments illustrate that the as-synthesized flower-like CdS with L-Histidine is more stable than CdS without L-Histidine in the hydrogen generation.

Shigella flexneri type III secreted effector OspF reveals new crosstalks of proinflammatory signaling pathways during bacterial infection.

PubMed

Reiterer, Veronika; Grossniklaus, Lars; Tschon, Therese; Kasper, Christoph Alexander; Sorg, Isabel; Arrieumerlou, Cécile

2011-07-01

Shigella flexneri type III secreted effector OspF harbors a phosphothreonine lyase activity that irreversibly dephosphorylates MAP kinases (MAPKs) p38 and ERK in infected epithelial cells and thereby, dampens innate immunity. Whereas this activity has been well characterized, the impact of OspF on other host signaling pathways that control inflammation was unknown. Here we report that OspF potentiates the activation of the MAPK JNK and the transcription factor NF-κB during S. flexneri infection. This unexpected effect of OspF was dependent on the phosphothreonine lyase activity of OspF on p38, and resulted from the disruption of a negative feedback loop regulation between p38 and TGF-beta activated kinase 1 (TAK1), mediated via the phosphorylation of TAK1-binding protein 1. Interestingly, potentiated JNK activation was not associated with enhanced c-Jun signaling as OspF also inhibits c-Jun expression at the transcriptional level. Altogether, our data reveal the impact of OspF on the activation of NF-κB, JNK and c-Jun, and demonstrate the existence of a negative feedback loop regulation between p38 and TAK1 during S. flexneri infection. Furthermore, this study validates the use of bacterial effectors as molecular tools to identify the crosstalks that connect important host signaling pathways induced upon bacterial infection. Copyright © 2011 Elsevier Inc. All rights reserved.

Immunoprecipitation of PDE2 phosphorylated and inactivated by an associated protein kinase.

PubMed

Bentley, J Kelley

2005-01-01

A PDE2A2-associated protein kinase phosphorylates PDE2A2 in vivo and in vitro to inhibit its catalytic activity. Rat brain PDE2A2 may be solubilized using nona (ethylene glycol) mono dodecyl ether (Lubrol 12A9). PDE2A2 exists in a complex with a protein kinase regulating its activity in an adenosine triphosphate-dependent manner. When native or recombinant PDE2 is immunoprecipitated from PC12 cells using an antibody to the amino terminus in a buffer containing Lubrol 12A9, protease inhibitors, and phosphatase inhibitors, a coimmunoprecipitating nerve growth factor-stimulated protein kinase acts to phosphorylate it. PDE2A2 phosphoryla-tion occurs optimally at pH 6.5 in a sodium 2-(4-morpholino)-ethane sulfonate buffer with 5 mM MgCl2 and 1 mM Na3VO4. I describe protocols for producing an antibody to an amino-terminal bacterial fusion protein encoding amino acids 1-251 of PDE2A2 as well as the use of this antibody in immunoprecipitating a PDE2: tyrosine protein-kinase complex from rat brain or PC12 cells.

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

PubMed Central

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

2006-01-01

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors. PMID:16477027

Neighbor-directed histidine N(τ) alkylation. A route to imidazolium-containing phosphopeptide macrocycles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Wen-Jian; Park, Jung-Eun; Grant, Robert

2015-07-07

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. These cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Furthermore, neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

DOE PAGES

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; ...

2014-12-30

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO 2/bicarbonate, and there is a positive correlation between the CO 2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His →more » Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO 2/bicarbonate, and there is a positive correlation between the CO 2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His →more » Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

PubMed

Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

PubMed Central

Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.

2014-12-30

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activitymore » in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

PubMed

Hsiao, Yi Yuong; Van, Ru Chuan; Hung, Hsiao Hui; Pan, Rong Long

2002-01-01

Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.

Influence of different histidine sources and zinc supplementation of broiler diets on dipeptide content and antioxidant status of blood and meat.

PubMed

Kopeć, W; Jamroz, D; Wiliczkiewicz, A; Biazik, E; Pudlo, A; Hikawczuk, T; Skiba, T; Korzeniowska, M

2013-01-01

1. The objective of this study was to investigate how a diet containing spray-dried blood cells (SDBC) (4%) with or without zinc (Zn) would affect the concentration of two histidine heterodipeptides and the antioxidant status of broiler blood and breast muscles. 2. The study was carried out on 920 male Flex chickens randomly assigned to 4 dietary treatments: I - control, II - diet I with SDBC, III - diet I with SDBC and supplemented with Zn and IV - diet I supplemented with L-histidine. Birds were raised on floor littered with wood shavings, given free access to water and fed ad libitum. Performance indices were measured on d 1, 21 and 42. 3. The activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase was analysed in plasma, erythrocytes and muscle tissue. The total antioxidant capacity of plasma and breast muscles was measured by 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, as well as by ferric reducing antioxidant power (FRAP). Carnosine/anserine content of meat and plasma were determined using HPLC. Diets and breast muscles were analysed for amino acid profile and selected microelement content. 4. Histidine supplementation of the diet increased glutathione peroxidase activity in plasma and superoxide dismutase activity in erythrocytes. Moreover, the addition of SDBC or pure histidine in the diet increased histidine dipeptide content and activated enzymatic and non-enzymatic antioxidant systems in chicken blood and muscles. However, it led to lower growth performance indices. 5. The enrichment of broiler diets with Zn increased the antioxidant potential and the activity of superoxide dismutase in plasma, which was independent of the histidine dipeptide concentration. Zn supplementation combined with SDBC in a broiler diet led to the increase of superoxide dismutase and glutathione peroxidase activity, but it did not affect the radical-scavenging or ferric iron reduction abilities of muscles.

The raf-like kinase ILK1 and the high affinity K+ transporter HAK5 are required for innate immunity and abiotic stress response

USDA-ARS?s Scientific Manuscript database

Plants combat bacterial infection by detecting conserved molecular signatures called pathogen-associated molecular patterns (PAMPs) and producing defensive compounds to restrict pathogen entry and reproduction. Numerous ion fluxes are activated within minutes of PAMP perception, including Ca2+ influ...

Innate immunity in rice

PubMed Central

Chen, Xuewei; Ronald, Pamela C.

2011-01-01

Advances in studies of rice innate immunity have led to the identification and characterization of host sensors encoding receptor kinases that perceive conserved microbial signatures. The non-RD domain, a newly recognized hallmark of these receptor kinases is highly expanded in rice (Oryza sativa) compared with Arabidopsis (Arabidopsis thaliana). Researchers have also identified a diverse array of microbial effectors from bacterial and fungal pathogens that triggers immune responses upon perception. These include both, effectors that indirectly target host Nucleotide binding site/Leucine rice repeat (NBS-LRR) proteins and transcription activator-like (TAL) effectors that directly bind promoters of host genes. Here we review the recognition and signaling events that govern rice innate immunity. PMID:21602092

Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage

PubMed Central

Qimron, Udi; Marintcheva, Boriana; Tabor, Stanley; Richardson, Charles C.

2006-01-01

Use of bacteriophages as a therapy for bacterial infection has been attempted over the last century. Such an endeavor requires the elucidation of basic aspects of the host–virus interactions and the resistance mechanisms of the host. Two recently developed bacterial collections now enable a genomewide search of the genetic interactions between Escherichia coli and bacteriophages. We have screened >85% of the E. coli genes for their ability to inhibit growth of T7 phage and >90% of the host genes for their ability to be used by the virus. In addition to identifying all of the known interactions, several other interactions have been identified. E. coli CMP kinase is essential for T7 growth, whereas overexpression of the E. coli uridine/cytidine kinase inhibits T7 growth. Mutations in any one of nine genes that encode enzymes for the synthesis of the E. coli lipopolysaccharide receptor for T7 adsorption leads to T7 resistance. Selection of T7 phage that can recognize these altered receptors has enabled the construction of phage to which the host is 100-fold less resistant. PMID:17135349

Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp

DOE PAGES

Liu, Kuanqing; Myers, Angela R.; Pisithkul, Tippapha; ...

2015-02-05

The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. In this paper, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A surveymore » of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). Finally, we propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.« less

Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poust, Sean; Yoon, Isu; Adams, Paul D.

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-likemore » subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.« less

Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

DOE PAGES

Poust, Sean; Yoon, Isu; Adams, Paul D.; ...

2014-10-06

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-likemore » subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.« less

Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCG.

PubMed

Sinha, Indrajit; Boon, Calvin; Dick, Thomas

2003-10-10

Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation.

MxaY regulates the lanthanide-mediated methanol dehydrogenase switch in Methylomicrobium buryatense

DOE PAGES

Chu, Frances; Beck, David A. C.; Lidstrom, Mary E.

2016-09-07

Many methylotrophs, microorganisms that consume carbon compounds lacking carbon–carbon bonds, use two different systems to oxidize methanol for energy production and biomass accumulation. The MxaFI methanol dehydrogenase (MDH) contains calcium in its active site, while the XoxF enzyme contains a lanthanide in its active site. The genes encoding the MDH enzymes are differentially regulated by the presence of lanthanides. In this study, we found that the histidine kinase MxaY controls the lanthanide-mediated switch in Methylomicrobium buryatense 5GB1C. MxaY controls the transcription of genes encoding MxaFI and XoxF at least partially by controlling the transcript levels of the orphan response regulatormore » MxaB. We identify a constitutively active version of MxaY, and identify the mutated residue that may be involved in lanthanide sensing. Finally, we find evidence to suggest that tight control of active MDH production is required for wild-type growth rates.« less

Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction.

PubMed

Casino, Patricia; Rubio, Vicente; Marina, Alberto

2009-10-16

The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity*

PubMed Central

Richard-Greenblatt, Melissa; Bach, Horacio; Adamson, John; Peña-Diaz, Sandra; Li, Wu; Steyn, Adrie J. C.; Av-Gay, Yossef

2015-01-01

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr213. The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages. PMID:26229105

Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

PubMed Central

Yee, Estella F.; Diensthuber, Ralph P.; Vaidya, Anand T.; Borbat, Peter P.; Engelhard, Christopher; Freed, Jack H.; Bittl, Robert; Möglich, Andreas; Crane, Brian R.

2015-01-01

Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications. PMID:26648256

SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition.

PubMed

Szaniawski, Matthew A; Spivak, Adam M; Cox, James E; Catrow, Jonathan L; Hanley, Timothy; Williams, Elizabeth S C P; Tremblay, Michel J; Bosque, Alberto; Planelles, Vicente

2018-05-15

Macrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted. IMPORTANCE Our experimental results demonstrate that SAMHD1 dephosphorylation at threonine-592 represents a central mechanism of HIV-1 restriction that is common to the three known families of IFNs. While IFN types I and II were potent inhibitors of HIV-1, type III IFN showed modest to undetectable activity. Regulation of SAMHD1 by IFNs involved changes in phosphorylation status but not in protein levels. Phosphorylation of SAMHD1 in macrophages occurred at least in part via CDK1. Tyrosine kinase inhibitors similarly induced SAMHD1 dephosphorylation, which protects macrophages from HIV-1 in a SAMHD1-dependent manner. SAMHD1 is a critical restriction factor regulating HIV-1 infection of macrophages. Copyright © 2018 Szaniawski et al.

Effect of histidine on sorafenib-induced vascular damage: Analysis using novel medaka fish model.

PubMed

Shinagawa-Kobayashi, Yoko; Kamimura, Kenya; Goto, Ryo; Ogawa, Kohei; Inoue, Ryosuke; Yokoo, Takeshi; Sakai, Norihiro; Nagoya, Takuro; Sakamaki, Akira; Abe, Satoshi; Sugitani, Soichi; Yanagi, Masahiko; Fujisawa, Koichi; Nozawa, Yoshizu; Koyama, Naoto; Nishina, Hiroshi; Furutani-Seiki, Makoto; Sakaida, Isao; Terai, Shuji

2018-02-05

Sorafenib (SFN) is an anti-angiogenic chemotherapeutic that prolongs survival of patients with hepatocellular carcinoma (HCC); its side effects, including vascular damages such as hand-foot syndrome (HFS), are a major cause of therapy discontinuation. We previously reported that maintenance of peripheral blood flow by intake of dried bonito broth (DBB) significantly prevented HFS and prolonged the administration period. The amino acids contained in DBB probably contribute to its effects, but the mechanism has not been clarified. We hypothesized that histidine, the largest component among the amino acids contained in DBB, has effects on SFN-induced vascular damage, and evaluated this possibility using a novel medaka fish model. The fli::GFP transgenic medaka fish model has a fluorescently visible systemic vasculature. We fed the fish with SFN with and without histidine to compare blood flow and vascular structure among the differently fed models. The vascular cross-sectional area of each fish was measured to determine vascular diameter changes. Our results demonstrated that SFN-fed medaka developed a narrower vascular diameter. In addition, this narrowing was counteracted by addition of histidine to the medaka diet. We observed no positive effect of histidine on regeneration of cut vessels or on cell growth of endothelial cells and HCC cell lines. We proved the efficacy of the medaka model to assess vascular changes after administration of specific chemicals. And our results suggest that SFN causes vascular damage by narrowing peripheral vessel diameter, and that histidine effectively counteracts these changes to maintain blood flow. Copyright © 2018 Elsevier Inc. All rights reserved.

Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

PubMed Central

Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

2015-01-01

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ. C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ has noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems. PMID:26489410

Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.

PubMed Central

Lev, S; Givol, D; Yarden, Y

1992-01-01

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor. Images PMID:1370584

Recombinant expression and characterization of L-asparaginase II from a moderately thermotolerant bacterial isolate.

PubMed

Vidya, Jalaja; Pandey, Ashok

2012-07-01

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; L-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver-Burk plot, and K(m) and V(max) were 0.89 mM and 0.18 U/mg, respectively.

Mutagenic and genotoxic potential of direct electric current in Escherichia coli and Salmonella thyphimurium strains.

PubMed

Gomes, Marina das Neves; Cardoso, Janine Simas; Leitão, Alvaro Costa; Quaresma, Carla Holandino

2016-05-01

Direct electric current has several therapeutic uses such as antibacterial and antiprotozoal action, tissues scarring and regeneration, as well as tumor treatment. This method has shown promising results in vivo and in vitro, with significant efficacy and almost no side effects. Considering lack of studies regarding direct electric current mutagenic and/or genotoxic effects, the present work evaluated both aspects by using five different bacterial experimental assays: survival of repair-deficient mutants, Salmonella-histidine reversion mutagenesis (Ames test), forward mutations to rifampicin resistance, phage reactivation, and lysogenic induction. In these experimental conditions, cells were submitted to an approach that allows evaluation of anodic, cathodic, and electro-ionic effects generated by 2 mA of direct electric current, with doses ranging from 0.36 to 3.60 Coulombs. Our results showed these doses did not induce mutagenic or genotoxic effects. © 2016 Wiley Periodicals, Inc.

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

PubMed Central

Munir, Annum

2016-01-01

ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. PMID:27895092

Glutathione affects the transport activity of Rhizobium leguminosarum 3841 and is essential for efficient nodulation.

PubMed

Cheng, Guojun; Karunakaran, Ramakrishnan; East, Alison K; Munoz-Azcarate, Olaya; Poole, Philip S

2017-04-01

As glutathione (GSH) plays an essential role in growth and symbiotic capacity of rhizobia, a glutathione synthetase (gshB) mutant of Rhizobium leguminosarum biovar viciae 3841 (Rlv3841) was characterised. It fails to efficiently utilise various compounds as a sole carbon source, including glucose, succinate, glutamine and histidine, and shows 60%-69% reduction in uptake rates of glucose, succinate and the non-metabolisable substrate α-amino isobutyric acid. The defect in glucose uptake can be overcome by addition of exogenous GSH, indicating GSH, but not its bacterial synthesis, is required for efficient transport. GSH is not involved in the regulation of the activity of Rlv3841's transporters via the global regulator of transport, PtsNTR. Although lack of GSH reduces transcription of the branched amino acid transporter, this was not the case for all uptake transport systems, for example, the amino acid permease. This suggests GSH alters activity and/or assembly of transport systems by an unknown mechanism. In interaction with plants, the gshB mutant is not only severely impaired in rhizosphere colonisation, but also shows a 50% reduction in dry weight of plants and nitrogen-fixation ability. This reveals that changes in GSH metabolism affect the bacterial-plant interactions required for symbiosis. © FEMS 2017.

A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA-induced IL-12 production and Th cell differentiation.

PubMed

Sugimoto, Kenji; Ohata, Mutsuhiro; Miyoshi, Jun; Ishizaki, Hiroyoshi; Tsuboi, Naotake; Masuda, Akio; Yoshikai, Yasunobu; Takamoto, Masaya; Sugane, Kazuo; Matsuo, Seiichi; Shimada, Yasuhiro; Matsuguchi, Tetsuya

2004-09-01

A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal-regulated kinase (ERK) activation and production of TNF-alpha and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2-independent manner. Peritoneal macrophages and bone marrow-derived DCs from Cot/Tpl2-/- mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2-/- macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter-binding protein (GA-12-binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2-/- mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA-guided vaccination.

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane.

PubMed

Legallais, C; Anspach, F B; Bueno, S M; Haupt, K; Vijayalakshmi, M A

1997-03-28

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG1 (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS of Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m2 histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 l of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.

Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by sup 15 N NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Dijk, A.A.; de Lange, L.C.M.; Robillard, G.T.

1990-09-04

The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with {sup 15}N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with {sup 15}N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N{delta}1 and N{epsilon}2 positions or at only the N{delta}1 position. {sup 15}N NMR spectra of two synthesized model compound, phosphoimidazole and phosphomethylimidazole, were also recorded. Themore » authors show that, prior to phosphorylation, the protonated His15 N{epsilon}2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N{delta}1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N{delta}1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.« less

Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abell, L.M.; O'Leary, M.H.

1988-08-09

The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30 a shows a carbon isotope effect k/sup 12//k/sup 13/ = 1.0334 +/- 0.0005 and a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9799 +/- 0.0006 at pH 4.8, 37/sup 0/C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D/sub 2/O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capablemore » of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.« less

Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens.

PubMed

Zink, Steven D; Van Slyke, Greta A; Palumbo, Michael J; Kramer, Laura D; Ciota, Alexander T

2015-10-27

Complex interactions between microbial residents of mosquitoes and arboviruses are likely to influence many aspects of vectorial capacity and could potentially have profound effects on patterns of arbovirus transmission. Such interactions have not been well studied for West Nile virus (WNV; Flaviviridae, Flavivirus) and Culex spp. mosquitoes. We utilized next-generation sequencing of 16S ribosomal RNA bacterial genes derived from Culex pipiens Linnaeus following WNV exposure and/or infection and compared bacterial populations and broad immune responses to unexposed mosquitoes. Our results demonstrate that WNV infection increases the diversity of bacterial populations and is associated with up-regulation of classical invertebrate immune pathways including RNA interference (RNAi), Toll, and Jak-STAT (Janus kinase-Signal Transducer and Activator of Transcription). In addition, WNV exposure alone, without the establishment of infection, results in similar alterations to microbial and immune signatures, although to a lesser extent. Multiple bacterial genera were found in greater abundance inWNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia.

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425

Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.

PubMed

Kumari, Uma; Vishwakarma, Rishi K; Sonawane, Prashant; Abbassi, Shakeel; Khan, Bashir M

2015-01-01

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2. Copyright © 2014 Elsevier B.V. All rights reserved.

The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

PubMed Central

Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

2015-01-01

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

Synthesis and activity of histidine-containing catalytic peptide dendrimers.

PubMed

Delort, Estelle; Nguyen-Trung, Nhat-Quang; Darbre, Tamis; Reymond, Jean-Louis

2006-06-09

Peptide dendrimers built by iteration of the diamino acid dendron Dap-His-Ser (His = histidine, Ser = Serine, Dap = diamino propionic acid) display a strong positive dendritic effect for the catalytic hydrolysis of 8-acyloxypyrene 1,3,6-trisulfonates, which proceeds with enzyme-like kinetics in aqueous medium (Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126, 15642-3). Thirty-two mutants of the original third generation dendrimer A3 ((Ac-His-Ser)8(Dap-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2) were prepared by manual synthesis or by automated synthesis with use of a Chemspeed PSW1100 peptide synthesizer. Dendrimer catalysis was specific for 8-acyloxypyrene 1,3,6-trisulfonates, and there was no activity with other types of esters. While dendrimers with hydrophobic residues at the core and histidine residues at the surface only showed weak activity, exchanging serine residues in dendrimer A3 against alanine (A3A), beta-alanine (A3B), or threonine (A3C) improved catalytic efficiency. Substrate binding was correlated with the total number of histidines per dendrimer, with an average of three histidines per substrate binding site. The catalytic rate constant kcat depended on the placement of histidines within the dendrimers and the nature of the other amino acid residues. The fastest catalyst was the threonine mutant A3C ((Ac-His-Thr)8(Dap-His-Thr)4(Dap-His-Thr)2Dap-His-Thr-NH2), with kcat = 1.3 min(-1), kcat/k(uncat) = 90'000, KM = 160 microM for 8-bytyryloxypyrene 1,3,6-trisulfonate, corresponding to a rate acceleration of 18'000 per catalytic site and a 5-fold improvement over the original sequence A3.

L-Histidine sensing by calcium sensing receptor inhibits voltage-dependent calcium channel activity and insulin secretion in β-cells

PubMed Central

Parkash, Jai; Asotra, Kamlesh

2011-01-01

Aims Our goal was to test the hypothesis that the histidine-induced activation of calcium sensing receptor (CaR) can regulate calcium channel activity of L-type voltage dependent calcium channel (VDCC) due to increased spatial interaction between CaR and VDCC in β-cells and thus modulate glucose-induced insulin secretion. Main methods Rat insulinoma (RINr1046-38) insulin-producing β-cells were cultured in RPMI-1640 medium on 25 mm diameter glass coverslips in six-well culture plates in a 5% CO2 incubator at 37°C. The intracellular calcium concentration, [Ca2+]i, was determined by ratio fluorescence microscopy using Fura-2AM. The spatial interactions between CaR and L-type VDCC in β-cells were measured by immunofluorescence confocal microscopy using a Nikon C1 laser scanning confocal microscope. The insulin release was determined by enzyme-linked immunosorbent assay (ELISA). Key findings The additions of increasing concentrations of L-histidine along with 10 mM glucose resulted in 57% decrease in [Ca2+]i. The confocal fluorescence imaging data showed 5.59 to 8.62-fold increase in colocalization correlation coefficient between CaR and VDCC in β-cells exposed to L-histidine thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. The insulin ELISA data showed 54% decrease in 1st phase of glucose-induced insulin secretion in β-cells exposed to increasing concentrations of L-histidine. Significance L-histidine-induced increased spatial interaction of CaR with VDCC can inhibit calcium channel activity of VDCC and consequently regulate glucose-induced insulin secretion by β-cells. The L-type VDCC could therefore be potential therapeutic target in diabetes. PMID:21219913

Effects of L-histidine depletion and L-tyrosine/L-phenylalanine depletion on sensory and motor processes in healthy volunteers

PubMed Central

van Ruitenbeek, P; Sambeth, A; Vermeeren, A; Young, SN; Riedel, WJ

2009-01-01

Background and purpose: Animal studies show that histamine plays a role in cognitive functioning and that histamine H3-receptor antagonists, which increase histaminergic function through presynaptic receptors, improve cognitive performance in models of clinical cognitive deficits. In order to test such new drugs in humans, a model for cognitive impairments induced by low histaminergic functions would be useful. Studies with histamine H1-receptor antagonists have shown limitations as a model. Here we evaluated whether depletion of L-histidine, the precursor of histamine, was effective in altering measures associated with histamine in humans and the behavioural and electrophysiological (event-related-potentials) effects. Experimental approach: Seventeen healthy volunteers completed a three-way, double-blind, crossover study with L-histidine depletion, L-tyrosine/L-phenylalanine depletion (active control) and placebo as treatments. Interactions with task manipulations in a choice reaction time task were studied. Task demands were increased using visual stimulus degradation and increased response complexity. In addition, subjective and objective measures of sedation and critical tracking task performance were assessed. Key results: Measures of sedation and critical tracking task performance were not affected by treatment. L-histidine depletion was effective and enlarged the effect of response complexity as measured with the response-locked lateralized readiness potential onset latency. Conclusions and implications: L-histidine depletion affected response- but not stimulus-related processes, in contrast to the effects of H1-receptor antagonists which were previously found to affect primarily stimulus-related processes. L-histidine depletion is promising as a model for histamine-based cognitive impairment. However, these effects need to be confirmed by further studies. PMID:19413574

Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation.

PubMed

Stratton, Amanda; Ericksen, Matthew; Harris, Travis V; Symmonds, Nick; Silverstein, Todd P

2017-02-01

The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his 40 and his 57 ) quickly and completely, with an IC 50 of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his 57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his 57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his 40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his 40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation. © 2017 The Protein Society.

Antioxidant status of turkey breast meat and blood after feeding a diet enriched with histidine.

PubMed

Kopec, W; Wiliczkiewicz, A; Jamroz, D; Biazik, E; Pudlo, A; Hikawczuk, T; Skiba, T; Korzeniowska, M

2016-01-01

The objective of this study was to investigate the effects of 1) spray dried blood cells rich in histidine and 2) pure histidine added to feed on the antioxidant status and concentration of carnosine related components in the blood and breast meat of female turkeys. The experiment was performed on 168 Big7 turkey females randomly assigned to 3 dietary treatments: control; control with the addition of 0.18% L-histidine (His); and control with the addition of spray dried blood cells (SDBC). Birds were raised for 103 d on a floor with sawdust litter, with drinking water and feed ad libitum. The antioxidant status of blood plasma and breast muscle was analyzed by ferric reducing ability (FRAP) and by 2,2-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals scavenging ability. The activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) was analyzed in the blood and breast meat, with the content of carnosine and anserine quantified by HPLC. Proximate analysis as well as amino acid profiling were carried out for the feed and breast muscles. Growth performance parameters also were calculated. Histidine supplementation of the turkey diet resulted in increased DPPH radical scavenging capacity in the breast muscles and blood, but did not result in higher histidine dipeptide concentrations. The enzymatic antioxidant system of turkey blood was affected by the diet with SDBC. In the plasma, the SDBC addition increased both SOD and GPx activity, and decreased GPx activity in the erythrocytes. Feeding turkeys with an SDBC containing diet increased BW and the content of isoleucine and valine in breast muscles. © 2015 Poultry Science Association Inc.

Mucosal reactive oxygen species decrease virulence by disrupting Campylobacter jejuni phosphotyrosine signaling

PubMed Central

Corcionivoschi, Nicolae; Alvarez, Luis A.; Sharp, Thomas H.; Strengert, Monika; Alemka, Abofu; Mantell, Judith; Verkade, Paul; Knaus, Ulla G.; Bourke, Billy

2013-01-01

Summary Reactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer membrane / periplasmic proteins including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling. PMID:22817987

Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens.

PubMed

Eldridge, Matthew J G; Shenoy, Avinash R

2015-02-01

Inflammasomes - molecular platforms for caspase-1 activation - have emerged as common hubs for a number of pathways that detect and respond to bacterial pathogens. Caspase-1 activation results in the secretion of bioactive IL-1β and IL-18 and pyroptosis, and thus launches a systemic immune and inflammatory response. In this review we discuss signal transduction leading to 'canonical' and 'non-canonical' activation of caspase-1 through the involvement of upstream caspases. Recent studies have identified a growing number of regulatory networks involving guanylate binding proteins, protein kinases, ubiquitylation and necroptosis related pathways that modulate inflammasome responses and immunity to bacterial infection. By being able to respond to extracellular, vacuolar and cytosolic bacteria, their cytosolic toxins or ligands for cell surface receptors, inflammasomes have emerged as important sentinels of infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

PubMed Central

Hawkins, Clare L; Pattison, David I; Davies, Michael J

2002-01-01

Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines. These results are consistent with the predictions of the kinetic model and suggest that histones are major targets for HOCl in the nucleosome. Furthermore, the resulting protein chloramines and the radicals derived from them may act as contributing agents in HOCl-mediated DNA oxidation. PMID:12010123

21 CFR 582.5361 - Histidine.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Histidine. 582.5361 Section 582.5361 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

21 CFR 582.5361 - Histidine.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Histidine. 582.5361 Section 582.5361 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

21 CFR 582.5361 - Histidine.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Histidine. 582.5361 Section 582.5361 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

21 CFR 582.5361 - Histidine.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Histidine. 582.5361 Section 582.5361 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

21 CFR 582.5361 - Histidine.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Histidine. 582.5361 Section 582.5361 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

A rapid and ultrasensitive SERRS assay for histidine and tyrosine based on azo coupling.

PubMed

Sui, Huimin; Wang, Yue; Yu, Zhi; Cong, Qian; Han, Xiao Xia; Zhao, Bing

2016-10-01

A simple and highly sensitive surface-enhanced resonance Raman scattering (SERRS)-based approach coupled with azo coupling reaction has been put forward for quantitative analysis of histidine and tyrosine. The SERRS-based assay is simple and rapid by mixing the azo reaction products with silver nanoparticles (AgNPs) for measurements within 2min. The limits of detection (LODs) of the method are as low as 4.33×10(-11) and 8.80×10(-11)M for histidine and tyrosine, respectively. Moreover, the SERRS fingerprint information specific to corresponding amino acids guarantees the selective detection for the target histidine and tyrosine. The results from serum indicated the potential application of the proposed approach into biological samples. Compared with the methods ever reported, the main advantages of this methodology are simpleness, rapidity without time-consuming separation or pretreatment steps, high sensitivity, selectivity and the potential for determination of other molecules containing imidazole or phenol groups. Copyright © 2016 Elsevier B.V. All rights reserved.

Human Aquaporin 4 Gating Dynamics under Perpendicularly-Oriented Electric-Field Impulses: A Molecular Dynamics Study

PubMed Central

Marracino, Paolo; Liberti, Micaela; Trapani, Erika; Burnham, Christian J.; Avena, Massimiliano; Garate, José-Antonio; Apollonio, Francesca; English, Niall J.

2016-01-01

Human aquaporin 4 has been studied using molecular dynamics (MD) simulations in the absence and presence of pulses of external static electric fields. The pulses were 10 ns in duration and 0.012–0.065 V/Å in intensity acting along both directions perpendicular to the pores. Water permeability and the dipolar response of all residues of interest (including the selectivity filter) within the pores have been studied. Results showed decreased levels of water osmotic permeability within aquaporin channels during orthogonally-oriented field impulses, although care must be taken with regard to statistical certainty. This can be explained observing enhanced “dipolar flipping” of certain key residues, especially serine 211, histidine 201, arginine 216, histidine 95 and cysteine 178. These residues are placed at the extracellular end of the pore (serine 211, histidine 201, and arginine 216) and at the cytoplasm end (histidine 95 and cysteine 178), with the key role in gating mechanism, hence influencing water permeability. PMID:27428954

Differentiating Amino Acid Residues and Side Chain Orientations in Peptides Using Scanning Tunneling Microscopy

PubMed Central

Claridge, Shelley A.; Thomas, John C.; Silverman, Miles A.; Schwartz, Jeffrey J.; Yang, Yanlian; Wang, Chen; Weiss, Paul S.

2014-01-01

Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structure at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer’s and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level. PMID:24219245

Evolution and tinkering: what do a protein kinase, a transcriptional regulator and chromosome segregation/cell division proteins have in common?

PubMed

Derouiche, Abderahmane; Shi, Lei; Kalantari, Aida; Mijakovic, Ivan

2016-02-01

In this study, we focus on functional interactions among multi-domain proteins which share a common evolutionary origin. The examples we develop are four Bacillus subtilis proteins, which all possess an ATP-binding Walker motif: the bacterial tyrosine kinase (BY-kinase) PtkA, the chromosome segregation protein Soj (ParA), the cell division protein MinD and a transcription regulator SalA. These proteins have arisen via duplication of the ancestral ATP-binding domain, which has undergone fusions with other functional domains in the process of divergent evolution. We point out that these four proteins, despite having very different physiological roles, engage in an unusually high number of binary functional interactions. Namely, MinD attracts Soj and PtkA to the cell pole, and in addition, activates the kinase function of PtkA. SalA also activates the kinase function of PtkA, and it gets phosphorylated by PtkA as well. The consequence of this phosphorylation is the activation of SalA as a transcriptional repressor. We hypothesize that these functional interactions remain preserved during divergent evolution and represent a constraint on the process of evolutionary "tinkering", brought about by fusions of different functional domains.

Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.

PubMed Central

Cuenda, A; Alonso, G; Morrice, N; Jones, M; Meier, R; Cohen, P; Nebreda, A R

1996-01-01

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6. Images PMID:8861944

Engineering cofactor and ligand binding in an artificial neuroglobin

NASA Astrophysics Data System (ADS)

Zhang, Lei

HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.

Computer Simulation Studies of Ion Channel Gating: Characteristics of the M2 Channel of Influenza-A Virus in a Phospholipid Bilayer

NASA Technical Reports Server (NTRS)

Schweighofer, Karl J.; Pohorille, Andrew; DeVincenzi, D. (Technical Monitor)

1999-01-01

The 25 amino acids long, transmembrane fragment of the Influenza virus M2 protein forms a homotetrameric channel that transports protons across lipid bilayers. It has been postulated that high efficiency and selectivity of this process is due to gating by four histidine residues that occlude the channel lumen in the closed state. Two mechanisms of gating have been postulated. In one mechanism, the proton is "shuttled" through the gate by attaching to the delta nitrogen atom on the extracellular side of the imidazole ring, followed by the release of the proton attached to the epsilon nitrogen atom on the opposite side. In the second mechanism, the four histidines move away from each other due to electrostatic repulsion upon protonation, thus opening the gate sufficiently that a wire of water molecules can penetrate the gate. Then, protons are transported by "hopping" along the wire. In this paper, both mechanisms are evaluated in a series of molecular dynamics simulations by investigating stability of different protonation states of the channel that are involved in these mechanisms. For the shuttle mechanism, these are states with all epsilon protonated histidines, one biprotonated residue or one histidine protonated in the delta position. For the gate opening mechanism, this is the state in which all four histidines are biprotonated. In addition, a state with two biprotonated histidines is considered. For each system, composed of the protein channel embedded in phospholipid bilayer located between two water lamellae, a molecular dynamics trajectory of approximately 1.3 ns (after equilibration) was obtained. It is found that the states involved in the shuttle mechanism are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfers involved in the shuttle. In contract, the fully biprotonated state, implicated in the gate opening mechanism, is not stable and the channel looses its structural integrity. If only two histidines are biprotonated the channel deforms but remains intact with the gate mostly closed. In summary, the results of this study lend support to the shuttle mechanism but not to the gate opening mechanism of proton gating in M2.