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Sample records for bacterial protein family

  1. A widespread family of bacterial cell wall assembly proteins

    PubMed Central

    Kawai, Yoshikazu; Marles-Wright, Jon; Cleverley, Robert M; Emmins, Robyn; Ishikawa, Shu; Kuwano, Masayoshi; Heinz, Nadja; Bui, Nhat Khai; Hoyland, Christopher N; Ogasawara, Naotake; Lewis, Richard J; Vollmer, Waldemar; Daniel, Richard A; Errington, Jeff

    2011-01-01

    Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR–Cps2A–Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall. PMID:21964069

  2. Bacterial 5S rRNA-binding proteins of the CTC family.

    PubMed

    Gongadze, G M; Korepanov, A P; Korobeinikova, A V; Garber, M B

    2008-12-01

    The presence of CTC family proteins is a unique feature of bacterial cells. In the CTC family, there are true ribosomal proteins (found in ribosomes of exponentially growing cells), and at the same time there are also proteins temporarily associated with the ribosome (they are produced by the cells under stress only and incorporate into the ribosome). One feature is common for these proteins - they specifically bind to 5S rRNA. In this review, the history of investigations of the best known representatives of this family is described briefly. Structural organization of the CTC family proteins and their occurrence among known taxonomic bacterial groups are discussed. Structural features of 5S rRNA and CTC protein are described that predetermine their specific interaction. Taking into account the position of a CTC protein and its intermolecular contacts in the ribosome, a possible role of its complex with 5S rRNA in ribosome functioning is discussed.

  3. Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

    PubMed Central

    Povolotsky, Tatyana Leonidovna; Orlova, Ekaterina; Tamang, Dorjee G.

    2010-01-01

    The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-010-9260-7) contains supplementary material, which is available to authorized users. PMID:20563570

  4. The OmpA family of proteins: roles in bacterial pathogenesis and immunity.

    PubMed

    Confer, Anthony W; Ayalew, Sahlu

    2013-05-03

    The OmpA family of outer membrane proteins is a group of genetically related, heat-modifiable, surface-exposed, porin proteins that are in high-copy number in the outer membrane of mainly Gram-negative bacteria. OmpA proteins are characterized by an N-terminal domain that forms an eight-stranded, anti-parallel β barrel, which is embedded in the outer membrane. The C-terminal domain is globular and located in the periplasmic space. Escherichia coli OmpA is the best characterized of the proteins. Other well-characterized OmpA-equivalent proteins from pathogenic bacteria include Pseudomonas aeruginosa OprF, Haemophilus influenzae P5, Klebsiella pneumoniae OmpA, and Chlamydia trachomatis major outer membrane protein (MOMP). OmpA from the veterinary pathogens Mannheimia haemolytica, Haemophilus parasuis, Leptospira interrogans, and Pasteurella multocida have been studied to a lesser extent. Among many of the pathogenic bacteria, OmpA proteins have important pathogenic roles including bacterial adhesion, invasion, or intracellular survival as well as evasion of host defenses or stimulators of pro-inflammatory cytokine production. These pathogenic roles are most commonly associated with central nervous system, respiratory and urogenital diseases. Alternatively, OmpA family proteins can serve as targets of the immune system with immunogenicity related to surface-exposed loops of the molecule. In several cases, OmpA proteins are under evaluation as potential vaccine candidates.

  5. The Bacterial Intimins and Invasins: A Large and Novel Family of Secreted Proteins

    PubMed Central

    Tsai, Jennifer C.; Yen, Ming-Ren; Castillo, Rostislav; Leyton, Denisse L.; Henderson, Ian R.; Saier, Milton H.

    2010-01-01

    Based on our in silico analyses we present a model for the biogenesis of these proteins. This study is the first of its kind to describe this unusual family of bacterial adhesins. PMID:21203509

  6. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family*

    PubMed Central

    Broussard, Tyler C.; Miller, Darcie J.; Jackson, Pamela; Nourse, Amanda; White, Stephen W.; Rock, Charles O.

    2016-01-01

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  7. SEDS proteins are a widespread family of bacterial cell wall polymerases.

    PubMed

    Meeske, Alexander J; Riley, Eammon P; Robins, William P; Uehara, Tsuyoshi; Mekalanos, John J; Kahne, Daniel; Walker, Suzanne; Kruse, Andrew C; Bernhardt, Thomas G; Rudner, David Z

    2016-09-29

    Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.

  8. SEDS proteins are a widespread family of bacterial cell wall polymerases

    PubMed Central

    Meeske, Alexander J.; Riley, Eammon P.; Robins, William P.; Uehara, Tsuyoshi; Mekelanos, John J.; Kahne, Daniel; Walker, Suzanne; Kruse, Andrew C.; Bernhardt, Thomas G.; Rudner, David Z.

    2016-01-01

    Summary Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan synthetic machinery called the Rod complex. We report that in Bacillus subtilis this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS family of proteins that play essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a new family of peptidoglycan polymerases. Thus, B. subtilis and likely most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development. PMID:27525505

  9. Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties.

    PubMed

    Uhrig, R Glen; Moorhead, Greg B

    2011-12-01

    Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

  10. REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua.

    PubMed

    Herrero, Salvador; Ansems, Marleen; Van Oers, Monique M; Vlak, Just M; Bakker, Petra L; de Maagd, Ruud A

    2007-11-01

    Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringiensis Cry1Ca toxin and non-exposed insects. Based on the SSH results, full cDNA sequences coding for four homologous proteins were obtained. Quantitative and semi-quantitative RT-PCR showed the increased expression of the genes coding for these proteins after exposure to different B. thuringiensis toxins as well as after infection with baculovirus. The proteins were named REPAT after their increased expression in Response to Pathogen. REPAT1, a member of this family, was recombinantly expressed using the baculovirus expression system, revealing the glycosylated nature of the protein. Recombinant baculoviruses expressing REPAT1 were used to infect larvae from S. exigua, showing that expression of REPAT1 was reducing the virulence of baculovirus to the infected larvae. Together, these results suggest a role for REPAT1 in mitigating pathological effects.

  11. Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins

    PubMed Central

    Reddy, Bhaskara L.; Saier, Milton H.

    2016-01-01

    We here report statistical analyses of 76 families of integral outer membrane pore-forming proteins (OMPPs) found in bacteria and eukaryotic organelles. 47 of these families fall into one superfamily (SFI) which segregate into fifteen phylogenetic clusters. Families with members of the same protein size, topology and substrate specificities often cluster together. Virtually all OMPP families include only proteins that form transmembrane pores. Nine such families, all of which cluster together in the SFI phylogenetic tree, contain both α- and β-structures, are multi domain, multi subunit systems, and transport macromolecules. Most other SFI OMPPs transport small molecules. SFII and SFV homologues derive from Actinobacteria while SFIII and SFIV proteins derive from chloroplasts. Three families of actinobacterial OMPPs and two families of eukaryotic OMPPs apparently consist primarily of α-helices (α-TMSs). Of the 71 families of (putative) β-barrel OMPPs, only twenty could not be assigned to a superfamily, and these derived primarily from Actinobacteria (1), chloroplasts (1), spirochaetes (8), and proteobacteria (10). Proteins were identified in which two or three full length OMPPs are fused together. Family characteristic are described and evidence agrees with a previous proposal suggesting that many arose by adjacent β-hairpin structural unit duplications. PMID:27064789

  12. Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

    PubMed Central

    2008-01-01

    Background Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins. Results Metazoan PBP-βL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-βL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-βL homologs comprise four alloparalogus protein lineages that derive from α-proteobacteria. Conclusion While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-βL proteins were conserved and are found in metazoans including humans. Metazoan PBP-βL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-βL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding. PMID:18226203

  13. Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

    PubMed

    Riley, Sean P; Bykowski, Tomasz; Cooley, Anne E; Burns, Logan H; Babb, Kelly; Brissette, Catherine A; Bowman, Amy; Rotondi, Matthew; Miller, M Clarke; DeMoll, Edward; Lim, Kap; Fried, Michael G; Stevenson, Brian

    2009-04-01

    The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

  14. Bacterial oligopeptide-binding proteins.

    PubMed

    Monnet, V

    2003-10-01

    This review focuses on bacterial oligopeptide-binding proteins, which form part of the oligopeptide transport system belonging to the ATP-binding cassette family of transporters. Depending on the bacterial species, these binding proteins (OppA) capture peptides ranging in size from 2 to 18 amino acids from the environment and pass them on to the other components of the oligopeptide transport system for internalisation. Bacteria have developed several strategies to produce these binding proteins, which are periplasmic in Gram- bacteria and membrane-anchored in Gram+, with a higher stoichiometry (probably necessary for efficient transport) than the other components in the transport system. The expression of OppA-encoding genes is clearly modulated by external factors, especially nitrogen compounds, but the mechanisms of regulation are not always clear. The best-understood roles played by OppAs are internalisation of peptides for nutrition and recycling of muropeptides. It has, however, recently become clear that OppAs are also involved in sensing the external medium via specific or non-specific peptides.

  15. The Nla6S protein of Myxococcus xanthus is the prototype for a new family of bacterial histidine kinases.

    PubMed

    Sarwar, Zaara; Garza, Anthony G

    2012-10-01

    Myxococcus xanthus has a large number of histidine kinase (HK) signal transduction proteins and many of these HKs are important for fruiting body development. Nla6S is an uncharacterized HK that lacks many of the conserved sequence motifs of typical HK proteins. In this study, we report that expression of the nla6S gene increases about sixfold during fruiting body development, that the Nla6S protein has the in vitro properties of HKs and that Nla6S is the prototype for a new family of HKs. To date, these Nla6-like HKs are found only in fruiting members of the Cystobacterineae suborder of the myxobacteria. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  17. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions.

  18. Key bacterial families (Clostridiaceae, Erysipelotrichaceae and Bacteroidaceae) are related to the digestion of protein and energy in dogs

    PubMed Central

    Maclean, Paul; Thomas, David G.; Cave, Nicholas J.; Young, Wayne

    2017-01-01

    Background Much of the recent research in companion animal nutrition has focussed on understanding the role of diet on faecal microbiota composition. To date, diet-induced changes in faecal microbiota observed in humans and rodents have been extrapolated to pets in spite of their very different dietary and metabolic requirements. This lack of direct evidence means that the mechanisms by which microbiota influences health in dogs are poorly understood. We hypothesised that changes in faecal microbiota correlate with physiological parameters including apparent macronutrient digestibility. Methods Fifteen adult dogs were assigned to two diet groups, exclusively fed either a premium kibbled diet (kibble; K; n = 8) or a raw red meat diet (meat; M; n = 7) for nine weeks. Apparent digestibility of macronutrients (protein, fat, gross energy and dry matter), faecal weight, faecal health scores, faecal VFA concentrations and faecal microbial composition were determined. Datasets were integrated using mixOmics in R. Results Faecal weight and VFA levels were lower and the apparent digestibility of protein and energy were higher in dogs on the meat diet. Diet significantly affected 27 microbial families and 53 genera in the faeces. In particular, the abundances of Bacteriodes, Prevotella, Peptostreptococcus and Faecalibacterium were lower in dogs fed the meat diet, whereas Fusobacterium, Lactobacillus and Clostridium were all more abundant. Discussion Our results show clear associations of specific microbial taxa with diet composition. For example, Clostridiaceae, Erysipelotrichaceae and Bacteroidaceae were highly correlated to parameters such as protein and fat digestibility in the dog. By understanding the relationship between faecal microbiota and physiological parameters we will gain better insights into the effects of diet on the nutrition of our pets. PMID:28265505

  19. Hsp90, Hsp60 and sHsp families of heat shock protein genes in channel catfish and their expression after bacterial infections.

    PubMed

    Xie, Yangjie; Song, Lin; Weng, Zhaohong; Liu, Shikai; Liu, Zhanjiang

    2015-06-01

    Heat shock proteins (Hsps) are a suite of highly conserved proteins whose expressions are generally induced by elevated temperature. However, many Hsps play important roles in both innate and adaptive immunity. On the basis of our previous work on Hsp40 and Hsp70 gene families in channel catfish (Ictalurus punctatus), the objective of this study was to characterize Hsp90, Hsp60, Hsp10, and small Hsp genes, and to investigate their expression profiles after bacterial infections. A total of 20 Hsp genes were identified and annotated in the channel catfish genome, including five Hsp90 genes, one Hsp60 gene, one Hsp10 gene, and 13 sHsp genes. Six Hsp genes were differentially expressed after Edwardsiella ictaluri infection, and 12 were differentially expressed after Flavobacterium columnare infection. Although expression of these genes exhibited both temporal and spatial regulation, the induction of Hsp genes was observed soon after bacterial infection, while the suppression of Hsp genes was observed at later time-points, suggesting their distinct roles in immune responses and disease defenses. A pathogen-specific expression pattern of Hsp90 was observed. After F. columnare infection, all Hsp90 genes were found up-regulated except Hsp90ab1, which was not significantly regulated. However, after E. ictaluri infection, only one Hsp90 gene was found significantly down-regulated. Both pathogen-specific and tissue-specific pattern of expression were observed with small Hsps after E. ictaluri and F. columnare bacterial infections. These results suggested that most of Hsp genes may play important roles in immune response and/or disease defense in channel catfish. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

    PubMed Central

    Milano, Teresa

    2016-01-01

    The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH) architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average) that is homologous to fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes like aspartate aminotransferase (AAT). These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs). Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups. PMID:27446613

  1. A growing family: the expanding universe of the bacterial cytoskeleton.

    PubMed

    Ingerson-Mahar, Michael; Gitai, Zemer

    2012-01-01

    Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes.

  2. SAP family proteins.

    PubMed

    Fujita, A; Kurachi, Y

    2000-03-05

    Thus far, five members including Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102, and PSD-93/chapsyn110 which belong to SAP family have been identified. Recent studies have revealed that these proteins play important roles in the localization and function of glutamate receptors and K(+) channels. Although most of them have been reported to be localized to the synapse, only one member, SAP97, is expressed also in the epithelial cells. In this review, we have summarized structural characters of SAP family proteins and discuss their functions in neurons and epithelial cells.

  3. Bacterial lipopolysaccharide increases tyrosine phosphorylation of zonula adherens proteins and opens the paracellular pathway in lung microvascular endothelia through TLR4, TRAF6, and src family kinase activation

    USDA-ARS?s Scientific Manuscript database

    Objective: LPS is a key mediator in vascular leak syndromes associated with Gram-negative bacterial infections and opens the pulmonary vascular endothelial paracellular pathway through protein tyrosine kinase (PTK) activation. We asked which PTKs and signaling molecules mediate LPS-induced endothel...

  4. Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins.

    PubMed

    Cirl, Christine; Wieser, Andreas; Yadav, Manisha; Duerr, Susanne; Schubert, Sören; Fischer, Hans; Stappert, Dominik; Wantia, Nina; Rodriguez, Nuria; Wagner, Hermann; Svanborg, Catharina; Miethke, Thomas

    2008-04-01

    Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.

  5. The family of bacterial ADP-ribosylating exotoxins.

    PubMed Central

    Krueger, K M; Barbieri, J T

    1995-01-01

    Pathogenic bacteria utilize a variety of virulence factors that contribute to the clinical manifestation of their pathogenesis. Bacterial ADP-ribosylating exotoxins (bAREs) represent one family of virulence factors that exert their toxic effects by transferring the ADP-ribose moiety of NAD onto specific eucaryotic target proteins. The observations that some bAREs ADP-ribosylate eucaryotic proteins that regulate signal transduction, like the heterotrimeric GTP-binding proteins and the low-molecular-weight GTP-binding proteins, has extended interest in bAREs beyond the bacteriology laboratory. Molecular studies have shown that bAREs possess little primary amino acid homology and have diverse quaternary structure-function organization. Underlying this apparent diversity, biochemical and crystallographic studies have shown that several bAREs have conserved active-site structures and possess a conserved glutamic acid within their active sites. PMID:7704894

  6. Rho-modifying bacterial protein toxins.

    PubMed

    Aktories, Klaus

    2015-12-01

    Rho proteins are targets of numerous bacterial protein toxins, which manipulate the GTP-binding proteins by covalent modifications, including ADP ribosylation, glycosylation, adenylylation, proteolytic cleavage and deamidation. Bacterial toxins are important virulence factors but are also potent and efficient pharmacological tools to study the physiological functions of their eukaryotic targets. Recent studies indicate that amazing variations exist in the molecular mechanisms by which toxins attack Rho proteins, which are discussed here. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Bacterial cell division proteins as antibiotic targets.

    PubMed

    den Blaauwen, Tanneke; Andreu, José M; Monasterio, Octavio

    2014-08-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of proteins and the availability of crystal structures has increased our knowledge on bacterial cell division considerably in this century. Consequently, bacterial cell division proteins are more and more recognized as potential new antibiotic targets. An international effort to find small molecules that inhibit the cell division initiating protein FtsZ has yielded many compounds of which some are promising as leads for preclinical use. The essential transglycosylase activity of peptidoglycan synthases has recently become accessible to inhibitor screening. Enzymatic assays for and structural information on essential integral membrane proteins such as MraY and FtsW involved in lipid II (the peptidoglycan building block precursor) biosynthesis have put these proteins on the list of potential new targets. This review summarises and discusses the results and approaches to the development of lead compounds that inhibit bacterial cell division.

  8. NACHT-LRR proteins (NLRs) in bacterial infection and immunity.

    PubMed

    Kufer, Thomas A; Fritz, Jörg H; Philpott, Dana J

    2005-08-01

    Eukaryotes have evolved systems to detect bacterial intrusion. Recognition of bacteria relies on the sensing of pathogen associated molecular patterns (PAMPs) by host pattern recognition molecules (PRMs), which include various families of leucine-rich repeat (LRR) bearing proteins in plants and animals. Detection of microbes often occurs outside the cell. Recent findings now indicate that mammals have also evolved strategies to recognize bacteria inside the cell via members of the NACHT-LRR protein family (NLRs). Here, we review the biology of these mammalian NLRs and the emerging view of their important, role not solely as PRMs but as signalling platforms and regulators of immunity.

  9. Engineered fluorescent proteins illuminate the bacterial periplasm.

    PubMed

    Dammeyer, Thorben; Tinnefeld, Philip

    2012-01-01

    The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  10. Thiol Dioxygenases: Unique Families of Cupin Proteins

    PubMed Central

    Simmons, C. R.; Karplus, P. A.; Dominy, J. E.

    2011-01-01

    Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a 6-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative

  11. Protein quality control in the bacterial periplasm

    PubMed Central

    Miot, Marika; Betton, Jean-Michel

    2004-01-01

    The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein. PMID:15132751

  12. Protein Aggregation Profile of the Bacterial Cytosol

    PubMed Central

    de Groot, Natalia S.; Ventura, Salvador

    2010-01-01

    Background Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity. Methodology/Principal Findings Here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes. Conclusions/Significance Overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms. PMID:20195530

  13. Bacterial protein acetylation: new discoveries unanswered questions.

    PubMed

    Wolfe, Alan J

    2016-05-01

    Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl-coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering.

  14. Insights from protein-protein interaction studies on bacterial pathogenesis.

    PubMed

    Gagarinova, Alla; Phanse, Sadhna; Cygler, Miroslaw; Babu, Mohan

    2017-09-01

    The threat bacterial pathogens pose to human health is increasing with the number and distribution of antibiotic-resistant bacteria, while the rate of discovery of new antimicrobials dwindles. Proteomics is playing key roles in understanding the molecular mechanisms of bacterial pathogenesis, and in identifying disease outcome determinants. The physical associations identified by proteomics can provide the means to develop pathogen-specific treatment methods that reduce the spread of antibiotic resistance and alleviate the negative effects of broad-spectrum antibiotics on beneficial bacteria. Areas covered: This review discusses recent trends in proteomics and introduces new and developing approaches that can be applied to the study of protein-protein interactions (PPIs) underlying bacterial pathogenesis. The approaches examined encompass options for mapping proteomes as well as stable and transient interactions in vivo and in vitro. We also explored the coverage of bacterial and human-bacterial PPIs, knowledge gaps in this area, and how they can be filled. Expert commentary: Identifying potential antimicrobial candidates is confounded by the complex molecular biology of bacterial pathogenesis and the lack of knowledge about PPIs underlying this process. Proteomics approaches can offer new perspectives for mechanistic insights and identify essential targets for guiding the discovery of next generation antimicrobials.

  15. A new family of bacterial DNA repair proteins annotated by the integration of non-homology, distant homology and structural bioinformatic methods.

    PubMed

    Mello, Luciane V; Rigden, Daniel J

    2012-11-02

    Different bioinformatics methods illuminate different aspects of protein function, from specific catalytic activities to broad functional categories. Here, a triple-pronged approach to predict function for a domain of unknown function, DUF2086, is applied. Distant homology to characterised enzymes and conservation of key residues suggest an oxygenase function. Modelling indicates that the substrate is most likely a nucleic acid. Finally, genomic context analysis linking DUF2086 to DNA repair, leads to a predicted activity of oxidative demethylation of damaged bases in DNA. The newly assigned activity is sporadically present in phyla not containing near relatives of the similarly active repair protein AlkB. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. The Pfam protein families database.

    PubMed

    Finn, Robert D; Tate, John; Mistry, Jaina; Coggill, Penny C; Sammut, Stephen John; Hotz, Hans-Rudolf; Ceric, Goran; Forslund, Kristoffer; Eddy, Sean R; Sonnhammer, Erik L L; Bateman, Alex

    2008-01-01

    Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden Markov models. The current release of Pfam (22.0) contains 9318 protein families. Pfam is now based not only on the UniProtKB sequence database, but also on NCBI GenPept and on sequences from selected metagenomics projects. Pfam is available on the web from the consortium members using a new, consistent and improved website design in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/), as well as from mirror sites in France (http://pfam.jouy.inra.fr/) and South Korea (http://pfam.ccbb.re.kr/).

  17. Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain.

    PubMed Central

    Tomme, P; Driver, D P; Amandoron, E A; Miller, R C; Antony, R; Warren, J; Kilburn, D G

    1995-01-01

    A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei. Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant. The hybrid was purified to homogeneity by affinity chromatography on cellulose. The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively. Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose. Both released predominantly cellobiose and cellotriose from amorphous cellulose. CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan. Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers. PMID:7635821

  18. Targeting the Bacterial Division Protein FtsZ.

    PubMed

    Hurley, Katherine A; Santos, Thiago M A; Nepomuceno, Gabriella M; Huynh, Valerie; Shaw, Jared T; Weibel, Douglas B

    2016-08-11

    Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanical properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial development. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of FtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors.

  19. The Pfam protein families database

    PubMed Central

    Finn, Robert D.; Mistry, Jaina; Tate, John; Coggill, Penny; Heger, Andreas; Pollington, Joanne E.; Gavin, O. Luke; Gunasekaran, Prasad; Ceric, Goran; Forslund, Kristoffer; Holm, Liisa; Sonnhammer, Erik L. L.; Eddy, Sean R.; Bateman, Alex

    2010-01-01

    Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is ∼100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11 912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/). PMID:19920124

  20. Bacterial proteins pinpoint a single eukaryotic root

    PubMed Central

    Derelle, Romain; Torruella, Guifré; Klimeš, Vladimír; Brinkmann, Henner; Kim, Eunsoo; Vlček, Čestmír; Lang, B. Franz; Eliáš, Marek

    2015-01-01

    The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between “Unikonta” and “Bikonta,” with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively. PMID:25646484

  1. Bacterial proteins and peptides in cancer therapy

    PubMed Central

    Chakrabarty, Ananda M; Bernardes, Nuno; Fialho, Arsenio M

    2014-01-01

    Cancer is one of the most deadly diseases worldwide. In the last three decades many efforts have been made focused on understanding how cancer grows and responds to drugs. The dominant drug-development paradigm has been the “one drug, one target.” Based on that, the two main targeted therapies developed to combat cancer include the use of tyrosine kinase inhibitors and monoclonal antibodies. Development of drug resistance and side effects represent the major limiting factors for their use in cancer treatment. Nowadays, a new paradigm for cancer drug discovery is emerging wherein multi-targeted approaches gain ground in cancer therapy. Therefore, to overcome resistance to therapy, it is clear that a new generation of drugs is urgently needed. Here, regarding the concept of multi-targeted therapy, we discuss the challenges of using bacterial proteins and peptides as a new generation of effective anti-cancer drugs. PMID:24875003

  2. Novel receptors for bacterial protein toxins.

    PubMed

    Schmidt, Gudula; Papatheodorou, Panagiotis; Aktories, Klaus

    2015-02-01

    While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin-host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella.

    PubMed

    Eckhard, Ulrich; Bandukwala, Hina; Mansfield, Michael J; Marino, Giada; Cheng, Jiujun; Wallace, Iain; Holyoak, Todd; Charles, Trevor C; Austin, John; Overall, Christopher M; Doxey, Andrew C

    2017-09-12

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per  ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.So far no enzymatic activity has been attributed to flagellin, the major component of bacterial flagella. Here the authors use bioinformatic analysis and identify a metallopeptidase insertion in flagellins from 74 bacterial species and show that recombinant flagellin and flagellar filaments have proteolytic activity.

  4. Supervised Protein Family Classification and New Family Construction

    PubMed Central

    Yi, Gangman; Thon, Michael R.

    2012-01-01

    Abstract The goal of protein family classification is to group proteins into families so that proteins within the same family have common function or are related by ancestry. While supervised classification algorithms are available for this purpose, most of these approaches focus on assigning unclassified proteins to known families but do not allow for progressive construction of new families from proteins that cannot be assigned. Although unsupervised clustering algorithms are also available, they do not make use of information from known families. By computing similarities between proteins based on pairwise sequence comparisons, we develop supervised classification algorithms that achieve improved accuracy over previous approaches while allowing for construction of new families. We show that our algorithm has higher accuracy rate and lower mis-classification rate when compared to algorithms that are based on the use of multiple sequence alignments and hidden Markov models, and our algorithm performs well even on families with very few proteins and on families with low sequence similarity. A software program implementing the algorithm (SClassify) is available online (http://faculty.cse.tamu.edu/shsze/sclassify). PMID:22876787

  5. New Functions for the Ancient DedA Membrane Protein Family

    PubMed Central

    Sikdar, Rakesh; Kumar, Sujeet; Boughner, Lisa A.

    2013-01-01

    The DedA protein family is a highly conserved and ancient family of membrane proteins with representatives in most sequenced genomes, including those of bacteria, archaea, and eukarya. The functions of the DedA family proteins remain obscure. However, recent genetic approaches have revealed important roles for certain bacterial DedA family members in membrane homeostasis. Bacterial DedA family mutants display such intriguing phenotypes as cell division defects, temperature sensitivity, altered membrane lipid composition, elevated envelope-related stress responses, and loss of proton motive force. The DedA family is also essential in at least two species of bacteria: Borrelia burgdorferi and Escherichia coli. Here, we describe the phylogenetic distribution of the family and summarize recent progress toward understanding the functions of the DedA membrane protein family. PMID:23086209

  6. Histone modifications induced by a family of bacterial toxins.

    PubMed

    Hamon, Mélanie Anne; Batsché, Eric; Régnault, Béatrice; Tham, To Nam; Seveau, Stéphanie; Muchardt, Christian; Cossart, Pascale

    2007-08-14

    Upon infection, pathogens reprogram host gene expression. In eukaryotic cells, genetic reprogramming is induced by the concerted activation/repression of transcription factors and various histone modifications that control DNA accessibility in chromatin. We report here that the bacterial pathogen Listeria monocytogenes induces a dramatic dephosphorylation of histone H3 as well as a deacetylation of histone H4 during early phases of infection. This effect is mediated by the major listerial toxin listeriolysin O in a pore-forming-independent manner. Strikingly, a similar effect also is observed with other toxins of the same family, such as Clostridium perfringens perfringolysin and Streptococcus pneumoniae pneumolysin. The decreased levels of histone modifications correlate with a reduced transcriptional activity of a subset of host genes, including key immunity genes. Thus, control of epigenetic regulation emerges here as an unsuspected function shared by several bacterial toxins, highlighting a common strategy used by intracellular and extracellular pathogens to modulate the host response early during infection.

  7. Protein family classification using sparse Markov transducers.

    PubMed

    Eskin, E; Grundy, W N; Singer, Y

    2000-01-01

    In this paper we present a method for classifying proteins into families using sparse Markov transducers (SMTs). Sparse Markov transducers, similar to probabilistic suffix trees, estimate a probability distribution conditioned on an input sequence. SMTs generalize probabilistic suffix trees by allowing for wild-cards in the conditioning sequences. Because substitutions of amino acids are common in protein families, incorporating wildcards into the model significantly improves classification performance. We present two models for building protein family classifiers using SMTs. We also present efficient data structures to improve the memory usage of the models. We evaluate SMTs by building protein family classifiers using the Pfam database and compare our results to previously published results.

  8. Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

    PubMed

    Kerk, David; Uhrig, R Glen; Moorhead, Greg B

    2013-01-01

    Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

  9. Discovery of an archetypal protein transport system in bacterial outer membranes.

    PubMed

    Selkrig, Joel; Mosbahi, Khedidja; Webb, Chaille T; Belousoff, Matthew J; Perry, Andrew J; Wells, Timothy J; Morris, Faye; Leyton, Denisse L; Totsika, Makrina; Phan, Minh-Duy; Celik, Nermin; Kelly, Michelle; Oates, Clare; Hartland, Elizabeth L; Robins-Browne, Roy M; Ramarathinam, Sri Harsha; Purcell, Anthony W; Schembri, Mark A; Strugnell, Richard A; Henderson, Ian R; Walker, Daniel; Lithgow, Trevor

    2012-04-01

    Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

  10. Learning about protein solubility from bacterial inclusion bodies

    PubMed Central

    Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

    2009-01-01

    The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed. PMID:19133126

  11. Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice

    PubMed Central

    Zhao, Yue; Shi, Jianjin; Shi, Xuyan; Wang, Yupeng; Wang, Fengchao

    2016-01-01

    Biochemical studies suggest that the NAIP family of NLR proteins are cytosolic innate receptors that directly recognize bacterial ligands and trigger NLRC4 inflammasome activation. In this study, we generated Naip5−/−, Naip1−/−, and Naip2−/− mice and showed that bone marrow macrophages derived from these knockout mice are specifically deficient in detecting bacterial flagellin, the type III secretion system needle, and the rod protein, respectively. Naip1−/−, Naip2−/−, and Naip5−/− mice also resist lethal inflammasome activation by the corresponding ligand. Furthermore, infections performed in the Naip-deficient macrophages have helped to define the major signal in Legionella pneumophila, Salmonella Typhimurium and Shigella flexneri that is detected by the NAIP/NLRC4 inflammasome. Using an engineered S. Typhimurium infection model, we demonstrate the critical role of NAIPs in clearing bacterial infection and protecting mice from bacterial virulence–induced lethality. These results provide definitive genetic evidence for the important physiological function of NAIPs in antibacterial defense and inflammatory damage–induced lethality in mice. PMID:27114610

  12. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  13. BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins.

    PubMed

    Kankainen, Matti; Ojala, Teija; Holm, Liisa

    2012-02-15

    Automated function prediction has played a central role in determining the biological functions of bacterial proteins. Typically, protein function annotation relies on homology, and function is inferred from other proteins with similar sequences. This approach has become popular in bacterial genomics because it is one of the few methods that is practical for large datasets and because it does not require additional functional genomics experiments. However, the existing solutions produce erroneous predictions in many cases, especially when query sequences have low levels of identity with the annotated source protein. This problem has created a pressing need for improvements in homology-based annotation. We present an automated method for the functional annotation of bacterial protein sequences. Based on sequence similarity searches, BLANNOTATOR accurately annotates query sequences with one-line summary descriptions of protein function. It groups sequences identified by BLAST into subsets according to their annotation and bases its prediction on a set of sequences with consistent functional information. We show the results of BLANNOTATOR's performance in sets of bacterial proteins with known functions. We simulated the annotation process for 3090 SWISS-PROT proteins using a database in its state preceding the functional characterisation of the query protein. For this dataset, our method outperformed the five others that we tested, and the improved performance was maintained even in the absence of highly related sequence hits. We further demonstrate the value of our tool by analysing the putative proteome of Lactobacillus crispatus strain ST1. BLANNOTATOR is an accurate method for bacterial protein function prediction. It is practical for genome-scale data and does not require pre-existing sequence clustering; thus, this method suits the needs of bacterial genome and metagenome researchers. The method and a web-server are available at http://ekhidna.biocenter.helsinki.fi/poxo/blannotator/.

  14. The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators.

    PubMed

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

    2015-07-01

    Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.

  15. The DSF Family of Cell–Cell Signals: An Expanding Class of Bacterial Virulence Regulators

    PubMed Central

    Ryan, Robert P.; An, Shi-qi; Allan, John H.; McCarthy, Yvonne; Dow, J. Maxwell

    2015-01-01

    Many pathogenic bacteria use cell–cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling. PMID:26181439

  16. The Assembly Motif of a Bacterial Small Multidrug Resistance Protein*

    PubMed Central

    Poulsen, Bradley E.; Rath, Arianna; Deber, Charles M.

    2009-01-01

    Multidrug transporters such as the small multidrug resistance (SMR) family of bacterial integral membrane proteins are capable of conferring clinically significant resistance to a variety of common therapeutics. As antiporter proteins of ∼100 amino acids, SMRs must self-assemble into homo-oligomeric structures for efflux of drug molecules. Oligomerization centered at transmembrane helix four (TM4) has been implicated in SMR assembly, but the full complement of residues required to mediate its self-interaction remains to be characterized. Here, we use Hsmr, the 110-residue SMR family member of the archaebacterium Halobacterium salinarum, to determine the TM4 residue motif required to mediate drug resistance and SMR self-association. Twelve single point mutants that scan the central portion of the TM4 helix (residues 85–104) were constructed and were tested for their ability to confer resistance to the cytotoxic compound ethidium bromide. Six residues were found to be individually essential for drug resistance activity (Gly90, Leu91, Leu93, Ile94, Gly97, and Val98), defining a minimum activity motif of 90GLXLIXXGV98 within TM4. When the propensity of these mutants to dimerize on SDS-PAGE was examined, replacements of all but Ile resulted in ∼2-fold reduction of dimerization versus the wild-type antiporter. Our work defines a minimum activity motif of 90GLXLIXXGV98 within TM4 and suggests that this sequence mediates TM4-based SMR dimerization along a single helix surface, stabilized by a small residue heptad repeat sequence. These TM4-TM4 interactions likely constitute the highest affinity locus for disruption of SMR function by directly targeting its self-assembly mechanism. PMID:19224913

  17. Exhaustive enumeration of protein domain families.

    PubMed

    Heger, Andreas; Holm, Liisa

    2003-05-02

    Domains are considered as the basic units of protein folding, evolution, and function. Decomposing each protein into modular domains is thus a basic prerequisite for accurate functional classification of biological molecules. Here, we present ADDA, an automatic algorithm for domain decomposition and clustering of all protein domain families. We use alignments derived from an all-on-all sequence comparison to define domains within protein sequences based on a global maximum likelihood model. In all, 90% of domain boundaries are predicted within 10% of domain size when compared with the manual domain definitions given in the SCOP database. A representative database of 249,264 protein sequences were decomposed into 450,462 domains. These domains were clustered on the basis of sequence similarities into 33,879 domain families containing at least two members with less than 40% sequence identity. Validation against family definitions in the manually curated databases SCOP and PFAM indicates almost perfect unification of various large domain families while contamination by unrelated sequences remains at a low level. The global survey of protein-domain space by ADDA confirms that most large and universal domain families are already described in PFAM and/or SMART. However, a survey of the complete set of mobile modules leads to the identification of 1479 new interesting domain families which shuffle around in multi-domain proteins. The data are publicly available at ftp://ftp.ebi.ac.uk/pub/contrib/heger/adda.

  18. The protein kinase C family.

    PubMed

    Azzi, A; Boscoboinik, D; Hensey, C

    1992-09-15

    Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-1,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.

  19. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

    PubMed Central

    Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

  20. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource.

  1. Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99)

    PubMed Central

    Ovchinnikova, Olga G.; Mallette, Evan; Koizumi, Akihiko; Lowary, Todd L.; Kimber, Matthew S.

    2016-01-01

    Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of β-Kdo residues links “group 2” capsular polysaccharides to (lyso)phosphatidylglycerol. β-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo–dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the β-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype β-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena. The β-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/β domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities. PMID:27199480

  2. Protein family classification using sparse markov transducers.

    PubMed

    Eskin, Eleazar; Noble, William Stafford; Singer, Yoram

    2003-01-01

    We present a method for classifying proteins into families based on short subsequences of amino acids using a new probabilistic model called sparse Markov transducers (SMT). We classify a protein by estimating probability distributions over subsequences of amino acids from the protein. Sparse Markov transducers, similar to probabilistic suffix trees, estimate a probability distribution conditioned on an input sequence. SMTs generalize probabilistic suffix trees by allowing for wild-cards in the conditioning sequences. Since substitutions of amino acids are common in protein families, incorporating wild-cards into the model significantly improves classification performance. We present two models for building protein family classifiers using SMTs. As protein databases become larger, data driven learning algorithms for probabilistic models such as SMTs will require vast amounts of memory. We therefore describe and use efficient data structures to improve the memory usage of SMTs. We evaluate SMTs by building protein family classifiers using the Pfam and SCOP databases and compare our results to previously published results and state-of-the-art protein homology detection methods. SMTs outperform previous probabilistic suffix tree methods and under certain conditions perform comparably to state-of-the-art protein homology methods.

  3. Bacterial expansins and related proteins from the world of microbes.

    PubMed

    Georgelis, Nikolaos; Nikolaidis, Nikolas; Cosgrove, Daniel J

    2015-05-01

    The discovery of microbial expansins emerged from studies of the mechanism of plant cell growth and the molecular basis of plant cell wall extensibility. Expansins are wall-loosening proteins that are universal in the plant kingdom and are also found in a small set of phylogenetically diverse bacteria, fungi, and other organisms, most of which colonize plant surfaces. They loosen plant cell walls without detectable lytic activity. Bacterial expansins have attracted considerable attention recently for their potential use in cellulosic biomass conversion for biofuel production, as a means to disaggregate cellulosic structures by nonlytic means ("amorphogenesis"). Evolutionary analysis indicates that microbial expansins originated by multiple horizontal gene transfers from plants. Crystallographic analysis of BsEXLX1, the expansin from Bacillus subtilis, shows that microbial expansins consist of two tightly packed domains: the N-terminal domain D1 has a double-ψ β-barrel fold similar to glycosyl hydrolase family-45 enzymes but lacks catalytic residues usually required for hydrolysis; the C-terminal domain D2 has a unique β-sandwich fold with three co-linear aromatic residues that bind β-1,4-glucans by hydrophobic interactions. Genetic deletion of expansin in Bacillus and Clavibacter cripples their ability to colonize plant tissues. We assess reports that expansin addition enhances cellulose breakdown by cellulase and compare expansins with distantly related proteins named swollenin, cerato-platanin, and loosenin. We end in a speculative vein about the biological roles of microbial expansins and their potential applications. Advances in this field will be aided by a deeper understanding of how these proteins modify cellulosic structures.

  4. Bacterial expansins and related proteins from the world of microbes

    DOE PAGES

    Georgelis, Nikolaos; Nikolaidis, Nikolas; Cosgrove, Daniel J.

    2015-04-02

    The discovery of microbial expansins emerged from studies of the mechanism of plant cell growth and the molecular basis of plant cell wall extensibility. Expansins are wall-loosening proteins that are universal in the plant kingdom and are also found in a small set of phylogenetically diverse bacteria, fungi, and other organisms, most of which colonize plant surfaces. They loosen plant cell walls without detectable lytic activity. Bacterial expansins have attracted considerable attention recently for their potential use in cellulosic biomass conversion for biofuel production, as a means to disaggregate cellulosic structures by nonlytic means (“amorphogenesis”). Evolutionary analysis indicates that microbialmore » expansins originated by multiple horizontal gene transfers from plants. Crystallographic analysis of BsEXLX1, the expansin from Bacillus subtilis, shows that microbial expansins consist of two tightly packed domains: the N-terminal domain D1 has a double-ψ β-barrel fold similar to glycosyl hydrolase family-45 enzymes but lacks catalytic residues usually required for hydrolysis; the C-terminal domain D2 has a unique β-sandwich fold with three co-linear aromatic residues that bind β-1,4-glucans by hydrophobic interactions. Genetic deletion of expansin in Bacillus and Clavibacter cripples their ability to colonize plant tissues. In this paper, we assess reports that expansin addition enhances cellulose breakdown by cellulase and compare expansins with distantly related proteins named swollenin, cerato-platanin, and loosenin. Finally, we end in a speculative vein about the biological roles of microbial expansins and their potential applications. Advances in this field will be aided by a deeper understanding of how these proteins modify cellulosic structures.« less

  5. Bacterial expansins and related proteins from the world of microbes

    SciTech Connect

    Georgelis, Nikolaos; Nikolaidis, Nikolas; Cosgrove, Daniel J.

    2015-04-02

    The discovery of microbial expansins emerged from studies of the mechanism of plant cell growth and the molecular basis of plant cell wall extensibility. Expansins are wall-loosening proteins that are universal in the plant kingdom and are also found in a small set of phylogenetically diverse bacteria, fungi, and other organisms, most of which colonize plant surfaces. They loosen plant cell walls without detectable lytic activity. Bacterial expansins have attracted considerable attention recently for their potential use in cellulosic biomass conversion for biofuel production, as a means to disaggregate cellulosic structures by nonlytic means (“amorphogenesis”). Evolutionary analysis indicates that microbial expansins originated by multiple horizontal gene transfers from plants. Crystallographic analysis of BsEXLX1, the expansin from Bacillus subtilis, shows that microbial expansins consist of two tightly packed domains: the N-terminal domain D1 has a double-ψ β-barrel fold similar to glycosyl hydrolase family-45 enzymes but lacks catalytic residues usually required for hydrolysis; the C-terminal domain D2 has a unique β-sandwich fold with three co-linear aromatic residues that bind β-1,4-glucans by hydrophobic interactions. Genetic deletion of expansin in Bacillus and Clavibacter cripples their ability to colonize plant tissues. In this paper, we assess reports that expansin addition enhances cellulose breakdown by cellulase and compare expansins with distantly related proteins named swollenin, cerato-platanin, and loosenin. Finally, we end in a speculative vein about the biological roles of microbial expansins and their potential applications. Advances in this field will be aided by a deeper understanding of how these proteins modify cellulosic structures.

  6. Bacterial expansins and related proteins from the world of microbes

    PubMed Central

    Georgelis, Nikolaos; Nikolaidis, Nikolas; Cosgrove, Daniel J.

    2015-01-01

    The discovery of microbial expansins emerged from studies of the mechanism of plant cell growth and the molecular basis of plant cell wall extensibility. Expansins are wall-loosening proteins that are universal in the plant kingdom and are also found in a small set of phylogenetically diverse bacteria, fungi, and other organisms, most of which colonize plant surfaces. They loosen plant cell walls without detectable lytic activity. Bacterial expansins have attracted considerable attention recently for their potential use in cellulosic biomass conversion for biofuel production, as a means to disaggregate cellulosic structures by non-lytic means (‘amorphogenesis’). Evolutionary analysis indicates that microbial expansins originated by multiple horizontal gene transfers from plants. Crystallographic analysis of BsEXLX1, the expansin from Bacillus subtilis, shows that microbial expansins consist of two tightly-packed domains: the N-terminal domain D1 has a double-ψ β-barrel fold similar to glycosyl hydrolase family-45 enzymes, but lacks catalytic residues usually required for hydrolysis; the C-terminal domain D2 has a unique β-sandwich fold with three co-linear aromatic residues that bind β-1,4-glucans by hydrophobic interactions. Genetic deletion of expansin in Bacillus and Clavibacter cripples their ability to colonize plant tissues. We assess reports that expansin addition enhances cellulose breakdown by cellulase and compare expansins with distantly related proteins named swollenin, cerato-platanin and loosenin. We end in a speculative vein about the biological roles of microbial expansins and their potential applications. Advances in this field will be aided by a deeper understanding of how these proteins modify cellulosic structures. PMID:25833181

  7. Bacterial chitinases and chitin-binding proteins as virulence factors.

    PubMed

    Frederiksen, Rikki F; Paspaliari, Dafni K; Larsen, Tanja; Storgaard, Birgit G; Larsen, Marianne H; Ingmer, Hanne; Palcic, Monica M; Leisner, Jørgen J

    2013-05-01

    Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

  8. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    PubMed Central

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-01-01

    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; however, the mechanism by which CotH affects germination is unclear. Here, 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. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

  9. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent 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; 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.

  10. 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

  11. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent 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; 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.

  12. Bacterial protein signals are associated with Crohn’s disease

    PubMed Central

    Juste, Catherine; Kreil, David P; Beauvallet, Christian; Guillot, Alain; Vaca, Sebastian; Carapito, Christine; Mondot, Stanislas; Sykacek, Peter; Sokol, Harry; Blon, Florence; Lepercq, Pascale; Levenez, Florence; Valot, Benoît; Carré, Wilfrid; Loux, Valentin; Pons, Nicolas; David, Olivier; Schaeffer, Brigitte; Lepage, Patricia; Martin, Patrice; Monnet, Véronique; Seksik, Philippe; Beaugerie, Laurent; Ehrlich, S Dusko; Gibrat, Jean-François; Van Dorsselaer, Alain; Doré, Joël

    2014-01-01

    Objective No Crohn’s disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. Design We first developed and validated a workflow—including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS—to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Results Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. Conclusions This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis. PMID:24436141

  13. Bacterial protein signals are associated with Crohn's disease.

    PubMed

    Juste, Catherine; Kreil, David P; Beauvallet, Christian; Guillot, Alain; Vaca, Sebastian; Carapito, Christine; Mondot, Stanislas; Sykacek, Peter; Sokol, Harry; Blon, Florence; Lepercq, Pascale; Levenez, Florence; Valot, Benoît; Carré, Wilfrid; Loux, Valentin; Pons, Nicolas; David, Olivier; Schaeffer, Brigitte; Lepage, Patricia; Martin, Patrice; Monnet, Véronique; Seksik, Philippe; Beaugerie, Laurent; Ehrlich, S Dusko; Gibrat, Jean-François; Van Dorsselaer, Alain; Doré, Joël

    2014-10-01

    No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  14. The lipocalin protein family: structure and function.

    PubMed Central

    Flower, D R

    1996-01-01

    The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds. PMID:8761444

  15. Insights into bacterial protein glycosylation in human microbiota.

    PubMed

    Zhu, Fan; Wu, Hui

    2016-01-01

    The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria- host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection.

  16. Insights into bacterial protein glycosylation in human microbiota

    PubMed Central

    Zhu, Fan; Wu, Hui

    2017-01-01

    The study of human microbiota is an emerging research topic. The past efforts have mainly centered on studying the composition and genomic landscape of bacterial species within the targeted communities. The interaction between bacteria and hosts is the pivotal event in the initiation and progression of infectious diseases. There is a great need to identify and characterize the molecules that mediate the bacteria-host interaction. Bacterial surface exposed proteins play an important role in the bacteria-host interaction. Numerous surface proteins are glycosylated, and the glycosylation is crucial for their function in mediating the bacterial interaction with hosts. Here we present an overview of surface glycoproteins from bacteria that inhabit three major mucosal environments across human body: oral, gut and skin. We describe the important enzymes involved in the process of protein glycosylation, and discuss how the process impacts the bacteria-host interaction. Emerging molecular details underlying glycosylation of bacterial surface proteins may lead to new opportunities for designing anti-infective small molecules, and developing novel vaccines in order to treat or prevent bacterial infection. PMID:26712033

  17. Characteristic motifs for families of allergenic proteins

    PubMed Central

    Ivanciuc, Ovidiu; Garcia, Tzintzuni; Torres, Miguel; Schein, Catherine H.; Braun, Werner

    2008-01-01

    The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver Motif-Mate (http://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins. PMID:18951633

  18. Recent insights into Pasteurella multocida toxin and other G-protein-modulating bacterial toxins.

    PubMed

    Wilson, Brenda A; Ho, Mengfei

    2010-08-01

    Over the past few decades, our understanding of the bacterial protein toxins that modulate G proteins has advanced tremendously through extensive biochemical and structural analyses. This article provides an updated survey of the various toxins that target G proteins, ending with a focus on recent mechanistic insights in our understanding of the deamidating toxin family. The dermonecrotic toxin from Pasteurella multocida (PMT) was recently added to the list of toxins that disrupt G-protein signal transduction through selective deamidation of their targets. The C3 deamidase domain of PMT has no sequence similarity to the deamidase domains of the dermonecrotic toxins from Escherichia coli (cytotoxic necrotizing factor [CNF]1-3), Yersinia (CNFY) and Bordetella (dermonecrotic toxin). The structure of PMT-C3 belongs to a family of transglutaminase-like proteins, with active site Cys-His-Asp catalytic triads distinct from E. coli CNF1.

  19. Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

    PubMed Central

    Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

    2009-01-01

    Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

  20. General Aspects and Recent Advances on Bacterial Protein Toxins

    PubMed Central

    Lemichez, Emmanuel; Barbieri, Joseph T.

    2013-01-01

    Bacterial pathogens produce protein toxins to influence host–pathogen interactions and tip the outcome of these encounters toward the benefit of the pathogen. Protein toxins modify host-specific targets through posttranslational modifications (PTMs) or noncovalent interactions that may inhibit or activate host cell physiology to benefit the pathogen. Recent advances have identified new PTMs and host targets for toxin action. Understanding the mechanisms of toxin action provides a basis to develop vaccines and therapies to combat bacterial pathogens and to develop new strategies to use toxin derivatives for the treatment of human disease. PMID:23378599

  1. Protein export through the bacterial Sec pathway.

    PubMed

    Tsirigotaki, Alexandra; De Geyter, Jozefien; Šoštaric, Nikolina; Economou, Anastassios; Karamanou, Spyridoula

    2017-01-01

    The general secretory (Sec) pathway comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane. Sec exportome polypeptides are synthesized as pre-proteins that have cleavable signal peptides fused to the exported mature domains. Recent advances have re-evaluated the interaction networks of pre-proteins with chaperones that are involved in pre-protein targeting from the ribosome to the SecYEG channel and have identified conformational signals as checkpoints for high-fidelity targeting and translocation. The recent structural and mechanistic insights into the channel and its ATPase motor SecA are important steps towards the elucidation of the allosteric crosstalk that mediates secretion. In this Review, we discuss recent biochemical, structural and mechanistic insights into the consecutive steps of the Sec pathway - sorting and targeting, translocation and release - in both co-translational and post-translational modes of export. The architecture and conformational dynamics of the SecYEG channel and its regulation by ribosomes, SecA and pre-proteins are highlighted. Moreover, we present conceptual models of the mechanisms and energetics of the Sec-pathway dependent secretion process in bacteria.

  2. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

    PubMed Central

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-01-01

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  3. Identification of ligands for bacterial sensor proteins.

    PubMed

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria.

  4. Protein oxidation: key to bacterial desiccation resistance?

    PubMed

    Fredrickson, James K; Li, Shu-mei W; Gaidamakova, Elena K; Matrosova, Vera Y; Zhai, Min; Sulloway, Heather M; Scholten, Johannes C; Brown, Mindy G; Balkwill, David L; Daly, Michael J

    2008-04-01

    For extremely ionizing radiation-resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the US Department of Energy's Hanford Site in Washington State. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of the genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms, which limit protein oxidation during dehydration.

  5. A systematic analysis of the RNA-targeting potential of secreted bacterial effector proteins.

    PubMed

    Tawk, Caroline; Sharan, Malvika; Eulalio, Ana; Vogel, Jörg

    2017-08-24

    Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins.

  6. CdnL, a member of the large CarD-like family of bacterial proteins, is vital for Myxococcus xanthus and differs functionally from the global transcriptional regulator CarD

    PubMed Central

    García-Moreno, Diana; Abellón-Ruiz, Javier; García-Heras, Francisco; Murillo, Francisco J.; Padmanabhan, S.; Elías-Arnanz, Montserrat

    2010-01-01

    CarD, a global transcriptional regulator in Myxococcus xanthus, interacts with CarG via CarDNter, its N-terminal domain, and with DNA via a eukaryotic HMGA-type C-terminal domain. Genomic analysis reveals a large number of standalone proteins resembling CarDNter. These constitute, together with the RNA polymerase (RNAP) interacting domain, RID, of transcription–repair coupling factors, the CarD_TRCF protein family. We show that one such CarDNter-like protein, M. xanthus CdnL, cannot functionally substitute CarDNter (or vice versa) nor interact with CarG. Unlike CarD, CdnL is vital for growth, and lethality due to its absence is not rescued by homologs from various other bacteria. In mycobacteria, with no endogenous DksA, the function of the CdnL homolog mirrors that of Escherichia coli DksA. Our finding that CdnL, like DksA, is indispensable in M. xanthus implies that they are not functionally redundant. Cells are normal on CdnL overexpression, but divide aberrantly on CdnL depletion. CdnL localizes to the nucleoid, suggesting piggyback recruitment by factors such as RNAP, which we show interacts with CdnL, CarDNter and RID. Our study highlights a complex network of interactions involving these factors and RNAP, and points to a vital role for M. xanthus CdnL in an essential DNA transaction that affects cell division. PMID:20371514

  7. Homologs of the Acinetobacter baumannii AceI Transporter Represent a New Family of Bacterial Multidrug Efflux Systems

    PubMed Central

    Liu, Qi; Henderson, Peter J. F.

    2015-01-01

    ABSTRACT Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. PMID:25670776

  8. Opa+ Neisseria gonorrhoeae Exhibits Reduced Survival in Human Neutrophils Via Src Family Kinase-Mediated Bacterial Trafficking Into Mature Phagolysosomes

    PubMed Central

    Johnson, M. Brittany; Ball, Louise M.; Daily, Kylene P.; Martin, Jennifer N.; Columbus, Linda; Criss, Alison K.

    2015-01-01

    Summary During gonorrheal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial content. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signaling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signaling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils’ full antimicrobial arsenal. PMID:25346239

  9. Host-microbe protein interactions during bacterial infection

    PubMed Central

    Schweppe, Devin K.; Harding, Christopher; Chavez, Juan D.; Wu, Xia; Ramage, Elizabeth; Singh, Pradeep K.; Manoil, Colin; Bruce, James E.

    2015-01-01

    Summary Interspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and interspecies complex structures has been more difficult to acquire. Using an unbiased chemical cross-linking/mass spectrometry approach, we identified interspecies protein-protein interactions in human lung epithelial cells infected with Acinetobacter baumannii. These efforts resulted in identification of 3076 total cross-linked peptide pairs and 46 interspecies protein-protein interactions. Most notably, the key A. baumannii virulence factor, OmpA, was identified cross-linked to host proteins involved in desmosomes, specialized structures that mediate host cell-to-cell adhesion. Co-immunoprecipitation and transposon mutant experiments were used to verify these interactions and demonstrate relevance for host cell invasion and acute murine lung infection. These results shed new light on A. baumannii-host protein interactions and their structural features and the presented approach is generally applicable to other systems. PMID:26548613

  10. The bacterial pathogen Listeria monocytogenes and the interferon family: type I, type II and type III interferons

    PubMed Central

    Dussurget, Olivier; Bierne, Hélène; Cossart, Pascale

    2014-01-01

    Interferons (IFNs) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. Although the importance of IFNs in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. This review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen Listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert IFN responses. PMID:24809023

  11. The bacterial pathogen Listeria monocytogenes and the interferon family: type I, type II and type III interferons.

    PubMed

    Dussurget, Olivier; Bierne, Hélène; Cossart, Pascale

    2014-01-01

    Interferons (IFNs) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. Although the importance of IFNs in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. This review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen Listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert IFN responses.

  12. Membrane composition influences the topology bias of bacterial integral membrane proteins.

    PubMed

    Bay, Denice C; Turner, Raymond J

    2013-02-01

    Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a "neutral" topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram+ compared to Gram- species and each lipid parameter demonstrated a linear inverse relationship.

  13. Bacterial protein toxins: current and potential clinical use.

    PubMed

    Fabbri, A; Travaglione, S; Falzano, L; Fiorentini, C

    2008-01-01

    Natural toxins are the product of a long-term evolution, and act on essential mechanisms in the most crucial and vital processes of living organisms. They can attack components of the protein synthesis machinery, actin polymerization, signal transduction pathways, intracellular trafficking of vesicles as well as immune and inflammatory responses. For this reason, toxins have increasingly being used as valuable tools for analysis of cellular physiology, and in the recent years, some of them are used medicinally for the treatment of human diseases. This review is devoted to protein toxins of bacterial origin, specifically those toxins that are currently used in therapy or those under study for their potential clinical applications. Bacterial protein toxins are all characterized by a specific mechanism of action that involves the central molecular pathways in the eukaryotic cell. Knowledge of their properties could be used for medical purposes.

  14. The MAGE protein family and cancer

    PubMed Central

    Weon, Jenny L.; Potts, Patrick Ryan

    2015-01-01

    The Melanoma Antigen Gene (MAGE) protein family is a large, highly conserved group of proteins that share a common MAGE homology domain. Intriguingly, many MAGE proteins are restricted in expression to reproductive tissues, but are aberrantly expressed in a wide-variety of cancer types. Originally discovered as antigens on tumor cells and developed as cancer immunotherapy targets, recent literature suggests a more prominent role for MAGEs in driving tumorigenesis. This review will highlight recent developments into the function of MAGEs as oncogenes, their mechanisms of action in regulation of ubiquitin ligases, and outstanding questions in the field. PMID:26342994

  15. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure

    PubMed Central

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-01-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (p<0.05) in CP 15% group among three CP levels. Relative abundance of Bacteroidetes phylum and bacterial genera including Leuconostoc, Bacillus, Atopostipes, Peptonphilus, Ruminococcaceae_uc, Bacteroides, and Pseudomonas was lower (p<0.05) in CP 15% than in CP 20% group. There was a positive correlation (p<0.05) between odorous compounds and bacterial genera: phenol, indole, iso-butyric acid, and iso-valeric acid with Atopostipes, p-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  16. Bacterial collagen-like proteins that form triple-helical structures.

    PubMed

    Yu, Zhuoxin; An, Bo; Ramshaw, John A M; Brodsky, Barbara

    2014-06-01

    A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35-39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

  17. Bacterial collagen-like proteins that form triple-helical structures

    PubMed Central

    Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara

    2014-01-01

    A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612

  18. Structural and Sequence Analysis of Imelysin-Like Proteins Implicated in Bacterial Iron Uptake

    PubMed Central

    Xu, Qingping; Rawlings, Neil D.; Farr, Carol L.; Chiu, Hsiu-Ju; Grant, Joanna C.; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Miller, Mitchell D.; Weekes, Dana; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2011-01-01

    Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution), have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function. PMID:21799754

  19. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  20. Fos family protein degradation by the proteasome.

    PubMed

    Gomard, Tiphanie; Jariel-Encontre, Isabelle; Basbous, Jihane; Bossis, Guillaume; Moquet-Torcy, Gabriel; Mocquet-Torcy, Gabriel; Piechaczyk, Marc

    2008-10-01

    c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

  1. FIGfams : yet another set of protein families.

    SciTech Connect

    Meyer, F.; Overbeek, R.; Rodriguez, A.; Mathematics and Computer Science; Univ. of Chicago; Fellowship for the Interpretation of Genomes

    2009-11-01

    We present FIGfams, a new collection of over 100,000 protein families that are the product of manual curation and close strain comparison. Using the Subsystem approach the manual curation is carried out, ensuring a previously unattained degree of throughput and consistency. FIGfams are based on over 950,000 manually annotated proteins and across many hundred Bacteria and Archaea. Associated with each FIGfam is a two-tiered, rapid, accurate decision procedure to determine family membership for new proteins. FIGfams are freely available under an open source license. These can be downloaded at ftp://ftp.theseed.org/FIGfams/. The web site for FIGfams is http://www.theseed.org/wiki/FIGfams/.

  2. Novel protein families in archaean genomes.

    PubMed Central

    Ouzonis, C; Kyrpides, N; Sander, C

    1995-01-01

    In a quest for novel functions in archaea, all archaean hypothetical open reading frames (ORFs), as annotated in the Swiss-Prot protein sequence database, were used to search the latest databases for the identification of characterized homologues. Of the 95 hypothetical archaean ORFs, 25 were found to be homologous to another hypothetical archaean ORF, while 36 were homologous to non-archaean proteins, of which as many as 30 were homologous to a characterized protein family. Thus the level of sequence similarity in this set reaches 64%, while the level of function assignment is only 32%. Of the ORFs with predicted functions, 12 homologies are reported here for the first time and represent nine new functions and one gene duplication at an acetyl-coA synthetase locus. The novel functions include components of the transcriptional and translational apparatus, such as ribosomal proteins, modification enzymes and a translation initiation factor. In addition, new enzymes are identified in archaea, such as cobyric acid synthase, dCTP deaminase and the first archaean homologues of a new subclass of ATP binding proteins found in fungi. Finally, it is shown that the putative laminin receptor family of eukaryotes and an archaean homologue belong to the previously characterized ribosomal protein family S2 from eubacteria. From the present and previous work, the major implication is that archaea seem to have a mode of expression of genetic information rather similar to eukaryotes, while eubacteria may have proceeded into unique ways of transcription and translation. In addition, with the detection of proteins in various metabolic and genetic processes in archaea, we can further predict the presence of additional proteins involved in these processes. PMID:7899076

  3. Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms.

    PubMed

    Zhang, Dapeng; Iyer, Lakshminarayan M; Aravind, L

    2012-10-01

    Gibberellic acids (GAs) are key plant hormones, regulating various aspects of growth and development, which have been at the center of the 'green revolution'. GRAS family proteins, the primary players in GA signaling pathways, remain poorly understood. Using sequence-profile searches, structural comparisons and phylogenetic analysis, we establish that the GRAS family first emerged in bacteria and belongs to the Rossmann fold methyltransferase superfamily. All bacterial and a subset of plant GRAS proteins are likely to function as small-molecule methylases. The remaining plant versions have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. We predict that GRAS proteins might either modify or bind small molecules such as GAs or their derivatives. aravind@ncbi.nlm.nih.gov Supplementary Material for this article is available at Bioinformatics online.

  4. Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms

    PubMed Central

    Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

    2012-01-01

    Summary: Gibberellic acids (GAs) are key plant hormones, regulating various aspects of growth and development, which have been at the center of the ‘green revolution’. GRAS family proteins, the primary players in GA signaling pathways, remain poorly understood. Using sequence-profile searches, structural comparisons and phylogenetic analysis, we establish that the GRAS family first emerged in bacteria and belongs to the Rossmann fold methyltransferase superfamily. All bacterial and a subset of plant GRAS proteins are likely to function as small-molecule methylases. The remaining plant versions have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. We predict that GRAS proteins might either modify or bind small molecules such as GAs or their derivatives. Contact: aravind@ncbi.nlm.nih.gov Supplementary Information: Supplementary Material for this article is available at Bioinformatics online. PMID:22829623

  5. Lethal protein produced in response to competition between sibling bacterial colonies.

    PubMed

    Be'er, Avraham; Ariel, Gil; Kalisman, Oren; Helman, Yael; Sirota-Madi, Alexandra; Zhang, H P; Florin, E-L; Payne, Shelley M; Ben-Jacob, Eshel; Swinney, Harry L

    2010-04-06

    Sibling Paenibacillus dendritiformis bacterial colonies grown on low-nutrient agar medium mutually inhibit growth through secretion of a lethal factor. Analysis of secretions reveals the presence of subtilisin (a protease) and a 12 kDa protein, termed sibling lethal factor (Slf). Purified subtilisin promotes the growth and expansion of P. dendritiformis colonies, whereas Slf is lethal and lyses P. dendritiformis cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa, termed dendritiformis sibling bacteriocin. The 20 kDa recombinant protein was produced and found to be inactive, but exposure to subtilisin resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modeling, show that subtilisin serves to regulate growth of the colony. Below a threshold concentration, subtilisin promotes colony growth and expansion. However, once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs of dendritiformis sibling bacteriocin in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.

  6. Physiological functions of MTA family of proteins.

    PubMed

    Sen, Nirmalya; Gui, Bin; Kumar, Rakesh

    2014-12-01

    Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

  7. On the Entropy of Protein Families

    NASA Astrophysics Data System (ADS)

    Barton, John P.; Chakraborty, Arup K.; Cocco, Simona; Jacquin, Hugo; Monasson, Rémi

    2016-03-01

    Proteins are essential components of living systems, capable of performing a huge variety of tasks at the molecular level, such as recognition, signalling, copy, transport, ... The protein sequences realizing a given function may largely vary across organisms, giving rise to a protein family. Here, we estimate the entropy of those families based on different approaches, including Hidden Markov Models used for protein databases and inferred statistical models reproducing the low-order (1- and 2-point) statistics of multi-sequence alignments. We also compute the entropic cost, that is, the loss in entropy resulting from a constraint acting on the protein, such as the mutation of one particular amino-acid on a specific site, and relate this notion to the escape probability of the HIV virus. The case of lattice proteins, for which the entropy can be computed exactly, allows us to provide another illustration of the concept of cost, due to the competition of different folds. The relevance of the entropy in relation to directed evolution experiments is stressed.

  8. TIGRFAMS: The TIGRFAMs database of protein families

    DOE Data Explorer

    TIGRFAMs are protein families based on Hidden Markov Models or HMMs. Use this page to see the curated seed alignmet for each TIGRFam, the full alignment of all family members and the cutoff scores for inclusion in each of the TIGRFAMs. Also use this page to search through the TIGRFAMs and HMMs for text in the TIGRFAMs Text Search or search for specific sequences in the TIGRFAMs Sequence Search.[Copied from the Overview at http://www.jcvi.org/cms/research/projects/tigrfams/overview/] See also TIGRFAMs ordered by the roles they play at http://cmr.jcvi.org/tigr-scripts/CMR/shared/EvidenceList.cgi?ev_type=TIGRFAM&order_type=role.

  9. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family.

    PubMed

    Cui, Jixin; Yao, Qing; Li, Shan; Ding, Xiaojun; Lu, Qiuhe; Mao, Haibin; Liu, Liping; Zheng, Ning; Chen, She; Shao, Feng

    2010-09-03

    A family of bacterial effectors including Cif homolog from Burkholderia pseudomallei (CHBP) and Cif from Enteropathogenic Escherichia coli (EPEC) adopt a functionally important papain-like hydrolytic fold. We show here that CHBP was a potent inhibitor of the eukaryotic ubiquitination pathway. CHBP acted as a deamidase that specifically and efficiently deamidated Gln40 in ubiquitin and ubiquitin-like protein NEDD8 both in vitro and during Burkholderia infection. Deamidated ubiquitin was impaired in supporting ubiquitin-chain synthesis. Cif selectively deamidated NEDD8, which abolished the activity of neddylated Cullin-RING ubiquitin ligases (CRLs). Ubiquitination and ubiquitin-dependent degradation of multiple CRL substrates were impaired by Cif in EPEC-infected cells. Mutations of substrate-contacting residues in Cif abolished or attenuated EPEC-induced cytopathic phenotypes of cell cycle arrest and actin stress fiber formation.

  10. Surfaces for competitive selective bacterial capture from protein solutions.

    PubMed

    Fang, Bing; Gon, Saugata; Nüsslein, Klaus; Santore, Maria M

    2015-05-20

    Active surfaces that form the basis for bacterial sensors for threat detection, food safety, or certain diagnostic applications rely on bacterial adhesion. However, bacteria capture from complex fluids on the active surfaces can be reduced by the competing adsorption of proteins and other large molecules. Such adsorption can also interfere with device performance. As a result, multiple upstream processing steps are frequently employed to separate macromolecules from any cells, which remain in the buffer. Here, we present an economical approach to capture bacteria, without competitive adsorption by proteins, on engineered surfaces that do not employ biomolecular recognition, antibodies, or other molecules with engineered sequences. The surfaces are based on polyethylene glycol (PEG) brushes that, on their own, repel both proteins and bacteria. These PEG brushes backfill the surface around sparsely adsorbed cationic polymer coils (here, poly-L-lysine (PLL)). The PLL coils are effectively embedded within the brush and produce locally cationic nanoscale regions that attract negatively charged regions of proteins or cells against the steric background repulsion from the PEG brush. By carefully designing the surfaces to include just enough PLL to capture bacteria, but not enough to capture proteins, we achieve sharp selectivity where S. aureus is captured from albumin- or fibrinogen-containing solutions, but free albumin or fibrinogen molecules are rejected from the surface. Bacterial adhesion on these surfaces is not reduced by competitive protein adsorption, in contrast to performance of more uniformly cationic surfaces. Also, protein adsorption to the bacteria does not interfere with capture, at least for the case of S. aureus, to which fibrinogen binds through a specific receptor.

  11. In situ protein folding and activation in bacterial inclusion bodies.

    PubMed

    Gonzalez-Montalban, Nuria; Natalello, Antonino; García-Fruitós, Elena; Villaverde, Antonio; Doglia, Silvia Maria

    2008-07-01

    Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

  12. Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria.

    PubMed

    Chakroun, Maissa; Banyuls, Núria; Bel, Yolanda; Escriche, Baltasar; Ferré, Juan

    2016-06-01

    Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

  13. Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

    PubMed Central

    Chakroun, Maissa; Banyuls, Núria; Bel, Yolanda; Escriche, Baltasar

    2016-01-01

    SUMMARY Entomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet. PMID:26935135

  14. Interactions of Bacterial Proteins with Host Eukaryotic Ubiquitin Pathways

    PubMed Central

    Perrett, Charlotte Averil; Lin, David Yin-Wei; Zhou, Daoguo

    2011-01-01

    Ubiquitination is a post-translational modification in which one or more 76 amino acid polypeptide ubiquitin molecules are covalently linked to the lysine residues of target proteins. Ubiquitination is the main pathway for protein degradation that governs a variety of eukaryotic cellular processes, including the cell-cycle, vesicle trafficking, antigen presentation, and signal transduction. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases including inflammatory and neurodegenerative disorders. Recent studies have revealed that viruses and bacterial pathogens exploit the host ubiquitination pathways to gain entry and to aid their survival/replication inside host cells. This review will summarize recent developments in understanding the biochemical and structural mechanisms utilized by bacterial pathogens to interact with the host ubiquitination pathways. PMID:21772834

  15. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  16. Repurposing bacterial toxins for intracellular delivery of therapeutic proteins.

    PubMed

    Beilhartz, Greg L; Sugiman-Marangos, Seiji N; Melnyk, Roman A

    2017-10-15

    Despite enormous efforts, achieving efficacious levels of proteins inside mammalian cells remains one of the greatest challenges in biologics-based drug discovery and development. The inability of proteins to readily cross biological membranes precludes access to the wealth of intracellular targets and applications that lie within mammalian cells. Existing methods of delivery commonly suffer from an inability to target specific cells and tissues, poor endosomal escape, and limited in vivo efficacy. The aim of the present commentary is to highlight the potential of certain classes of bacterial toxins, which naturally deliver a large protein into the cytosolic compartment of target cells after binding a host cell-surface receptor with high affinity, as robust protein delivery platforms. We review the progress made in recent years toward demonstrating the utility of these systems at delivering a wide variety of protein cargo, with special attention paid to three distinct toxin-based platforms. We contend that with recent advances in protein deimmunization strategies, bacterial toxins are poised to introduce biologics into the inner sanctum of cells and treat a wealth of heretofore untreatable diseases with a new generation of therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    PubMed

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery.

  18. Subcellular localization of the Schlafen protein family.

    PubMed

    Neumann, Brent; Zhao, Liang; Murphy, Kathleen; Gonda, Thomas J

    2008-05-23

    Although the first members of the Schlafen gene family were first described almost 10 years ago, the precise molecular/biochemical functions of the proteins they encode still remain largely unknown. Roles in cell growth, haematopoietic cell differentiation, and T cell development/maturation have, with some experimental support, been postulated, but none have been conclusively verified. Here, we have determined the subcellular localization of Schlafens 1, 2, 4, 5, 8, and 9, representing all three of the murine subgroups. We show that the proteins from subgroups I and II localize to the cytoplasm, while the longer forms in subgroup III localize exclusively to the nuclear compartment. We also demonstrate upregulation of Schlafen2 upon differentiation of haematopoietic cells and show this endogenous protein localizes to the cytoplasm. Thus, we propose the different subgroups of Schlafen proteins are likely to have functionally distinct roles, reflecting their differing localizations within the cell.

  19. Subcellular localization of the Schlafen protein family

    SciTech Connect

    Neumann, Brent; Zhao Liang; Murphy, Kathleen; Gonda, Thomas J.

    2008-05-23

    Although the first members of the Schlafen gene family were first described almost 10 years ago, the precise molecular/biochemical functions of the proteins they encode still remain largely unknown. Roles in cell growth, haematopoietic cell differentiation, and T cell development/maturation have, with some experimental support, been postulated, but none have been conclusively verified. Here, we have determined the subcellular localization of Schlafens 1, 2, 4, 5, 8, and 9, representing all three of the murine subgroups. We show that the proteins from subgroups I and II localize to the cytoplasm, while the longer forms in subgroup III localize exclusively to the nuclear compartment. We also demonstrate upregulation of Schlafen2 upon differentiation of haematopoietic cells and show this endogenous protein localizes to the cytoplasm. Thus, we propose the different subgroups of Schlafen proteins are likely to have functionally distinct roles, reflecting their differing localizations within the cell.

  20. SUMOylation of Myc-Family Proteins

    PubMed Central

    Sabò, Arianna; Doni, Mirko; Amati, Bruno

    2014-01-01

    Myc-family proteins are key controllers of the metabolic and proliferative status of the cell, and are subjected to a complex network of regulatory events that guarantee their efficient and fast modulation by extracellular stimuli. Hence, unbalances in regulatory mechanisms leading to altered Myc levels or activities are often reported in cancer cells. Here we show that c- and N-Myc are conjugated to SUMO proteins at conserved lysines in their C-terminal domain. No obvious effects of SUMOylation were detected on bulk N-Myc stability or activities, including the regulation of transcription, proliferation or apoptosis. N-Myc SUMOylation could be induced by cellular stresses, such as heat shock and proteasome inhibition, and in all instances concerned a small fraction of the N-Myc protein. We surmise that, as shown for other substrates, SUMOylation may be part of a quality-control mechanism acting on misfolded Myc proteins. PMID:24608896

  1. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  2. Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome.

    PubMed

    Hadizadeh, Nastaran; Johnson, Reid C; Marko, John F

    2016-06-15

    Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (kexch) of ≈10(4) M(-1) s(-1), comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

    PubMed Central

    Hadizadeh, Nastaran; Johnson, Reid C.

    2016-01-01

    ABSTRACT Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or “facilitated dissociation.” Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an “exchange” rate constant (kexch) of ≈104 M−1 s−1, comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo. IMPORTANCE Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function. PMID:27044624

  4. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

    PubMed Central

    Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

    2005-01-01

    Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  5. Survey of bacterial proteins released in cheese: a proteomic approach.

    PubMed

    Gagnaire, Valérie; Piot, Michel; Camier, Bénédicte; Vissers, Johannes P C; Jan, Gwénaël; Léonil, Joëlle

    2004-07-15

    During the ripening of Emmental cheese, the bacterial ecosystem confers its organoleptic characteristics to the evolving curd both by the action of the living cells, and through the release of numerous proteins, including various types of enzymes into the cheese when the cells lyse. In Emmental cheese these proteins can be released from thermophilic lactic acid bacteria used as starters like Lactobacillus helveticus, Lb delbruecki subsp. lactis and Streptococcus salivarius subsp. thermophilus and ripening bacteria such as Propionibacterium freudenreichii. The aim of this study was to obtain a proteomic view of the different groups of proteins within the cheese using proteomic tools to create a reference map. A methodology was therefore developed to reduce the complexity of cheese matrix prior to 2D-PAGE analysis. The aqueous phase of cheese was prefractionated by size exclusion chromatography, bacterial and milk proteins were separated and subsequently characterised by mass spectrometry, prior to peptide mass fingerprint and sequence homology database search. Five functional groups of proteins were identified involved in: (i) proteolysis, (ii) glycolysis, (iii) stress response, (iv) DNA and RNA repair and (v) oxidoreduction. The results revealed stress responses triggered by thermophilic lactic acid bacteria and Propionibacterium strains at the end of ripening. Information was also obtained regarding the origin and nature of the peptidases released into the cheese, thus providing a greater understanding of casein degradation mechanisms during ripening. Different peptidases arose from St thermophilus and Lb helveticus, suggesting that streptococci are involved in peptide degradation in addition to the proteolytic activity of lactobacilli.

  6. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures.

    PubMed

    Slinger, Betty L; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M

    2015-12-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition.

  7. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures

    PubMed Central

    Slinger, Betty L.; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M.

    2015-01-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  8. A versatile nano display platform from bacterial spore coat proteins

    PubMed Central

    Wu, I-Lin; Narayan, Kedar; Castaing, Jean-Philippe; Tian, Fang; Subramaniam, Sriram; Ramamurthi, Kumaran S.

    2015-01-01

    Dormant bacterial spores are encased in a thick protein shell, the ‘coat', which contains ∼70 different proteins. The coat protects the spore from environmental insults, and is among the most durable static structures in biology. Owing to extensive cross-linking among coat proteins, this structure has been recalcitrant to detailed biochemical analysis, so molecular details of how it assembles are largely unknown. Here, we reconstitute the basement layer of the coat atop spherical membranes supported by silica beads to create artificial spore-like particles. We report that these synthetic spore husk-encased lipid bilayers (SSHELs) assemble and polymerize into a static structure, mimicking in vivo basement layer assembly during sporulation in Bacillus subtilis. In addition, we demonstrate that SSHELs may be easily covalently modified with small molecules and proteins. We propose that SSHELs may be versatile display platforms for drugs and vaccines in clinical settings, or for enzymes that neutralize pollutants for environmental remediation. PMID:25854653

  9. The family of LSU-like proteins

    PubMed Central

    Sirko, Agnieszka; Wawrzyńska, Anna; Rodríguez, Milagros Collados; Sęktas, Pawel

    2015-01-01

    The plant response to sulfur deficiency includes extensive metabolic changes which can be monitored at various levels (transcriptome, proteome, metabolome) even before the first visible symptoms of sulfur starvation appear. Four members of the plant-specific LSU (response to Low SUlfur) gene family occur in Arabidopsis thaliana (LSU1-4). Variable numbers of LSU genes occur in other plant species but they were studied only in Arabidopsis and tobacco. Three out of four of the Arabidopsis LSU genes are induced by sulfur deficiency. The LSU-like genes in tobacco were characterized as UP9 (UPregulated by sulfur deficit 9). LSU-like proteins do not have characteristic domains that provide clues to their function. Despite having only moderate primary sequence conservation they share several common features including small size, a coiled–coil secondary structure and short conserved motifs in specific positions. Although the precise function of LSU-like proteins is still unknown there is some evidence that members of the LSU family are involved in plant responses to environmental challenges, such as sulfur deficiency, and possibly in plant immune responses. Various bioinformatic approaches have identified LSU-like proteins as important hubs for integration of signals from environmental stimuli. In this paper we review a variety of published data on LSU gene expression, the properties of lsu mutants and features of LSU-like proteins in the hope of shedding some light on their possible role in plant metabolism. PMID:25628631

  10. The family of LSU-like proteins.

    PubMed

    Sirko, Agnieszka; Wawrzyńska, Anna; Rodríguez, Milagros Collados; Sęktas, Pawel

    2014-01-01

    The plant response to sulfur deficiency includes extensive metabolic changes which can be monitored at various levels (transcriptome, proteome, metabolome) even before the first visible symptoms of sulfur starvation appear. Four members of the plant-specific LSU (response to Low SUlfur) gene family occur in Arabidopsis thaliana (LSU1-4). Variable numbers of LSU genes occur in other plant species but they were studied only in Arabidopsis and tobacco. Three out of four of the Arabidopsis LSU genes are induced by sulfur deficiency. The LSU-like genes in tobacco were characterized as UP9 (UPregulated by sulfur deficit 9). LSU-like proteins do not have characteristic domains that provide clues to their function. Despite having only moderate primary sequence conservation they share several common features including small size, a coiled-coil secondary structure and short conserved motifs in specific positions. Although the precise function of LSU-like proteins is still unknown there is some evidence that members of the LSU family are involved in plant responses to environmental challenges, such as sulfur deficiency, and possibly in plant immune responses. Various bioinformatic approaches have identified LSU-like proteins as important hubs for integration of signals from environmental stimuli. In this paper we review a variety of published data on LSU gene expression, the properties of lsu mutants and features of LSU-like proteins in the hope of shedding some light on their possible role in plant metabolism.

  11. Targeting functional motifs of a protein family.

    PubMed

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β-lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β-lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β-lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  12. Targeting functional motifs of a protein family

    NASA Astrophysics Data System (ADS)

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  13. A remorin protein interacts with symbiotic receptors and regulates bacterial infection.

    PubMed

    Lefebvre, Benoit; Timmers, Ton; Mbengue, Malick; Moreau, Sandra; Hervé, Christine; Tóth, Katalin; Bittencourt-Silvestre, Joana; Klaus, Dörte; Deslandes, Laurent; Godiard, Laurence; Murray, Jeremy D; Udvardi, Michael K; Raffaele, Sylvain; Mongrand, Sebastien; Cullimore, Julie; Gamas, Pascal; Niebel, Andreas; Ott, Thomas

    2010-02-02

    Remorin proteins have been hypothesized to play important roles during cellular signal transduction processes. Induction of some members of this multigene family has been reported during biotic interactions. However, no roles during host-bacteria interactions have been assigned to remorin proteins until now. We used root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti to study the roles of a remorin that is specifically induced during nodulation. Here we show that this oligomeric remorin protein attaches to the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm. It interacts with the core set of symbiotic receptors that are essential for perception of bacterial signaling molecules, and thus might represent a plant-specific scaffolding protein.

  14. Protein–protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins

    PubMed Central

    Kleanthous, Colin; Rassam, Patrice; Baumann, Christoph G

    2015-01-01

    It has until recently been unclear whether outer membrane proteins (OMPs) of Gram-negative bacteria are organized or distributed randomly. Studies now suggest promiscuous protein–protein interactions (PPIs) between β-barrel OMPs in Escherichia coli govern their local and global dynamics, engender spatiotemporal patterning of the outer membrane into micro-domains and are the basis of β-barrel protein turnover. We contextualize these latest advances, speculate on areas of bacterial cell biology that might be influenced by the organization of OMPs into supramolecular assemblies, and highlight the new questions and controversies this revised view of the bacterial outer membrane raises. PMID:26629934

  15. A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

    PubMed Central

    Rao, Lang; Xue, Yanfen; Zheng, Yingying; Lu, Jian R.; Ma, Yanhe

    2013-01-01

    Background Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. Methodology/Principal Findings Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. Conclusion EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these

  16. Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

    PubMed Central

    2012-01-01

    A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics. PMID:22686540

  17. N-linked protein glycosylation in a bacterial system.

    PubMed

    Nothaft, Harald; Liu, Xin; McNally, David J; Szymanski, Christine M

    2010-01-01

    N-Linked protein glycosylation is conserved throughout the three domains of life and influences protein function, stability, and protein complex formation. N-Linked glycosylation is an essential process in Eukaryotes; however, although N-glycosylation affects multiple cellular processes in Archaea and Bacteria, it is not needed for cell survival. Methods for the analyses of N-glycosylation in eukaryotes are well established, but comparable techniques for the analyses of the pathways in Bacteria and Archaea are needed. In this chapter we describe new methods for the detection and analyses of N-linked, and the recently discovered free oligosaccharides (fOS), from whole cell lysates of Campylobacter jejuni using non-specific pronase E digestion and permethylation followed by mass spectrometry. We also describe the expression and immunodetection of the model N-glycoprotein, AcrA, fused to a hexa-histidine tag to follow protein glycosylation in C. jejuni. This chapter concludes with the recent demonstration that high-resolution magic angle spinning NMR of intact bacterial cells provides a rapid, non-invasive method for analyzing fOS in C. jejuni in vivo. This combination of techniques provides a powerful tool for the exploration, quantification, and structural analyses of N-linked and free oligosaccharides in the bacterial system.

  18. Correlated rigid modes in protein families

    NASA Astrophysics Data System (ADS)

    Striegel, D. A.; Wojtowicz, D.; Przytycka, T. M.; Periwal, V.

    2016-04-01

    A great deal of evolutionarily conserved information is contained in genomes and proteins. Enormous effort has been put into understanding protein structure and developing computational tools for protein folding, and many sophisticated approaches take structure and sequence homology into account. Several groups have applied statistical physics approaches to extracting information about proteins from sequences alone. Here, we develop a new method for sequence analysis based on first principles, in information theory, in statistical physics and in Bayesian analysis. We provide a complete derivation of our approach and we apply it to a variety of systems, to demonstrate its utility and its limitations. We show in some examples that phylogenetic alignments of amino-acid sequences of families of proteins imply the existence of a small number of modes that appear to be associated with correlated global variation. These modes are uncovered efficiently in our approach by computing a non-perturbative effective potential directly from the alignment. We show that this effective potential approaches a limiting form inversely with the logarithm of the number of sequences. Mapping symbol entropy flows along modes to underlying physical structures shows that these modes arise due to correlated compensatory adjustments. In the protein examples, these occur around functional binding pockets.

  19. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    PubMed Central

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  20. Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

    PubMed Central

    Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

    2011-01-01

    In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

  1. Affinity binding of proteins to the modified bacterial cellulose nanofibers.

    PubMed

    Bakhshpour, Monireh; Tamahkar, Emel; Andaç, Müge; Denizli, Adil

    2017-05-01

    The potential of the modified bacterial cellulose (BC) nanofibers was determined bearing metal ion coordination interactions to enhance the protein adsorption and binding capacity. Thus, a household synthesized metal chelating monomer, namely N-methacryloyl-l-histidine methylester (MAH), and a commercial metal chelating monomer, namely 4-vinylimidazole (VIm), were used to complex with metal ions Cu(II) and Ni(II) respectively for the synthesis of the modified BC nanofibers. The modified nanofibers were characterized by FT-IR, SEM and EDX measurements. The protein adsorption tests were carried out using hemoglobin as a model protein and it was determined that the maximum adsorption capacity of hemoglobin onto the modified BC nanofibers was found as 47.40mg/g. The novel strategy for the preparation of metal chelated nanofibers was developed. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Physiological Functions of APP Family Proteins

    PubMed Central

    Müller, Ulrike C.; Zheng, Hui

    2012-01-01

    Biochemical and genetic evidence establishes a central role of the amyloid precursor protein (APP) in Alzheimer disease (AD) pathogenesis. Biochemically, deposition of the β-amyloid (Aβ) peptides produced from proteolytic processing of APP forms the defining pathological hallmark of AD; genetically, both point mutations and duplications of wild-type APP are linked to a subset of early onset of familial AD (FAD) and cerebral amyloid angiopathy. As such, the biological functions of APP and its processing products have been the subject of intense investigation, and the past 20+ years of research have met with both excitement and challenges. This article will review the current understanding of the physiological functions of APP in the context of APP family members. PMID:22355794

  3. Assembly of Fe/S proteins in bacterial systems: Biochemistry of the bacterial ISC system.

    PubMed

    Blanc, B; Gerez, C; Ollagnier de Choudens, S

    2015-06-01

    Iron/sulfur clusters are key cofactors in proteins involved in a large number of conserved cellular processes, including gene expression, DNA replication and repair, ribosome biogenesis, tRNA modification, central metabolism and respiration. Fe/S proteins can perform a wide range of functions, from electron transfer to redox and non-redox catalysis. In all living organisms, Fe/S proteins are first synthesized in an apo-form. However, as the Fe/S prosthetic group is required for correct folding and/or protein stability, Fe/S clusters are inserted co-translationally or immediately after translation by specific assembly machineries. These systems have been extensively studied over the last decade, both in prokaryotes and eukaryotes. The present review covers the basic principles of the bacterial housekeeping Fe/S biogenesis ISC system, and related recent molecular advances. Some of the most exciting recent highlights relating to this system include structural and functional characterization of binary and ternary complexes involved in Fe/S cluster formation on the scaffold protein IscU. These advances enhance our understanding of the Fe/S cluster assembly mechanism by revealing essential interactions that could never be determined with isolated proteins and likely are closer to an in vivo situation. Much less is currently known about the molecular mechanism of the Fe/S transfer step, but a brief account of the protein-protein interactions involved is given. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  4. Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection

    PubMed Central

    Derebe, Mehabaw G; Zlatkov, Clare M; Gattu, Sureka; Ruhn, Kelly A; Vaishnava, Shipra; Diehl, Gretchen E; MacMillan, John B; Williams, Noelle S; Hooper, Lora V

    2014-01-01

    Retinol plays a vital role in the immune response to infection, yet proteins that mediate retinol transport during infection have not been identified. Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization, but their exact functions remain unclear. Here we show that mouse and human SAAs are retinol binding proteins. Mouse and human SAAs bound retinol with nanomolar affinity, were associated with retinol in vivo, and limited the bacterial burden in tissues after acute infection. We determined the crystal structure of mouse SAA3 at a resolution of 2 Å, finding that it forms a tetramer with a hydrophobic binding pocket that can accommodate retinol. Our results thus identify SAAs as a family of microbe-inducible retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and suggest how retinol is circulated during infection. DOI: http://dx.doi.org/10.7554/eLife.03206.001 PMID:25073702

  5. Protein export through the bacterial flagellar type III export pathway.

    PubMed

    Minamino, Tohru

    2014-08-01

    For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  6. Sequence analysis of the AAA protein family.

    PubMed Central

    Beyer, A.

    1997-01-01

    The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases. PMID:9336829

  7. A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

    PubMed Central

    Yan, Michael B.; Tran, Minh; Wright, Nathan; Luzader, Deborah H.; Kendall, Melissa M.; Ruiz‐Perez, Fernando; Nataro, James P.

    2016-01-01

    Summary AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners. PMID:27038276

  8. A large family of anti-activators accompanying XylS/AraC family regulatory proteins.

    PubMed

    Santiago, Araceli E; Yan, Michael B; Tran, Minh; Wright, Nathan; Luzader, Deborah H; Kendall, Melissa M; Ruiz-Perez, Fernando; Nataro, James P

    2016-07-01

    AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

  9. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells.

    PubMed

    Popa, Crina M; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding.

  10. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  11. Rho-modifying bacterial protein toxins from Photorhabdus species.

    PubMed

    Jank, Thomas; Lang, Alexander E; Aktories, Klaus

    2016-06-15

    Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by N-acetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Fuel of the Bacterial Flagellar Type III Protein Export Apparatus.

    PubMed

    Minamino, Tohru; Kinoshita, Miki; Namba, Keiichi

    2017-01-01

    The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.

  13. Functional neighbors: inferring relationships between nonhomologous protein families using family-specific packing motifs.

    PubMed

    Bandyopadhyay, Deepak; Huan, Jun; Liu, Jinze; Prins, Jan; Snoeyink, Jack; Wang, Wei; Tropsha, Alexander

    2010-09-01

    We describe a new approach for inferring the functional relationships between nonhomologous protein families by looking at statistical enrichment of alternative function predictions in classification hierarchies such as Gene Ontology (GO) and Structural Classification of Proteins (SCOP). Protein structures are represented by robust graph representations, and the fast frequent subgraph mining algorithm is applied to protein families to generate sets of family-specific packing motifs, i.e., amino acid residue-packing patterns shared by most family members but infrequent in other proteins. The function of a protein is inferred by identifying in it motifs characteristic of a known family. We employ these family-specific motifs to elucidate functional relationships between families in the GO and SCOP hierarchies. Specifically, we postulate that two families are functionally related if one family is statistically enriched by motifs characteristic of another family, i.e., if the number of proteins in a family containing a motif from another family is greater than expected by chance. This function-inference method can help annotate proteins of unknown function, establish functional neighbors of existing families, and help specify alternate functions for known proteins.

  14. Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.

    PubMed Central

    Ilan, O; Bloch, Y; Frankel, G; Ullrich, H; Geider, K; Rosenshine, I

    1999-01-01

    In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics. PMID:10369665

  15. Learning generative models for protein fold families.

    PubMed

    Balakrishnan, Sivaraman; Kamisetty, Hetunandan; Carbonell, Jaime G; Lee, Su-In; Langmead, Christopher James

    2011-04-01

    We introduce a new approach to learning statistical models from multiple sequence alignments (MSA) of proteins. Our method, called GREMLIN (Generative REgularized ModeLs of proteINs), learns an undirected probabilistic graphical model of the amino acid composition within the MSA. The resulting model encodes both the position-specific conservation statistics and the correlated mutation statistics between sequential and long-range pairs of residues. Existing techniques for learning graphical models from MSA either make strong, and often inappropriate assumptions about the conditional independencies within the MSA (e.g., Hidden Markov Models), or else use suboptimal algorithms to learn the parameters of the model. In contrast, GREMLIN makes no a priori assumptions about the conditional independencies within the MSA. We formulate and solve a convex optimization problem, thus guaranteeing that we find a globally optimal model at convergence. The resulting model is also generative, allowing for the design of new protein sequences that have the same statistical properties as those in the MSA. We perform a detailed analysis of covariation statistics on the extensively studied WW and PDZ domains and show that our method out-performs an existing algorithm for learning undirected probabilistic graphical models from MSA. We then apply our approach to 71 additional families from the PFAM database and demonstrate that the resulting models significantly out-perform Hidden Markov Models in terms of predictive accuracy.

  16. A knot in the protein structure: probing the near-infrared fluorescent protein iRFP designed from bacterial phytochrome

    PubMed Central

    Stepanenko, Olesya V.; Bublikov, Gregory S.; Stepanenko, Olga V.; Shcherbakova, Daria M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.; Kuznetsova, Irina M.

    2014-01-01

    Substantial interest to a family of the bacterial phytochrome photoreceptors (BphPs) is caused by development of near-infrared fluorescent proteins and biosensors, molecularly engineered from BphPs. The near-infrared fluorescent proteins have allowed bioimaging of deep tissues and whole organs noninvasively in living animals. BphPs and derived near-infrared fluorescent proteins contain a structural element, called knot, in their polypeptide chains. Formation of knot structures in proteins was denied for a long time. Here, we studied denaturation and renaturation processes of the near-infrared fluorescent probe, iRFP engineered from RpBphP2, which utilizes a heme-derived tetrapyrrole compound biliverdin as a chromophore. iRFP contains a unique figure-of-eight knot. The denaturation and renaturation curves of the iRFP apoform coincided well, suggesting its efficient refolding. However, the iRFP holoform exhibited the irreversible unfolding and aggregation, associated with the bound chromophore. The knot structure in the apoform did not prevent its subsequent binding of biliverdin, resulting in the functional iRFP holoform. We suggest that the irreversibility of protein unfolding is caused by posttranslational protein modifications, such as chromophore binding, rather than the presence of the knot. These results are important for future design of BphP-based near-infrared probes and add important features to the fundamental problem of protein folding. PMID:24628916

  17. Bacterial flagellar capping proteins adopt diverse oligomeric states

    PubMed Central

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H; Beckett, Dorothy; Wintrode, Patrick L; Sundberg, Eric J

    2016-01-01

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies. DOI: http://dx.doi.org/10.7554/eLife.18857.001 PMID:27664419

  18. Bacterial flagellar capping proteins adopt diverse oligomeric states.

    PubMed

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H; Beckett, Dorothy; Wintrode, Patrick L; Sundberg, Eric J

    2016-09-24

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.

  19. Bacterial flagellar capping proteins adopt diverse oligomeric states

    SciTech Connect

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A.; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H.; Beckett, Dorothy; Wintrode, Patrick L.; Sundberg, Eric J.

    2016-09-24

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD fromPseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find thatPseudomonasFliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.

  20. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  1. Linkage, mobility, and selfishness in the MazF family of bacterial toxins: a snapshot of bacterial evolution.

    PubMed

    Chopra, Nikita; Saumitra; Pathak, Abhinandan; Bhatnagar, Rakesh; Bhatnagar, Sonika

    2013-01-01

    Prokaryotic MazF family toxins cooccur with cognate antitoxins having divergent DNA-binding folds and can be of chromosomal or plasmid origin. Sequence similarity search was carried out to identify the Toxin-Antitoxin (TA) operons of MazF family followed by sequence analysis and phylogenetic studies. The genomic DNA upstream of the TA operons was searched for the presence of regulatory motifs. The MazF family toxins showed a conserved hydrophobic pocket in a multibinding site and are present in pathogenic bacteria. The toxins of the MazF family are associated with four main types of cognate antitoxin partners and cluster as a subfamily on the branches of the phylogenetic tree. This indicates that transmission of the entire operon is the dominant mode of inheritance. The plasmid borne TA modules were interspersed between the chromosomal TA modules of the same subfamily, compatible with a frequent interchange of TA genes between the chromosome and the plasmid akin to that observed for antibiotic resistance gens. The split network of the MazF family toxins showed the AbrB-linked toxins as a hub of horizontal gene transfer. Distinct motifs are present in the upstream region of each subfamily. The presence of MazF family TA modules in pathogenic bacteria and identification of a conserved binding pocket are significant for the development of novel antibacterials to disrupt the TA interaction. However, the role of TAs in stress resistance needs to be established. Phylogenetic studies provide insight into the evolution of MazF family TAs and effect on the bacterial genome.

  2. Two novel human members of an emerging mammalian gene family related to mono-ADP-ribosylating bacterial toxins

    SciTech Connect

    Koch-Nolte, F.; Haag, F.; Braren, R.

    1997-02-01

    Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designated ART3 and ART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32-41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and by in situ hybridization, we have mapped the two genes to human chromosomes 4p14-p15.l and 12q13.2- q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the {open_quotes}tip of an iceberg,{close_quote} i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation. 35 refs., 4 figs.

  3. Identification of protein secretion systems in bacterial genomes.

    PubMed

    Abby, Sophie S; Cury, Jean; Guglielmini, Julien; Néron, Bertrand; Touchon, Marie; Rocha, Eduardo P C

    2016-03-16

    Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems' components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SS(iii) and T9SS were restricted to Bacteroidetes, and T6SS(ii) to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems.

  4. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts.

    PubMed

    Yong, Ann-Li; Ooh, Keng-Fei; Ong, Hean-Chooi; Chai, Tsun-Thai; Wong, Fai-Chu

    2015-11-01

    In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. UGT-29 protein expression and localization during bacterial infection in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Wong, Rui-Rui; Lee, Song-Hua; Nathan, Sheila

    2014-09-01

    The nematode Caenorhabditis elegans is routinely used as an animal model to delineate complex molecular mechanisms involved in the host response to pathogen infection. Following up on an earlier study on host-pathogen interaction, we constructed a ugt-29::GFP transcriptional fusion transgenic worm strain to examine UGT-29 protein expression and localization upon bacterial infection. UGT-29 orthologs can be found in higher organisms including humans and is proposed as a member of the UDP-Glucoronosyl Transferase family of proteins which are involved in phase II detoxification of compounds detrimental to the host organism. Under uninfected conditions, UGT-29::GFP fusion protein was highly expressed in the C. elegans anterior pharynx and intestine, two major organs involved in detoxification. We further evaluated the localization of the enzyme in worms infected with the bacterial pathogen, Burkholderia pseudomallei. The infected ugt-29::GFP transgenic strain exhibited increased fluorescence in the pharynx and intestine with pronounced fluorescence also extending to body wall muscle. This transcriptional fusion GFP transgenic worm is a convenient and direct tool to provide information on UGT detoxification enzyme gene expression and could be a useful tool for a number of diverse applications.

  6. Graded Structural Polymorphism in a Bacterial Thermosensor Protein.

    PubMed

    Narayan, Abhishek; Campos, Luis A; Bhatia, Sandhya; Fushman, David; Naganathan, Athi N

    2017-01-18

    Thermosensing is critical for the expression of virulence genes in pathogenic bacteria that infect warm-blooded hosts. Proteins of the Hha-family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of a large number of genes upon temperature shifts. However, there is little mechanistic evidence at the molecular level as to how changes in temperature are transduced into structural changes and hence the functional outcome. In this study, we delineate the conformational behavior of Cnu, a putative molecular thermosensor, employing diverse spectroscopic, calorimetric and hydrodynamic measurements. We find that Cnu displays probe-dependent unfolding in equilibrium, graded increase in structural fluctuations and temperature-dependent swelling of the dimensions of its native ensemble within the physiological range of temperatures, features that are indicative of a highly malleable native ensemble. Site-specific fluorescence and NMR experiments in combination with multiple computational approaches-statistical mechanical model, coarse-grained and all-atom MD simulations-reveal that the fourth helix of Cnu acts as a unique thermosensing module displaying varying degrees of order and orientation in response to temperature modulations while undergoing a continuous unfolding transition. Our combined experimental-computational study unravels the folding-functional landscape of a natural thermosensor protein, the molecular origins of its unfolding complexity, highlights the role of functional constraints in determining folding-mechanistic behaviors, and the design principles orchestrating the signal transduction roles of the Hha protein family.

  7. Structural and functional insight into the universal stress protein family

    PubMed Central

    Tkaczuk, Karolina L; A Shumilin, Igor; Chruszcz, Maksymilian; Evdokimova, Elena; Savchenko, Alexei; Minor, Wladek

    2013-01-01

    We present the crystal structures of two universal stress proteins (USP) from Archaeoglobus fulgidus and Nitrosomonas europaea in both apo- and ligand-bound forms. This work is the first complete synthesis of the structural properties of 26 USP available in the Protein Data Bank, over 75% of which were determined by structure genomics centers with no additional information provided. The results of bioinformatic analyses of all available USP structures and their sequence homologs revealed that these two new USP structures share overall structural similarity with structures of USPs previously determined. Clustering and cladogram analyses, however, show how they diverge from other members of the USP superfamily and show greater similarity to USPs from organisms inhabiting extreme environments. We compared them with other archaeal and bacterial USPs and discuss their similarities and differences in context of structure, sequential motifs, and potential function. We also attempted to group all analyzed USPs into families, so that assignment of the potential function to those with no experimental data available would be possible by extrapolation. PMID:23745136

  8. Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection.

    PubMed Central

    Barillas-Mury, C; Han, Y S; Seeley, D; Kafatos, F C

    1999-01-01

    A new insect member of the STAT family of transcription factors (Ag-STAT) has been cloned from the human malaria vector Anopheles gambiae. The domain involved in DNA interaction and the SH2 domain are well conserved. Ag-STAT is most similar to Drosophila D-STAT and to vertebrate STATs 5 and 6, constituting a proposed ancient class A of the STAT family. The mRNA is expressed at all developmental stages, and the protein is present in hemocytes, pericardial cells, midgut, skeletal muscle and fat body cells. There is no evidence of transcriptional activation following bacterial challenge. However, bacterial challenge results in nuclear translocation of Ag-STAT protein in fat body cells and induction of DNA-binding activity that recognizes a STAT target site. In vitro treatment with pervanadate (vanadate and H2O2) translocates Ag-STAT to the nucleus in midgut epithelial cells. This is the first evidence of direct participation of the STAT pathway in immune responses in insects. PMID:10022838

  9. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    SciTech Connect

    Volkman, Brian Finley

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  10. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    PubMed

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-04-16

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well.

  11. Essential bacterial helicases that counteract the toxicity of recombination proteins

    PubMed Central

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-01-01

    PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previously isolated mutations in B.subtilis recF, recL, recO and recR, which belong to the same complementation group, all suppress the lethality of a pcrA mutation. Similarly, recF, recO or recR mutations suppress the lethality of the Escherichia coli rep uvrD double mutant. We conclude that RecFOR proteins are toxic in cells devoid of PcrA in Gram-positive bacteria, or Rep and UvrD in Gram-negative bacteria, and propose that the RecFOR proteins interfere with an essential cellular process, possibly replication, when DExx helicases PcrA, or Rep and UvrD are absent. PMID:12065426

  12. Molybdenum Site Structure of MOSC Family Proteins

    PubMed Central

    2015-01-01

    Mo K-edge X-ray absorption spectroscopy has been used to probe as-isolated structures of the MOSC family proteins pmARC-1 and HMCS-CT. The Mo K-edge near-edge spectrum of HMCS-CT is shifted ∼2.5 eV to lower energy compared to the pmARC-1 spectrum, which indicates that as-isolated HMCS-CT is in a more reduced state than pmARC-1. Extended X-ray absorption fine structure analysis indicates significant structural differences between pmARC-1 and HMCS-CT, with the former being a dioxo site and the latter possessing only a single terminal oxo ligand. The number of terminal oxo donors is consistent with pmARC-1 being in the MoVI oxidation state and HMCS-CT in the MoIV state. These structures are in accord with oxygen-atom-transfer reactivity for pmARC-1 and persulfide bond cleavage chemistry for HMCS-CT. PMID:25166909

  13. HMM Logos for visualization of protein families

    PubMed Central

    Schuster-Böckler, Benjamin; Schultz, Jörg; Rahmann, Sven

    2004-01-01

    Background Profile Hidden Markov Models (pHMMs) are a widely used tool for protein family research. Up to now, however, there exists no method to visualize all of their central aspects graphically in an intuitively understandable way. Results We present a visualization method that incorporates both emission and transition probabilities of the pHMM, thus extending sequence logos introduced by Schneider and Stephens. For each emitting state of the pHMM, we display a stack of letters. The stack height is determined by the deviation of the position's letter emission frequencies from the background frequencies. The stack width visualizes both the probability of reaching the state (the hitting probability) and the expected number of letters the state emits during a pass through the model (the state's expected contribution). A web interface offering online creation of HMM Logos and the corresponding source code can be found at the Logos web server of the Max Planck Institute for Molecular Genetics . Conclusions We demonstrate that HMM Logos can be a useful tool for the biologist: We use them to highlight differences between two homologous subfamilies of GTPases, Rab and Ras, and we show that they are able to indicate structural elements of Ras. PMID:14736340

  14. The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

    PubMed Central

    Das, Debanu; Finn, Robert D; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

    2010-01-01

    Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam. PMID:20836087

  15. Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

    PubMed Central

    Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

    2015-01-01

    Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

  16. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis.

    PubMed

    Laddomada, Federica; Miyachiro, Mayara M; Dessen, Andréa

    2016-04-28

    The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the "divisome") and/or cell wall elongation (the "elongasome"), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies.

  17. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

    PubMed Central

    Laddomada, Federica; Miyachiro, Mayara M.; Dessen, Andréa

    2016-01-01

    The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the “divisome”) and/or cell wall elongation (the “elongasome”), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies. PMID:27136593

  18. Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria.

    PubMed

    Nguyen, Hien-Anh; El Khoury, Takla; Guiral, Sébastien; Laaberki, Maria-Halima; Candusso, Marie-Pierre; Galisson, Frédéric; Foucher, Anne-Emmanuelle; Kesraoui, Salsabil; Ballut, Lionel; Vallet, Sylvain; Orelle, Cédric; Zucchini, Laure; Martin, Juliette; Page, Adeline; Attieh, Jihad; Aghajari, Nushin; Grangeasse, Christophe; Jault, Jean-Michel

    2017-10-13

    Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0Å) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a ∆ydiB mutant was more sensitive to paraquat treatment than the wild type, with ~13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Dock protein family in brain development and neurological disease.

    PubMed

    Shi, Lei

    2013-11-01

    The family of dedicator of cytokinesis (Dock), a protein family that belongs to the atypical Rho guanine nucleotide exchange factors (GEFs) for Rac and/or Cdc42 GTPases, plays pivotal roles in various processes of brain development. To date, 11 members of Docks have been identified in the mammalian system. Emerging evidence has suggested that members of the Dock family are associated with several neurodegenerative and neuropsychiatric diseases, including Alzheimer disease and autism spectrum disorders. This review summarizes recent advances on the understanding of the roles of the Dock protein family in normal and diseased processes in the nervous system. Furthermore, interacting proteins and the molecular regulation of Docks are discussed.

  20. Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses

    PubMed Central

    Venclovas, Česlovas

    2012-01-01

    Motivation: Eukaryote-infecting nucleo-cytoplasmic large DNA viruses (NCLDVs) feature some of the largest genomes in the viral world. These viruses typically do not strongly depend on the host DNA replication systems. In line with this observation, a number of essential DNA replication proteins, such as DNA polymerases, primases, helicases and ligases, have been identified in the NCLDVs. One other ubiquitous component of DNA replisomes is the single-stranded DNA-binding (SSB) protein. Intriguingly, no NCLDV homologs of canonical OB-fold-containing SSB proteins had previously been detected. Only in poxviruses, one of seven NCLDV families, I3 was identified as the SSB protein. However, whether I3 is related to any known protein structure has not yet been established. Results: Here, we addressed the case of ‘missing’ canonical SSB proteins in the NCLDVs and also probed evolutionary origins of the I3 family. Using advanced computational methods, in four NCLDV families, we detected homologs of the bacteriophage T7 SSB protein (gp2.5). We found the properties of these homologs to be consistent with the SSB function. Moreover, we implicated specific residues in single-stranded DNA binding. At the same time, we found no evolutionary link between the T7 gp2.5-like NCLDV SSB homologs and the poxviral SSB protein (I3). Instead, we identified a distant relationship between I3 and small protein B (SmpB), a bacterial RNA-binding protein. Thus, apparently, the NCLDVs have the two major distinct sets of SSB proteins having bacteriophage and bacterial origins, respectively. Contact: venclovas@ibt.lt Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23097418

  1. Small Alarmone Synthetases as novel bacterial RNA-binding proteins.

    PubMed

    Hauryliuk, Vasili; Atkinson, Gemma C

    2017-08-18

    The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017). RelQ's activities as an enzyme and as a RNA-binding protein are mutually incompatible: binding of single-stranded RNA potently inhibits (p)ppGpp synthesis in a sequence-specific manner, and RelQ's enzymatic activity destabilizes the RNA:RelQ complex. RelQ's allosteric regulator, pppGpp, destabilizes RNA binding and activates RelQ's enzymatic activity. Since SAS enzymes are widely distributed in bacteria, and, as it has been discovered recently, are also mobilized by phages (Dedrick et al. 2017), RNA binding to SAS is could be a wide-spread mechanism. The initial discovery raises numerous questions regarding RNA-binding function of the SAS enzymes: What is the molecular mechanism underlying the incompatibility of RNA:SAS complex formation with pppGpp binding and (p)ppGpp synthesis? What are the RNA targets in living cells? What is the regulatory output of the system - (p)ppGpp synthesis, modulation of RNA structure and function, or both?

  2. A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

    PubMed Central

    Schmid, Michael C; Scheidegger, Florine; Dehio, Michaela; Balmelle-Devaux, Nadège; Schulein, Ralf; Guye, Patrick; Chennakesava, Cuddapah S; Biedermann, Barbara; Dehio, Christoph

    2006-01-01

    The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium. PMID:17121462

  3. Keeping it in the family: Coevolution of latrunculid sponges and their dominant bacterial symbionts.

    PubMed

    Matcher, Gwynneth F; Waterworth, Samantha C; Walmsley, Tara A; Matsatsa, Tendayi; Parker-Nance, Shirley; Davies-Coleman, Michael T; Dorrington, Rosemary A

    2017-04-01

    The Latrunculiidae are a family of cold water sponges known for their production of bioactive pyrroloiminoquinone alkaloids. Previously it was shown that the bacterial community associated with a Tsitsikamma sponge species comprises unusual bacterial taxa and is dominated by a novel Betaproteobacterium. Here, we have characterized the bacterial communities associated with six latrunculid species representing three genera (Tsitsikamma, Cyclacanthia, and Latrunculia) as well as a Mycale species, collected from Algoa Bay on the South African southeast coast. The bacterial communities of all seven sponge species were dominated by a single Betaproteobacterium operational taxonomic unit (OTU0.03 ), while a second OTU0.03 was dominant in the Mycale sp. The Betaproteobacteria OTUs from the different latrunculid sponges are closely related and their phylogenetic relationship follows that of their hosts. We propose that the latrunculid Betaproteobacteria OTUs are members of a specialized group of sponge symbionts that may have coevolved with their hosts. A single dominant Spirochaetae OTU0.03 was present in the Tsitsikamma and Cyclacanthia sponge species, but absent from the Latrunculia and Mycale sponges. This study sheds new light on the interactions between latrunculid sponges and their bacterial communities and may point to the potential involvement of dominant symbionts in the biosynthesis of the bioactive secondary metabolites. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  4. The MetaFam Server: a comprehensive protein family resource.

    PubMed

    Silverstein, K A; Shoop, E; Johnson, J E; Kilian, A; Freeman, J L; Kunau, T M; Awad, I A; Mayer, M; Retzel, E F

    2001-01-01

    MetaFam is a comprehensive relational database of protein family information. This web-accessible resource integrates data from several primary sequence and secondary protein family databases. By pooling together the information from these disparate sources, MetaFam is able to provide the most complete protein family sets available. Users are able to explore the interrelationships among these primary and secondary databases using a powerful graphical visualization tool, MetaFamView. Additionally, users can identify corresponding sequence entries among the sequence databases, obtain a quick summary of corresponding families (and their sequence members) among the family databases, and even attempt to classify their own unassigned sequences. Hypertext links to the appropriate source databases are provided at every level of navigation. Global family database statistics and information are also provided. Public access to the data is available at http://metafam.ahc.umn.edu/.

  5. Structural relationship between a bacterial developmental protein and eukaryotic PP2C protein phosphatases.

    PubMed

    Adler, E; Donella-Deana, A; Arigoni, F; Pinna, L A; Stragler, P

    1997-01-01

    Bacillus subtilis SpoIIE is a Ser protein phosphatase whose action on the phosphoprotein SpoIIAA triggers the cell type-specific activation of a sporulation transcription factor. Here we report that SpoIIE displays sequence similarity to the PP2C family of eukaryotic Ser/Thr protein phosphatases, and that residues common to these proteins are required for the function of both SpoIIE and TPD1, a yeast PP2C. These findings suggest that SpoIIE and the PP2C protein phosphatases are structurally related, and reveal a striking formal similarity between the SpoIIAA regulatory circuit and that of mammalian mitochondrial pyruvate dehydrogenase. This similarity may reflect an evolutionarily conserved mechanism of biological regulation based on the interplay of His protein kinase-like Ser kinases and PP2C-like protein phosphatases.

  6. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  7. Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins*

    PubMed Central

    Yi, Chae-ryun; Allen, John E.; Russo, Brian; Lee, Soo Young; Heindl, Jason E.; Baxt, Leigh A.; Herrera, Bobby Brooke; Kahoud, Emily; MacBeath, Gavin; Goldberg, Marcia B.

    2014-01-01

    Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain. PMID:25124035

  8. Involvement of PCH family proteins in cytokinesis and actin distribution.

    PubMed

    Lippincott, J; Li, R

    2000-04-15

    Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

  9. RTX proteins: a highly diverse family secreted by a common mechanism

    PubMed Central

    Linhartová, Irena; Bumba, Ladislav; Mašín, Jiří; Basler, Marek; Osička, Radim; Kamanová, Jana; Procházková, Kateřina; Adkins, Irena; Hejnová-Holubová, Jana; Sadílková, Lenka; Morová, Jana; Šebo, Peter

    2010-01-01

    Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest. PMID:20528947

  10. Yeast Mitochondria as a Model System to Study the Biogenesis of Bacterial β-Barrel Proteins.

    PubMed

    Ulrich, Thomas; Oberhettinger, Philipp; Autenrieth, Ingo B; Rapaport, Doron

    2015-01-01

    Beta-barrel proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The evolutionary conservation in the biogenesis of these proteins allows mitochondria to assemble bacterial β-barrel proteins in their functional form. In this chapter, we describe exemplarily how the capacity of yeast mitochondria to process the trimeric autotransporter YadA can be used to study the role of bacterial periplasmic chaperones in this process.

  11. Genomic analysis of membrane protein families: abundance and conserved motifs

    PubMed Central

    Liu, Yang; Engelman, Donald M; Gerstein, Mark

    2002-01-01

    Background Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. Results Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. Conclusions We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID:12372142

  12. An Evolutionary Strategy for All-Atom Folding of the 60-Amino-Acid Bacterial Ribosomal Protein L20

    PubMed Central

    Schug, A.; Wenzel, W.

    2006-01-01

    We have investigated an evolutionary algorithm for de novo all-atom folding of the bacterial ribosomal protein L20. We report results of two simulations that converge to near-native conformations of this 60-amino-acid, four-helix protein. We observe a steady increase of “native content” in both simulated ensembles and a large number of near-native conformations in their final populations. We argue that these structures represent a significant fraction of the low-energy metastable conformations, which characterize the folding funnel of this protein. These data validate our all-atom free-energy force field PFF01 for tertiary structure prediction of a previously inaccessible structural family of proteins. We also compare folding simulations of the evolutionary algorithm with the basin-hopping technique for the Trp-cage protein. We find that the evolutionary algorithm generates a dynamic memory in the simulated population, which leads to faster overall convergence. PMID:16565067

  13. The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

    PubMed

    Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

    2016-12-01

    Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    PubMed

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Interactome of E. piscicida and grouper liver proteins reveals strategies of bacterial infection and host immune response

    PubMed Central

    Li, Hui; Zhu, Qing-feng; Peng, Xuan-xian; Peng, Bo

    2017-01-01

    The occurrence of infectious diseases is related to heterogeneous protein interactions between a host and a microbe. Therefore, elucidating the host-pathogen interplay is essential. We previously revealed the protein interactome between Edwardsiella piscicida and fish gill cells, and the present study identified the protein interactome between E. piscicida and E. drummondhayi liver cells. E. drummondhayi liver cells and bacterial pull-down approaches were used to identify E. piscicida outer membrane proteins that bind to liver cells and fish liver cell proteins that interact with bacterial cells, respectively. Eight bacterial proteins and 11 fish proteins were characterized. Heterogeneous protein-protein interactions between these bacterial cells and fish liver cells were investigated through far-Western blotting and co-immunoprecipitation. A network was constructed based on 42 heterogeneous protein-protein interactions between seven bacterial proteins and 10 fish proteins. A comparison of the new interactome with the previously reported interactome showed that four bacterial proteins overlapped, whereas all of the identified fish proteins were new, suggesting a difference between bacterial tricks for evading host immunity and the host strategy for combating bacterial infection. Furthermore, these bacterial proteins were found to regulate the expression of host innate immune-related proteins. These findings indicate that the interactome contributes to bacterial infection and host immunity. PMID:28045121

  16. Mu-8: visualizing differences between proteins and their families

    PubMed Central

    2014-01-01

    Background A complete understanding of the relationship between the amino acid sequence and resulting protein function remains an open problem in the biophysical sciences. Current approaches often rely on diagnosing functionally relevant mutations by determining whether an amino acid frequently occurs at a specific position within the protein family. However, these methods do not account for the biophysical properties and the 3D structure of the protein. We have developed an interactive visualization technique, Mu-8, that provides researchers with a holistic view of the differences of a selected protein with respect to a family of homologous proteins. Mu-8 helps to identify areas of the protein that exhibit: (1) significantly different bio-chemical characteristics, (2) relative conservation in the family, and (3) proximity to other regions that have suspect behavior in the folded protein. Methods Our approach quantifies and communicates the difference between a reference protein and its family based on amino acid indices or principal components of amino acid index classes, while accounting for conservation, proximity amongst residues, and overall 3D structure. Results We demonstrate Mu-8 in a case study with data provided by the 2013 BioVis contest. When comparing the sequence of a dysfunctional protein to its functional family, Mu-8 reveals several candidate regions that may cause function to break down. PMID:25237392

  17. Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family

    PubMed Central

    Heinz, Eva; Stubenrauch, Christopher J.; Grinter, Rhys; Croft, Nathan P.; Purcell, Anthony W.; Strugnell, Richard A.; Dougan, Gordon; Lithgow, Trevor

    2016-01-01

    The bacterial cell surface proteins intimin and invasin are virulence factors that share a common domain structure and bind selectively to host cell receptors in the course of bacterial pathogenesis. The β-barrel domains of intimin and invasin show significant sequence and structural similarities. Conversely, a variety of proteins with sometimes limited sequence similarity have also been annotated as “intimin-like” and “invasin” in genome datasets, while other recent work on apparently unrelated virulence-associated proteins ultimately revealed similarities to intimin and invasin. Here we characterize the sequence and structural relationships across this complex protein family. Surprisingly, intimins and invasins represent a very small minority of the sequence diversity in what has been previously the “intimin/invasin protein family”. Analysis of the assembly pathway for expression of the classic intimin, EaeA, and a characteristic example of the most prevalent members of the group, FdeC, revealed a dependence on the translocation and assembly module as a common feature for both these proteins. While the majority of the sequences in the grouping are most similar to FdeC, a further and widespread group is two-partner secretion systems that use the β-barrel domain as the delivery device for secretion of a variety of virulence factors. This comprehensive analysis supports the adoption of the “inverse autotransporter protein family” as the most accurate nomenclature for the family and, in turn, has important consequences for our overall understanding of the Type V secretion systems of bacterial pathogens. PMID:27190006

  18. DUF538 protein super family is predicted to be the potential homologue of bactericidal/permeability-increasing protein in plant system.

    PubMed

    Gholizadeh, Ashraf; Kohnehrouz, Samira Baghban

    2013-03-01

    DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.

  19. Histone-Like Proteins of the Dinoflagellate Crypthecodinium cohnii Have Homologies to Bacterial DNA-Binding Proteins

    PubMed Central

    Wong, J. T. Y.; New, D. C.; Wong, J. C. W.; Hung, V. K. L.

    2003-01-01

    The dinoflagellates have very large genomes encoded in permanently condensed and histoneless chromosomes. Sequence alignment identified significant similarity between the dinoflagellate chromosomal histone-like proteins of Crypthecodinium cohnii (HCCs) and the bacterial DNA-binding and the eukaryotic histone H1 proteins. Phylogenetic analysis also supports the origin of the HCCs from histone-like proteins of bacteria. PMID:12796310

  20. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  1. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  2. Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration.

    PubMed

    Simon, Jörg; Kern, Melanie

    2008-10-01

    Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

  3. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  4. Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

    PubMed Central

    Vita, Nicolas; Landolfi, Gianpiero; Baslé, Arnaud; Platsaki, Semeli; Lee, Jaeick; Waldron, Kevin J.; Dennison, Christopher

    2016-01-01

    Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 1017 M−1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria. PMID:27991525

  5. Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

    NASA Astrophysics Data System (ADS)

    Vita, Nicolas; Landolfi, Gianpiero; Baslé, Arnaud; Platsaki, Semeli; Lee, Jaeick; Waldron, Kevin J.; Dennison, Christopher

    2016-12-01

    Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1-2) × 1017 M-1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.

  6. The KP4 killer protein gene family

    USDA-ARS?s Scientific Manuscript database

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  7. S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

    PubMed

    Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

    2016-11-01

    Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Comparison of the structural basis for thermal stability between archaeal and bacterial proteins.

    PubMed

    Ding, Yanrui; Cai, Yujie; Han, Yonggang; Zhao, Bingqiang

    2012-01-01

    In this study, the structural basis for thermal stability in archaeal and bacterial proteins was investigated. There were many common factors that confer resistance to high temperature in both archaeal and bacterial proteins. These factors include increases in the Lys content, the bends and blanks of secondary structure, the Glu content of salt bridge; decreases in the number of main-side chain hydrogen bond and exposed surface area, and changes in the bends and blanks of amino acids. Certainly, the utilization of charged amino acids to form salt bridges is a primary factor. In both heat-resistant archaeal and bacterial proteins, most Glu and Asp participate in the formation of salt bridges. Other factors may influence either archaeal or bacterial protein thermostability, which includes the more frequent occurrence of shorter 3(10)-helices and increased hydrophobicity in heat-resistant archaeal proteins. However, there were increases in average helix length, the Glu content in salt bridges, temperature factors and decreases in the number of main-side chain hydrogen bonds, uncharged-uncharged hydrogen bonds, hydrophobicity, and buried and exposed polar surface area in heat-resistant bacterial proteins. Evidently, there are few similarities and many disparities between the heat-resistant mechanisms of archaeal and bacterial proteins.

  9. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles

    PubMed Central

    Zhong, Cuncong; Yooseph, Shibu

    2016-01-01

    Analyses of metagenome data (MG) and metatranscriptome data (MT) are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM) framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR) gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE) genes. Finally, HMM-GRASPx was used to

  10. Genome Pool Strategy for Structural Coverage of Protein Families

    PubMed Central

    Jaroszewski, Lukasz; Slabinski, Lukasz; Wooley, John; Deacon, Ashley M.; Lesley, Scott A.; Wilson, Ian. A.; Godzik, Adam

    2010-01-01

    As noticed by generations of structural biologists, closely homologous proteins may have substantially different crystallization properties and propensities. These observations can be used to systematically introduce additional dimensionality into crystallization trials by targeting homologous proteins from multiple genomes in a “genome pool” strategy. Through extensive use of our recently introduced “crystallization feasibility score” (Slabinski et al., 2007a), we can explain that the genome pool strategy works well because the crystallization feasibility scores are surprisingly broad within families of homologous proteins, with most families containing a range of optimal to very difficult targets. We also show that some families can be regarded as relatively “easy”, where a significant number of proteins are predicted to have optimal crystallization features, and others are “very difficult”, where almost none are predicted to result in a crystal structure. Thus, the outcome of such variable distributions of such crystallizability' preferences leads to uneven structural coverage of known families, with “easier” or “optimal” families having several times more solved structures than “very difficult” ones. Nevertheless, this latter category can be successfully targeted by increasing the number of genomes that are used to select targets from a given family. On average, adding 10 new genomes to the “genome pool” provides more promising targets for 7 “very difficult” families. In contrast, our crystallization feasibility score does not indicate that any specific microbial genomes can be readily classified as “easier” or “very difficult” with respect to providing suitable candidates for crystallization and structure determination. Finally, our analyses show that specific physicochemical properties of the protein sequence favor successful outcomes for structure determination and, hence, the group of proteins with known 3D

  11. Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD

    PubMed Central

    Mouratou, Barbara; Schaeffer, Francis; Guilvout, Ingrid; Tello-Manigne, Diana; Pugsley, Anthony P.; Alzari, Pedro M.; Pecorari, Frédéric

    2007-01-01

    We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced. PMID:17984049

  12. TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis.

    PubMed

    Hatakeyama, Shigetsugu

    2017-01-21

    Tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activities, have various functions in cellular processes including intracellular signaling, development, apoptosis, protein quality control, innate immunity, autophagy, and carcinogenesis. The ubiquitin system is one of the systems for post-translational modifications, which play crucial roles not only as markers for degradation of target proteins by the proteasome but also as regulators of protein-protein interactions and of the activation of enzymes. Accumulating evidence has shown that TRIM family proteins have unique, important roles and that their dysregulation causes several diseases classified as cancer, immunological disease, or developmental disorders. In this review we focus on recent emerging topics on TRIM proteins in the regulation of autophagy, innate immunity, and carcinogenesis.

  13. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  14. Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance

    DTIC Science & Technology

    2008-06-06

    Biowarfare potential further jus- tifies the development of countermeasures against this highly virulent class of viruses . Methods Virus , cells, plasmids...BioMed CentralVirology Journal ssOpen AcceResearch Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of...recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for

  15. Dock protein family in brain development and neurological disease

    PubMed Central

    Shi, Lei

    2013-01-01

    The family of dedicator of cytokinesis (Dock), a protein family that belongs to the atypical Rho guanine nucleotide exchange factors (GEFs) for Rac and/or Cdc42 GTPases, plays pivotal roles in various processes of brain development. To date, 11 members of Docks have been identified in the mammalian system. Emerging evidence has suggested that members of the Dock family are associated with several neurodegenerative and neuropsychiatric diseases, including Alzheimer disease and autism spectrum disorders. This review summarizes recent advances on the understanding of the roles of the Dock protein family in normal and diseased processes in the nervous system. Furthermore, interacting proteins and the molecular regulation of Docks are discussed. PMID:24563715

  16. A bacterial ice-binding protein from the Vostok ice core.

    PubMed

    Raymond, James A; Christner, Brent C; Schuster, Stephan C

    2008-09-01

    Bacterial and yeast isolates recovered from a deep Antarctic ice core were screened for proteins with ice-binding activity, an indicator of adaptation to icy environments. A bacterial strain recovered from glacial ice at a depth of 3,519 m, just above the accreted ice from Subglacial Lake Vostok, was found to produce a 54 kDa ice-binding protein (GenBank EU694412) that is similar to ice-binding proteins previously found in sea ice diatoms, a snow mold, and a sea ice bacterium. The protein has the ability to inhibit the recrystallization of ice, a phenotype that has clear advantages for survival in ice.

  17. The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis.

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Kinoshita, Miki; Aldridge, Phillip D; Namba, Keiichi

    2014-12-22

    For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

  18. 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

  19. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

    PubMed Central

    Ribardo, Deborah A.; Brennan, Caitlin A.; Ruby, Edward G.; Jensen, Grant J.; Hendrixson, David R.

    2016-01-01

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. PMID:26976588

  20. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.

    PubMed

    Beeby, Morgan; Ribardo, Deborah A; Brennan, Caitlin A; Ruby, Edward G; Jensen, Grant J; Hendrixson, David R

    2016-03-29

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

  1. Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ.

    PubMed Central

    Osteryoung, K W; Stokes, K D; Rutherford, S M; Percival, A L; Lee, W Y

    1998-01-01

    The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process. PMID:9836740

  2. Unique core genomes of the bacterial family vibrionaceae: insights into niche adaptation and speciation

    PubMed Central

    2012-01-01

    Background The criteria for defining bacterial species and even the concept of bacterial species itself are under debate, and the discussion is apparently intensifying as more genome sequence data is becoming available. However, it is still unclear how the new advances in genomics should be used most efficiently to address this question. In this study we identify genes that are common to any group of genomes in our dataset, to determine whether genes specific to a particular taxon exist and to investigate their potential role in adaptation of bacteria to their specific niche. These genes were named unique core genes. Additionally, we investigate the existence and importance of unique core genes that are found in isolates of phylogenetically non-coherent groups. These groups of isolates, that share a genetic feature without sharing a closest common ancestor, are termed genophyletic groups. Results The bacterial family Vibrionaceae was used as the model, and we compiled and compared genome sequences of 64 different isolates. Using the software orthoMCL we determined clusters of homologous genes among the investigated genome sequences. We used multilocus sequence analysis to build a host phylogeny and mapped the numbers of unique core genes of all distinct groups of isolates onto the tree. The results show that unique core genes are more likely to be found in monophyletic groups of isolates. Genophyletic groups of isolates, in contrast, are less common especially for large groups of isolate. The subsequent annotation of unique core genes that are present in genophyletic groups indicate a high degree of horizontally transferred genes. Finally, the annotation of the unique core genes of Vibrio cholerae revealed genes involved in aerotaxis and biosynthesis of the iron-chelator vibriobactin. Conclusion The presented work indicates that genes specific for any taxon inside the bacterial family Vibrionaceae exist. These unique core genes encode conserved metabolic functions

  3. Expansion of the Protein Repertoire in Newly Explored Environments: Human Gut Microbiome Specific Protein Families

    PubMed Central

    Ellrott, Kyle; Jaroszewski, Lukasz; Li, Weizhong; Wooley, John C.; Godzik, Adam

    2010-01-01

    The microbes that inhabit particular environments must be able to perform molecular functions that provide them with a competitive advantage to thrive in those environments. As most molecular functions are performed by proteins and are conserved between related proteins, we can expect that organisms successful in a given environmental niche would contain protein families that are specific for functions that are important in that environment. For instance, the human gut is rich in polysaccharides from the diet or secreted by the host, and is dominated by Bacteroides, whose genomes contain highly expanded repertoire of protein families involved in carbohydrate metabolism. To identify other protein families that are specific to this environment, we investigated the distribution of protein families in the currently available human gut genomic and metagenomic data. Using an automated procedure, we identified a group of protein families strongly overrepresented in the human gut. These not only include many families described previously but also, interestingly, a large group of previously unrecognized protein families, which suggests that we still have much to discover about this environment. The identification and analysis of these families could provide us with new information about an environment critical to our health and well being. PMID:20532204

  4. Functional domains and motifs of bacterial type III effector proteins and their roles in infection.

    PubMed

    Dean, Paul

    2011-11-01

    A key feature of the virulence of many bacterial pathogens is the ability to deliver effector proteins into eukaryotic cells via a dedicated type three secretion system (T3SS). Many bacterial pathogens, including species of Chlamydia, Xanthomonas, Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia and Yersinia, depend on the T3SS to cause disease. T3SS effectors constitute a large and diverse group of virulence proteins that mimic eukaryotic proteins in structure and function. A salient feature of bacterial effectors is their modular architecture, comprising domains or motifs that confer an array of subversive functions within the eukaryotic cell. These domains/motifs therefore represent a fascinating repertoire of molecular determinants with important roles during infection. This review provides a snapshot of our current understanding of bacterial effector domains and motifs where a defined role in infection has been demonstrated.

  5. [Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

    PubMed

    Minamino, Tohru

    2015-01-01

    The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

  6. IDENTIFICATION OF NICOTINAMIDE MONONUCLEOTIDE DEAMIDASE OF THE BACTERIAL PYRIDINE NUCLEOTIDE CYCLE REVEALS A NOVEL BROADLY CONSERVED AMIDOHYDROLASE FAMILY

    SciTech Connect

    Galeazzi, Luca; Bocci, Paolo; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret F.; Reed, Samantha B.; Osterman, Andrei; Rodionov, Dmitry A.; Sorci, Leonardo; Raffaelli, Nadia

    2011-09-27

    The pyridine nucleotide cycle (PNC) is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial PNC was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in E. coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and non functional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

  7. Identification of Nicotinamide Mononucleotide Deamidase of the Bacterial Pyridine Nucleotide Cycle Reveals a Novel Broadly Conserved Amidohydrolase Family*

    PubMed Central

    Galeazzi, Luca; Bocci, Paola; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret; Reed, Samantha; Osterman, Andrei L.; Rodionov, Dmitry A.; Sorci, Leonardo; Raffaelli, Nadia

    2011-01-01

    The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase. PMID:21953451

  8. Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family.

    PubMed

    Galeazzi, Luca; Bocci, Paola; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret; Reed, Samantha; Osterman, Andrei L; Rodionov, Dmitry A; Sorci, Leonardo; Raffaelli, Nadia

    2011-11-18

    The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

  9. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system.

    PubMed

    Peng, Ying; Wang, Xiangru; Shou, Jin; Zong, Bingbing; Zhang, Yanyan; Tan, Jia; Chen, Jing; Hu, Linlin; Zhu, Yongwei; Chen, Huanchun; Tan, Chen

    2016-05-27

    Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells.

  10. Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

    PubMed

    Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

    2013-11-01

    Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

  11. Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously

    PubMed Central

    Cho, Hongbaek; Wivagg, Carl N.; Kapoor, Mrinal; Barry, Zachary; Rohs, Patricia D.A.; Suh, Hyunsuk; Marto, Jarrod A.; Garner, Ethan C.; Bernhardt, Thomas G.

    2016-01-01

    Multi-protein complexes organized by cytoskeletal proteins are essential for cell wall biogenesis in most bacteria. Current models of the wall assembly mechanism assume class A penicillin-binding proteins (aPBPs), the targets of penicillin-like drugs, function as the primary cell wall polymerases within these machineries. Here, we use an in vivo cell wall polymerase assay in Escherichia coli combined with measurements of the localization dynamics of synthesis proteins to investigate this hypothesis. We find that aPBP activity is not necessary for glycan polymerization by the cell elongation machinery as is commonly believed. Instead, our results indicate that cell wall synthesis is mediated by two distinct polymerase systems, SEDS-family proteins working within the cytoskeletal machines and aPBP enzymes functioning outside of these complexes. These findings thus necessitate a fundamental change in our conception of the cell wall assembly process in bacteria. PMID:27643381

  12. The schlafen family of proteins and their regulation by interferons.

    PubMed

    Mavrommatis, Evangelos; Fish, Eleanor N; Platanias, Leonidas C

    2013-04-01

    The Schlafen (SLFN) family of proteins includes several mouse and human members. There is emerging evidence that members of this family of proteins are involved in important functions, such as the control of cell proliferation, induction of immune responses, and the regulation of viral replication. These proteins span across all species with great diversity, with 10 murine and 5 human isoforms. Recent work has established that mouse and human SLFN proteins are regulated by interferons (IFNs). Several Slfn genes were shown to be induced as classical interferon-stimulated genes, and emerging evidence suggests that these proteins play important roles in the growth inhibitory and antineoplastic effects of IFNs. In the current review, the known properties of mouse and human SLFNs are reviewed, and the implications of their emerging functions are discussed.

  13. The Schlafen Family of Proteins and Their Regulation by Interferons

    PubMed Central

    Mavrommatis, Evangelos; Fish, Eleanor N.

    2013-01-01

    The Schlafen (SLFN) family of proteins includes several mouse and human members. There is emerging evidence that members of this family of proteins are involved in important functions, such as the control of cell proliferation, induction of immune responses, and the regulation of viral replication. These proteins span across all species with great diversity, with 10 murine and 5 human isoforms. Recent work has established that mouse and human SLFN proteins are regulated by interferons (IFNs). Several Slfn genes were shown to be induced as classical interferon-stimulated genes, and emerging evidence suggests that these proteins play important roles in the growth inhibitory and antineoplastic effects of IFNs. In the current review, the known properties of mouse and human SLFNs are reviewed, and the implications of their emerging functions are discussed. PMID:23570387

  14. BCL-2 family proteins as regulators of mitochondria metabolism.

    PubMed

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  15. Calcineurin homologous protein: a multifunctional Ca2+-binding protein family

    PubMed Central

    Vadnagara, Komal; Moe, Orson W.; Babich, Victor

    2012-01-01

    The calcineurin homologous protein (CHP) belongs to an evolutionarily conserved Ca2+-binding protein subfamily. The CHP subfamily is composed of CHP1, CHP2, and CHP3, which in vertebrates share significant homology at the protein level with each other and between other Ca2+-binding proteins. The CHP structure consists of two globular domains containing from one to four EF-hand structural motifs (calcium-binding regions composed of two helixes, E and F, joined by a loop), the myristoylation, and nuclear export signals. These structural features are essential for the function of the three members of the CHP subfamily. Indeed, CHP1–CHP3 have multiple and diverse essential functions, ranging from the regulation of the plasma membrane Na+/H+ exchanger protein function, to carrier vesicle trafficking and gene transcription. The diverse functions attributed to the CHP subfamily rendered an understanding of its action highly complex and often controversial. This review provides a comprehensive and organized examination of the properties and physiological roles of the CHP subfamily with a view to revealing a link between CHP diverse functions. PMID:22189947

  16. Disorder and function: a review of the dehydrin protein family

    PubMed Central

    Graether, Steffen P.; Boddington, Kelly F.

    2014-01-01

    Dehydration proteins (dehydrins) are group 2 members of the late embryogenesis abundant (LEA) protein family. The protein architecture of dehydrins can be described by the presence of three types of conserved sequence motifs that have been named the K-, Y-, and S-segments. By definition, a dehydrin must contain at least one copy of the lysine-rich K-segment. Abiotic stresses such as drought, cold, and salinity cause the upregulation of dehydrin mRNA and protein levels. Despite the large body of genetic and protein evidence of the importance of these proteins in stress response, the in vivo protective mechanism is not fully known. In vitro experimental evidence from biochemical assays and localization experiments suggests multiple roles for dehydrins, including membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species. Membrane binding by dehydrins is likely to be as a peripheral membrane protein, since the protein sequences are highly hydrophilic and contain many charged amino acids. Because of this, dehydrins in solution are intrinsically disordered proteins, that is, they have no well-defined secondary or tertiary structure. Despite their disorder, dehydrins have been shown to gain structure when bound to ligands such as membranes, and to possibly change their oligomeric state when bound to ions. We review what is currently known about dehydrin sequences and their structures, and examine the various ligands that have been shown to bind to this family of proteins. PMID:25400646

  17. Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites.

    PubMed

    Albillos, Agustín; de-la-Hera, Antonio; Alvarez-Mon, Melchor

    2004-05-15

    Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

  18. Arabidopsis FNRL protein is an NADPH-dependent chloroplast oxidoreductase resembling bacterial ferredoxin-NADP(+) reductases.

    PubMed

    Koskela, Minna M; Dahlström, Käthe M; Goñi, Guillermina; Lehtimäki, Nina; Nurmi, Markus; Velazquez-Campoy, Adrian; Hanke, Guy; Bölter, Bettina; Salminen, Tiina A; Medina, Milagros; Mulo, Paula

    2017-08-18

    Plastidic ferredoxin-NADP(+) oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of FAD dependent oxidoreductases. In the present study, we show that the Arabidopsis thaliana At1g15140 (FERREDOXIN-NADP(+) OXIDOREDUCTASE -LIKE, FNRL) is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with kcat = 3.24 ± 0.17 s(-1) , Km(NADPH) = 1.6 ± 0.3 μM and kcat / Km(NADPH) = 2.0 ± 0.4 μM(-1) s(-1) . Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1 (AtFd1), which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. Additionally, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded β-sheet and one α-helix, and a C-terminal NADP(+) -binding α/β domain with a five-stranded β-sheet with a pair of α-helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs. This article is protected by copyright. All rights reserved.

  19. Haemophilus ducreyi targets Src family protein tyrosine kinases to inhibit phagocytic signaling.

    PubMed

    Mock, Jason R; Vakevainen, Merja; Deng, Kaiping; Latimer, Jo L; Young, Jennifer A; van Oers, Nicolai S C; Greenberg, Steven; Hansen, Eric J

    2005-12-01

    Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.

  20. The Amt/Mep/Rh family of ammonium transport proteins.

    PubMed

    Andrade, Susana L A; Einsle, Oliver

    2007-01-01

    The Amt/Mep/Rh family of integral membrane proteins comprises ammonium transporters of bacteria, archaea and eukarya, as well as the Rhesus proteins found in animals. They play a central role in the uptake of reduced nitrogen for biosynthetic purposes, in energy metabolism, or in renal excretion. Recent structural information on two prokaryotic Amt proteins has significantly contributed to our understanding of this class, but basic questions concerning the transport mechanism and the nature of the transported substrate, NH3 or [NH4(+)], remain to be answered. Here we review functional and structural studies on Amt proteins and discuss the bioenergetic issues raised by the various mechanistic proposals present in the literature.

  1. Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

    PubMed Central

    Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

    2013-01-01

    Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

  2. Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family

    PubMed Central

    Pérez-Pulido, Antonio J.; Reynaud, Emmanuel G.; Andrade-Navarro, Miguel A.

    2017-01-01

    Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis. PMID:28072865

  3. Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family.

    PubMed

    Mier, Pablo; Pérez-Pulido, Antonio J; Reynaud, Emmanuel G; Andrade-Navarro, Miguel A

    2017-01-01

    Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis.

  4. Analysis of Known Bacterial Protein Vaccine Antigens Reveals Biased Physical Properties and Amino Acid Composition

    PubMed Central

    Mayers, Carl; Rowe, Sonya; Miller, Julie; Lingard, Bryan; Hayward, Sarah; Titball, Richard W.

    2003-01-01

    Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location. PMID:18629010

  5. Metagenomic mining for thermostable esterolytic enzymes uncovers a new family of bacterial esterases

    PubMed Central

    Zarafeta, Dimitra; Moschidi, Danai; Ladoukakis, Efthymios; Gavrilov, Sergey; Chrysina, Evangelia D.; Chatziioannou, Aristotelis; Kublanov, Ilya; Skretas, Georgios; Kolisis, Fragiskos N.

    2016-01-01

    Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Here, we have identified a new esterolytic enzyme by screening a metagenomic sample collected from a hot spring in Kamchatka, Russia. Biochemical characterization of the new esterase, termed EstDZ2, revealed that it is highly active against medium chain fatty acid esters at temperatures between 25 and 60 °C and at pH values 7–8. The new enzyme is moderately thermostable with a half-life of more than six hours at 60 °C, but exhibits exquisite stability against high concentrations of organic solvents. Phylogenetic analysis indicated that EstDZ2 is likely an Acetothermia enzyme that belongs to a new family of bacterial esterases, for which we propose the index XV. One distinctive feature of this new family, is the presence of a conserved GHSAG catalytic motif. Multiple sequence alignment, coupled with computational modelling of the three-dimensional structure of EstDZ2, revealed that the enzyme lacks the largest part of the “cap” domain, whose extended structure is characteristic for the closely related Family IV esterases. Thus, EstDZ2 appears to be distinct from known related esterolytic enzymes, both in terms of sequence characteristics, as well as in terms of three-dimensional structure. PMID:27991516

  6. Systems Proteomics View of the Endogenous Human Claudin Protein Family

    PubMed Central

    Liu, Fei; Koval, Michael; Ranganathan, Shoba; Fanayan, Susan; Hancock, William S.; Lundberg, Emma K.; Beavis, Ronald C.; Lane, Lydie; Duek, Paula; McQuade, Leon; Kelleher, Neil L.; Baker, Mark S.

    2016-01-01

    Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein–protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation. PMID:26680015

  7. Protein Adsorption and Its Role in Bacterial Film Development

    DTIC Science & Technology

    1989-06-27

    only the secondary antibody conjugated to alkaline phosphatase was used. Combined Amino Acids as Measured by HPLC We are interested in a simple, direct...specific assay for chitin that relies on the lectin, wheat germ agglutinin (WGA). Lectins are a general class of proteins that bind to carbohydrates. The...protein; 2) a new method for measuring combined amino acids (includes proteins) in seawater was shown to measure higher concentration than the old

  8. [Pedigree survey in a family with hereditary protein S deficiency].

    PubMed

    Huang, K Y; Kong, L Q; Wu, Z; Wen, X; Zhao, J; Zhang, H C; Xu, Y; Long, X J; Kang, Y

    2016-09-24

    Objective: To observe the clinical feature of familiar hereditary protein S deficiency(HPSD), and to explore the related gene mutations. Methods: A total of seven family members were enrolled in this study and examined during the June to September 2015. Medical histories of the families were analyzed to detect HPSD according to the diagnostic criteria. PROS1 genes of the proband and her family were analyzed. DNA was extracted from peripheral blood. The 15 exons and their intron-exon boundaries of PROS1 were amplified with PCR, and the PCR products were sequenced and analyzed to identify potential mutations. Medical histories from the family members died prior this study were also obtained. Results: Four out of 7 family members of 2 generations were diagnosed as HPSD. The proband suffered from pulmonary embolism, her elder brother suffered from cerebral infarction and her niece suffered from deep vein thrombosis. A missense mutation at the 1063 bp of cDNA(c.1063C>T)was detected in the exon 10 of PROS1, which resulted in arginine 355 to cysteine replacement in the first ball domain of laminin of the protein S(p.R355C). Conclusion: HPSD is an autosomal dominant genetic disease, patients often suffer from recurring vein thrombosis and pulmonary embolism. A missense mutation(c.1063C>T, p. R355C)of PROS1 was discovered in this Chinese family with HPSD, thus, this mutation might be the genetic basis responsible for these family members with HPSD .

  9. DAZ Family Proteins, Key Players for Germ Cell Development.

    PubMed

    Fu, Xia-Fei; Cheng, Shun-Feng; Wang, Lin-Qing; Yin, Shen; De Felici, Massimo; Shen, Wei

    2015-01-01

    DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.

  10. A Comparison of Rosetta Stones in Adapter Protein Families

    PubMed Central

    Kumar, Hulikal Shivashankara Santosh; Kumar, Vadlapudi

    2016-01-01

    The inventory of proteins used in different kingdoms appears surprisingly similar in all sequenced eukaryotic genome. Protein domains represent the basic evolutionary units that form proteins. Domain duplication and shuffling by recombination are probably the most important forces driving protein evolution and hence the complexity of the proteome. While the duplication of whole genes as well as domain encoding exons increases the abundance of domains in the proteome, domain shuffling increases versatility, i.e. the number of distinct contexts in which a domain can occur. In this study we considered five important adapter domain families namely WD40, KELCH, Ankyrin, PDZ and Pleckstrin Homology (PH domain) family for the comparison of Domain versatility, Abundance and domain sharing between them. We used ecological statistics methods such as Jaccard’s Similarity Index (JSI), Detrended Correspondence Analysis, k-Means clustering for the domain distribution data. We found high propensity of domain sharing between PH and PDZ. We found higher abundance of only few selected domains in PH, PDZ, ANK and KELCH families. We also found WD40 family with high versatility and less redundant domain occurrence, with less domain sharing. Hence, the assignments of functions to more orphan WD40 proteins that will help in the identification of suitable drug targets. PMID:28246462

  11. Current overview of allergens of plant pathogenesis related protein families.

    PubMed

    Sinha, Mau; Singh, Rashmi Prabha; Kushwaha, Gajraj Singh; Iqbal, Naseer; Singh, Avinash; Kaushik, Sanket; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2014-01-01

    Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.

  12. Current Overview of Allergens of Plant Pathogenesis Related Protein Families

    PubMed Central

    Sinha, Mau; Singh, Rashmi Prabha; Kushwaha, Gajraj Singh; Iqbal, Naseer; Singh, Avinash; Kaushik, Sanket; Sharma, Sujata; Singh, Tej P.

    2014-01-01

    Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens. PMID:24696647

  13. Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

    PubMed Central

    Rosander, Anna; Bjerketorp, Joakim; Frykberg, Lars

    2003-01-01

    Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins. PMID:14569614

  14. An Ancient Family of RNA-Binding Proteins: Still Important!

    PubMed

    Wells, Melissa L; Perera, Lalith; Blackshear, Perry J

    2017-04-01

    RNA-binding proteins are important modulators of mRNA stability, a crucial process that determines the ultimate cellular levels of mRNAs and their encoded proteins. The tristetraprolin (TTP) family of RNA-binding proteins appeared early in the evolution of eukaryotes, and has persisted in modern eukaryotes. The domain structures and biochemical functions of family members from widely divergent lineages are remarkably similar, but their mRNA 'targets' can be very different, even in closely related species. Recent gene knockout studies in species as distantly related as plants, flies, yeasts, and mice have demonstrated crucial roles for these proteins in a wide variety of physiological processes. Inflammatory and hematopoietic phenotypes in mice have suggested potential therapeutic approaches for analogous human disorders.

  15. General Protein Diffusion Barriers create Compartments within Bacterial Cells

    PubMed Central

    Schlimpert, Susan; Klein, Eric A.; Briegel, Ariane; Hughes, Velocity; Kahnt, Jörg; Bolte, Kathrin; Maier, Uwe G.; Brun, Yves V.; Jensen, Grant J.; Gitai, Zemer; Thanbichler, Martin

    2013-01-01

    SUMMARY In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell cycle-dependent manner. Their presence is critical for cellular fitness, as they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins. PMID:23201141

  16. Multiple oligomeric structures of a bacterial small heat shock protein

    PubMed Central

    Mani, Nandini; Bhandari, Spraha; Moreno, Rodolfo; Hu, Liya; Prasad, B. V. Venkataram; Suguna, Kaza

    2016-01-01

    Small heat shock proteins are ubiquitous molecular chaperones that form the first line of defence against the detrimental effects of cellular stress. Under conditions of stress they undergo drastic conformational rearrangements in order to bind to misfolded substrate proteins and prevent cellular protein aggregation. Owing to the dynamic nature of small heat shock protein oligomers, elucidating the structural basis of chaperone action and oligomerization still remains a challenge. In order to understand the organization of sHSP oligomers, we have determined crystal structures of a small heat shock protein from Salmonella typhimurium in a dimeric form and two higher oligomeric forms: an 18-mer and a 24-mer. Though the core dimer structure is conserved in all the forms, structural heterogeneity arises due to variation in the terminal regions. PMID:27053150

  17. Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome

    PubMed Central

    De Santi, Concetta; Willassen, Nils Peder

    2016-01-01

    Background The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. Results MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes. PMID:27433797

  18. A knot in the protein structure - probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome.

    PubMed

    Stepanenko, Olesya V; Bublikov, Grigory S; Stepanenko, Olga V; Shcherbakova, Daria M; Verkhusha, Vladislav V; Turoverov, Konstantin K; Kuznetsova, Irina M

    2014-05-01

    The possibility of engineering near-infrared fluorescent proteins and biosensors from bacterial phytochrome photoreceptors (BphPs) has led to substantial interest in this family of proteins. The near-infrared fluorescent proteins have allowed non-invasive bio-imaging of deep tissues and whole organs in living animals. BphPs and derived near-infrared fluorescent proteins contain a structural element, called a knot, in their polypeptide chains. The formation of knot structures in proteins was refuted for a long time. Here, we studied the denaturation and renaturation processes of the near-infrared fluorescent probe iRFP, engineered from RpBphP2, which utilizes a heme-derived tetrapyrrole compound biliverdin as a chromophore. iRFP contains a unique figure-of-eight knot. The denaturation and renaturation curves of the iRFP apoform coincided well, suggesting efficient refolding. However, the iRFP holoform exhibited irreversible unfolding and aggregation associated with the bound chromophore. The knot structure in the apoform did not prevent subsequent binding of biliverdin, resulting in the functional iRFP holoform. We suggest that the irreversibility of protein unfolding is caused by post-translational protein modifications, such as chromophore binding, rather than the presence of the knot. These results are essential for future design of BphP-based near-infrared probes, and add important features to our knowledge of protein folding.

  19. A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family

    PubMed Central

    Yao, Qing; Lu, Qiuhe; Wan, Xiaobo; Song, Feng; Xu, Yue; Hu, Mo; Zamyatina, Alla; Liu, Xiaoyun; Huang, Niu; Zhu, Ping; Shao, Feng

    2014-01-01

    A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC–TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex. DOI: http://dx.doi.org/10.7554/eLife.03714.001 PMID:25310236

  20. Comparative genomics of the Rab protein family in Apicomplexan parasites

    PubMed Central

    Langsley, Gordon; van Noort, Vera; Carret, Céline; Meissner, Markus; de Villiers, Etienne P.; Bishop, Richard; Pain, Arnab

    2008-01-01

    Rab genes encode a subgroup of small GTP-binding proteins within the ras super-family that regulate targeting and fusion of transport vesicles within the secretory and endocytic pathways. These genes are of particular interest in the protozoan phylum Apicomplexa, since a family of Rab GTPases has been described for Plasmodium and most putative secretory pathway proteins in Apicomplexa have conventional predicted signal peptides. Moreover, peptide motifs have now been identified within a large number of secreted Plasmodium proteins that direct their targeting to the red blood cell cytosol, the apicoplast, the food vacuole and Maurer's clefs; in contrast, motifs that direct proteins to secretory organelles (rhoptries, micronemes and microspheres) have yet to be defined. The nature of the vesicle in which these proteins are transported to their destinations remains unknown and morphological structures equivalent to the endoplasmic reticulum and trans-Golgi stacks typical of other eukaryotes cannot be visualised in Apicomplexa. Since Rab GTPases regulate vesicular traffic in all eukaryotes, and this traffic in intracellular parasites could regulate import of nutrient and drugs and export of antigens, host cell modulatory proteins and lactate we compare and contrast here the Rab families of Apicomplexa. PMID:18468471

  1. Systematic analysis of the twin cx(9)c protein family.

    PubMed

    Longen, Sebastian; Bien, Melanie; Bihlmaier, Karl; Kloeppel, Christine; Kauff, Frank; Hammermeister, Miriam; Westermann, Benedikt; Herrmann, Johannes M; Riemer, Jan

    2009-10-23

    The Mia40-Erv1 disulfide relay system is of high importance for mitochondrial biogenesis. Most so far identified substrates of this machinery contain either two cysteine-x(3)-cysteine (twin Cx(3)C) or two cysteine-x(9)-cysteine (twin Cx(9)C) motifs. While the first group is composed of well-characterized components of the mitochondrial import machinery, the molecular function of twin Cx(9)C proteins still remains unclear. To systematically characterize this protein family, we performed a database search to identify the full complement of Cx(9)C proteins in yeast. Thereby, we identified 14 potential family members, which, with one exception, are conserved among plants, fungi, and animals. Among these, three represent novel proteins, which we named Cmc2 to 4 (for Cx(9)C motif-containing protein) and which we demonstrated to be dependent for import on the Mia40-Erv1 disulfide relay. By testing deletion mutants of all 14 proteins for function of the respiratory chain, we found a critical function of most of these proteins for the assembly or stability of respiratory chain complexes. Our data suggest that already early during the evolution of eukaryotic cells, a multitude of twin Cx(9)C proteins developed, which exhibit largely nonredundant roles critical for the biogenesis of enzymes of the respiratory chain in mitochondria.

  2. Six Subgroups and Extensive Recent Duplications Characterize the Evolution of the Eukaryotic Tubulin Protein Family

    PubMed Central

    Findeisen, Peggy; Mühlhausen, Stefanie; Dempewolf, Silke; Hertzog, Jonny; Zietlow, Alexander; Carlomagno, Teresa; Kollmar, Martin

    2014-01-01

    Tubulins belong to the most abundant proteins in eukaryotes providing the backbone for many cellular substructures like the mitotic and meiotic spindles, the intracellular cytoskeletal network, and the axonemes of cilia and flagella. Homologs have even been reported for archaea and bacteria. However, a taxonomically broad and whole-genome-based analysis of the tubulin protein family has never been performed, and thus, the number of subfamilies, their taxonomic distribution, and the exact grouping of the supposed archaeal and bacterial homologs are unknown. Here, we present the analysis of 3,524 tubulins from 504 species. The tubulins formed six major subfamilies, α to ζ. Species of all major kingdoms of the eukaryotes encode members of these subfamilies implying that they must have already been present in the last common eukaryotic ancestor. The proposed archaeal homologs grouped together with the bacterial TubZ proteins as sister clade to the FtsZ proteins indicating that tubulins are unique to eukaryotes. Most species contained α- and/or β-tubulin gene duplicates resulting from recent branch- and species-specific duplication events. This shows that tubulins cannot be used for constructing species phylogenies without resolving their ortholog–paralog relationships. The many gene duplicates and also the independent loss of the δ-, ε-, or ζ-tubulins, which have been shown to be part of the triplet microtubules in basal bodies, suggest that tubulins can functionally substitute each other. PMID:25169981

  3. Identification of secreted bacterial proteins by noncanonical amino acid tagging.

    PubMed

    Mahdavi, Alborz; Szychowski, Janek; Ngo, John T; Sweredoski, Michael J; Graham, Robert L J; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K; Tirrell, David A

    2014-01-07

    Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

  4. Chromophore/protein interaction in bacterial sensory rhodopsin and bacteriorhodopsin.

    PubMed Central

    Spudich, J L; McCain, D A; Nakanishi, K; Okabe, M; Shimizu, N; Rodman, H; Honig, B; Bogomolni, R A

    1986-01-01

    Retinal analogues with altered conjugated double bond systems or altered stereochemistry were incorporated into the phototaxis receptor sensory rhodopsin (SR) and the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium halobium. Wavelength shifts in absorption ("opsin shifts") due to analogue interaction with the protein microenvironment demonstrate that the same overall electrostatic and steric properties of the retinal binding-site structures exist in both proteins despite their different functions. pi-Electron calculations from the opsin shifts lead to a new description of protein charge distribution that applies to the binding sites of both SR and BR. The new data extends the previously proposed external point charge model for BR to include an ion-pair protein/chromophore interaction near the beta-ionone moiety. The new data modifies the previously proposed external point-charge model, the derivation of which involved an experimentally erroneous opsin shift for one of the BR analogues. PMID:2937462

  5. An Ribonuclease T2 Family Protein Modulates Acinetobacter baumannii Abiotic Surface Colonization

    PubMed Central

    Jacobs, Anna C.; Blanchard, Catlyn E.; Catherman, Seana C.; Dunman, Paul M.; Murata, Yoshihiko

    2014-01-01

    Acinetobacter baumannii is an emerging bacterial pathogen of considerable medical concern. The organism's transmission and ability to cause disease has been associated with its propensity to colonize and form biofilms on abiotic surfaces in health care settings. To better understand the genetic determinants that affect biomaterial attachment, we performed a transposon mutagenesis analysis of abiotic surface-colonization using A. baumannii strain 98-37-09. Disruption of an RNase T2 family gene was found to limit the organism's ability to colonize polystyrene, polypropylene, glass, and stainless steel surfaces. DNA microarray analyses revealed that in comparison to wild type and complemented cells, the RNase T2 family mutant exhibited reduced expression of 29 genes, 15 of which are predicted to be associated with bacterial attachment and surface-associated motility. Motility assays confirmed that RNase T2 mutant displays a severe motility defect. Taken together, our results indicate that the RNase T2 family protein identified in this study is a positive regulator of A. baumannii's ability to colonize inanimate surfaces and motility. Moreover, the enzyme may be an effective target for the intervention of biomaterial colonization, and consequently limit the organism's transmission within the hospital setting. PMID:24489668

  6. Methyl-accepting protein associated with bacterial sensory phodopsin I

    SciTech Connect

    Spudich, E.N.; Hasselbacher, C.A. ); Spudich, J.L. )

    1988-09-01

    In vivo radiolabeling of Halaobacterium halobium phototaxis mutants and revertants with L-(methyl-{sup 3}H) methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in photoaxis. The lability of the radiolabeled bands to mild base treatment indicated the the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing photoaxis receptors in H. halobium. It was absent in a strain the contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based in the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By ({sup 3}H)retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced ({sup 3}H) retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the siginal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.

  7. Methyl-accepting protein associated with bacterial sensory rhodopsin I.

    PubMed Central

    Spudich, E N; Hasselbacher, C A; Spudich, J L

    1988-01-01

    In vivo radiolabeling of Halobacterium halobium phototaxis mutants and revertants with L-[methyl-3H] methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in phototaxis. The lability of the radiolabeled bands to mild base treatment indicated that the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing phototaxis receptors in H. halobium. It was absent in a strain that contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based on the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By [3H]retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced [3H]retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the signal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I. Images PMID:3410829

  8. Probing the cellular effects of bacterial effector proteins with the Yersinia toolbox.

    PubMed

    Wölke, Stefan; Heesemann, Jürgen

    2012-04-01

    The type 3 secretion system (T3SS) is a powerful bacterial nanomachine that is able to modify the host cellular immune defense in favor of the pathogen by injection of effector proteins. In this regard, cellular Rho GTPases such as Rac1, RhoA or Cdc42 are targeted by a large group of T3SS effectors by mimicking cellular guanine exchange factors or GTPase-activating proteins. However, functional analysis of one type of T3SS effector that is translocated by bacterial pathogens is challenging because the T3SS effector repertoire can comprise a large number of proteins with redundant or interfering functions. Therefore, we developed the Yersinia toolbox to either analyze singular effector proteins of Yersinia spp. or different bacterial species in the context of bacterial T3SS injection into cells. Here, we focus on the WxxxE guanine exchange factor mimetic proteins IpgB1, IpgB2 and Map, which activate Rac1, RhoA or Cdc42, respectively, as well as the Rho GTPase inactivators YopE (a GTPase-activating mimetic protein) and YopT (cysteine protease), to generate a toolbox module for Rho GTPase manipulation.

  9. Human erythrocyte membrane proteins of zone 4.5 exist as families of related proteins.

    PubMed

    Whitfield, C F; Coleman, D B; Kay, M M; Shiffer, K A; Miller, J; Goodman, S R

    1985-01-01

    An analysis of the polypeptide composition of zone 4.5 of human erythrocyte membranes has been done by immunoautoradiographic and two-dimensional peptide mapping techniques. Results of these studies demonstrated that the Coomassie blue profile was constant, with 14 well-resolved bands present. Zone 4.5 polypeptides existed as at least four families of two or more components with closely related polypeptide backbones. The families could be distinguished on the basis of their extraction characteristics, immunological cross-reactivity, and two-dimensional peptide maps. One family was related to protein 4.1, one family was related to band 3, and two families were independent and not similar to other larger membrane proteins. The data show that all of the visualized bands in zone 4.5 do not have the same protein composition and that several closely related forms of some polypeptides are present.

  10. 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.

  11. APP Protein Family Signaling at the Synapse: Insights from Intracellular APP-Binding Proteins.

    PubMed

    Guénette, Suzanne; Strecker, Paul; Kins, Stefan

    2017-01-01

    Understanding the molecular mechanisms underlying amyloid precursor protein family (APP/APP-like proteins, APLP) function in the nervous system can be achieved by studying the APP/APLP interactome. In this review article, we focused on intracellular APP interacting proteins that bind the YENPTY internalization motif located in the last 15 amino acids of the C-terminal region. These proteins, which include X11/Munc-18-interacting proteins (Mints) and FE65/FE65Ls, represent APP cytosolic binding partners exhibiting different neuronal functions. A comparison of FE65 and APP family member mutant mice revealed a shared function for APP/FE65 protein family members in neurogenesis and neuronal positioning. Accumulating evidence also supports a role for membrane-associated APP/APLP proteins in synapse formation and function. Therefore, it is tempting to speculate that APP/APLP C-terminal interacting proteins transmit APP/APLP-dependent signals at the synapse. Herein, we compare our current knowledge of the synaptic phenotypes of APP/APLP mutant mice with those of mice lacking different APP/APLP interaction partners and discuss the possible downstream effects of APP-dependent FE65/FE65L or X11/Mint signaling on synaptic vesicle release, synaptic morphology and function. Given that the role of X11/Mint proteins at the synapse is well-established, we propose a model highlighting the role of FE65 protein family members for transduction of APP/APLP physiological function at the synapse.

  12. Meeting report - Arf and Rab family G proteins.

    PubMed

    Casanova, James E; Hsu, Victor W; Jackson, Catherine L; Kahn, Richard A; Roy, Craig R; Stow, Jennifer L; Wandinger-Ness, Angela; Sztul, Elizabeth

    2013-12-01

    A FASEB Summer Research Conference entitled 'Arf and Rab family G proteins' was held in July 2013 at Snowmass Village, Snowmass, Colorado. Arfs and Rabs are two families of GTPases that control membrane trafficking in eukaryotic cells, and increasing evidence indicates that their functions are tightly coordinated. Because many workers in this field have focused on only one family, this meeting was designed to integrate our understanding of the two families. The conference was organized by Elizabeth Sztul (University of Alabama, Birmingham, USA) and Jim Casanova (University of Virginia, Charlottesville, USA), and provided an opportunity for approximately 90 scientists to communicate their work and discuss future directions for the field. The talks highlighted the structural, functional and regulatory properties of Arf and Rab GTPases and the need to develop coordinated approaches to investigate them. Here, we present the major themes that emerged from the meeting.

  13. Behind the lines–actions of bacterial type III effector proteins in plant cells

    PubMed Central

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

  14. Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance

    SciTech Connect

    Daly, Michael J.; Gaidamakova, E.; Matrosova, V.; Vasilenko, A.; Zhai, M.; leapman, Richard D.; Lai, Barry; Ravel, Bruce; Li, Shu-Mei W.; Kemner, Kenneth M.; Fredrickson, Jim K.

    2007-04-02

    In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of manganese-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications which introduce carbonyl groups. Conditions which inhibited Mn-accumulation or Mn redox-cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence (XRF) microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR resistant and sensitive bacteria, our findings support that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

  15. Reversals and collisions optimize protein exchange in bacterial swarms

    NASA Astrophysics Data System (ADS)

    Amiri, Aboutaleb; Harvey, Cameron; Buchmann, Amy; Christley, Scott; Shrout, Joshua D.; Aranson, Igor S.; Alber, Mark

    2017-03-01

    Swarming groups of bacteria coordinate their behavior by self-organizing as a population to move over surfaces in search of nutrients and optimal niches for colonization. Many open questions remain about the cues used by swarming bacteria to achieve this self-organization. While chemical cue signaling known as quorum sensing is well-described, swarming bacteria often act and coordinate on time scales that could not be achieved via these extracellular quorum sensing cues. Here, cell-cell contact-dependent protein exchange is explored as a mechanism of intercellular signaling for the bacterium Myxococcus xanthus. A detailed biologically calibrated computational model is used to study how M. xanthus optimizes the connection rate between cells and maximizes the spread of an extracellular protein within the population. The maximum rate of protein spreading is observed for cells that reverse direction optimally for swarming. Cells that reverse too slowly or too fast fail to spread extracellular protein efficiently. In particular, a specific range of cell reversal frequencies was observed to maximize the cell-cell connection rate and minimize the time of protein spreading. Furthermore, our findings suggest that predesigned motion reversal can be employed to enhance the collective behavior of biological synthetic active systems.

  16. Site-directed lipid modification of IgG-binding protein by intracellular bacterial lipoprotein process.

    PubMed

    Shigematsu, H; Ebihara, T; Yanagida, Y; Haruyama, T; Kobatake, E; Aizawa, M

    1999-09-24

    IgG-binding protein was genetically expressed and lipid-modified in a site-directed manner in Escherichia coli. The DNA sequence encoding the signal peptide and the nine N-terminal amino acid residues of the major lipoprotein of E. coli (lpp) was fused to the sequence of B-domain which was one of the IgG binding domains of Staphylococcal Protein A (SpA). The N-terminal cysteine residue of the resulting protein was enzymatically linked with lipids in the bacterial membrane. The lipid-modified protein was translocated at the bacterial membrane in a manner similar to native bacterial lipoprotein, and it was purified with IgG-Sepharose by affinity chromatography. The lipid modified proteins (lppB1 and lppB5) showed a similar IgG binding activity to unmodified proteins, which was estimated by competitive ELISA. Proteoliposomes of lipid modified proteins were prepared in an elegant fashion so that the IgG binding site should be properly oriented on the surface of an individual liposome by anchoring the lipid-tail into the hydrophobic layer of the liposome membrane. As compared with the unmodified one, the lipid modified protein incorporated into the proteoliposome exhibited higher IgG binding activity.

  17. Mechanisms of the sialidase and trans-sialidase activities of bacterial sialyltransferases from glycosyltransferase family 80.

    PubMed

    Mehr, Kevin; Withers, Stephen G

    2016-04-01

    Many important biological functions are mediated by complex glycan structures containing the nine-carbon sugar sialic acid (Sia) at terminal, non-reducing positions. Sia are introduced onto glycan structures by enzymes known as sialyltransferases (STs). Bacterial STs from the glycosyltransferase family GT80 are a group of well-studied enzymes used for the synthesis of sialylated glycan structures. While highly efficient at sialyl transfer, these enzymes also demonstrate sialidase and trans-sialidase activities for which there is some debate surrounding the corresponding enzymatic mechanisms. Here we propose a mechanism for STs from the glycosyltransferase family GT80 in which sialidase and trans-sialidase activities occur through reverse sialylation of CMP. The resulting CMP-Sia is then enzymatically hydrolyzed or used as a donor in subsequent ST reactions resulting in sialidase and trans-sialidase activities, respectively. We provide evidence for this mechanism by demonstrating that CMP is required for sialidase and trans-sialidase activities and that its removal with phosphatase ablates activity. We also confirm the formation of CMP-Sia using a coupled enzyme assay. A clear understanding of the sialidase and trans-sialidase mechanisms for this class of enzymes allows for more effective use of these enzymes in the synthesis of glycoconjugates.

  18. Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family.

    PubMed

    Hsueh, Yi-Ching; Flinner, Nadine; Gross, Lucia E; Haarmann, Raimund; Mirus, Oliver; Sommer, Maik S; Schleiff, Enrico

    2017-08-01

    Proteins of the Omp85 family chaperone the membrane insertion of β-barrel-shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear-encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal membrane-embedded β-barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β-barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75-V, which is consistent with the phylogenetic clustering of P39 in the Toc75-V rather than the Toc75-III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75-III, Toc75-V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391-1401. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

  19. A database of protein structure families with common folding motifs.

    PubMed

    Holm, L; Ouzounis, C; Sander, C; Tuparev, G; Vriend, G

    1992-12-01

    The availability of fast and robust algorithms for protein structure comparison provides an opportunity to produce a database of three-dimensional comparisons, called families of structurally similar proteins (FSSP). The database currently contains an extended structural family for each of 154 representative (below 30% sequence identity) protein chains. Each data set contains: the search structure; all its relatives with 70-30% sequence identity, aligned structurally; and all other proteins from the representative set that contain substructures significantly similar to the search structure. Very close relatives (above 70% sequence identity) rarely have significant structural differences and are excluded. The alignments of remote relatives are the result of pairwise all-against-all structural comparisons in the set of 154 representative protein chains. The comparisons were carried out with each of three novel automatic algorithms that cover different aspects of protein structure similarity. The user of the database has the choice between strict rigid-body comparisons and comparisons that take into account interdomain motion or geometrical distortions; and, between comparisons that require strictly sequential ordering of segments and comparisons, which allow altered topology of loop connections or chain reversals. The data sets report the structurally equivalent residues in the form of a multiple alignment and as a list of matching fragments to facilitate inspection by three-dimensional graphics. If substructures are ignored, the result is a database of structure alignments of full-length proteins, including those in the twilight zone of sequence similarity.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  1. Ferritin family proteins and their use in bionanotechnology

    PubMed Central

    He, Didi; Marles-Wright, Jon

    2015-01-01

    Ferritin family proteins are found in all kingdoms of life and act to store iron within a protein cage and to protect the cell from oxidative damage caused by the Fenton reaction. The structural and biochemical features of the ferritins have been widely exploited in bionanotechnology applications: from the production of metal nanoparticles; as templates for semi-conductor production; and as scaffolds for vaccine design and drug delivery. In this review we first discuss the structural properties of the main ferritin family proteins, and describe how their organisation specifies their functions. Second, we describe materials science applications of ferritins that rely on their ability to sequester metal within their cavities. Finally, we explore the use of ferritin as a container for drug delivery and as a scaffold for the production of vaccines. PMID:25573765

  2. Molecular modeling of pathogenesis-related proteins of family 5.

    PubMed

    Thompson, Claudia E; Fernandes, Cláudia L; de Souza, Osmar N; Salzano, Francisco M; Bonatto, Sandro L; Freitas, Loreta B

    2006-01-01

    The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of alpha-amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for each protein.

  3. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  4. REVIEW ARTICLE: DNA protein interactions and bacterial chromosome architecture

    NASA Astrophysics Data System (ADS)

    Stavans, Joel; Oppenheim, Amos

    2006-12-01

    Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

  5. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems

    PubMed Central

    Feeley, Eric M.; Pilla-Moffett, Danielle M.; Zwack, Erin E.; Piro, Anthony S.; Finethy, Ryan; Kolb, Joseph P.; Martinez, Jennifer; Brodsky, Igor E.; Coers, Jörn

    2017-01-01

    Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as “patterns of pathogenesis” associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3–deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3–dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program. PMID:28193861

  6. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems.

    PubMed

    Feeley, Eric M; Pilla-Moffett, Danielle M; Zwack, Erin E; Piro, Anthony S; Finethy, Ryan; Kolb, Joseph P; Martinez, Jennifer; Brodsky, Igor E; Coers, Jörn

    2017-02-28

    Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella-containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia-containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella-containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

  7. A novel family of small proteins that affect plant development

    SciTech Connect

    John Charles Walker

    2011-04-29

    The DVL genes represent a new group of plant proteins that influence plant growth and development. Overexpression of DVL1, and other members of the DVL family, causes striking phenotypic changes. The DVL proteins share sequence homology in their C-terminal half. Point mutations in the C-terminal domain show it is necessary and deletion studies demonstrate the C-terminal domain is sufficient to confer the overexpression phenotypes. The phenotypes observed, and the conservation of the protein sequence in the plant kingdom, does suggest the DVL proteins have a role in modulating plant growth and development. Our working hypothesis is the DVL proteins function as regulators of cellular signaling pathways that control growth and development.

  8. The APOBEC Protein Family: United by Structure, Divergent in Function.

    PubMed

    Salter, Jason D; Bennett, Ryan P; Smith, Harold C

    2016-07-01

    The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

  9. IQGAPs in Cancer: A Family of Scaffold Proteins Underlying Tumorigenesis

    PubMed Central

    White, Colin D.; Brown, Matthew D.; Sacks, David B.

    2009-01-01

    The IQGAP family comprises three proteins in humans. The best characterized is IQGAP1, which participates in protein-protein interactions and integrates diverse signaling pathways. IQGAP2 and IQGAP3 harbor all the domains identified in IQGAP1, but their biological roles are poorly defined. Proteins that bind IQGAP1 include Cdc42 and Rac1, E-cadherin, β-catenin, calmodulin and components of the mitogen-activated protein kinase pathway, all of which are involved in cancer. Here, we summarize the biological functions of IQGAPs that may contribute to neoplasia. Additionally, we review published data which implicate IQGAPs in cancer and tumorigenesis. The cumulative evidence suggests IQGAP1 is an oncogene while IQGAP2 may be a tumor suppressor. PMID:19433088

  10. Site specific protein O-glucosylation with bacterial toxins.

    PubMed

    Sun, Y; Willis, L M; Batchelder, H R; Nitz, M

    2016-10-27

    Using a MALDI-MS based assay, the kinetic parameters for peptide glucosylation using the C. difficile toxin B glycosyltransferase domain were determined. The minimum consensus sequence for glucosylation was YXXTXFXXY and the optimal peptide found was YAPTVFDAY. Using this sequence, homogenous glucosylated proteins could be readily produced.

  11. Identification of novel members of the bacterial azoreductase family in Pseudomonas aeruginosa.

    PubMed

    Crescente, Vincenzo; Holland, Sinead M; Kashyap, Sapna; Polycarpou, Elena; Sim, Edith; Ryan, Ali

    2016-03-01

    Azoreductases are a family of diverse enzymes found in many pathogenic bacteria as well as distant homologues being present in eukarya. In addition to having azoreductase activity, these enzymes are also suggested to have NAD(P)H quinone oxidoreductase (NQO) activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play a role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterized recombinant enzymes following expression of a group of putative azoreductase genes from P. aeruginosa expressed in Escherichia coli. The enzymes include members of the arsenic-resistance protein H (ArsH), tryptophan repressor-binding protein A (WrbA), modulator of drug activity B (MdaB) and YieF families. The ArsH, MdaB and YieF family members all show azoreductase and NQO activities. In contrast, WrbA is the first enzyme to show NQO activity but does not reduce any of the 11 azo compounds tested under a wide range of conditions. These studies will allow further investigation of the possible role of these enzymes in the pathogenesis of P. aeruginosa.

  12. 'Drugs from bugs': bacterial effector proteins as promising biological (immune-) therapeutics.

    PubMed

    Rüter, Christian; Hardwidge, Philip R

    2014-02-01

    Immune system malfunctions cause many of the most severe human diseases. The immune system has evolved primarily to control bacterial, viral, fungal, and parasitic infections. In turn, over millions of years of coevolution, microbial pathogens have evolved various mechanisms to control and modulate the host immune system for their own benefit and survival. For example, many bacterial pathogens use virulence proteins to modulate and exploit target cell mechanisms. Our understanding of these bacterial strategies opens novel possibilities to exploit 'microbial knowledge' to control excessive immune reactions. Gaining access to strategies of microbial pathogens could lead to potentially huge benefits for the therapy of inflammatory diseases. Most work on bacterial pathogen effector proteins has the long-term aim of neutralizing the infectious capabilities of the pathogen. However, attenuated pathogens and microbial products have been used for over a century with overwhelming success in the form of vaccines to induce specific immune responses that protect against the respective infectious diseases. In this review, we focus on bacterial effector and virulence proteins capable of modulating and suppressing distinct signaling pathways with potentially desirable immune-modulating effects for treating unrelated inflammatory diseases.

  13. Inducible expression of transmembrane proteins on bacterial magnetic particles in Magnetospirillum magneticum AMB-1.

    PubMed

    Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

    2010-02-01

    Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.

  14. Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1▿

    PubMed Central

    Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

    2010-01-01

    Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs. PMID:20038711

  15. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    PubMed Central

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  16. Evolutionary hierarchy of vertebrate-like heterotrimeric G protein families.

    PubMed

    Krishnan, Arunkumar; Mustafa, Arshi; Almén, Markus Sällman; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

    2015-10-01

    Heterotrimeric G proteins perform a crucial role as molecular switches controlling various cellular responses mediated by G protein-coupled receptor (GPCR) signaling pathway. Recent data have shown that the vertebrate-like G protein families are found across metazoans and their closest unicellular relatives. However, an overall evolutionary hierarchy of vertebrate-like G proteins, including gene family annotations and in particular mapping individual gene gain/loss events across diverse holozoan lineages is still incomplete. Here, with more expanded invertebrate taxon sampling, we have reconstructed phylogenetic trees for each of the G protein classes/families and provide a robust classification and hierarchy of vertebrate-like heterotrimeric G proteins. Our results further extend the evidence that the common ancestor (CA) of holozoans had at least five ancestral Gα genes corresponding to all major vertebrate Gα classes and contain a total of eight genes including two Gβ and one Gγ. Our results also indicate that the GNAI/O-like gene likely duplicated in the last CA of metazoans to give rise to GNAI- and GNAO-like genes, which are conserved across invertebrates. Moreover, homologs of GNB1-4 paralogon- and GNB5 family-like genes are found in most metazoans and that the unicellular holozoans encode two ancestral Gβ genes. Similarly, most bilaterian invertebrates encode two Gγ genes which include a representative of the GNG gene cluster and a putative homolog of GNG13. Interestingly, our results also revealed key evolutionary events such as the Drosophila melanogaster eye specific Gβ subunit that is found conserved in most arthropods and several previously unidentified species specific expansions within Gαi/o, Gαs, Gαq, Gα12/13 classes and the GNB1-4 paralogon. Also, we provide an overall proposed evolutionary scenario on the expansions of all G protein families in vertebrate tetraploidizations. Our robust classification/hierarchy is essential to further

  17. A novel family of plant nuclear envelope-associated proteins.

    PubMed

    Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

    2016-10-01

    This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

  18. The HMG-1 box protein family: classification and functional relationships.

    PubMed Central

    Baxevanis, A D; Landsman, D

    1995-01-01

    The abundant and highly-conserved nucleoproteins comprising the high mobility group-1/2 (HMG-1/2) family contains two homologous basic domains of about 75 amino acids. These basic domains, termed HMG-1 boxes, are highly structured and facilitate HMG-DNA interactions. Many proteins that regulate various cellular functions involving DNA binding and whose target DNA sequences share common structural characteristics have been identified as having an HMG-1 box; these proteins include the RNA polymerase I transcription factor UBF, the mammalian testis-determining factor SRY and the mitochondrial transcription factors ABF2 and mtTF1, among others. The sequences of 121 HMG-1 boxes have been compiled and aligned in accordance with thermodynamic results from homology model building (threading) experiments, basing the alignment on structure rather than by using traditional sequence homology methods. The classification of a representative subset of these proteins was then determined using standard least-squares distance methods. The proteins segregate into two groups, the first consisting of HMG-1/2 proteins and the second consisting of proteins containing the HMG-1 box but which are not canonical HMG proteins. The proteins in the second group further segregate based on their function, their ability to bind specific sequences of DNA, or their ability to recognize discrete non-B-DNA structures. The HMG-1 box provides an excellent example of how a specific protein motif, with slight alteration, can be used to recognize DNA in a variety of functional contexts. Images PMID:7784217

  19. Nme protein family evolutionary history, a vertebrate perspective

    PubMed Central

    Desvignes, Thomas; Pontarotti, Pierre; Fauvel, Christian; Bobe, Julien

    2009-01-01

    Background The Nme family, previously known as Nm23 or NDPK, is involved in various molecular processes including tumor metastasis and some members of the family, but not all, exhibit a Nucleoside Diphosphate Kinase (NDPK) activity. Ten genes are known in humans, in which some members have been extensively studied. In non-mammalian species, the Nme protein family has received, in contrast, far less attention. The picture of the vertebrate Nme family remains thus incomplete and orthology relationships with mammalian counterparts were only partially characterized. The present study therefore aimed at characterizing the Nme gene repertoire in vertebrates with special interest for teleosts, and providing a comprehensive overview of the Nme gene family evolutionary history in vertebrates. Results In the present study, we present the evolutionary history of the Nme family in vertebrates and characterize the gene family repertoire for the first time in several non-mammalian species. Our observations show that vertebrate Nme genes can be separated in two evolutionary distinct groups. Nme1, Nme2, Nme3, and Nme4 belong to Group I while vertebrate Nme5, Nme6, Nme7, Nme8, and Nme9 belong to Group II. The position of Nme10 is in contrast more debatable due to its very specific evolutionary history. The present study clearly indicates that Nme5, Nme6, Nme7, and Nme8 originate from duplication events that occurred before the chordate radiation. In contrast, Nme genes of the Group I have a very different evolutionary history as our results suggest that they all arise from a common gene present in the chordate ancestor. In addition, expression patterns of all zebrafish nme transcripts were studied in a broad range of tissues by quantitative PCR and discussed in the light of the function of their mammalian counterparts. Conclusion This work offers an evolutionary framework that will pave the way for future studies on vertebrate Nme proteins and provides a unified vertebrate Nme

  20. The APSES family proteins in fungi: Characterizations, evolution and functions.

    PubMed

    Zhao, Yong; Su, Hao; Zhou, Jing; Feng, Huihua; Zhang, Ke-Qin; Yang, Jinkui

    2015-08-01

    The APSES protein family belongs to transcriptional factors of the basic helix-loop-helix (bHLH) class, the originally described members (APSES: Asm1p, Phd1p, Sok2p, Efg1p and StuAp) are used to designate this group of proteins, and they have been identified as key regulators of fungal development and other biological processes. APSES proteins share a highly conserved DNA-binding domain (APSES domain) of about 100 amino acids, whose central domain is predicted to form a typical bHLH structure. Besides APSES domain, several APSES proteins also contain additional domains, such as KilA-N and ankyrin repeats. In recent years, an increasing number of APSES proteins have been identified from diverse fungi, and they involve in numerous biological processes, such as sporulation, cellular differentiation, mycelial growth, secondary metabolism and virulence. Most fungi, including Aspergillus fumigatus, Aspergillus nidulans, Candida albicans, Fusarium graminearum, and Neurospora crassa, contain five APSES proteins. However, Cryptococcus neoformans only contains two APSES proteins, and Saccharomyces cerevisiae contains six APSES proteins. The phylogenetic analysis showed the APSES domains from different fungi were grouped into four clades (A, B, C and D), which is consistent with the result of homologous alignment of APSES domains using DNAman. The roles of APSES proteins in clade C have been studied in detail, while little is known about the roles of other APSES proteins in clades A, B and D. In this review, the biochemical properties and functional domains of APSES proteins are predicted and compared, and the phylogenetic relationship among APSES proteins from various fungi are analyzed based on the APSES domains. Moreover, the functions of APSES proteins in different fungi are summarized and discussed.

  1. Reviewing the current classification of inhibitor of growth family proteins

    PubMed Central

    Unoki, Motoko; Kumamoto, Kensuke; Takenoshita, Seiichi; Harris, Curtis C.

    2009-01-01

    Inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors for more than a decade. Recent emerging results using siRNA and knockout mice are expanding the previous understanding of this protein family. The results of ING1 knockout mouse experiments revealed that ING1 has a protective effect on apoptosis. Our recent results showed that ING2 is overexpressed in colorectal cancer, and induces colon cancer cell invasion through an MMP13-dependent pathway. Knockdown of ING2 by siRNA induces premature senescence in normal human fibroblast cells, and apoptosis or cell cycle arrest in various adherent cancer cells. Taken together, these results suggest that ING2 may also have roles in cancer progression and/or malignant transformation under some conditions. Additionally, knockdown of ING4 and ING5 by siRNA shows an inhibitory effect on the transition from G2/M to G1 phase and DNA replication, respectively, suggesting that these proteins may play roles during cell proliferation in some context. ING family proteins may play dual roles, similar to transforming growth factor-β, which has tumor suppressor-like functions in normal epithelium and also oncogenic functions in invasive metastatic cancers. In the present article, we briefly review ING history and propose a possible interpretation of discrepancies between past and recent data. PMID:19432890

  2. TIM-family proteins inhibit HIV-1 release

    PubMed Central

    Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

    2014-01-01

    Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

  3. Protein localization and dynamics within a bacterial organelle

    PubMed Central

    Hughes, H. Velocity; Huitema, Edgar; Pritchard, Sean; Keiler, Kenneth C.; Brun, Yves V.; Viollier, Patrick H.

    2010-01-01

    Protein localization mechanisms dictate the functional and structural specialization of cells. Of the four polar surface organelles featured by the dimorphic bacterium Caulobacter crescentus, the stalk, a cylindrical extension of all cell envelope layers, is the least well characterized at the molecular level. Here we apply a powerful experimental scheme that integrates genetics with high-throughput localization to discover StpX, an uncharacterized bitopic membrane protein that modulates stalk elongation and is sequestered to the stalk. In stalkless mutants StpX is dispersed. Two populations of StpX were discernible within the stalk with different mobilities: an immobile one near the stalk base and a mobile one near the stalk tip. Molecular anatomy provides evidence that (i) the StpX transmembrane domain enables access to the stalk organelle, (ii) the N-terminal periplasmic domain mediates retention in the stalk, and (iii) the C-terminal cytoplasmic domain enhances diffusion within the stalk. Moreover, the accumulation of StpX and an N-terminally truncated isoform is differentially coordinated with the cell cycle. Thus, at the submicron scale the localization and the mobility of a protein are precisely regulated in space and time and are important for the correct organization of a subcellular compartment or organelle such as the stalk. PMID:20212131

  4. Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins.

    PubMed

    Andreini, Claudia; Banci, Lucia; Rosato, Antonio

    2016-04-01

    Organisms from all kingdoms of life use iron-sulfur proteins (FeS-Ps) in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of FeS-Ps. Sixty-one percent of human FeS-Ps bind Fe4S4 clusters, whereas 39% bind Fe2S2 clusters. However, this relative ratio varies significantly depending on the specific cellular compartment. We compared the portfolio of human FeS-Ps to 12 other eukaryotes and to about 700 prokaryotes. The comparative analysis of the organization of the prokaryotic homologues of human FeS-Ps within operons allowed us to reconstruct the human functional networks involving the conserved FeS-Ps common to prokaryotes and eukaryotes. These functional networks have been maintained during evolution and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps. Purine metabolism is another process including several FeS-Ps, in which BOLA proteins possibly have a regulatory role. The analysis of the co-occurrence of human FeS-Ps with other proteins highlighted numerous links between the iron-sulfur cluster machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of reactive oxygen species within the biogenesis and degradation of FeS-Ps.

  5. A polymeric bacterial protein activates dendritic cells via TLR4.

    PubMed

    Berguer, Paula M; Mundiñano, Juliana; Piazzon, Isabel; Goldbaum, Fernando A

    2006-02-15

    The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.

  6. Self-Regulation and Interplay of Rsm Family Proteins Modulate the Lifestyle of Pseudomonas putida

    PubMed Central

    2016-01-01

    ABSTRACT In the plant-beneficial bacterium Pseudomonas putida KT2440, three genes have been identified that encode posttranscriptional regulators of the CsrA/RsmA family. Their regulatory roles in the motile and sessile lifestyles of P. putida have been investigated by generating single-, double-, and triple-null mutants and by overexpressing each protein (RsmA, RsmE, and RsmI) in different genetic backgrounds. The rsm triple mutant shows reduced swimming and swarming motilities and increased biofilm formation, whereas overexpression of RsmE or RsmI results in reduced bacterial attachment. However, biofilms formed on glass surfaces by the triple mutant are more labile than those of the wild-type strain and are easily detached from the surface, a phenomenon that is not observed on plastic surfaces. Analysis of the expression of adhesins and exopolysaccharides in the different genetic backgrounds suggests that the biofilm phenotypes are due to alterations in the composition of the extracellular matrix and in the timing of synthesis of its elements. We have also studied the expression patterns of Rsm proteins and obtained data that indicate the existence of autoregulation mechanisms. IMPORTANCE Proteins of the CsrA/RsmA family function as global regulators in different bacteria. More than one of these proteins is present in certain species. In this study, all of the RsmA homologs in P. putida are characterized and globally taken into account to investigate their roles in controlling bacterial lifestyles and the regulatory interactions among them. The results offer new perspectives on how biofilm formation is modulated in this environmentally relevant bacterium. PMID:27422830

  7. N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

    PubMed Central

    Nothaft, Harald; Zheng, Jing

    2013-01-01

    Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

  8. CpsA, a LytR-CpsA-Psr Family Protein in Mycobacterium marinum, Is Required for Cell Wall Integrity and Virulence

    PubMed Central

    Wang, Qinglan; Zhu, Lin; Jones, Victoria; Wang, Chuan; Hua, Yifei; Shi, Xujun; Feng, Xia; Jackson, Mary; Niu, Chen

    2015-01-01

    LytR-CpsA-Psr family proteins play an important role in bacterial cell wall integrity. Although the pathogenic relevance of LytR-CpsA-Psr family proteins has been studied in a few bacterial pathogens, their function in mycobacteria remains uncharacterized. In this work, a transposon insertion mutant (cpsA::Tn) of Mycobacterium marinum was studied. We found that inactivation of CpsA altered bacterial colony morphology, sliding motility, cell surface hydrophobicity, and cell wall permeability. Besides, the cpsA mutant exhibited a decreased arabinogalactan content, indicating that CpsA plays a role in cell wall assembly. Moreover, the mutant shows impaired growth within macrophage cell lines and is severely attenuated in zebrafish larvae and adult zebrafish. Taken together, our results indicated that CpsA, a previously uncharacterized protein, is important for mycobacterial cell wall integrity and is required for mycobacterial virulence. PMID:25939506

  9. Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families.

    PubMed

    Igreja, Susana; Chahal, Harvinder S; King, Peter; Bolger, Graeme B; Srirangalingam, Umasuthan; Guasti, Leonardo; Chapple, J Paul; Trivellin, Giampaolo; Gueorguiev, Maria; Guegan, Katie; Stals, Karen; Khoo, Bernard; Kumar, Ajith V; Ellard, Sian; Grossman, Ashley B; Korbonits, Márta

    2010-08-01

    Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15-40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and beta-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6+/-11.2 years) than AIP mutation-negative patients (40.4+/-14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein-protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants.

  10. A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis

    PubMed Central

    2014-01-01

    Background A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. Results Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. Conclusions Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063). PMID:24581117

  11. Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family

    SciTech Connect

    Lee, Saeyoung; Park, Eun-Hye; Ko, Hyeok-Jin; Bang, Won Gi; Kim, Hye-Yeon; Kim, Kyoung Heon; Choi, In-Geol

    2015-11-13

    The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/β fold class, exhibiting an open twisted β-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad, is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr{sup 136} and Thr{sup 330}) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys{sup 84} as the catalytic base to polarize the Ser{sup 187} nucleophile in the catalytic triad. - Highlights: • We determined the first structure of a bacterial aryl acylamidase (EC 3.5.1.13). • Structure revealed spatially distinct architecture of the substrate-binding pocket. • Hydrogen-bonding with Tyr{sup 136} and Thr{sup 330} mediates ligand-binding and substrate.

  12. Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

    PubMed

    Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2011-11-01

    A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

  13. A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement

    PubMed Central

    Ravagnani, Adriana; Finan, Christopher L; Young, Michael

    2005-01-01

    Background In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf) is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria). The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria) and obtain information about how they may control bacterial growth and resuscitation. Results In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes. Conclusions The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope. PMID:15774001

  14. Biochemical and Structural Analyses of a Bacterial Endo-β-1,2-Glucanase Reveal A New Glycoside Hydrolase Family.

    PubMed

    Abe, Koichi; Nakajima, Masahiro; Yamashita, Tetsuro; Matsunaga, Hiroki; Kamisuki, Shinji; Nihira, Takanori; Takahashi, Yuta; Sugimoto, Naohisa; Miyanaga, Akimasa; Nakai, Hiroyuki; Arakawa, Takatoshi; Fushinobu, Shinya; Taguchi, Hayao

    2017-03-07

    β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an endo-β-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-β-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis. The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥ 5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs) and thus the enzyme defines a novel GH family, GHxxx. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represents a critical advance toward elucidating structure-function relationships of GH enzymes.

  15. Genes and proteins involved in bacterial magnetic particle formation.

    PubMed

    Matsunaga, Tadashi; Okamura, Yoshiko

    2003-11-01

    Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.

  16. Vaccinia Virus N1l Protein Resembles a B Cell Lymphoma-2 (Bcl-2) Family Protein

    SciTech Connect

    Aoyagi, M.; Zhai, D.; Jin, C.; Aleshin, A.E.; Stec, B.; Reed, J.C.; Liddington, R.C.; /Burnham Inst.

    2007-07-03

    Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.

  17. ProML--the protein markup language for specification of protein sequences, structures and families.

    PubMed

    Hanisch, Daniel; Zimmer, Ralf; Lengauer, Thomas

    2002-01-01

    We propose a specification language ProML for protein sequences, structures, and families based on the open XML standard. The language allows for portable, system-independent, machine-parsable and human-readable representation of essential features of proteins. The language is of immediate use for several bioinformatics applications: we discuss clustering of proteins into families and the representation of the specific shared features of the respective clusters. Moreover, we use ProML for specification of data used in fold recognition bench-marks exploiting experimentally derived distance constraints.

  18. The Crystal Structure of Rv0813c from Mycobacterium tuberculosis Reveals a New Family of Fatty Acid-Binding Protein-Like Proteins in Bacteria▿

    PubMed Central

    Shepard, William; Haouz, Ahmed; Graña, Martin; Buschiazzo, Alejandro; Betton, Jean-Michel; Cole, Stewart T.; Alzari, Pedro M.

    2007-01-01

    The gene Rv0813c from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is conserved within the order Actinomycetales but absent elsewhere. The crystal structure of Rv0813c reveals a new family of proteins that resemble the fatty acid-binding proteins (FABPs) found in eukaryotes. Rv0813c adopts the 10-stranded β-barrel fold typical of FABPs but lacks the double-helix insert that covers the entry to the binding site in the eukaryotic proteins. The barrel encloses a deep cavity, at the bottom of which a small cyclic ligand was found to bind to the hydroxyl group of Tyr192. This residue is part of a conserved Arg-X-Tyr motif much like the triad that binds the carboxylate group of fatty acids in FABPs. Most of the residues forming the internal surface of the cavity are conserved in homologous protein sequences found in CG-rich prokaryotes, strongly suggesting that Rv0813c is a member of a new family of bacterial FABP-like proteins that may have roles in the recognition, transport, and/or storage of small molecules in the bacterial cytosol. PMID:17172346

  19. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    PubMed Central

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  20. In silico modeling of the yeast protein and protein family interaction network

    NASA Astrophysics Data System (ADS)

    Goh, K.-I.; Kahng, B.; Kim, D.

    2004-03-01

    Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

  1. Solution structure of Arabidopsis thaliana protein At5g39720.1, a member of the AIG2-like protein family

    SciTech Connect

    Lytle, Betsy L.; Peterson, Francis C.; Tyler, Ejan M.; Newman, Carrie L.; Vinarov, Dmitriy A.; Markley, John L.; Volkman, Brian F.

    2006-06-01

    The solution structure of A. thaliana protein At5g39720.1 reported here is the first for a member of the AIG2-like family (PF06094). The three-dimensional structure shows similarity to those determined for members of the uncharacterized Pfam family UPF0131. The three-dimensional structure of Arabidopsis thaliana protein At5g39720.1 was determined by NMR spectroscopy. It is the first representative structure of Pfam family PF06094, which contains protein sequences similar to that of AIG2, an A. thaliana protein of unknown function induced upon infection by the bacterial pathogen Pseudomonas syringae. The At5g39720.1 structure consists of a five-stranded β-barrel surrounded by two α-helices and a small β-sheet. A long flexible α-helix protrudes from the structure at the C-terminal end. A structural homology search revealed similarity to three members of Pfam family UPF0131. Conservation of residues in a hydrophilic cavity able to bind small ligands in UPF0131 proteins suggests that this may also serve as an active site in AIG2-like proteins.

  2. Rv0216, a Conserved Hypothetical Protein from Myocbacterium Tuberculosis that is Essential for Bacterial Survival During Infection, has a Double Hotdog Fold

    SciTech Connect

    Castell,A.; Johansson, P.; Unge, T.; Jones, T.; Backbro, K.

    2005-01-01

    The Mycobacterium tuberculosis genome contains about 4000 genes, of which approximately a third code for proteins of unknown function or are classified as conserved hypothetical proteins. We have determined the three-dimensional structure of one of these, the rv0216 gene product, which has been shown to be essential for M. tuberculosis growth in vivo. The structure exhibits the greatest similarity to bacterial and eukaryotic hydratases that catalyse the R-specific hydration of 2-enoyl coenzyme A. However, only part of the catalytic machinery is conserved in Rv0216 and it showed no activity for the substrate crotonyl-CoA. The structure of Rv0216 allows us to assign new functional annotations to a family of seven other M. tuberculosis proteins, a number if which are essential for bacterial survival during infection and growth.

  3. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

    PubMed Central

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-01-01

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

  4. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs.

    PubMed

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-10-24

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4(+) cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.

  5. Protein engineering of bacterial histidine kinase receptor systems.

    PubMed

    Xie, Wei; Blain, Katherine Y; Kuo, Mario Meng-Chiang; Choe, Senyon

    2010-07-01

    Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signal transduction in prokaryotes in which the two essential components are a sensor histidine kinase (HK) receptor and a response regulator (RR). Despite great efforts in structural and functional characterization of signal perception mechanisms, the exact signaling mechanisms remain elusive for many TCSs. Mimicking the natural TCS signaling pathways, chimeric receptor kinases and response regulators have been constructed through the process of swapping modular domains of related TCSs. To design chimeras with new signaling pathways, domains from different proteins that have little relationship at the primary structural level but carrying desirable functional properties can be conjoined to engineer novel TCSs. These chimeras maintain the ability to respond to environmental stimulants by regulating protein phosphorylation to produce downstream output signals. Depending on the nature of external signals, chimeric TCSs can serve as a novel tool not only to examine the natural signaling mechanisms in TCSs, but also for industrial and clinical applications.

  6. [Immunodiffusion analysis of plasma proteins in the canine family].

    PubMed

    Baranov, O K; Iurishina, N A; Savina, M A

    1976-01-01

    Immunodiffusion studies have been made on the plasma of 9 species (Vulpes vulpes, V. corsak, Alopex lagopus, Canis aureus, C. lupus, C. familiaris, C. dingo, Nyctereutes procynoides, Fennecus zerde) from the family of Canidae using milk antisera. Unlike rabbit antisera used earlier, milk antisera make it possible to detect more significant antigenic divergency with respect to 5 alpha- and beta-globulins. These globulins seem to have a higher evolution rate of antigenic mosaics as compared to other plasma proteins in the family investigated. The family Canidae serologically may be divided into two main groups: 1) the genus Canis which includes the wolf, domestic dog, dingo, jackal and 2) species which significantly differ from the former (the fox, polar fox, dog fox, fennec). In relation to these two groups, the raccoon dog occupies special position.

  7. Cbln and C1q family proteins: new transneuronal cytokines.

    PubMed

    Yuzaki, M

    2008-06-01

    The C1q family is characterized by a C-terminal conserved global C1q domain, which is structurally very similar to the tumor necrosis factor homology domain. Although some C1q family members are expressed in the central nervous system, their functions have not been well characterized. Cbln1, a member of the Cbln subfamily of the C1q family, is predominantly expressed in cerebellar granule cells. Interestingly, Cbln1 was recently shown to play two unique roles at excitatory synapses formed between cerebellar granule cells and Purkinje cells: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytosis pathway. Since other Cbln subfamily members, Cbln2-Cbln4, are expressed in various regions of developing and mature brains, Cbln subfamily proteins may generally serve as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions.

  8. The Nck family of adapter proteins: regulators of actin cytoskeleton.

    PubMed

    Buday, László; Wunderlich, Livius; Tamás, Peter

    2002-09-01

    SH2/SH3 domain-containing adapter proteins, such as the Nck family, play a major role in regulating tyrosine kinase signalling. They serve to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. Initially, it was not clear why cells from nematodes to vertebrates contain redundant and closely related SH2/SH3 adapters, such as Grb2, Crk and Nck. Recent evidence suggests that their biological roles are clearly different, whereas, for example, Grb2 connects activated receptor tyrosine kinases to Sos and Ras, leading to cell proliferation. The proteins of Nck family are implicated in organisation of actin cytoskeleton, cell movement or axon guidance in flies. In this review, the author attempts to summarise signalling pathways in which Nck plays a critical role.

  9. [Structure and function analysis of Arabidopsis thaliana SRO protein family].

    PubMed

    Li, Bao-Zhu; Zhao, Xiang; Zhao, Xiao-Liang; Peng, Lei

    2013-10-01

    Many biotic and abiotic stresses can cause oxidative stress in plants. The identification of components involved in plant response to oxidative stress has attracted wide attention. The members of AtSRO family, including AtRCD1, AtSRO1, and AtSRO5, regulate plants' response to oxidative stress. AtSROs participate in plant normal growth and development, and play important roles in plant response to stresses, such as drought, salt, heavy metal, and so on. In addition, AtSROs possess some special domains, including PARP and RST. It is speculated that AtSROs may function in regulating protein transcription, adjustment, and modification. This review highlights some recent progresses, such as basic situation of AtSROs, effects of AtSRO family proteins on plant growth and response to abiotic stress, which will provide a theoretical basis for further studying on biological functions of AtSRO.

  10. Investigating Structure and Dynamics of Atg8 Family Proteins.

    PubMed

    Weiergräber, O H; Schwarten, M; Strodel, B; Willbold, D

    2017-01-01

    Atg8 family members were the first autophagy-related proteins to be investigated in structural detail and continue to be among the best-understood molecules of the pathway. In this review, we will first provide a concise outline of the major methods that are being applied for structural characterization of these proteins and the complexes they are involved in. This includes a discussion of the strengths and limitations associated with each method, along with guidelines for successful adoption to a specific problem. Subsequently, we will present examples illustrating the application of these techniques, with a particular focus on the complementarity of information they provide. © 2017 Elsevier Inc. All rights reserved.

  11. Argonaute Family Protein Expression in Normal Tissue and Cancer Entities

    PubMed Central

    Bruckmann, Astrid; Hauptmann, Judith; Deutzmann, Rainer; Meister, Gunter; Bosserhoff, Anja Katrin

    2016-01-01

    The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease. PMID:27518285

  12. Web-based software for rapid "top-down" proteomic identification of protein biomarkers with implications for bacterial identification

    USDA-ARS?s Scientific Manuscript database

    We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption/ionization (MALDI), mass-isolated and fragmented using a time-of-flight/time-of-flight (TOF-TOF)...

  13. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response

    PubMed Central

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  14. Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

    PubMed Central

    Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

    2013-01-01

    Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

  15. Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila.

    PubMed Central

    abu Kwaik, Y; Fields, B S; Engleberg, N C

    1994-01-01

    Legionella pneumophila is ingested by both human macrophages and amoebae, and it multiplies within similar endocytic compartments in both eukaryotic species. Inhibitors of eukaryotic protein synthesis, such as cycloheximide and emetine, had no effect on the uptake of L. pneumophila by macrophages but completely abolished ingestion by the amoeba Hartmannella vermiformis. Therefore, host cell protein synthesis is required for the bacterium to infect the amoeba but not human macrophages. To identify proteins expressed by H. vermiformis upon contact with L. pneumophila, we radiolabeled amoebal proteins after contact with bacteria in bacteriostatic concentrations of tetracycline to inhibit bacterial protein synthesis. We analyzed protein expression by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found that 33 amoebal proteins were induced; 12 of these were not detected in resting amoebae. Eleven other amoebal proteins were repressed; four of them became undetectable. In contrast, no phenotypic changes were observed in H. vermiformis upon contact with Escherichia coli or heat-killed L. pneumophila. An isogenic, avirulent variant of L. pneumophila, incapable of infecting either macrophages or amoebae, induced a different pattern of protein expression upon contact with H. vermiformis. Our data showed that amoebae manifested a specific phenotypic response upon contact with virulent L. pneumophila. This phenotypic modulation may be necessary for uptake of the bacteria into an endocytic compartment that permits bacterial survival and multiplication. Images PMID:8168950

  16. A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

    PubMed Central

    Ittig, Simon J.; Schmutz, Christoph; Kasper, Christoph A.; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M. Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R.; Nigg, Erich A.

    2015-01-01

    Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. PMID:26598622

  17. The latent transforming growth factor beta binding protein (LTBP) family.

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

    The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers. PMID:11104663

  18. The ADF/cofilin family: actin-remodeling proteins.

    PubMed

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.

  19. Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology.

    PubMed

    Gatti-Lafranconi, Pietro; Natalello, Antonino; Ami, Diletta; Doglia, Silvia Maria; Lotti, Marina

    2011-07-01

    Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies. The recent discovery that aggregated proteins can retain native-like conformation and biological activity has opened the way for a dramatic change in the means by which intracellular aggregation is approached and exploited. This paper summarizes recent studies towards the direct use of inclusion bodies in biotechnology and for the detection of bottlenecks in the folding pathways of specific proteins. We also review the major biophysical methods available for revealing fine structural details of aggregated proteins and which information can be obtained through these techniques. © 2011 The Authors Journal compilation © 2011 FEBS.

  20. Molecular evolution of the ependymin protein family: a necessary update

    PubMed Central

    Suárez-Castillo, Edna C; García-Arrarás, José E

    2007-01-01

    Background Ependymin (Epd), the predominant protein in the cerebrospinal fluid of teleost fishes, was originally associated with neuroplasticity and regeneration. Ependymin-related proteins (Epdrs) have been identified in other vertebrates, including amphibians and mammals. Recently, we reported the identification and characterization of an Epdr in echinoderms, showing that there are ependymin family members in non-vertebrate deuterostomes. We have now explored multiple databases to find Epdrs in different metazoan species. Using these sequences we have performed genome mapping, molecular phylogenetic analyses using Maximum Likelihood and Bayesian methods, and statistical tests of tree topologies, to ascertain the phylogenetic relationship among ependymin proteins. Results Our results demonstrate that ependymin genes are also present in protostomes. In addition, as a result of the putative fish-specific genome duplication event and posterior divergence, the ependymin family can be divided into four groups according to their amino acid composition and branching pattern in the gene tree: 1) a brain-specific group of ependymin sequences that is unique to teleost fishes and encompasses the originally described ependymin; 2) a group expressed in non-brain tissue in fishes; 3) a group expressed in several tissues that appears to be deuterostome-specific, and 4) a group found in invertebrate deuterostomes and protostomes, with a broad pattern of expression and that probably represents the evolutionary origin of the ependymins. Using codon-substitution models to statistically assess the selective pressures acting over the ependymin protein family, we found evidence of episodic positive Darwinian selection and relaxed selective constraints in each one of the postduplication branches of the gene tree. However, purifying selection (with among-site variability) appears to be the main influence on the evolution of each subgroup within the family. Functional divergence among the

  1. Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization

    PubMed Central

    Wang, Yong; Tao, Xia-Fang; Su, Zhi-Xi; Liu, A-Ke; Liu, Tian-Lei; Sun, Ling; Yao, Qin; Chen, Ke-Ping; Gu, Xun

    2016-01-01

    Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5′-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5′-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories. PMID:27980385

  2. Flexibility in targeting and insertion during bacterial membrane protein biogenesis

    SciTech Connect

    Bloois, Edwin van; Hagen-Jongman, Corinne M. ten; Luirink, Joen

    2007-10-26

    The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.

  3. A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

    PubMed

    Ma, Cheng; Hao, Zhenyu; Huysmans, Gerard; Lesiuk, Amelia; Bullough, Per; Wang, Yingying; Bartlam, Mark; Phillips, Simon E; Young, James D; Goldman, Adrian; Baldwin, Stephen A; Postis, Vincent L G

    2015-01-01

    Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a

  4. Formyl-methionine as a degradation signal at the N-termini of bacterial proteins

    PubMed Central

    Piatkov, Konstantin I.; Vu, Tri T. M.; Hwang, Cheol-Sang; Varshavsky, Alexander

    2015-01-01

    In bacteria, all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. The fMet residue is cotranslationally deformylated by a ribosome-associated deformylase. The formylation of N-terminal Met in bacterial proteins is not strictly essential for either translation or cell viability. Moreover, protein synthesis by the cytosolic ribosomes of eukaryotes does not involve the formylation of N-terminal Met. What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the similarity of the formyl and acetyl groups, their identical locations in N terminally formylated (Nt formylated) and Nt-acetylated proteins, and the recently discovered proteolytic function of Nt-acetylation in eukaryotes might also signify a proteolytic role of Nt formylation in bacteria. We addressed this hypothesis about fMet based degradation signals, termed fMet/N-degrons, using specific E. coli mutants, pulse-chase degradation assays, and protein reporters whose deformylation was altered, through site-directed mutagenesis, to be either rapid or relatively slow. Our findings strongly suggest that the formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. We suggest that the faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N degrons, in contrast to the cotranslational design of analogous Ac/N degrons in eukaryotes. PMID:26866044

  5. A method for in vivo identification of bacterial small RNA-binding proteins.

    PubMed

    Osborne, Jonathan; Djapgne, Louise; Tran, Bao Quoc; Goo, Young Ah; Oglesby-Sherrouse, Amanda G

    2014-12-01

    Small bacterial regulatory RNAs (sRNAs) have gained immense appreciation over the last decade for their roles in mediating posttranscriptional gene regulation of numerous physiological processes. Several proteins contribute to sRNA stability and regulation, most notably the Hfq RNA-binding protein. However, not all sRNAs rely on Hfq for their stability. It is therefore likely that other proteins contribute to the stability and function of certain bacterial sRNAs. Here, we describe a methodology for identifying in vivo-binding proteins of sRNAs, developed using the iron-responsive PrrF and PrrH sRNAs of Pseudomonas aeruginosa. RNA was isolated from iron-depleted cultures, which were irradiated to cross-link nucleoprotein complexes. Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins. This method identified Hfq as a potential PrrF- and PrrH-binding protein. Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo. Hfq binding to the PrrF and PrrH sRNAs was validated by electrophoretic mobility shift assays with purified Hfq protein from P. aeruginosa. As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

  6. Molecular evolution of the EGF-CFC protein family.

    PubMed

    Ravisankar, V; Singh, Taran P; Manoj, Narayanan

    2011-08-15

    The epidermal growth factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, characterized by the highly conserved EGF and CFC domains, are extracellular membrane associated growth factor-like glycoproteins. These proteins are essential components of the Nodal signaling pathway during early vertebrate embryogenesis. Homologs of the EGF-CFC family have also been implicated in tumorigenesis in humans. Yet, little is known about the mode of molecular evolution in this family. Here we investigate the origin, extent of conservation and evolutionary relationships of EGF-CFC proteins across the metazoa. The results suggest that the first appearance of the EGF-CFC gene occurred in the ancestor of the deuterostomes. Phylogenetic analysis supports the classification of the family into distinct subfamilies that appear to have evolved through lineage-specific duplication and divergence. Site-specific analyses of evolutionary rate shifts between the two major mammalian paralogous subfamilies, Cripto and Cryptic, reveal critical amino acid sites that may account for the observed functional divergence. Furthermore, estimates of functional divergence suggest that rapid change of evolutionary rates at sites located mainly in the CFC domain may contribute towards distinct functional properties of the two paralogs. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Calcium channel gamma subunits: a functionally diverse protein family.

    PubMed

    Chen, Ren-Shiang; Deng, Tzyy-Chyn; Garcia, Thomas; Sellers, Zachary M; Best, Philip M

    2007-01-01

    The calcium channel gamma subunits comprise an eight-member protein family that share a common topology consisting of four transmembrane domains and intracellular N- and C-termini. Although the first gamma subunit was identified as an auxiliary subunit of a voltage-dependent calcium channel, a review of phylogenetic, bioinformatic, and functional studies indicates that they are a functionally diverse protein family. A cluster containing gamma1 and gamma6 conforms to the original description of the protein family as they seem to act primarily as subunits of calcium channels expressed in muscle. Members of a second cluster (gamma2, gamma3, gamma4, gamma8) function as regulators of AMPA receptor localization and function in the brain and are collectively known as TARPs. The function of members of the third cluster (gamma5, gamma7) remains unclear. Our analysis shows that the members of each cluster contain conserved regulatory motifs that help to differentiate the groups. However, the physiological significance of these motifs in many cases remains to be demonstrated.

  8. Characterization of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Mutations in Familial Isolated Pituitary Adenoma Families

    PubMed Central

    Igreja, Susana; Chahal, Harvinder S; King, Peter; Bolger, Graeme B; Srirangalingam, Umasuthan; Guasti, Leonardo; Chapple, J Paul; Trivellin, Giampaolo; Gueorguiev, Maria; Guegan, Katie; Stals, Karen; Khoo, Bernard; Kumar, Ajith V; Ellard, Sian; Grossman, Ashley B; Korbonits, Márta

    2010-01-01

    Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation-negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc. PMID:20506337

  9. UBXD Proteins: A Family of Proteins with Diverse Functions in Cancer

    PubMed Central

    Rezvani, Khosrow

    2016-01-01

    The UBXD family is a diverse group of UBX (ubiquitin-regulatory X) domain-containing proteins in mammalian cells. Members of this family contain a UBX domain typically located at the carboxyl-terminal of the protein. In contrast to the UBX domain shared by all members of UBXD family, the amino-terminal domains are diverse and appear to carry out different roles in a subcellular localization-dependent manner. UBXD proteins are principally associated with the endoplasmic reticulum (ER), where they positively or negatively regulate the ER-associated degradation machinery (ERAD). The distinct protein interaction networks of UBXD proteins allow them to have specific functions independent of the ERAD pathway in a cell type- and tissue context-dependent manner. Recent reports have illustrated that a number of mammalian members of the UBXD family play critical roles in several proliferation and apoptosis pathways dysregulated in selected types of cancer. This review covers recent advances that elucidate the therapeutic potential of selected members of the UBXD family that can contribute to tumor growth. PMID:27754413

  10. Exploring Bacterial Organelle Interactomes: A Model of the Protein-Protein Interaction Network in the Pdu Microcompartment

    PubMed Central

    Jorda, Julien; Liu, Yu; Bobik, Thomas A.; Yeates, Todd O.

    2015-01-01

    Bacterial microcompartments (MCPs) are protein-bound organelles that carry out diverse metabolic pathways in a wide range of bacteria. These supramolecular assemblies consist of a thin outer protein shell, reminiscent of a viral capsid, which encapsulates sequentially acting enzymes. The most complex MCP elucidated so far is the propanediol utilizing (Pdu) microcompartment. It contains the reactions for degrading 1,2-propanediol. While several experimental studies on the Pdu system have provided hints about its organization, a clear picture of how all the individual components interact has not emerged yet. Here we use co-evolution-based methods, involving pairwise comparisons of protein phylogenetic trees, to predict the protein-protein interaction (PPI) network governing the assembly of the Pdu MCP. We propose a model of the Pdu interactome, from which selected PPIs are further inspected via computational docking simulations. We find that shell protein PduA is able to serve as a “universal hub” for targeting an array of enzymes presenting special N-terminal extensions, namely PduC, D, E, L and P. The varied N-terminal peptides are predicted to bind in the same cleft on the presumptive luminal face of the PduA hexamer. We also propose that PduV, a protein of unknown function with remote homology to the Ras-like GTPase superfamily, is likely to localize outside the MCP, interacting with the protruding β-barrel of the hexameric PduU shell protein. Preliminary experiments involving a bacterial two-hybrid assay are presented that corroborate the existence of a PduU-PduV interaction. This first systematic computational study aimed at characterizing the interactome of a bacterial microcompartment provides fresh insight into the organization of the Pdu MCP. PMID:25646976

  11. A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP.

    PubMed

    Spanò, Stefania; Gao, Xiang; Hannemann, Sebastian; Lara-Tejero, María; Galán, Jorge E

    2016-02-10

    Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. The PIN-FORMED (PIN) protein family of auxin transporters

    PubMed Central

    2009-01-01

    Summary The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies. PMID:20053306

  13. IQGAP1 Interaction with RHO Family Proteins Revisited

    PubMed Central

    Nouri, Kazem; Fansa, Eyad K.; Amin, Ehsan; Dvorsky, Radovan; Gremer, Lothar; Willbold, Dieter; Schmitt, Lutz; Timson, David J.; Ahmadian, Mohammad R.

    2016-01-01

    IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses. PMID:27815503

  14. The innate immune protein Nod2 binds directly to MDP, a bacterial cell wall fragment.

    PubMed

    Grimes, Catherine Leimkuhler; Ariyananda, Lushanti De Zoysa; Melnyk, James E; O'Shea, Erin K

    2012-08-22

    Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn's disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.

  15. Bacterial SET domain proteins and their role in eukaryotic chromatin modification

    PubMed Central

    Alvarez-Venegas, Raúl

    2014-01-01

    It has been shown by many researchers that SET-domain containing proteins modify chromatin structure and, as expected, genes coding for SET-domain containing proteins have been found in all eukaryotic genomes sequenced to date. However, during the last years, a great number of bacterial genomes have been sequenced and an important number of putative genes involved in histone post-translational modifications (histone PTMs) have been identified in many bacterial genomes. Here, I aim at presenting an overview of SET domain genes that have been identified in numbers of bacterial genomes based on similarity to SET domains of eukaryotic histone methyltransferases. I will argue in favor of the hypothesis that SET domain genes found in extant bacteria are of bacterial origin. Then, I will focus on the available information on pathogen and symbiont SET-domain containing proteins and their targets in eukaryotic organisms, and how such histone methyltransferases allow a pathogen to inhibit transcriptional activation of host defense genes. PMID:24765100

  16. The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues.

    PubMed

    Bork, P; Brown, N P; Hegyi, H; Schultz, J

    1996-07-01

    A thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs. These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE. Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.

  17. Bacterial filtration efficiency of green soy protein based nanofiber air filter.

    PubMed

    Lubasova, D; Netravali, A; Parker, J; Ingel, B

    2014-07-01

    High bacterial filtration efficiency (BFE) filters, based on nanofibers derived from blends of grain proteins and poly-ethylene-oxide (PEO), were produced by an electrospinning process. Specifically, polymer blends consisting of purified soy flour/PEO with a ratio of 7/3 were spun into nanofibers and characterized. A new laboratory based experimental apparatus for testing BFE was designed and used to test BFE of bacterial aerosols consisting of Escherichia coli (E. coli). Performances of soy protein based nanofiber filters with nanofiber mass varying from 1 to 5 g/m2 as well as a nanofiber filters prepared from pure PEO were compared. The results showed that BFE values for filters containing 5 g/m2 protein based nanofibers and PEO nanofiber filter were 100 and 81.5%, respectively. The results also indicated that the BFE increased as the protein content in the nanofiber filter increased. These novel protein based nanofiber filters have demonstrated a clear potential for effective removal and retention of E. coli bacteria during air-filtration. These filters can be effectively deployed in environments such as hospitals and senior residential areas to reduce bacterial infections.

  18. A phage protein that inhibits the bacterial ATPase required for type IV pilus assembly.

    PubMed

    Chung, In-Young; Jang, Hye-Jeong; Bae, Hee-Won; Cho, You-Hee

    2014-08-05

    Type IV pili (TFPs) are required for bacterial twitching motility and for phage infection in the opportunistic human pathogen Pseudomonas aeruginosa. Here we describe a phage-encoded protein, D3112 protein gp05 (hereafter referred to as Tip, representing twitching inhibitory protein), whose expression is necessary and sufficient to mediate the inhibition of twitching motility. Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. Based on these observations and the fact that many P. aeruginosa phages require TFPs for infection, Tip-mediated twitching inhibition may represent a generalized strategy for superinfection exclusion. Moreover, because TFPs are required for full virulence, PilB may be an attractive target for the development of novel antiinfectives.

  19. Detection of bacterial protein toxins by solid phase magnetic immunocapture and mass spectrometry.

    PubMed

    Pocsfalvi, Gabriella; Schlosser, Gitta

    2011-01-01

    Bacterial protein toxins are involved in a number of infectious and foodborne diseases and are considered as potential biological warfare agents as well. Their sensitive multiplex detection in complex environmental, food, and biological samples are an important although challenging task. Solid-phase immunoaffinity capture provides an efficient way to enrich and purify a wide range of proteins from complex mixtures. We have shown that staphylococcal enterotoxins, for example, can be efficiently enriched by means of magnetic immunocapture using antibody functionalized paramagnetic beads. The method was successfully interfaced by the on-beads and off-beads detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry at the protein level and by the off-beads nano-electrospray ionization-MS/MS detection at the enzyme digests level, enabling thus the unambiguous identification of the toxin. The method is applicable to any bacterial toxin to which an antibody is available.

  20. Vav Family Proteins Couple to Diverse Cell Surface Receptors

    PubMed Central

    Moores, Sheri L.; Selfors, Laura M.; Fredericks, Jessica; Breit, Timo; Fujikawa, Keiko; Alt, Frederick W.; Brugge, Joan S.; Swat, Wojciech

    2000-01-01

    Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFκB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways. PMID:10938113

  1. Channel crossing: how are proteins shipped across the bacterial plasma membrane?

    PubMed Central

    Collinson, Ian; Corey, Robin A.; Allen, William J.

    2015-01-01

    The structure of the first protein-conducting channel was determined more than a decade ago. Today, we are still puzzled by the outstanding problem of protein translocation—the dynamic mechanism underlying the consignment of proteins across and into membranes. This review is an attempt to summarize and understand the energy transducing capabilities of protein-translocating machines, with emphasis on bacterial systems: how polypeptides make headway against the lipid bilayer and how the process is coupled to the free energy associated with ATP hydrolysis and the transmembrane protein motive force. In order to explore how cargo is driven across the membrane, the known structures of the protein-translocation machines are set out against the background of the historic literature, and in the light of experiments conducted in their wake. The paper will focus on the bacterial general secretory (Sec) pathway (SecY-complex), and its eukaryotic counterpart (Sec61-complex), which ferry proteins across the membrane in an unfolded state, as well as the unrelated Tat system that assembles bespoke channels for the export of folded proteins. PMID:26370937

  2. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    PubMed

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.

  3. Mass Spectrometry and Tandem Mass Spectrometry for Protein Biomarker Discovery and Bacterial Speciation

    NASA Astrophysics Data System (ADS)

    Fox, Alvin; Fox, Karen

    After culture, MALDI-MS protein profiling, for species characterization, is widely used. DNA-based identification of bacterial species (with or without prior culture) often involves PCR and/or sequencing. 16S rRNA sequence cataloging is the gold standard but discrimination is often only at the genus level. This chapter discusses protein marker discovery and chemotaxonomy for threat agents using MS and MS/MS. Characterization of small acid soluble proteins (SASPs) of Bacillus anthracis and related species are used for illustrative purposes. The ultimate goal of our studies is universal applicability with species-level certainty in these identifications including biodetection without culture.

  4. Predicting gram-positive bacterial protein subcellular localization based on localization motifs.

    PubMed

    Hu, Yinxia; Li, Tonghua; Sun, Jiangming; Tang, Shengnan; Xiong, Wenwei; Li, Dapeng; Chen, Guanyan; Cong, Peisheng

    2012-09-07

    The subcellular localization of proteins is closely related to their functions. In this work, we propose a novel approach based on localization motifs to improve the accuracy of predicting subcellular localization of Gram-positive bacterial proteins. Our approach performed well on a five-fold cross validation with an overall success rate of 89.5%. Besides, the overall success rate of an independent testing dataset was 97.7%. Moreover, our approach was tested using a new experimentally-determined set of Gram-positive bacteria proteins and achieved an overall success rate of 96.3%.

  5. Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds*

    PubMed Central

    Wang, Dai; Zetterström, Caroline E.; Gabrielsen, Mads; Beckham, Katherine S. H.; Tree, Jai J.; Macdonald, Sarah E.; Byron, Olwyn; Mitchell, Tim J.; Gally, David L.; Herzyk, Pawel; Mahajan, Arvind; Uvell, Hanna; Burchmore, Richard; Smith, Brian O.; Elofsson, Mikael; Roe, Andrew J.

    2011-01-01

    A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157. PMID:21724850

  6. Molecular imprinted polymer with cloned bacterial protein template enriches authentic target in cell extract.

    PubMed

    Zhao, Zhuo; Wang, Chunhong; Guo, Minjie; Shi, Linqi; Fan, Yunge; Long, Yi; Mi, Huaifeng

    2006-05-15

    Here we describe a new method for preparing a protein-imprinted polymer with a cloned bacterial protein template, which recognizes/adsorbs authentic target protein present at a relatively low level in cell extract. In this work, cloned pig cyclophilin 18 (pCyP18) was used as a template. The template protein was selectively assembled with memory molecules from their library, which consists of numerous limited length polymer chains with randomly distributed recognition sites and immobilizing sites. These assemblies of protein and memory molecules were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, binding sites that were complementary to the target protein in size, shape and the position of recognition groups were exposed, and their confirmation was preserved by the cross-linked structure. The synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 300 times.

  7. Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

    PubMed Central

    Soler, Nicolas; Marguet, Evelyne; Cortez, Diego; Desnoues, Nicole; Keller, Jenny; van Tilbeurgh, Herman; Sezonov, Guennadi; Forterre, Patrick

    2010-01-01

    Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication. PMID:20403814

  8. Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family

    PubMed Central

    Hübscher, Judith; Lüthy, Lucas; Berger-Bächi, Brigitte; Stutzmann Meier, Patricia

    2008-01-01

    Background The bacterial cell wall is the target of many antibiotics and cell envelope constituents are critical to host-pathogen interactions. To combat resistance development and virulence, a detailed knowledge of the individual factors involved is essential. Members of the LytR-CpsA-Psr family of cell envelope-associated attenuators are relevant for β-lactam resistance, biofilm formation, and stress tolerance, and they are suggested to play a role in cell wall maintenance. However, their precise function is still unknown. This study addresses the occurrence as well as sequence-based characteristics of the LytR-CpsA-Psr proteins. Results A comprehensive list of LytR-CpsA-Psr proteins was established, and their phylogenetic distribution and clustering into subgroups was determined. LytR-CpsA-Psr proteins were present in all Gram-positive organisms, except for the cell wall-deficient Mollicutes and one strain of the Clostridiales. In contrast, the majority of Gram-negatives did not contain LytR-CpsA-Psr family members. Despite high sequence divergence, the LytR-CpsA-Psr domains of different subclusters shared a highly similar, predicted mixed a/β-structure, and conserved charged residues. PhoA fusion experiments, using MsrR of Staphylococcus aureus, confirmed membrane topology predictions and extracellular location of its LytR-CpsA-Psr domain. Conclusion The LytR-CpsA-Psr domain is unique to bacteria. The presence of diverse subgroups within the LytR-CpsA-Psr family might indicate functional differences, and could explain variations in phenotypes of respective mutants reported. The identified conserved structural elements and amino acids are likely to be important for the function of the domain and will help to guide future studies of the LytR-CpsA-Psr proteins. PMID:19099556

  9. Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

    PubMed Central

    2010-01-01

    Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST) method uses all-against-all substructure comparison to determine Substructural Clusters (SCs). SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH) framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated, statistically rigorous procedure

  10. Bioinformatic analysis of the TonB protein family.

    PubMed

    Chu, Byron C H; Peacock, R Sean; Vogel, Hans J

    2007-06-01

    TonB is a protein prevalent in a large number of Gram-negative bacteria that is believed to be responsible for the energy transduction component in the import of ferric iron complexes and vitamin B(12) across the outer membrane. We have analyzed all the TonB proteins that are currently contained in the Entrez database and have identified nine different clusters based on its conserved 90-residue C-terminal domain amino acid sequence. The vast majority of the proteins contained a single predicted cytoplasmic transmembrane domain; however, nine of the TonB proteins encompass a approximately 290 amino acid N-terminal extension homologous to the MecR1 protein, which is composed of three additional predicted transmembrane helices. The periplasmic linker region, which is located between the N-terminal domain and the C-terminal domain, is extremely variable both in length (22-283 amino acids) and in proline content, indicating that a Pro-rich domain is not a required feature for all TonB proteins. The secondary structure of the C-terminal domain is found to be well preserved across all families, with the most variable region being between the second alpha-helix and the third beta-strand of the antiparallel beta-sheet. The fourth beta-strand found in the solution structure of the Escherichia coli TonB C-terminal domain is not a well conserved feature in TonB proteins in most of the clusters. Interestingly, several of the TonB proteins contained two C-terminal domains in series. This analysis provides a framework for future structure-function studies of TonB, and it draws attention to the unusual features of several TonB proteins.

  11. Evolutionary conservation and diversification of Rh family genes and proteins

    PubMed Central

    Huang, Cheng-Han; Peng, Jianbin

    2005-01-01

    Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{3}/{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document}, whereas others think that Rh proteins transport CO2 and Amt proteins NH3. In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution. PMID:16227429

  12. Evolutionary conservation and diversification of Rh family genes and proteins.

    PubMed

    Huang, Cheng-Han; Peng, Jianbin

    2005-10-25

    Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, NH(3)/NH(4)(+), whereas others think that Rh proteins transport CO(2) and Amt proteins NH(3). In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution.

  13. Bacterial protein interaction networks: puzzle stones from solved complex structures add to a clearer picture.

    PubMed

    Terradot, Laurent; Noirot-Gros, Marie-Francoise

    2011-06-01

    Global scale studies of protein-protein interaction (PPI) networks have considerably expanded our view of how proteins act in the cell. In particular, bacterial "interactome" surveys have revealed that proteins can sometimes interact with a large number of protein partners and connect different cellular processes. More targeted, pathway-orientated PPI studies have also helped to propose functions for unknown proteins based on the "guilty by association" principle. However, given the immense repertoire of PPIs generated and the variability of PPI networks, more studies are required to understand the role(s) of these interactions in the cell. With the availability of bioinformatic analysis tools, transcriptomics and co-expression experiments for a given interaction, interactomes are being deciphered. More recently, functional and structural studies have been derived from these PPI networks. In this review, we will give a number of examples of how combining functional and structural studies into PPI networks has contributed to understanding the functions of some of these interactions. We discuss how interactomes now represent a unique opportunity to determine the structures of bacterial protein complexes on a large scale by the integration of multiple technologies. This journal is © The Royal Society of Chemistry 2011

  14. Clinical Prognosis in Neonatal Bacterial Meningitis: The Role of Cerebrospinal Fluid Protein

    PubMed Central

    Zhao, Dongying; Ren, Fang; Luo, Zhongcheng; Zhang, Yongjun

    2015-01-01

    Neonates are at high risk of meningitis and of resulting neurologic complications. Early recognition of neonates at risk of poor prognosis would be helpful in providing timely management. From January 2008 to June 2014, we enrolled 232 term neonates with bacterial meningitis admitted to 3 neonatology departments in Shanghai, China. The clinical status on the day of discharge from these hospitals or at a postnatal age of 2.5 to 3 months was evaluated using the Glasgow Outcome Scale (GOS). Patients were classified into two outcome groups: good (167 cases, 72.0%, GOS = 5) or poor (65 cases, 28.0%, GOS = 1–4). Neonates with good outcome had less frequent apnea, drowsiness, poor feeding, bulging fontanelle, irritability and more severe jaundice compared to neonates with poor outcome. The good outcome group also had less pneumonia than the poor outcome group. Besides, there were statistically significant differences in hemoglobin, mean platelet volume, platelet distribution width, C-reaction protein, procalcitonin, cerebrospinal fluid (CSF) glucose and CSF protein. Multivariate logistic regression analyses suggested that poor feeding, pneumonia and CSF protein were the predictors of poor outcome. CSF protein content was significantly higher in patients with poor outcome. The best cut-offs for predicting poor outcome were 1,880 mg/L in CSF protein concentration (sensitivity 70.8%, specificity 86.2%). After 2 weeks of treatment, CSF protein remained higher in the poor outcome group. High CSF protein concentration may prognosticate poor outcome in neonates with bacterial meningitis. PMID:26509880

  15. WASP Family Proteins: Their Evolution and Its Physiological Implications

    PubMed Central

    Veltman, Douwe M.

    2010-01-01

    WASP family proteins control actin polymerization by activating the Arp2/3 complex. Several subfamilies exist, but their regulation and physiological roles are not well understood, nor is it even known if all subfamilies have been identified. Our extensive search reveals few novel WASP family proteins. The WASP, WASH, and SCAR/WAVE subfamilies are evolutionarily ancient, with WASH the most universally present, whereas WHAMM/JMY first appears in invertebrates. An unusual Dictyostelium WASP homologue that has lost the WH1 domain has retained its function in clathrin-mediated endocytosis, demonstrating that WASPs can function with a remarkably diverse domain topology. The WASH and SCAR/WAVE regulatory complexes are much more rigidly maintained; their domain topology is highly conserved, and all subunits are present or lost together, showing that the complexes are ancient and functionally interdependent. Finally, each subfamily has a distinctive C motif, indicating that this motif plays a specific role in each subfamily's function, unlike the generic V and A motifs. Our analysis identifies which features are universally conserved, and thus essential, and which are branch-specific modifications. It also shows the WASP family is more widespread and diverse than currently appreciated and unexpectedly biases the physiological role of the Arp2/3 complex toward vesicle traffic. PMID:20573979

  16. Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

    PubMed Central

    2011-01-01

    Many bacterial species contain intracellular nano- and micro-compartments consisting of self-assembling proteins that form protein-only shells. These structures are built up by combinations of a reduced number of repeated elements, from 60 repeated copies of one unique structural element self-assembled in encapsulins of 24 nm to 10,000-20,000 copies of a few protein species assembled in a organelle of around 100-150 nm in cross-section. However, this apparent simplicity does not correspond to the structural and functional sophistication of some of these organelles. They package, by not yet definitely solved mechanisms, one or more enzymes involved in specific metabolic pathways, confining such reactions and sequestering or increasing the inner concentration of unstable, toxics or volatile intermediate metabolites. From a biotechnological point of view, we can use the self assembling properties of these particles for directing shell assembling and enzyme packaging, mimicking nature to design new applications in biotechnology. Upon appropriate engineering of the building blocks, they could act as a new family of self-assembled, protein-based vehicles in Nanomedicine to encapsulate, target and deliver therapeutic cargoes to specific cell types and/or tissues. This would provide a new, intriguing platform of microbial origin for drug delivery. PMID:22046962

  17. Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

    PubMed Central

    Noda, Yoichi; Hara, Takehiro; Ishii, Minako; Yoda, Koji

    2014-01-01

    ABSTRACT The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases. PMID:24585773

  18. Bacterial TIR-containing proteins and host innate immune system evasion.

    PubMed

    Rana, Rohini R; Zhang, Minghao; Spear, Abigail M; Atkins, Helen S; Byrne, Bernadette

    2013-02-01

    The innate immune system provides the first line of host defence against invading pathogens. Key to upregulation of the innate immune response are Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) and trigger a signaling pathway culminating in the production of inflammatory mediators. Central to this TLR signaling pathway are heterotypic protein-protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms. Recent work has identified a potentially novel evasion mechanism involving bacterial TIR domain proteins. Such domains have been identified in a wide range of pathogenic bacteria, and there is evidence to suggest that they interfere directly with the TLR signaling pathway and thus inhibit the activation of NF-κB. The individual TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis, Brucella sp, uropathogenic E. coli and Yersinia pestis have been analyzed in detail. The individual bacterial TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of the bacterial TIR domains.

  19. Super-resolution imaging of the bacterial cytokinetic protein FtsZ.

    PubMed

    Jennings, Phoebe C; Cox, Guy C; Monahan, Leigh G; Harry, Elizabeth J

    2011-06-01

    The idea of a bacterial cytoskeleton arose just 10 years ago with the identification of the cell division protein, FtsZ, as a tubulin homolog. FtsZ plays a pivotal role in bacterial division, and is present in virtually all prokaryotes and in some eukaryotic organelles. The earliest stage of bacterial cell division is the assembly of FtsZ into a Z ring at the division site, which subsequently constricts during cytokinesis. FtsZ also assembles into dynamic helical structures along the bacterial cell, which are thought to act as precursors to the Z ring via a cell cycle-mediated FtsZ polymer remodelling. The fine structures of the FtsZ helix and ring are unknown but crucial for identifying the molecular details of Z ring assembly and its regulation. We now reveal using STED microscopy that the FtsZ helical structure in cells of the gram positive bacterium, Bacillus subtilis, is a highly irregular and discontinuous helix of FtsZ; very different to the smooth cable-like appearance observed by conventional fluorescence optics. STED also identifies a novel FtsZ helical structure of smaller pitch that is invisible to standard optical methods, identifying a possible third intermediate in the pathway to Z ring assembly, which commits bacterial cells to divide.

  20. The Origin and Evolution of the Plant Cell Surface: Algal Integrin-Associated Proteins and a New Family of Integrin-Like Cytoskeleton-ECM Linker Proteins

    PubMed Central

    Becker, Burkhard; Doan, Jean Michel; Wustman, Brandon; Carpenter, Eric J.; Chen, Li; Zhang, Yong; Wong, Gane K.-S.; Melkonian, Michael

    2015-01-01

    The extracellular matrix of scaly green flagellates consists of small organic scales consisting of polysaccharides and scale-associated proteins (SAPs). Molecular phylogenies have shown that these organisms represent the ancestral stock of flagellates from which all green plants (Viridiplantae) evolved. The molecular characterization of four different SAPs is presented. Three SAPs are type-2 membrane proteins with an arginine/alanine-rich short cytoplasmic tail and an extracellular domain that is most likely of bacterial origin. The fourth protein is a filamin-like protein. In addition, we report the presence of proteins similar to the integrin-associated proteins α-actinin (in transcriptomes of glaucophytes and some viridiplants), LIM-domain proteins, and integrin-associated kinase in transcriptomes of viridiplants, glaucophytes, and rhodophytes. We propose that the membrane proteins identified are the predicted linkers between scales and the cytoskeleton. These proteins are present in many green algae but are apparently absent from embryophytes. These proteins represent a new protein family we have termed gralins for green algal integrins. Gralins are absent from embryophytes. A model for the evolution of the cell surface proteins in Plantae is discussed. PMID:25977459

  1. [Identification of proteins interacting with the circadian clock protein PER1 in tumors using bacterial two-hybrid system technique].

    PubMed

    Zhang, Yu; Yao, Youlin; Jiang, Siyuan; Lu, Yilu; Liu, Yunqiang; Tao, Dachang; Zhang, Sizhong; Ma, Yongxin

    2015-04-01

    To identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique. Human cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed. Fourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins. Identification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.

  2. Structural basis for protein–protein interactions in the 14-3-3 protein family

    PubMed Central

    Yang, Xiaowen; Lee, Wen Hwa; Sobott, Frank; Papagrigoriou, Evangelos; Robinson, Carol V.; Grossmann, J. Günter; Sundström, Michael; Doyle, Declan A.; Elkins, Jonathan M.

    2006-01-01

    The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein–protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners. PMID:17085597

  3. Role of Key Salt Bridges in Thermostability of G. thermodenitrificans EstGtA2: Distinctive Patterns within the New Bacterial Lipolytic Enzyme Family XV

    PubMed Central

    Charbonneau, David M.; Beauregard, Marc

    2013-01-01

    Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15th family (family XV) and suggest criteria for the assignation of protein sequences to the N’ subfamily. Five selected salt bridges, hallmarks of the N’ subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K→A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing Tm by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N’ subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for

  4. All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition

    PubMed Central

    Kinomoto, Masanobu; Kanno, Takayuki; Shimura, Mari; Ishizaka, Yukihito; Kojima, Asato; Kurata, Takeshi; Sata, Tetsutaro; Tokunaga, Kenzo

    2007-01-01

    Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements. PMID:17439959

  5. [The importance of ADAM family proteins in malignant tumors].

    PubMed

    Walkiewicz, Katarzyna; Gętek, Monika; Muc-Wierzgoń, Małgorzata; Kokot, Teresa; Nowakowska-Zajdel, Ewa

    2016-02-11

    Increasing numbers of reports about the role of adamalysins (ADAM) in malignant tumors are being published. To date, more than 30 representatives of this group, out of which about 20 occur in humans, have been described. The ADAM family is a homogeneous group of proteins which regulate, from the stage of embryogenesis, a series of processes such as cell migration, adhesion, and cell fusion. Half of them have proteolytic activity and are involved in the degradation of the extracellular matrix and the disintegration of certain protein complexes, thereby regulating the bioavailability of various growth factors. Many of these functions have a direct role in the processes of carcinogenesis and promoting the growth of tumor, which affect some signaling pathways, including those related to insulin-like growth factors (IGF1, IGF2), vascular growth factor (VEGF), tumor necrosis factor α (TNFα) and the EGFR/HER pathway. Another branch of studies is the evaluation of the possibility of using members of ADAM family proteins in the diagnosis, especially in breast, colon and non- small cell lung cancer. The detection of concentrations of adamalysin in serum, urine and pleural aspirates might contribute to the development of methods of early diagnosis of cancer and monitoring the therapy. However, both the role of adamalysins in the development and progression of tumors and their importance as a diagnostic and predictive further research still need to be checked on large groups of patients.

  6. Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins

    PubMed Central

    Hassan, Karl A.; Jackson, Scott M.; Penesyan, Anahit; Patching, Simon G.; Tetu, Sasha G.; Eijkelkamp, Bart A.; Brown, Melissa H.; Henderson, Peter J. F.; Paulsen, Ian. T.

    2013-01-01

    Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other γ- or β-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [14C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters. PMID:24277845

  7. Development of efficient expression system for protein display on bacterial magnetic particles.

    PubMed

    Yoshino, Tomoko; Matsunaga, Tadashi

    2005-12-30

    Bacterial magnetic particles (BMPs) are utilized for various biomedical applications because they are easily manipulated by magnets, and functional proteins are easily displayed on BMPs. To establish highly expressed protein display on BMPs, strong promoters were identified using Magnetospirillum magneticum AMB-1 genome and proteome databases. Initially, several proteins highly expressed in AMB-1 were identified, and the upstream DNA sequences of the open-reading frames were evaluated using a luciferase-reporter gene assay to compare promoter activities. Consequently, luminescence intensity was 400 times higher due to the novel promoter identified in this study than the magA promoter previously used. Subsequently, efficient protein display on BMPs was performed using the newly identified promoter sequences. This developed display system will facilitate the assembly of various functional proteins onto BMPs to create novel magnetic nanoparticles.

  8. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.

    PubMed

    Rapoport, Tom A

    2007-11-29

    A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.

  9. A novel protein tightly bound to bacterial magnetic particles in Magnetospirillum magneticum strain AMB-1.

    PubMed

    Arakaki, Atsushi; Webb, John; Matsunaga, Tadashi

    2003-03-07

    Magnetic bacteria synthesize magnetite crystals with species-dependent morphologies. The molecular mechanisms that control nano-sized magnetite crystal formation and the generation of diverse morphologies are not well understood. From the analysis of magnetite crystal-associated proteins, several low molecular mass proteins tightly bound to bacterial magnetite were obtained from Magnetospirillum magneticum strain AMB-1. These proteins showed common features in their amino acid sequences, which contain hydrophobic N-terminal and hydrophilic C-terminal regions. The C-terminal regions in Mms5, Mms6, Mms7, and Mms13 contain dense carboxyl and hydroxyl groups that bind iron ions. Nano-sized magnetic particles similar to those in magnetic bacteria were prepared by chemical synthesis of magnetite in the presence of the acidic protein Mms6. These proteins may be directly involved in biological magnetite crystal formation in magnetic bacteria.

  10. [Bacterial SXT/R391 family from integrating conjugative elements--a review].

    PubMed

    Luo, Peng; Hu, Chaoqun

    2014-05-04

    SXT/R391 family has the most abundant types and members in integrating conjugative elements (ICE). SXT/ R391 elements are comprised of conservative core genes and genes in variable regions. The functions of conservative core genes of SXT/R391 include integration and excision, self-transfer through conjugation, and regulation of its expression. The genes in the variable regions often encode for drug and heavy metal resistances, forming of biofilm, adjustment of bacterial motility, and toxin-antitoxin systems that prevent SXT/R391 deletion from hosts. Some genes in variable region of SXT/R391 also encode for restriction-modification system, helicase, and endonuclease. The activity of SXT/R391 is positively regulated by activator SetCD, and negatively regulated by repressor SetR. SXT/R391 cannot be easily deleted from the primary donors in the process of transfer. SXT/R391 prevent the acquirement of closely related and homogeneous elements but cannot prevent the acquirement of heterogenetic ICE, which leads to the generation of hybrid ICE under the action of recombination system encoded by SXT/R391 themselves. SXT/R391 have high transferable frequency and wide host range, and until now more than 40 different SXT/R391 elements have been discovered in various bacteria, especially in Vibrio species, which mainly distribute in coastal areas in Asia and Africa. It suggests that marine environments are likely the main reservoir for SXT/R391 and these elements probably spread from marine environmental strains to clinical strains, under increasing selective pressure. Due to the hazard caused by the prevalence and the transfer of SXT/R391, medical microbiologist and health departments should be fully alert to the spread of the elements.

  11. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes.

    PubMed

    Thaller, M C; Lombardi, G; Berlutti, F; Schippa, S; Rossolini, G M

    1995-01-01

    The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

  12. Muscarinic receptor family interacting proteins: role in receptor function.

    PubMed

    Borroto-Escuela, Dasiel O; Correia, Patrícia A; Romero-Fernandez, Wilber; Narvaez, Manuel; Fuxe, Kjell; Ciruela, Francisco; Garriga, Pere

    2011-02-15

    G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

  13. Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

    PubMed

    Chen, Chaoxiang; Zhang, Xiang; Zhang, Shuyue; Zhu, Shaobin; Xu, Jingyi; Zheng, Yan; Han, Jinyan; Zeng, Jin-Zhang; Yan, Xiaomei

    2015-12-15

    Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.

  14. Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis.

    PubMed

    Steelman, Zachary; Meng, Zhaokai; Traverso, Andrew J; Yakovlev, Vladislav V

    2015-05-01

    Bacterial meningitis is a disease of pronounced clinical significance, especially in the developing world. Immediate treatment with antibiotics is essential, and no single test can provide a conclusive diagnosis. It is well established that elevated total protein in cerebrospinal fluid (CSF) is associated with bacterial meningitis. Brillouin spectroscopy is a widely used optical technique for noninvasive determination of the elastic moduli of materials. We found that elevated protein levels in CSF alter the fluid elasticity sufficiently to be measurable by Brillouin spectroscopy, with model healthy and diseased fluids distinguishable to marked significance (P = 0.014), which increases with sample concentration by dialysis. Typical raw output of a 2-stage VIPA Brillouin spectrometer: inelastically scattered Brillouin peaks (arrows) and elastically scattered incident radiation (center cross).

  15. [The roles of epigenetics and protein post-translational modifications in bacterial antibiotic resistance].

    PubMed

    Xie, Longxiang; Yu, Zhaoxiao; Guo, Siyao; Li, Ping; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2015-08-01

    The increasing antibiotic resistance is now threatening to take us back to a pre-antibiotic era. Bacteria have evolved diverse resistance mechanisms, on which in-depth research could help the development of new strategies to control antibiotic-resistant infections. Epigenetic alterations and protein post-translational modifications (PTMs) play important roles in multiple cellular processes such as metabolism, signal transduction, protein degradation, DNA replication regulation and stress response. Recent studies demonstrated that epigenetics and PTMs also play vital roles in bacterial antibiotic resistance. In this review, we summarize the regulatory roles of epigenetic factors including DNA methylation and regulatory RNAs as well as PTMs such as phosphorylation and succinylation in bacterial antibiotic resistance, which may provide innovative perspectives on selecting antibacterial targets and developing antibiotics.

  16. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

    PubMed

    Kim, Jinoh; Thanabalasuriar, Ajitha; Chaworth-Musters, Tessa; Fromme, J Chris; Frey, Elizabeth A; Lario, Paula I; Metalnikov, Pavel; Rizg, Keyrillos; Thomas, Nikhil A; Lee, Sau Fung; Hartland, Elizabeth L; Hardwidge, Philip R; Pawson, Tony; Strynadka, Natalie C; Finlay, B Brett; Schekman, Randy; Gruenheid, Samantha

    2007-09-13

    Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.

  17. A novel plant major intrinsic protein in Physcomitrella patens most similar to bacterial glycerol channels.

    PubMed

    Gustavsson, Sofia; Lebrun, Anne-Sophie; Nordén, Kristina; Chaumont, François; Johanson, Urban

    2005-09-01

    A gene encoding a novel fifth type of major intrinsic protein (MIP) in plants has been identified in the moss Physcomitrella patens. Phylogenetic analyses show that this protein, GlpF-like intrinsic protein (GIP1;1), is closely related to a subclass of glycerol transporters in bacteria that in addition to glycerol are highly permeable to water. A likely explanation of the occurrence of this bacterial-like MIP in P. patens is horizontal gene transfer. The expressed P. patens GIP1;1 gene contains five introns and encodes a unique C-loop extension of approximately 110 amino acid residues that has no obvious similarity with any other known protein. Based on alignments and structural comparisons with other MIPs, GIP1;1 is suggested to have retained the permeability for glycerol but not for water. Studies on heterologously expressed GIP1;1 in Xenopus laevis oocytes confirm the predicted substrate specificity. Interestingly, proteins of one of the plant-specific subgroups of MIPs, the NOD26-like intrinsic proteins, are also facilitating the transport of glycerol and have previously been suggested to have evolved from a horizontally transferred bacterial gene. Further studies on localization and searches for GIP1;1 homologs in other plants will clarify the function and significance of this new plant MIP.

  18. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    PubMed

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  19. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  20. The RNPP family of quorum-sensing proteins in Gram-positive bacteria.

    PubMed

    Rocha-Estrada, Jorge; Aceves-Diez, Angel E; Guarneros, Gabriel; de la Torre, Mayra

    2010-07-01

    Quorum sensing is one of several mechanisms that bacterial cells use to interact with each other and coordinate certain physiological processes in response to cell density. This mechanism is mediated by extracellular signaling molecules; once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed), facilitating the expression of quorum sensing-dependent genes. Gram-positive bacteria mostly use oligo-peptides as signaling molecules. These cells have a special kind of quorum-sensing systems in which the receptor protein interacts directly with its cognate signaling peptide. The receptors are either Rap phosphatases or transcriptional regulators and integrate the protein family RNPP, from Rap, Npr, PlcR, and PrgX. These quorum-sensing systems control several microbial processes, like sporulation, virulence, biofilm formation, conjugation, and production of extracellular enzymes. Insights of the mechanism of protein-signaling peptide binding as well as the molecular interaction among receptor protein, signaling peptide, and target DNA have changed some earlier perceptions. In spite of the increased knowledge and the potential biotechnological applications of these quorum-sensing systems, few examples on engineering for biotechnological applications have been published. Real applications will arise only when researchers working in applied microbiology and biotechnology are aware of the importance of quorum-sensing systems for health and bioprocess applications.

  1. 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

  2. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

    PubMed

    Bickel, Perry E; Tansey, John T; Welte, Michael A

    2009-06-01

    The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

  3. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores

    PubMed Central

    Bickel, Perry E.; Tansey, John T.; Welte, Michael A.

    2009-01-01

    Summary The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  4. Targeting human SET1/MLL family of proteins

    PubMed Central

    Blazer, Levi; Eram, Mohammad S.; Barsyte‐Lovejoy, Dalia; Arrowsmith, Cheryl H.; Hajian, Taraneh

    2017-01-01

    Abstract The SET1 family of proteins, and in particular MLL1, are essential regulators of transcription and key mediators of normal development and disease. Here, we summarize the detailed characterization of the methyltransferase activity of SET1 complexes and the role of the key subunits, WDR5, RbBP5, ASH2L, and DPY30. We present new data on full kinetic characterization of human MLL1, MLL3, SET1A, and SET1B trimeric, tetrameric, and pentameric complexes to elaborate on substrate specificities and compare our findings with what has been reported before. We also review exciting recent work identifying potent inhibitors of oncogenic MLL1 function through disruption of protein–protein interactions within the MLL1 complex. PMID:28160335

  5. Palmitoylation of POTE family proteins for plasma membrane targeting

    SciTech Connect

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-11-23

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

  6. Genealogy of an ancient protein family: the Sirtuins, a family of disordered members

    PubMed Central

    2013-01-01

    Background Sirtuins genes are widely distributed by evolution and have been found in eubacteria, archaea and eukaryotes. While prokaryotic and archeal species usually have one or two sirtuin homologs, in humans as well as in eukaryotes we found multiple versions and in mammals this family is comprised of seven different homologous proteins being all NAD-dependent de-acylases. 3D structures of human SIRT2, SIRT3, and SIRT5 revealed the overall conformation of the conserved core domain but they were unable to give a structural information about the presence of very flexible and dynamically disordered regions, the role of which is still structurally and functionally unclear. Recently, we modeled the 3D-structure of human SIRT1, the most studied member of this family, that unexpectedly emerged as a member of the intrinsically disordered proteins with its long disordered terminal arms. Despite clear similarities in catalytic cores between the human sirtuins little is known of the general structural characteristics of these proteins. The presence of disorder in human SIRT1 and the propensity of these proteins in promoting molecular interactions make it important to understand the underlying mechanisms of molecular recognition that reasonably should involve terminal segments. The mechanism of recognition, in turn, is a prerequisite for the understanding of any functional activity. Aim of this work is to understand what structural properties are shared among members of this family in humans as well as in other organisms. Results We have studied the distribution of the structural features of N- and C-terminal segments of sirtuins in all known organisms to draw their evolutionary histories by taking into account average length of terminal segments, amino acid composition, intrinsic disorder, presence of charged stretches, presence of putative phosphorylation sites, flexibility, and GC content of genes. Finally, we have carried out a comprehensive analysis of the putative

  7. The aquaporin family of water channel proteins in clinical medicine.

    PubMed

    Lee, M D; King, L S; Agre, P

    1997-05-01

    The aquaporins are a family of membrane channel proteins that serve as selective pores through which water crosses the plasma membranes of many human tissues and cell types. The sites where aquaporins are expressed implicate these proteins in renal water reabsorption, cerebrospinal fluid secretion and reabsorption, generation of pulmonary secretions, aqueous humor secretion and reabsorption, lacrimation, and multiple other physiologic processes. Determination of the aquaporin gene sequences and their chromosomal locations has provided insight into the structure and pathophysiologic roles of these proteins, and primary and secondary involvement of aquaporins is becoming apparent in diverse clinical disorders. Aquaporin-1 (AQP1) is expressed in multiple tissues including red blood cells, and the Colton blood group antigens represent a polymorphism on the AQP1 protein. AQP2 is restricted to renal collecting ducts and has been linked to congenital nephrogenic diabetes insipidus in humans and to lithium-induced nephrogenic diabetes insipidus and fluid retention from congestive heart failure in rat models. Congenital cataracts result from mutations in the mouse gene encoding the lens homolog Aqp0 (Mip). The present understanding of aquaporin physiology is still incomplete; identification of additional members of the aquaporin family will affect future studies of multiple disorders of water distribution throughout the body. In some tissues, the aquaporins may participate in the transepithelial movement of fluid without being rate limiting, so aquaporins may be involved in clinical disorders without being causative. As outlined in this review, our challenge is to identify disease states in which aquaporins are involved, to define the aquaporins' roles mechanistically, and to search for ways to exploit this information therapeutically.

  8. Sequence diversity of the Trypanosoma cruzi complement regulatory protein family.

    PubMed

    Beucher, M; Norris, K A

    2008-02-01

    As a central component of innate immunity, complement activation is a critical mechanism of containment and clearance of microbial pathogens in advance of the development of acquired immunity. Several pathogens restrict complement activation through the acquisition of host proteins that regulate complement activation or through the production of their own complement regulatory molecules (M. K. Liszewski, M. K. Leung, R. Hauhart, R. M. Buller, P. Bertram, X. Wang, A. M. Rosengard, G. J. Kotwal, and J. P. Atkinson, J. Immunol. 176:3725-3734, 2006; J. Lubinski, L. Wang, D. Mastellos, A. Sahu, J. D. Lambris, and H. M. Friedman, J. Exp. Med. 190:1637-1646, 1999). The infectious stage of the protozoan parasite Trypanosoma cruzi produces a surface-anchored complement regulatory protein (CRP) that functions to inhibit alternative and classical pathway complement activation (K. A. Norris, B. Bradt, N. R. Cooper, and M. So, J. Immunol. 147:2240-2247, 1991). This study addresses the genomic complexity of the T. cruzi CRP and its relationship to the T. cruzi supergene family comprising active trans-sialidase (TS) and TS-like proteins. The TS superfamily consists of several functionally distinct subfamilies that share a characteristic sialidase domain at their amino termini. These TS families include active TS, adhesions, CRPs, and proteins of unknown functions (G. A. Cross and G. B. Takle, Annu. Rev. Microbiol. 47:385-411, 1993). A sequence comparison search of GenBank using BLASTP revealed several full-length paralogs of CRP. These proteins share significant homology at their amino termini and a strong spatial conservation of cysteine residues. Alternative pathway complement regulation was confirmed for CRP paralogs with 58% (low) and 83% (high) identity to AAB49414. CRPs are functionally similar to the microbial and mammalian proteins that regulate complement activation. Sequence alignment of mammalian complement control proteins to CRP showed that these sequences are

  9. Recombinant expression and purification of "virus-like" bacterial encapsulin protein cages.

    PubMed

    Rurup, W Frederik; Cornelissen, Jeroen J L M; Koay, Melissa S T

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules and organelles, the rate of migration can be used as a tool for purification. Here we describe a detailed protocol for the purification of recently discovered virus-like assemblies called bacterial encapsulins from Thermotoga maritima and Brevibacterium linens.

  10. Natural Products at Work: Structural Insights into Inhibition of the Bacterial Membrane Protein MraY.

    PubMed

    Koppermann, Stefan; Ducho, Christian

    2016-09-19

    Natural(ly) fit: The X-ray crystal structure of the bacterial membrane protein MraY in complex with its natural product inhibitor muraymycin D2 is discussed. MraY catalyzes one of the membrane-associated steps in peptidoglycan biosynthesis and, therefore, represents a promising target for novel antibiotics. Structural insights derived from the protein-inhibitor complex might now pave the way for the development of new antimicrobial drugs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T.; Ihalin, Riikka

    2013-01-01

    Aggregatibacteractinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1

  12. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    PubMed

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T; Ihalin, Riikka

    2013-01-01

    Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1

  13. Identification of a Bacteria-Specific Binding Protein from the Sequenced Bacterial Genome.

    PubMed

    Kong, Minsuk; Ryu, Sangryeol

    2016-01-01

    Novel and specific recognition elements are of central importance in the development of a pathogen detection method. Here, we describe a simple method for identifying the cell-wall binding domain (CBD) from a sequenced bacterial genome employing homology search for phage lysin genes. A putative CBD (CPF369_CBD) was identified from a genome of Clostridium perfringens type strain ATCC 13124, and its function was studied with the CBDGFP fusion protein recombinantly expressed in Escherichia coli. Fluorescence microscopy showed the specific binding of the fusion protein to C. perfringens cells, which demonstrates the potential of this method for the identification of novel bioprobes for specific detection of pathogenic bacteria.

  14. ChIP-seq for genome-scale analysis of bacterial DNA-binding proteins.

    PubMed

    Bonocora, Richard P; Wade, Joseph T

    2015-01-01

    Protein-DNA interactions are central to many basic biological processes, including transcription regulation, DNA replication, and DNA repair. Chromatin Immunoprecipitation (ChIP) is used to determine the position and strength of protein-DNA interactions in vivo. Coupling ChIP with microarrays (ChIP-chip), and more recently with deep sequencing (ChIP-seq), has allowed genome-wide profiling of DNA binding events in vivo. In this chapter we outline the steps to generate ChIP-seq libraries from bacterial samples and briefly discuss basic analysis of the data.

  15. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

    PubMed Central

    Hueck, Christoph J.

    1998-01-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  16. Type III protein secretion systems in bacterial pathogens of animals and plants.

    PubMed

    Hueck, C J

    1998-06-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  17. Adducin family proteins possess different nuclear export potentials.

    PubMed

    Liu, Chia-Mei; Hsu, Wen-Hsin; Lin, Wan-Yi; Chen, Hong-Chen

    2017-05-10

    The adducin (ADD) family proteins, namely ADD1, ADD2, and ADD3, are actin-binding proteins that play important roles in the stabilization of membrane cytoskeleton and cell-cell junctions. All the ADD proteins contain a highly conserved bipartite nuclear localization signal (NLS) at the carboxyl termini, but only ADD1 can localize to the nucleus. The reason for this discrepancy is not clear. To avoid the potential effect of cell-cell junctions on the distribution of ADD proteins, HA epitope-tagged ADD proteins and mutants were transiently expressed in NIH3T3 fibroblasts and their distribution in the cytoplasm and nucleus was examined by immunofluorescence staining. Several nuclear proteins were identified to interact with ADD1 by mass spectrometry, which were further verified by co-immunoprecipitation. In this study, we found that ADD1 was detectable both in the cytoplasm and nucleus, whereas ADD2 and ADD3 were detected only in the cytoplasm. However, ADD2 and ADD3 were partially (~40%) sequestered in the nucleus by leptomycin B, a CRM1/exportin1 inhibitor. Upon the removal of leptomycin B, ADD2 and ADD3 re-distributed to the cytoplasm. These results indicate that ADD2 and ADD3 possess functional NLS and are quickly transported to the cytoplasm upon entering the nucleus. Indeed, we found that ADD2 and ADD3 possess much higher potential to counteract the activity of the NLS derived from Simian virus 40 large T-antigen than ADD1. All the ADD proteins appear to contain multiple nuclear export signals mainly in their head and neck domains. However, except for the leucine-rich motif ((377)FEALMRMLDWLGYRT(391)) in the neck domain of ADD1, no other classic nuclear export signal was identified in the ADD proteins. In addition, the nuclear retention of ADD1 facilitates its interaction with RNA polymerase II and zinc-finger protein 331. Our results suggest that ADD2 and ADD3 possess functional NLS and shuttle between the cytoplasm and nucleus. The discrepancy in the

  18. Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

    NASA Astrophysics Data System (ADS)

    Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

    2009-09-01

    Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

  19. Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins.

    PubMed Central

    Aderem, A A; Keum, M M; Pure, E; Cohn, Z A

    1986-01-01

    We demonstrate stimulus-dependent incorporation of exogenously added [3H]myristic acid into specific macrophage proteins. In control unstimulated cells an 18-kDa protein is the major acylated species. In cells incubated with bacterial lipopolysaccharide (LPS), or its monoacyl glucosamine phosphate derivative, fatty acid is incorporated into proteins with molecular mass of 68 kDa and a doublet of approximately 42-45 kDa. Phorbol 12-myristate 13-acetate (PMA) or a phagocytic stimulus (zymosan) promotes the acylation of a similar array of proteins. However, PMA and zymosan also promote the myristoylation of unique proteins of 92 and 50 kDa. The fatty acid associated with each of the acylated proteins is myristic acid. The myristate is probably linked to the proteins through amide bonds, since it is not released by treatment with hydroxylamine. Palmitate and arachidonate are not incorporated into proteins in the same manner. Temporal analysis revealed that LPS-induced proteins are myristoylated by 30 min, while the 50-kDa protein myristoylated in response to PMA is labeled later. Most myristoylated proteins appear to be associated with the membrane fraction. Macrophages from C3H/HeJ mice, which do not respond to LPS, do not show any LPS-dependent protein acylation. Interestingly, zymosan and PMA induce the myristoylation of the 50-kDa protein in C3H/HeJ macrophages, but not the acylation of the 68-kDa and 42-kDa doublet species. We suggest that myristoylation of specific proteins is an intermediary in the capacity of LPS, PMA, and zymosan to alter macrophage functions such as arachidonic acid metabolism. Images PMID:3461461

  20. Chemosensitization of Prostate Cancer by Modulating Bcl-2 Family Proteins

    PubMed Central

    Karnak, David; Xu, Liang

    2010-01-01

    A major challenge in oncology is the development of chemoresistance. This often occurs as cancer progresses and malignant cells acquire mechanisms to resist insults that would normally induce apoptosis. The onset of androgen independence in advanced prostate cancer is a prime example of this phenomenon. Overexpression of the pro-survival/anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are hallmarks of this transition. Here we outline the evolution of therapeutics designed to either limit the source or disrupt the interactions of these pro-survival proteins. By either lessening the stoichiometric abundance of Bcl-2/xL/Mcl-1 in reference to their pro-apoptotic foils or freeing these pro-apoptotic proteins from their grip, these treatments aim to sensitize cells to chemotherapy by priming cells for death. DNA anti-sense and RNA interference have been effectively employed to decrease Bcl-2 family mRNA and protein levels in cell culture models of advanced prostate cancer. However, clinical studies are lagging due to in vivo delivery challenges. The burgeoning field of nanoparticle delivery holds great promise in helping to overcome the challenge of administering highly labile nucleic acid based therapeutics. On another front, small molecule inhibitors that block the hetero-dimerization of pro-survival with pro-apoptotic proteins have significant clinical advantages and have advanced farther in clinical trials with promising early results. Most recently, a peptide has been discovered that can convert Bcl-2 from a pro-survival to a pro-apoptotic protein. The future may lie in targeting multiple steps of the apoptotic pathway, including Bcl-2/xL/Mcl-1, to debilitate the survival capacity of cancer cells and make chemotherapy induced death their only option. PMID:20298153

  1. The Golgin Family of Coiled-Coil Tethering Proteins

    PubMed Central

    Witkos, Tomasz M.; Lowe, Martin

    2016-01-01

    The golgins are a family of predominantly coiled-coil proteins that are localized to the Golgi apparatus. Golgins are present in all eukaryotes, suggesting an evolutionary conserved function. Golgins are anchored to the Golgi membrane by their carboxy terminus and are predicted to adopt an extended conformation that projects into the surrounding cytoplasm. This arrangement is ideal for the capture or tethering of nearby membranes or cytoskeletal elements. Golgin-mediated tethering is thought to be important for vesicular traffic at the Golgi apparatus, the maintenance of Golgi architecture, as well as the positioning of the Golgi apparatus within cells. In addition to acting as tethers, some golgins can also sequester various factors at the Golgi membrane, allowing for the spatiotemporal regulation of downstream cellular functions. Although it is now established that golgins are membrane and cytoskeleton tethers, the mechanisms underlying tethering remain poorly defined. Moreover, the importance of golgin-mediated tethering in a physiological context remains to be fully explored. This review will describe our current understanding of golgin function, highlighting recent progress that has been made, and goes on to discuss outstanding questions and potential avenues for future research with regard to this family of conserved Golgi-associated proteins. PMID:26793708

  2. Docking validation resources: protein family and ligand flexibility experiments.

    PubMed

    Mukherjee, Sudipto; Balius, Trent E; Rizzo, Robert C

    2010-11-22

    A database consisting of 780 ligand-receptor complexes, termed SB2010, has been derived from the Protein Databank to evaluate the accuracy of docking protocols for regenerating bound ligand conformations. The goal is to provide easily accessible community resources for development of improved procedures to aid virtual screening for ligands with a wide range of flexibilities. Three core experiments using the program DOCK, which employ rigid (RGD), fixed anchor (FAD), and flexible (FLX) protocols, were used to gauge performance by several different metrics: (1) global results, (2) ligand flexibility, (3) protein family, and (4) cross-docking. Global spectrum plots of successes and failures vs rmsd reveal well-defined inflection regions, which suggest the commonly used 2 Å criteria is a reasonable choice for defining success. Across all 780 systems, success tracks with the relative difficulty of the calculations: RGD (82.3%) > FAD (78.1%) > FLX (63.8%). In general, failures due to scoring strongly outweigh those due to sampling. Subsets of SB2010 grouped by ligand flexibility (7-or-less, 8-to-15, and 15-plus rotatable bonds) reveal that success degrades linearly for FAD and FLX protocols, in contrast to RGD, which remains constant. Despite the challenges associated with FLX anchor orientation and on-the-fly flexible growth, success rates for the 7-or-less (74.5%) and, in particular, the 8-to-15 (55.2%) subset are encouraging. Poorer results for the very flexible 15-plus set (39.3%) indicate substantial room for improvement. Family-based success appears largely independent of ligand flexibility, suggesting a strong dependence on the binding site environment. For example, zinc-containing proteins are generally problematic, despite moderately flexible ligands. Finally, representative cross-docking examples, for carbonic anhydrase, thermolysin, and neuraminidase families, show the utility of family-based analysis for rapid identification of particularly good or bad

  3. Analysis of membrane proteins in metagenomics: networks of correlated environmental features and protein families.

    PubMed

    Patel, Prianka V; Gianoulis, Tara A; Bjornson, Robert D; Yip, Kevin Y; Engelman, Donald M; Gerstein, Mark B

    2010-07-01

    Recent metagenomics studies have begun to sample the genomic diversity among disparate habitats and relate this variation to features of the environment. Membrane proteins are an intuitive, but thus far overlooked, choice in this type of analysis as they directly interact with the environment, receiving signals from the outside and transporting nutrients. Using global ocean sampling (GOS) data, we found nearly approximately 900,000 membrane proteins in large-scale metagenomic sequence, approximately a fifth of which are completely novel, suggesting a large space of hitherto unexplored protein diversity. Using GPS coordinates for the GOS sites, we extracted additional environmental features via interpolation from the World Ocean Database, the National Center for Ecological Analysis and Synthesis, and empirical models of dust occurrence. This allowed us to study membrane protein variation in terms of natural features, such as phosphate and nitrate concentrations, and also in terms of human impacts, such as pollution and climate change. We show that there is widespread variation in membrane protein content across marine sites, which is correlated with changes in both oceanographic variables and human factors. Furthermore, using these data, we developed an approach, protein families and environment features network (PEN), to quantify and visualize the correlations. PEN identifies small groups of covarying environmental features and membrane protein families, which we call "bimodules." Using this approach, we find that the affinity of phosphate transporters is related to the concentration of phosphate and that the occurrence of iron transporters is connected to the amount of shipping, pollution, and iron-containing dust.

  4. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae.

    PubMed

    Antúnez, Karina; Anido, Matilde; Evans, Jay D; Zunino, Pablo

    2010-03-24

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms. Copyright 2009 Elsevier B.V. All rights reserved.

  5. Proteins on the catwalk: modelling the structural domains of the CCN family of proteins.

    PubMed

    Holbourn, Kenneth P; Perbal, Bernard; Ravi Acharya, K

    2009-03-01

    The CCN family of proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) are multifunctional mosaic proteins that play keys roles in crucial areas of physiology such as angiogenesis, skeletal development tumourigenesis, cell proliferation, adhesion and survival. This expansive repertoire of functions comes through a modular structure of 4 discrete domains that act both independently and in concert. How these interactions with ligands and with neighbouring domains lead to the biological effects is still to be explored but the molecular structure of the domains is likely to play an important role in this. In this review we have highlighted some of the key features of the individual domains of CCN family of proteins based on their biological effects using a homology modelling approach.

  6. Structure of a bacterial microcompartment shell protein bound to a cobalamin cofactor

    PubMed Central

    Thompson, Michael C.; Crowley, Christopher S.; Kopstein, Jeffrey; Bobik, Thomas A.; Yeates, Todd O.

    2014-01-01

    The EutL shell protein is a key component of the ethanolamine-utilization microcompartment, which serves to compartmentalize ethanolamine degradation in diverse bacteria. The apparent function of this shell protein is to facilitate the selective diffusion of large cofactor molecules between the cytoplasm and the lumen of the microcompartment. While EutL is implicated in molecular-transport phenomena, the details of its function, including the identity of its transport substrate, remain unknown. Here, the 2.1 Å resolution X-ray crystal structure of a EutL shell protein bound to cobalamin (vitamin B12) is presented and the potential relevance of the observed protein–ligand interaction is briefly discussed. This work represents the first structure of a bacterial microcompartment shell protein bound to a potentially relevant cofactor molecule. PMID:25484204

  7. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

    PubMed Central

    Shen, Hsin-Hui; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

    2015-01-01

    In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here, we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines. PMID:25341963

  8. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

    NASA Astrophysics Data System (ADS)

    Shen, Hsin-Hui; Leyton, Denisse L.; Shiota, Takuya; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

    2014-10-01

    In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

  9. The Cbln family of proteins interact with multiple signaling pathways.

    PubMed

    Wei, Peng; Pattarini, Roberto; Rong, Yongqi; Guo, Hong; Bansal, Parmil K; Kusnoor, Sheila V; Deutch, Ariel Y; Parris, Jennifer; Morgan, James I

    2012-06-01

    Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic β-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.

  10. Bacterial infection of osteoblasts induces interleukin-1beta and interleukin-18 transcription but not protein synthesis.

    PubMed

    Marriott, Ian; Hughes, Francis M; Bost, Kenneth L

    2002-10-01

    A growing body of evidence has shown that bacterially challenged bone-forming osteoblasts are a significant source of an array of cytokines and chemokines that can support immune responses during bone disease. In the present study, Staphylococcus aureus and Salmonella, two common pathogens of bone, were investigated for their ability to induce production of two related inflammatory cytokines, interleukin-1beta (IL-1beta) and IL18, in osteoblasts. Cultured mouse osteoblasts were found to respond rapidly to either bacterial challenge by upregulation in the levels of mRNA encoding both IL-1beta and IL-18. Surprisingly, this mRNA expression did not translate into intracellular accumulation of IL-1beta or IL-18 precursor proteins or secretion of mature cytokines, despite the presence of detectable caspase-1 activity in these cells. These studies demonstrate that although osteoblasts can secrete a number of key proinflammatory mediators in response to bacterial pathogens, IL-1beta and IL-18 are not among this number. We suggest that osteoblasts are an unlikely source of these cytokines during the progression of bacterial infection of bone.

  11. Bacterial Cytotoxins Target Rho GTPases

    NASA Astrophysics Data System (ADS)

    Schmidt, Gudula; Aktories, Klaus

    1998-06-01

    Low molecular mass GTPases of the Rho family, which are involved in the regulation of the actin cytoskeleton and in various signal transduction processes, are the eukaryotic targets of bacterial protein toxins. The toxins covalently modify Rho proteins by ADP ribosylation, glucosylation, and deamidation, thereby inactivating and activating the GTPases.

  12. The impact of grafted modification of silicone surfaces with quantum-sized materials on protein adsorption and bacterial adhesion.

    PubMed

    Nune, C; Xu, W; Misra, R D K

    2012-12-01

    The majority of the infections associated with the biomedical devices including cardiovascular implants and catheters are instigated by the adhesion of bacteria including staphylococcus aureus, which is subsequently followed by biofilm formation. Keeping in mind the detrimental effect of bacterial adhesion, the objective of the study is to probe the impact of grafted modification of silicone surfaces with quantum-sized carbon on biofilm formation. Also, explored is the effect of protein adsorption on modified surface and its subsequent influence on bacterial adhesion. We compare and contrast the architecture and foot print of protein adsorption on unmodified and modified model silicone surfaces on bacterial adhesion. The study underscores that protein adsorption on quantum-sized carbon-grafted surface acts as a repellant for bacterial adhesion because of steric repulsion between the negatively charged protein and bacteria. Thus, we establish here the efficacy of modified surfaces in preventing biofilm formation.

  13. Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production.

    PubMed

    He, Ya-Wen; Wu, Ji'en; Cha, Jae-Soon; Zhang, Lian-Hui

    2010-07-09

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production. Xoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 microM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after inoculation. When grown in

  14. Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production

    PubMed Central

    2010-01-01

    Background Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production. Results Xoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 μM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after

  15. Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

    PubMed

    Smith, Roxanne P; Paxman, Jason J; Scanlon, Martin J; Heras, Begoña

    2016-07-16

    Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.

  16. Object-adapted trapping and shape-tracking to probe a bacterial protein chain motor

    NASA Astrophysics Data System (ADS)

    Roth, Julian; Koch, Matthias; Rohrbach, Alexander

    2015-03-01

    The helical bacterium Spiroplasma is a motile plant and anthropod pathogen which swims by propagating pairs of kinks along its cell body. As a well suited model system for bacterial locomotion, understanding the cell's molecular motor is of vital interest also regarding the combat of bacterial diseases. The extensive deformations related to these kinks are caused by a contractile cytoskeletal protein ribbon representing a linear motor in contrast to common rotary motors as, e.g., flagella. We present new insights into the working of this motor through experiments with object-adapted optical traps and shape-tracking techniques. We use the given laser irradiation from the optical trap to hinder bacterial energy (ATP) production through the production of O2 radicals. The results are compared with experiments performed under the influence of an O2-Scavenger and ATP inhibitors, respectively. Our results show clear dependences of the kinking properties on the ATP concentration inside the bacterium. The experiments are supported by a theoretical model which we developed to describe the switching of the ribbon's protein subunits.

  17. Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria

    PubMed Central

    Zhu, Yingying; Lin, Xisha; Zhao, Fan; Shi, Xuebin; Li, He; Li, Yingqiu; Zhu, Weiyun; Xu, Xinglian; Lu, Chunbao; Zhou, Guanghong

    2015-01-01

    Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host. PMID:26463271

  18. Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria.

    PubMed

    Zhu, Yingying; Lin, Xisha; Zhao, Fan; Shi, Xuebin; Li, He; Li, Yingqiu; Zhu, Weiyun; Xu, Xinglian; Li, Chunbao; Lu, Chunbao; Zhou, Guanghong

    2015-10-14

    Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host.

  19. The cholesterol-dependent cytolysin family of gram-positive bacterial toxins.

    PubMed

    Heuck, Alejandro P; Moe, Paul C; Johnson, Benjamin B

    2010-01-01

    The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.

  20. Members of the salivary gland surface protein (SGS) family are major immunogenic components of mosquito saliva.

    PubMed

    King, Jonas G; Vernick, Kenneth D; Hillyer, Julián F

    2011-11-25

    Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.

  1. Phytochemicals as inhibitors of bacterial cell division protein FtsZ: coumarins are promising candidates.

    PubMed

    Duggirala, Sridevi; Nankar, Rakesh P; Rajendran, Selvakumar; Doble, Mukesh

    2014-09-01

    Naturally occurring phytochemicals with reported antibacterial activity were screened for their ability to inhibit the bacterial cell division protein Escherichia coli FtsZ. Among the representative compounds, coumarins inhibit the GTPase and polymerization activities of this protein effectively. Further screening with ten coumarin analogs we identified two promising candidates, scopoletin and daphnetin. The former is found to inhibit the GTPase activity of the protein in a noncompetitive manner. Docking of these coumarins with the modeled protein indicate that they bind to T7 loop, which is different from the GTP-binding site (active site), thereby supporting the experimental data. Lowest binding energy is obtained with scopoletin. 3D QSAR indicates the need for groups such as hydroxyl, diethyl, or dimethyl amino in the 7th carbon for enhanced activity. None of the coumarins exhibited cytotoxicity against NIH/3T3 and human embryonic kidney cell lines. The length of Bacillus subtilis increases in the presence of these compounds probably due to the lack of septum formation. Results of this study indicate the role of coumarins in halting the first step of bacterial cell division process.

  2. Bacterial fermentation of cheese whey for production of a ruminant feed supplement rich in curde protein.

    PubMed Central

    Reddy, C A; Henderson, H E; Erdman, M D

    1976-01-01

    A simple and efficient process for the production of a ruminant feed supplement, rich in crude protein (defined as total N X 6.25), by bacterial fermentation of cheese whey has been developed. The lactose in unpasteurized whey is fermented to lactate acid by Lactobacillus bulgaricus at a temperature of 43 degrees C and pH 5.5. The lactic acid produced is continually neutralized with ammonia to form ammonium lactate. The fermented product is concentrated by evaporation to a solids content of about 70% and adjusted to pH 6.8 with additional ammonia. The concentrated product contains about 55% crude protein. Approximately 6 to 8% of the crude protein is derived from bacterial cells. 17% from whey proteins, and 75 to 77% from ammonium lactate. The efficiency of conversion of lactose to lactic acid usually exceeds 95%. The fermentation time is greatly reduced upon the addition of 0.2% yeast extract or 0.1% corn steep liquor as a source of growth factors. Whey containing lactose at concentrations up to 7% can be fermented efficiently, but at higher concentrations lactose is fermented incompletely. The process has been scaled up to a pilot plant level, and 40 tons of concentrated product were produced fro animal feeding trials, without ever encountering putrefactive spoilage. PMID:12720

  3. Biophysical analysis of the interaction of the serum protein human β2GPI with bacterial lipopolysaccharide

    PubMed Central

    Gries, Anna; Prassl, Ruth; Fukuoka, Satoshi; Rössle, Manfred; Kaconis, Yani; Heinbockel, Lena; Gutsmann, Thomas; Brandenburg, Klaus

    2014-01-01

    There are several human serum proteins for which no clear role is yet known. Among these is the abundant serum protein beta2-glycoprotein-I (β2GPI), which is known to bind to negatively charged phospholipids as well as to bacterial lipopolysaccharides (LPS), and was therefore proposed to play a role in the immune response. To understand the details of these interactions, a biophysical analysis of the binding of β2GPI to LPS and phosphatidylserine (PS) was performed. The data indicate only a moderate tendency of the protein (1) to influence the LPS-induced cytokine production in vitro, (2) to react exothermally with LPS in a non-saturable way, and (3) to change its local microenvironment upon LPS association. Additionally, we found that the protein binds more strongly to phosphatidylserine (PS) than to LPS. Furthermore, β2GPI converts the LPS bilayer aggregates into a stronger multilamellar form, and reduces the fluidity of the hydrocarbon moiety of LPS due to a rigidification of the acyl chains. From these data it can be concluded that β2GPI plays a role as an immune-modulating agent, but there is much less evidence for a role in immune defense against bacterial toxins such as LPS. PMID:24918058

  4. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean

    PubMed Central

    Zhou, Yuan; Yang, Yan; Zhou, Xinjian; Chi, Yingjun; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    Proteins containing the FxxxVQxhTG or VQ motif interact with WRKY transcription factors. Although VQ proteins have been reported in several plants, knowledge about their structures, functions and evolution is still very limited. Here, we report structural and functional analysis of the VQ protein family from soybean. Like Arabidopsis homologues, soybean VQ proteins bind only Group I and IIc WRKY proteins and a substantial number of their genes are responsive to stress-associated phytohormones. Overexpression of some soybean VQ genes in Arabidopsis had strong effects on plant growth, development, disease resistance and heat tolerance. Phylogenetic analysis, sequence alignment and site-directed mutagenesis revealed that the region immediately upstream of the FxxxVQxhTG motif also affects binding to WRKY proteins. Consistent with a larger WRKY-binding VQ domain, soybean VQ22 protein from cultivated soybean contains a 4-amino acid deletion in the region preceding its VQ motif that completely abolishes its binding to WRKY proteins. By contrast, the 4-amino acid deletion is absent in the VQ22 protein from wild soybean species (Glycine soja). Overexpression of wild soybean VQ22 in cultivated soybean inhibited growth, particularly after cold treatment. Thus, the mutation of soybean VQ22 is associated with advantageous phenotypes and may have been positively selected during evolution. PMID:27708406

  5. Infection Strategies of Bacterial and Viral Pathogens through Pathogen–Human Protein–Protein Interactions

    PubMed Central

    Durmuş Tekir, Saliha; Çakir, Tunahan; Ülgen, Kutlu Ö

    2012-01-01

    Since ancient times, even in today’s modern world, infectious diseases cause lots of people to die. Infectious organisms, pathogens, cause diseases by physical interactions with human proteins. A thorough analysis of these interspecies interactions is required to provide insights about infection strategies of pathogens. Here we analyzed the most comprehensive available pathogen–human protein interaction data including 23,435 interactions, targeting 5,210 human proteins. The data were obtained from the newly developed pathogen–host interaction search tool, PHISTO. This is the first comprehensive attempt to get a comparison between bacterial and viral infections. We investigated human proteins that are targeted by bacteria and viruses to provide an overview of common and special infection strategies used by these pathogen types. We observed that in the human protein interaction network the proteins targeted by pathogens have higher connectivity and betweenness centrality values than those proteins not interacting with pathogens. The preference of interacting with hub and bottleneck proteins is found to be a common infection strategy of all types of pathogens to manipulate essential mechanisms in human. Compared to bacteria, viruses tend to interact with human proteins of much higher connectivity and centrality values in the human network. Gene Ontology enrichment analysis of the human proteins targeted by pathogens indicates crucial clues about the infection mechanisms of bacteria and viruses. As the main infection strategy, bacteria interact with human proteins that function in immune response to disrupt human defense mechanisms. Indispensable viral strategy, on the other hand, is the manipulation of human cellular processes in order to use that transcriptional machinery for their own genetic material transcription. A novel observation about pathogen–human systems is that the human proteins targeted by both pathogens are enriched in the regulation of

  6. Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins.

    PubMed

    Badgandi, Hemant B; Hwang, Sun-Hee; Shimada, Issei S; Loriot, Evan; Mukhopadhyay, Saikat

    2017-03-06

    The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein-coupled receptors (GPCRs) and the polycystic kidney disease-causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia. Using minimal ciliary localization sequences from GPCRs and fibrocystin (also implicated in polycystic kidney disease), we demonstrate these motifs to be sufficient and TULP3 dependent for ciliary trafficking. We propose a three-step model for TULP3/TUB-mediated ciliary trafficking, including the capture of diverse membrane cargo by the tubby domain in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent manner, ciliary delivery by intraflagellar transport complex A binding to the TULP3/TUB N terminus, and subsequent release into PI(4,5)P2-deficient ciliary membrane. © 2017 Badgandi et al.

  7. Non-apoptotic functions of BCL-2 family proteins.

    PubMed

    Gross, Atan; Katz, Samuel G

    2017-02-24

    The BCL-2 family proteins are major regulators of the apoptosis process, but the mechanisms by which they regulate this process are only partially understood. It is now well documented that these proteins play additional non-apoptotic roles that are likely to be related to their apoptotic roles and to provide important clues to cracking their mechanisms of action. It seems that these non-apoptotic roles are largely related to the activation of cellular survival pathways designated to maintain or regain cellular survival, but, if unsuccessful, will switch over into a pro-apoptotic mode. These non-apoptotic roles span a wide range of processes that include the regulation of mitochondrial physiology (metabolism, electron transport chain, morphology, permeability transition), endoplasmic reticulum physiology (calcium homeostasis, unfolded protein response (UPR)), nuclear processes (cell cycle, DNA damage response (DDR)), whole-cell metabolism (glucose and lipid), and autophagy. Here we review all these different non-apoptotic roles, make an attempt to link them to the apoptotic roles, and present many open questions for future research directions in this fascinating field.Cell Death and Differentiation advance online publication, 24 February 2017; doi:10.1038/cdd.2017.22.

  8. Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish, Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes.

    PubMed

    Haygood, M G

    1990-01-01

    Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a lux AB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase alpha subunit gene luxA) of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G + C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.

  9. Synthetic interaction between the TipN polarity factor and an AcrAB-family efflux pump implicates cell polarity in bacterial drug resistance.

    PubMed

    Kirkpatrick, Clare L; Viollier, Patrick H

    2014-05-22

    Quinolone antibiotics are clinically important drugs that target bacterial DNA replication and chromosome segregation. Although the AcrAB-family efflux pumps generally protect bacteria from such drugs, the physiological role of these efflux systems and their interplay with other cellular events are poorly explored. Here, we report an intricate relationship between antibiotic resistance and cell polarity in the model bacterium Caulobacter crescentus. We show that a polarity landmark protein, TipN, identified by virtue of its ability to direct flagellum placement to the new cell pole, protects cells from toxic misregulation of an AcrAB efflux pump through a cis-encoded nalidixic acid-responsive transcriptional repressor. Alongside the importance of polarity in promoting the inheritance and activity of virulence functions including motility, we can now ascribe to it an additional role in drug resistance that is distinct from classical efflux mechanisms.

  10. Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter.

    PubMed

    Bozzi, Aaron T; Bane, Lukas B; Weihofen, Wilhelm A; Singharoy, Abhishek; Guillen, Eduardo R; Ploegh, Hidde L; Schulten, Klaus; Gaudet, Rachelle

    2016-12-06

    The widely conserved natural resistance-associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a (absent from a previous Nramp structure). Mapping our cysteine accessibility scanning results onto this structure, we identified the metal-permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations that impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal-permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism.

    PubMed

    Wu, L F; Ize, B; Chanal, A; Quentin, Y; Fichant, G

    2000-04-01

    The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.

  12. Interaction of Alzheimer's beta -amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade.

    PubMed

    Taru, Hidenori; Iijima, Ko-Ichi; Hase, Momoko; Kirino, Yutaka; Yagi, Yoshimasa; Suzuki, Toshiharu

    2002-05-31

    We have isolated a novel protein based on its association with Drosophila APP-like protein (APPL), a homolog of the beta-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novel APPL-interacting protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH(2)-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease.

  13. The r1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome.

    PubMed

    Meyer, Richard

    2009-03-01

    The mobilization proteins of the broad-host-range plasmid R1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. Such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal DNA when R1162 is present. The R1162-dependent transfer of chromosomal DNA, initiated from one such potential site in Pectobacterium atrosepticum, is shown here. A second active site was identified in Escherichia coli, where it is also shown that large amounts of DNA are transferred. This transfer probably reflects the combined activity of the multiple cryptic origins in the chromosome. Transfer of chromosomal DNA due to the presence of a plasmid in the cytoplasm describes a previously unrecognized potential for the exchange of bacterial DNA.

  14. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  15. Chirality Switching by Martensitic Transformation in Protein Cylindrical Crystals: Application to Bacterial Flagella

    NASA Astrophysics Data System (ADS)

    Komai, Ricardo Kiyohiro

    Martensitic transformations provide unique engineering properties that, when designed properly, become important parts of new technology. Martensitic transformations have been studied for many years in traditional alloys (iron, steel, titanium, etc.), however there is still much to be learned in regards to these transformations in biological materials. Olson and Hartman showed in 1982 that these transformations are also observed in bacterial flagella and T4 bacteriophage viral sheaths, allowing for propulsion of bacteria in a fluid environment and, for the virus, is responsible for the infection mechanism. This work demonstrates, using the bacterial flagella as an example, that these transformations can be modelled using thermodynamic methods that are also used to model the transformations in alloys. This thesis work attempts to explain the transformations that occur in bacterial flagella, which are capable of small strain, highly reversible martensitic transformations. The first stress/temperature phase diagrams of these flagella were created by adding the mechanical energy of the transformation of the flagella to limited chemical thermodynamics information of the transformation. Mechanical energy is critical to the transformation process because the bacterial body applies a torque to the radius of the flagella. Finally, work has begun and will be completed in regards to understanding the kinetics of the transformation of the flagella. The motion of the transformation interface can be predicted by using a Landau-Ginzburg model. The crystallography of the transformation in bacterial flagella is also being computed to determine the invariant lines of transformation that occur within this cylindrical crystal. This work has shown that it is possible to treat proteins in a similar manner that alloys are treated when using thermodynamic modelling. Much can be learned from translating what is known regarding phase transformations in hard material systems to soft, organic

  16. Yeast as a tool for studying proteins of the Bcl-2 family

    PubMed Central

    Polčic, Peter; Jaká, Petra; Mentel, Marek

    2015-01-01

    Permeabilization of the outer mitochondrial membrane that leads to the release of cytochrome c and several other apoptogenic proteins from mitochondria into cytosol represents a commitment point of apoptotic pathway in mammalian cells. This crucial event is governed by proteins of the Bcl-2 family. Molecular mechanisms, by which Bcl-2 family proteins permeabilize mitochondrial membrane, remain under dispute. Although yeast does not have apparent homologues of these proteins, when mammalian members of Bcl-2 family are expressed in yeast, they retain their activity, making yeast an attractive model system, in which to study their action. This review focuses on using yeast expressing mammalian proteins of the Bcl-2 family as a tool to investigate mechanisms, by which these proteins permeabilize mitochondrial membranes, mechanisms, by which pro- and antiapoptotic members of this family interact, and involvement of other cellular components in the regulation of programmed cell death by Bcl-2 family proteins. PMID:28357280

  17. Yeast as a tool for studying proteins of the Bcl-2 family.

    PubMed

    Polčic, Peter; Jaká, Petra; Mentel, M