Science.gov

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. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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/).

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

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

    PubMed Central

    2012-01-01

    Background 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. Results 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. Conclusions 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

  7. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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/.

  1. 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.

  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. 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. 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.

  5. 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.

  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. 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

  8. 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.

  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. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Annotation extension through protein family annotation coherence metrics

    PubMed Central

    Bastos, Hugo P.; Clarke, Luka A.; Couto, Francisco M.

    2013-01-01

    Protein functional annotation consists in associating proteins with textual descriptors elucidating their biological roles. The bulk of annotation is done via automated procedures that ultimately rely on annotation transfer. Despite a large number of existing protein annotation procedures the ever growing protein space is never completely annotated. One of the facets of annotation incompleteness derives from annotation uncertainty. Often when protein function cannot be predicted with enough specificity it is instead conservatively annotated with more generic terms. In a scenario of protein families or functionally related (or even dissimilar) sets this leads to a more difficult task of using annotations to compare the extent of functional relatedness among all family or set members. However, we postulate that identifying sub-sets of functionally coherent proteins annotated at a very specific level, can help the annotation extension of other incompletely annotated proteins within the same family or functionally related set. As an example we analyse the status of annotation of a set of CAZy families belonging to the Polysaccharide Lyase class. We show that through the use of visualization methods and semantic similarity based metrics it is possible to identify families and respective annotation terms within them that are suitable for possible annotation extension. Based on our analysis we then propose a semi-automatic methodology leading to the extension of single annotation terms within these partially annotated protein sets or families. PMID:24130572

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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...

  6. 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.

  7. The KP4 killer protein gene family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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

  9. 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

  10. 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

  11. 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.

  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. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. [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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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)

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. DNA-binding properties of ARID family proteins

    PubMed Central

    Patsialou, Antonia; Wilsker, Deborah; Moran, Elizabeth

    2005-01-01

    The ARID (A–T Rich Interaction Domain) is a helix–turn–helix motif-based DNA-binding domain, conserved in all eukaryotes and diagnostic of a family that includes 15 distinct human proteins with important roles in development, tissue-specific gene expression and proliferation control. The 15 human ARID family proteins can be divided into seven subfamilies based on the degree of sequence identity between individual members. Most ARID family members have not been characterized with respect to their DNA-binding behavior, but it is already apparent that not all ARIDs conform to the pattern of binding AT-rich sequences. To understand better the divergent characteristics of the ARID proteins, we undertook a survey of DNA-binding properties across the entire ARID family. The results indicate that the majority of ARID subfamilies (i.e. five out of seven) bind DNA without obvious sequence preference. DNA-binding affinity also varies somewhat between subfamilies. Site-specific mutagenesis does not support suggestions made from structure analysis that specific amino acids in Loop 2 or Helix 5 are the main determinants of sequence specificity. Most probably, this is determined by multiple interacting differences across the entire ARID structure. PMID:15640446

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. [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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. '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.

  1. 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.

  2. Graphical models of residue coupling in protein families.

    PubMed

    Thomas, John; Ramakrishnan, Naren; Bailey-Kellogg, Chris

    2008-01-01

    Many statistical measures and algorithmic techniques have been proposed for studying residue coupling in protein families. Generally speaking, two residue positions are considered coupled if, in the sequence record, some of their amino acid type combinations are significantly more common than others. While the proposed approaches have proven useful in finding and describing coupling, a significant missing component is a formal probabilistic model that explicates and compactly represents the coupling, integrates information about sequence,structure, and function, and supports inferential procedures for analysis, diagnosis, and prediction.We present an approach to learning and using probabilistic graphical models of residue coupling. These models capture significant conservation and coupling constraints observable ina multiply-aligned set of sequences. Our approach can place a structural prior on considered couplings, so that all identified relationships have direct mechanistic explanations. It can also incorporate information about functional classes, and thereby learn a differential graphical model that distinguishes constraints common to all classes from those unique to individual classes. Such differential models separately account for class-specific conservation and family-wide coupling, two different sources of sequence covariation. They are then able to perform interpretable functional classification of new sequences, explaining classification decisions in terms of the underlying conservation and coupling constraints. We apply our approach in studies of both G protein-coupled receptors and PDZ domains, identifying and analyzing family-wide and class-specific constraints, and performing functional classification. The results demonstrate that graphical models of residue coupling provide a powerful tool for uncovering, representing, and utilizing significant sequence structure-function relationships in protein families.

  3. 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

  4. 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

  5. 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.

  6. C21orf57 is a human homologue of bacterial YbeY proteins.

    PubMed

    Ghosal, Anubrata; Köhrer, Caroline; Babu, Vignesh M P; Yamanaka, Kinrin; Davies, Bryan W; Jacob, Asha I; Ferullo, Daniel J; Gruber, Charley C; Vercruysse, Maarten; Walker, Graham C

    2017-03-11

    The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.

  7. 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.

  8. 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

  9. 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.

  10. [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.

  11. 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

  12. 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.

  13. Web-based software for rapid "top-down" proteomic identification of protein biomarkers with implications for bacterial identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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)...

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  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. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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%.

  1. 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.

  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. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  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. Yeast as a tool for studying proteins of the Bcl-2 family.

    PubMed

    Polčic, Peter; Jaká, Petra; Mentel, Marek

    2015-03-02

    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.

  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. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. The Overlap of Small Molecule and Protein Binding Sites within Families of Protein Structures

    PubMed Central

    Davis, Fred P.; Sali, Andrej

    2010-01-01

    Protein–protein interactions are challenging targets for modulation by small molecules. Here, we propose an approach that harnesses the increasing structural coverage of protein complexes to identify small molecules that may target protein interactions. Specifically, we identify ligand and protein binding sites that overlap upon alignment of homologous proteins. Of the 2,619 protein structure families observed to bind proteins, 1,028 also bind small molecules (250–1000 Da), and 197 exhibit a statistically significant (p<0.01) overlap between ligand and protein binding positions. These “bi-functional positions”, which bind both ligands and proteins, are particularly enriched in tyrosine and tryptophan residues, similar to “energetic hotspots” described previously, and are significantly less conserved than mono-functional and solvent exposed positions. Homology transfer identifies ligands whose binding sites overlap at least 20% of the protein interface for 35% of domain–domain and 45% of domain–peptide mediated interactions. The analysis recovered known small-molecule modulators of protein interactions as well as predicted new interaction targets based on the sequence similarity of ligand binding sites. We illustrate the predictive utility of the method by suggesting structural mechanisms for the effects of sanglifehrin A on HIV virion production, bepridil on the cellular entry of anthrax edema factor, and fusicoccin on vertebrate developmental pathways. The results, available at http://pibase.janelia.org, represent a comprehensive collection of structurally characterized modulators of protein interactions, and suggest that homologous structures are a useful resource for the rational design of interaction modulators. PMID:20140189

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. The macro domain protein family: structure, functions, and their potential therapeutic implications.

    PubMed

    Han, Weidong; Li, Xiaolei; Fu, Xiaobing

    2011-01-01

    Macro domains are ancient, highly evolutionarily conserved domains that are widely distributed throughout all kingdoms of life. The 'macro fold' is roughly 25kDa in size and is composed of a mixed α-β fold with similarity to the P loop-containing nucleotide triphosphate hydrolases. They function as binding modules for metabolites of NAD(+), including poly(ADP-ribose) (PAR), which is synthesized by PAR polymerases (PARPs). Although there is a high degree of sequence similarity within this family, particularly for residues that might be involved in catalysis or substrates binding, it is likely that the sequence variation that does exist among macro domains is responsible for the specificity of function of individual proteins. Recent findings have indicated that macro domain proteins are functionally promiscuous and are implicated in the regulation of diverse biological functions, such as DNA repair, chromatin remodeling and transcriptional regulation. Significant advances in the field of macro domain have occurred in the past few years, including biological insights and the discovery of novel signaling pathways. To provide a framework for understanding these recent findings, this review will provide a comprehensive overview of the known and proposed biochemical, cellular and physiological roles of the macro domain family. Recent data that indicate a critical role of macro domain regulation for the proper progression of cellular differentiation programs will be discussed. In addition, the effect of dysregulated expression of macro domain proteins will be considered in the processes of tumorigenesis and bacterial pathogenesis. Finally, a series of observations will be highlighted that should be addressed in future efforts to develop macro domains as effective therapeutic targets.

  10. MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia.

    PubMed

    Ouyang, Yi-Bing; Giffard, Rona G

    2014-11-01

    The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNAs), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins.

  11. PATtyFams: Protein Families for the Microbial Genomes in the PATRIC Database

    PubMed Central

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-01-01

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). This new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods. PMID:26903996

  12. PATtyFams: Protein families for the microbial genomes in the PATRIC database

    SciTech Connect

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-02-08

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.

  13. The secretory carrier membrane protein family: structure and membrane topology.

    PubMed

    Hubbard, C; Singleton, D; Rauch, M; Jayasinghe, S; Cafiso, D; Castle, D

    2000-09-01

    Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane

  14. Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

    PubMed Central

    Bassegoda, Arnau; Pastor, F. I. Javier

    2012-01-01

    Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases. PMID:22226953

  15. 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.

  16. Acyl-CoA Dehydrogenases: Dynamic History of Protein Family Evolution

    PubMed Central

    Mohsen, Al-Walid

    2014-01-01

    The acyl-CoA dehydrogenases (ACADs) are enzymes that catalyze the α,β-dehydrogenation of acyl-CoA esters in fatty acid and amino acid catabolism. Eleven ACADs are now recognized in the sequenced human genome, and several homologs have been reported from bacteria, fungi, plants, and nematodes. We performed a systematic comparative genomic study, integrating homology searches with methods of phylogenetic reconstruction, to investigate the evolutionary history of this family. Sequence analyses indicate origin of the family in the common ancestor of Archaea, Bacteria, and Eukaryota, illustrating its essential role in the metabolism of early life. At least three ACADs were already present at that time: ancestral glutaryl-CoA dehydrogenase (GCD), isovaleryl-CoA dehydrogenase (IVD), and ACAD10/11. Two gene duplications were unique to the eukaryotic domain: one resulted in the VLCAD and ACAD9 paralogs and another in the ACAD10 and ACAD11 paralogs. The overall patchy distribution of specific ACADs across the tree of life is the result of dynamic evolution that includes numerous rounds of gene duplication and secondary losses, interdomain lateral gene transfer events, alteration of cellular localization, and evolution of novel proteins by domain acquisition. Our finding that eukaryotic ACAD species are more closely related to bacterial ACADs is consistent with endosymbiotic origin of ACADs in eukaryotes and further supported by the localization of all nine previously studied ACADs in mitochondria. PMID:19639238

  17. Deciphering the molecular and functional basis of Dbl family proteins: a novel systematic approach toward classification of selective activation of the Rho family proteins.

    PubMed

    Jaiswal, Mamta; Dvorsky, Radovan; Ahmadian, Mohammad Reza

    2013-02-08

    The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.

  18. All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly.

    PubMed Central

    Bergdoll, M.; Eltis, L. D.; Cameron, A. D.; Dumas, P.; Bolin, J. T.

    1998-01-01

    The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions. PMID:10082363

  19. 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

  20. 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

  1. 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.

  2. The first structure from the SOUL/HBP family of heme-binding proteins, murine P22HBP.

    PubMed

    Dias, Jorge S; Macedo, Anjos L; Ferreira, Gloria C; Peterson, Francis C; Volkman, Brian F; Goodfellow, Brian J

    2006-10-20

    Murine p22HBP, a 22-kDa monomer originally identified as a cytosolic heme-binding protein ubiquitously expressed in various tissues, has 27% sequence identity to murine SOUL, a heme-binding hexamer specifically expressed in the retina. In contrast to murine SOUL, which binds one heme per subunit via coordination of the Fe(III)-heme to a histidine, murine p22HBP binds one heme molecule per subunit with no specific axial ligand coordination of the Fe(III)-heme. Using intrinsic protein fluorescence quenching, the values for the dissociation constants of p22HBP for hemin and protoporphyrin-IX were determined to be in the low nanomolar range. The three-dimensional structure of murine p22HBP, the first for a protein from the SOUL/HBP family, was determined by NMR methods to consist of a 9-stranded distorted beta-barrel flanked by two long alpha-helices. Although homologous domains have been found in three bacterial proteins, two of which are transcription factors, the fold determined for p22HBP corresponds to a novel alpha plus beta fold in a eukaryotic protein. Chemical shift mapping localized the tetrapyrrole binding site to a hydrophobic cleft formed by residues from helix alphaA and an extended loop. In an attempt to assess the structural basis for tetrapyrrole binding in the SOUL/HBP family, models for the p22HBP-protoporphyrin-IX complex and the SOUL protein were generated by manual docking and automated methods.

  3. Phylogenetic analysis of the Argonaute protein family in platyhelminths.

    PubMed

    Zheng, Yadong

    2013-03-01

    Argonaute proteins (AGOs) are mediators of gene silencing via recruitment of small regulatory RNAs to induce translational regression or degradation of targeted molecules. Platyhelminths have been reported to express microRNAs but the diversity of AGOs in the phylum has not been explored. Phylogenetic relationships of members of this protein family were studied using data from six platyhelminth genomes. Phylogenetic analysis showed that all cestode and trematode AGOs, along with some triclad planarian AGOs, were grouped into the Ago subfamily and its novel sister clade, here referred to as Cluster 1. These were very distant from Piwi and Class 3 subfamilies. By contrast, a number of planarian Piwi-like AGOs formed a novel sister clade to the Piwi subfamily. Extensive sequence searching revealed the presence of an additional locus for AGO2 in the cestode Echinococcus granulosus and exon expansion in this species and E. multilocularis. The current study suggests the absence of the Piwi subfamily and Class 3 AGOs in cestodes and trematodes and the Piwi-like AGO expansion in a free-living triclad planarian and the occurrence of exon expansion prior to or during the evolution of the most-recent common ancestor of the Echinococcus species studied.

  4. A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

    PubMed Central

    Korthout, H A; de Boer, A H

    1994-01-01

    The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants. PMID:7827499

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Genome-enabled selection doubles the accuracy of predicted breeding values for bacterial cold water disease resistance compared to traditional family-based selection in rainbow trout aquaculture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have shown previously that bacterial cold water disease (BCWD) resistance in rainbow trout can be improved using traditional family-based selection, but progress has been limited to exploiting only between-family genetic variation. Genomic selection (GS) is a new alternative enabling exploitation...

  11. A new family of highly variable proteins in the Chlamydophila pneumoniae genome

    PubMed Central

    Rocha, Eduardo P. C.; Pradillon, Olivier; Bui, Hung; Sayada, Chalom; Denamur, Erick

    2002-01-01

    Chlamydiaceae are obligate intracellular bacterial pathogens characterized by a wide range of vertebrate host, tissue tropism and spectrum of diseases. To get insights into the biological mechanisms involved in these differences, we have put forward a computational and experimental procedure to identify the genome recombination hotspots, as frequent sequence variation allows rapid adaptation to environmental changes. We find a larger potential for recombination in Chlamydophila pneumoniae genomes as compared with Chlamydia trachomatis or Chlamydia muridarum. Such potential is mostly concentrated in a family of seven previously uncharacterized species-specific elements that we named ppp for C.pneumoniae polymorphic protein genes, which have the potential to vary by homologous recombination and slipped-mispair. Experimen tally, we show that these sequences are indeed highly polymorphic among a collection of nine C.pneumoniae strains of very diverse geographical and pathological origins, mainly by slippage of a poly(C) tract. We also show that most elements are transcribed during infection. In silico analyses suggest that Ppps correspond to outer membrane proteins. Given their species specificity, their putative location in the outer membrane and their extreme polymorphism, Ppps are most likely to be important in the pathogenesis of C.pneumoniae and could represent targets for future vaccine development. PMID:12384581

  12. 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.

  13. The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

    PubMed Central

    Das, Debanu; Finn, Robert D.; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; 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; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins. PMID:20944221

  14. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

  15. Small G-protein Signaling in Neuronal Plasticity and Memory Formation: the Specific Role of Ras Family Proteins

    PubMed Central

    Ye, Xiaojing; Carew, Thomas J.

    2010-01-01

    Small G-proteins are an extensive family of proteins that bind and hydrolyze GTP. They are ubiquitous inside cells, regulating a wide range of cellular processes. Recently, many studies have examined the role of small G-proteins, particularly the Ras family of G-proteins, in memory formation. Once thought to be primarily involved in the transduction of a variety of extracellular signals during development, it is now clear that Ras family proteins also play critical roles in molecular processing underlying neuronal and behavioral plasticity. We here review a number of recent studies that explore how the signaling of Ras family proteins contributes to memory formation. Understanding these signaling processes is of fundamental importance both from a basic scientific perspective, with the goal of providing mechanistic insights into a critical aspect of cognitive behavior, and from a clinical perspective, with the goal of providing effective therapies for a range of disorders involving cognitive impairments. PMID:21040840

  16. The head protein D of bacterial virus lambda is related to eukaryotic chromosomal proteins.

    PubMed Central

    Witkiewicz, H; Schweiger, M

    1982-01-01

    Bacteriophage lambda structural head protein D has physiochemical properties in common with eukaryotic chromosomal proteins. It has a low affinity for hydroxylapatite, it is heat stable and acid soluble. Moreover, it cross-reacts immunologically with histones H2A and H2B. The deduced primary structure of the D protein shows striking homology to calf chromosomal high mobility group HMG-14 protein. There are two clusters of four ( LSAK , ASDE ) and one of three (APA) identical amino acid residues. Additionally the cluster ETK of protein D occurs three times in HMG-14 and 14 single identical residues are present. A mechanism for an alternative to a nucleosomal mode of nuclear DNA condensation and a possible function of HMG proteins are discussed. Images Fig. 1. Fig. 2. Fig. 3. PMID:6233140

  17. 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.

  18. 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

  19. Spermadhesins: a new protein family. Facts, hypotheses and perspectives.

    PubMed

    Töpfer-Petersen, E; Romero, A; Varela, P F; Ekhlasi-Hundrieser, M; Dostàlovà, Z; Sanz, L; Calvete, J J

    1998-01-01

    Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN-1, AQN-2, PSP-I and PSP-II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand-binding abilities, e.g. carbohydrates, sulfated glycosaminoglycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins bind the zona pellucida glycoproteins in a cation-dependent manner with a Kd in a low micromolar range, and AWN, AQN-1 and AQN-3 display similar binding affinity for glycoproteins containing Gal beta(1-3)-GalNAc and Gal beta(1-4)-GlcNAc sequences in O-linked and N-linked oligosaccharides, respectively. During sperm passage through the epididymis AQN-3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid-bound spermadhesins. AWN and AQN-3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to initial binding and recognition between sperm and egg. The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate recognition domains (CRD). However, they belong to the superfamily of proteins with a CUB domain with a predicted all-beta structure. The crystal structure of the heterodimeric complex of the spermadhesins PSP-I/PSP-II has been solved, showing

  20. Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein*

    PubMed Central

    Rosas-Acosta, Germán; Wilson, Van G.

    2012-01-01

    SUMMARY The E1 protein, a papillomavirus (PV)-encoded origin-binding helicase essential for PV DNA replication, is post-translationally modified by sumoylation. As this modification is essential for the nuclear accumulation of the bovine PV E1 (BPV E1), factors modulating the sumoylation of E1 could ultimately alter the outcome of a papillomavirus infection. Therefore, we systematically tested the known sumoylation enhancing factors (E3 SUMO ligases), namely RanBP2 and PIAS family proteins, to determine their ability to bind to E1 and stimulate its sumoylation, using in vitro assays. We found that RanBP2 bound to BPV E1 but failed to bind to the E1 from a human PV (HPV11 E1), and lacked any sumoylation enhancing activity for both BPV E1 and HPV11 E1. In contrast, proteins of the PIAS family (except for PIASy) bound to both BPV E1 and HPV11 E1 and stimulated their sumoylation, with PIASxβ (Miz1) exerting the largest stimulatory effect. The structural integrity of the RING finger domain of the PIAS proteins was required for their E3 SUMO ligase activity on PV E1 sumoylation, but was dispensable for their PV E1 binding activity. Furthermore, the sumoylation enhancing activity exerted by the PIAS proteins on BPV E1 was more pronounced than on HPV11 E1, and appeared to favor SUMO1 versus SUMO2 as the SUMO modifier. Altogether, this study identifies PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirus infections. PMID:15582666

  1. The protein-protein interaction network of the human Sirtuin family.

    PubMed

    Sharma, Ankush; Costantini, Susan; Colonna, Giovanni

    2013-10-01

    Protein-protein interaction networks are useful for studying human diseases and to look for possible health care through a holistic approach. Networks are playing an increasing and important role in the understanding of physiological processes such as homeostasis, signaling, spatial and temporal organizations, and pathological conditions. In this article we show the complex system of interactions determined by human Sirtuins (Sirt) largely involved in many metabolic processes as well as in different diseases. The Sirtuin family consists of seven homologous Sirt-s having structurally similar cores but different terminal segments, being rather variable in length and/or intrinsically disordered. Many studies have determined their cellular location as well as biological functions although molecular mechanisms through which they act are actually little known therefore, the aim of this work was to define, explore and understand the Sirtuin-related human interactome. As a first step, we have integrated the experimentally determined protein-protein interactions of the Sirtuin-family as well as their first and second neighbors to a Sirtuin-related sub-interactome. Our data showed that the second-neighbor network of Sirtuins encompasses 25% of the entire human interactome, and exhibits a scale-free degree distribution and interconnectedness among top degree nodes. Moreover, the Sirtuin sub interactome showed a modular structure around the core comprising mixed functions. Finally, we extracted from the Sirtuin sub-interactome subnets related to cancer, aging and post-translational modifications for information on key nodes and topological space of the subnets in the Sirt family network.

  2. Protein-protein interaction network prediction by using rigid-body docking tools: application to bacterial chemotaxis.

    PubMed

    Matsuzaki, Yuri; Ohue, Masahito; Uchikoga, Nobuyuki; Akiyama, Yutaka

    2014-01-01

    Core elements of cell regulation are made up of protein-protein interaction (PPI) networks. However, many parts of the cell regulatory systems include unknown PPIs. To approach this problem, we have developed a computational method of high-throughput PPI network prediction based on all-to-all rigid-body docking of protein tertiary structures. The prediction system accepts a set of data comprising protein tertiary structures as input and generates a list of possible interacting pairs from all the combinations as output. A crucial advantage of this docking based method is in providing predictions of protein pairs that increases our understanding of biological pathways by analyzing the structures of candidate complex structures, which gives insight into novel interaction mechanisms. Although such exhaustive docking calculation requires massive computational resources, recent advancements in the computational sciences have made such large-scale calculations feasible. In this study we applied our prediction method to a pathway reconstruction problem of bacterial chemotaxis by using two different rigid-body docking tools with different scoring models. We found that the predicted interactions were different between the results from the two tools. When the positive predictions from both of the docking tools were combined, all the core signaling interactions were correctly predicted with the exception of interactions activated by protein phosphorylation. Large-scale PPI prediction using tertiary structures is an effective approach that has a wide range of potential applications. This method is especially useful for identifying novel PPIs of new pathways that control cellular behavior.

  3. Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins.

    PubMed

    Altenhofer, Pia; Schierhorn, Angelika; Fricke, Beate

    2006-10-01

    2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.

  4. Overexpression of the Eggplant (Solanum melongena) NAC Family Transcription Factor SmNAC Suppresses Resistance to Bacterial Wilt

    PubMed Central

    Na, Chen; Shuanghua, Wu; Jinglong, Fu; Bihao, Cao; Jianjun, Lei; Changming, Chen; Jin, Jiang

    2016-01-01

    Bacterial wilt (BW) is a serious disease that affects eggplant (Solanum melongena) production. Although resistance to this disease has been reported, the underlying mechanism is unknown. In this study, we identified a NAC family transcription factor (SmNAC) from eggplant and characterized its expression, its localization at the tissue and subcellular levels, and its role in BW resistance. To this end, transgenic eggplant lines were generated in which the expression of SmNAC was constitutively up regulated or suppressed using RNAi. The results indicated that overexpression of SmNAC decreases resistance to BW. Moreover, SmNAC overexpression resulted in the reduced accumulation of the plant immune signaling molecule salicylic acid (SA) and reduced expression of ICS1 (a gene that encode isochorismate synthase 1, which is involved in SA biosynthesis). We propose that reduced SA content results in increased bacterial wilt susceptibility in the transgenic lines. Our results provide important new insights into the regulatory mechanisms of bacterial wilt resistance in eggplant. PMID:27528282

  5. Overexpression of the Eggplant (Solanum melongena) NAC Family Transcription Factor SmNAC Suppresses Resistance to Bacterial Wilt.

    PubMed

    Na, Chen; Shuanghua, Wu; Jinglong, Fu; Bihao, Cao; Jianjun, Lei; Changming, Chen; Jin, Jiang

    2016-08-16

    Bacterial wilt (BW) is a serious disease that affects eggplant (Solanum melongena) production. Although resistance to this disease has been reported, the underlying mechanism is unknown. In this study, we identified a NAC family transcription factor (SmNAC) from eggplant and characterized its expression, its localization at the tissue and subcellular levels, and its role in BW resistance. To this end, transgenic eggplant lines were generated in which the expression of SmNAC was constitutively up regulated or suppressed using RNAi. The results indicated that overexpression of SmNAC decreases resistance to BW. Moreover, SmNAC overexpression resulted in the reduced accumulation of the plant immune signaling molecule salicylic acid (SA) and reduced expression of ICS1 (a gene that encode isochorismate synthase 1, which is involved in SA biosynthesis). We propose that reduced SA content results in increased bacterial wilt susceptibility in the transgenic lines. Our results provide important new insights into the regulatory mechanisms of bacterial wilt resistance in eggplant.

  6. Subcellular localization of Gram-negative bacterial proteins using sparse learning.

    PubMed

    Zheng, Zhonglong; Yang, Jie

    2010-04-01

    One of the main challenges faced by biological applications is to predict protein subcellular localization in an automatic fashion accurately. To achieve this in these applications, a wide variety of machine learning methods have been proposed in recent years. Most of them focus on finding the optimal classification scheme and less of them take the simplifying the complexity of biological system into account. Traditionally such bio-data are analyzed by first performing a feature selection before classification. Motivated by CS (Compressive Sensing), we propose a method which performs locality preserving projection with a sparseness criterion such that the feature selection and dimension reduction are merged into one analysis. The proposed sparse method decreases the complexity of biological system, while increases protein subcellular localization accuracy. Experimental results are quite encouraging, indicating that the aforementioned sparse method is quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gram-negative bacterial proteins.

  7. Membrane proteases in the bacterial protein secretion and quality control pathway.

    PubMed

    Dalbey, Ross E; Wang, Peng; van Dijl, Jan Maarten

    2012-06-01

    Proteolytic cleavage of proteins that are permanently or transiently associated with the cytoplasmic membrane is crucially important for a wide range of essential processes in bacteria. This applies in particular to the secretion of proteins and to membrane protein quality control. Major progress has been made in elucidating the structure-function relationships of many of the responsible membrane proteases, including signal peptidases, signal peptide hydrolases, FtsH, the rhomboid protease GlpG, and the site 1 protease DegS. These enzymes employ very different mechanisms to cleave substrates at the cytoplasmic and extracytoplasmic membrane surfaces or within the plane of the membrane. This review highlights the different ways that bacterial membrane proteases degrade their substrates, with special emphasis on catalytic mechanisms and substrate delivery to the respective active sites.

  8. Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

    PubMed

    Weiss, Jamie L; Evans, Nicholas A; Ahmed, Tanweer; Wrigley, Jonathan D J; Khan, Shukria; Wright, Charles; Keen, Jeffrey N; Holzenburg, Andreas; Findlay, John B C

    2005-03-01

    We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

  9. Adhesion family of G protein-coupled receptors and cancer.

    PubMed

    Lin, Hsi-Hsien

    2012-01-01

    The adhesion-class G protein-coupled receptors (adhesion-GPCRs) constitute the second largest GPCR sub-family in humans. Adhesion-GPCRs are defined by the chimeric structure of an unusually large extracellular cell-adhesion domain and a GPCR-like seven-pass transmembrane domain. Adhesion-GPCRs are hence expected to display both cellular adhesion and signaling functions in many biological systems. Adhesion-GPCRs are normally expressed in the central nervous, immune, and reproductive systems in a cell type- or tissue-restricted fashion. However, aberrant expression of distinct adhesion-GPCR molecules has been identified in various human cancers with some of the receptors closely associated with cancer development. Tumor-associated adhesion-GPCRs are thought to involve in tumorigenesis by affecting the growth of tumor cells, angiogenesis, tumor cell migration, invasion and metastasis either positively or negatively. Furthermore, some adhesion-GPCRs are considered potential biomarkers for specific types of cancers. In this review article, the expressional characteristics and functional role of cancer-associated adhesion-GPCRs are discussed in depth.

  10. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  11. Protein Mis-Termination Initiates Genetic Diseases, Cancers, and Restricts Bacterial Genome Expansion.

    PubMed

    Wong, Tit-Yee; Schwartzbach, Steve D

    2015-01-01

    Protein termination is an important cellular process. Protein termination relies on the stop-codons in the mRNA interacting properly with the releasing factors on the ribosome. One third of inherited diseases, including cancers, are associated with the mutation of the stop-codons. Many pathogens and viruses are able to manipulate their stop-codons to express their virulence. The influence of stop-codons is not limited to the primary reading frame of the genes. Stop-codons in the second and third reading frames are referred as premature stop signals (PSC). Stop-codons and PSCs together are collectively referred as stop-signals. The ratios of the stop-signals (referred as translation stop-signals ratio or TSSR) of genetically related bacteria, despite their great differences in gene contents, are much alike. This nearly identical Genomic-TSSR value of genetically related bacteria may suggest that bacterial genome expansion is limited by their unique stop-signals bias. We review the protein termination process and the different types of stop-codon mutation in plants, animals, microbes, and viruses, with special emphasis on the role of PSCs in directing bacterial evolution in their natural environments. Knowing the limit of genomic boundary could facilitate the formulation of new strategies in controlling the spread of diseases and combat antibiotic-resistant bacteria.

  12. Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein.

    PubMed

    Müller, Ina; Gernold, Marina; Schneider, Bernd; Geider, Klaus

    2012-01-01

    Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.

  13. Differential expression of genes encoding calmodulin-binding proteins in response to bacterial pathogens and inducers of defense responses.

    PubMed

    Ali, Gul Shad; Reddy, Vaka S; Lindgren, Peter B; Jakobek, Judy L; Reddy, A S N

    2003-04-01

    Calmodulin (CaM) plays an important role in sensing and transducing changes in cellular Ca2+ concentration in response to several biotic and abiotic stresses. Although CaM is implicated in plant-pathogen interactions, its molecular targets and their role in defense signaling pathway(s) are poorly understood. To elucidate the signaling pathways that link CaM to defense responses, we screened a cDNA library constructed from bean leaves undergoing a hypersensitive response (HR) with radiolabeled CaM isoforms. A total of 26 putative CBPs were identified. Sequencing of the cDNAs revealed that they represent 8 different genes. They are homologues of previously identified CaM-binding proteins (CBPs) in other systems. However, some CBPs are novel members of known CBP families. The proteins encoded by these clones bound CaM in a Ca2+-dependent manner. To determine if these CBPs are involved in plant defense responses, we analyzed their expression in bean leaves inoculated with compatible, incompatible and nonpathogenic bacterial strains. Expression of three CBPs including an isoform of cyclic nucleotide-gated channels (PvCNGC-A) and two hypothetical proteins (PvCBP60-C and PvCBP60-D) was induced whereas the expression of two other isoforms of CNGCs (PvCNGC-B and PvCNGC-C) was repressed in response to incompatible pathogens. The expression of the rest, a small auxin up RNA (PvSAUR1) and two hypothetical proteins (PvCBP60-A and PvCBP60-B), was not changed. The expression of most of the pathogen-regulated genes was also affected by salicylic acid, jasmonic acid, hydrogen peroxide and a fungal elicitor, which are known to induce defense responses. Our results strongly suggest that at least five bean CBPs are involved in plant defense responses.

  14. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    PubMed

    Krinsky, Nitzan; Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  15. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    PubMed Central

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  16. Internal organization of large protein families: relationship between the sequence, structure, and function-based clustering.

    PubMed

    Cai, Xiao-Hui; Jaroszewski, Lukasz; Wooley, John; Godzik, Adam

    2011-08-01

    The protein universe can be organized in families that group proteins sharing common ancestry. Such families display variable levels of structural and functional divergence, from homogenous families, where all members have the same function and very similar structure, to very divergent families, where large variations in function and structure are observed. For practical purposes of structure and function prediction, it would be beneficial to identify sub-groups of proteins with highly similar structures (iso-structural) and/or functions (iso-functional) within divergent protein families. We compared three algorithms in their ability to cluster large protein families and discuss whether any of these methods could reliably identify such iso-structural or iso-functional groups. We show that clustering using profile-sequence and profile-profile comparison methods closely reproduces clusters based on similarities between 3D structures or clusters of proteins with similar biological functions. In contrast, the still commonly used sequence-based methods with fixed thresholds result in vast overestimates of structural and functional diversity in protein families. As a result, these methods also overestimate the number of protein structures that have to be determined to fully characterize structural space of such families. The fact that one can build reliable models based on apparently distantly related templates is crucial for extracting maximal amount of information from new sequencing projects.

  17. Bacterial Obg proteins: GTPases at the nexus of protein and DNA synthesis.

    PubMed

    Kint, Cyrielle; Verstraeten, Natalie; Hofkens, Johan; Fauvart, Maarten; Michiels, Jan

    2014-08-01

    Obg proteins (also known as ObgE, YhbZ and CgtA) are conserved P-loop GTPases, essential for growth in bacteria. Like other GTPases, Obg proteins cycle between a GTP-bound ON and a GDP-bound OFF state, thereby controlling cellular processes. Interestingly, the in vitro biochemical properties of Obg proteins suggest that they act as sensors for the cellular GDP/GTP pools and adjust their activity according to the cellular energy status. Obg proteins have been attributed a host of cellular functions, including roles in essential cellular processes (DNA replication, ribosome maturation) and roles in different stress adaptation pathways (stringent response, sporulation, general stress response). This review summarizes the current knowledge on Obg activity and function. Furthermore, we present a model that integrates the different functions of Obg by assigning it a fundamental role in cellular physiology, at the hub of protein and DNA synthesis. In particular, we believe that Obg proteins might provide a connection between different global pathways in order to fine-tune cellular processes in response to a given energy status.

  18. Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

    PubMed

    Huang, Guangrui; Huang, Shengfeng; Yan, Xinyu; Yang, Ping; Li, Jun; Xu, Weiya; Zhang, Lingling; Wang, Ruihua; Yu, Yingcai; Yuan, Shaochun; Chen, Shangwu; Luo, Guangbin; Xu, Anlong

    2014-09-16

    Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

  19. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

    PubMed

    Gennaris, Alexandra; Ezraty, Benjamin; Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

    2015-12-17

    The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

  20. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

    PubMed Central

    Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

    2015-01-01

    The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts 1-3. Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both R- and S- diastereoisomers of Met-O, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting Met residues from oxidation should prompt search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum (ER). PMID:26641313

  1. d-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling*

    PubMed Central

    Fura, Jonathan M.; Kearns, Daniel; Pires, Marcos M.

    2015-01-01

    Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural d-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural d-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural d-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural d-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural d-amino acids into bacterial peptidoglycan. PMID:26499795

  2. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols

    SciTech Connect

    Wise, A.A.; Kuske, C.R.

    2000-01-01

    The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. The authors have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.

  3. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    SciTech Connect

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-05-27

    Highlights: {yields} Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. {yields} Monoubiquitination sites of Tob are identified by mass spectrometry. {yields} The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  4. VH3 family antibodies bind domain D of staphylococcal protein A.

    PubMed

    Roben, P W; Salem, A N; Silverman, G J

    1995-06-15

    Staphylococcal protein A (SpA) is a 45-kDa bacterial membrane protein that can interact with either Fc gamma, a constant region portion of IgG, or with the Fab portion that also mediates conventional Ag binding. In recent reports, SpA has been shown to specifically interact with Fab derived from the VH3 family and is little affected by VH CDR3, JH, or light chain usage. To identify a site on SpA responsible for VH3 Fab binding, we cloned and expressed in Escherichia coli the 61 amino acid sequence of SpA that represents domain D, and this small protein exhibited both the VH3 Fab and Fc gamma binding specificities. Surface plasmon resonance measurements demonstrated that domain D and native SpA had the strongest binding interactions with an IgM-kappa encoded by the germline configuration of the VH3 gene VH26c. In contrast, the apparent affinities for Fc gamma binding were at least fivefold weaker. A variant of domain D was also created that is devoid of the three-codon insertion that distinguishes domain D from all other domains in SpA. Although this deletion did not significantly affect the VH3 Fab-mediated SpA binding activity, it did improve the affinity of Fc gamma binding by an order of magnitude. These observations characterize a site on SpA responsible for binding interactions with B cell Ag receptors that are highly analogous to that of superantigens for T cell receptors.

  5. Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

    PubMed Central

    2012-01-01

    Background Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. Results In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the

  6. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  7. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  8. Multi-color imaging of the bacterial nucleoid and division proteins with blue, orange, and near-infrared fluorescent proteins

    PubMed Central

    Wu, Fabai; Van Rijn, Erwin; Van Schie, Bas G. C.; Dekker, Cees

    2015-01-01

    Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange (TagRFP-T, mKO2), and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified in vivo for their introduction into bacteria; (2) spectral crosstalk and inter-variant interactions between FPs should be carefully examined for multi-color imaging; and (3) successful genomic fusion to the 5′-end of a gene strongly depends on the translational read-through of the inserted coding sequence. PMID:26136737

  9. Bcl-2-related protein family gene expression during oligodendroglial differentiation.

    PubMed

    Itoh, Takayuki; Itoh, Aki; Pleasure, David

    2003-06-01

    Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.

  10. Localization of a bacterial group II intron-encoded protein in human cells.

    PubMed

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

    2015-08-05

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

  11. Localization of a bacterial group II intron-encoded protein in human cells

    PubMed Central

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

    2015-01-01

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

  12. A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy

    PubMed Central

    Khweek, Arwa Abu; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A.; Tazi, Mia; Hassan, Hoda; Majumdar, Neal; Doran, Andrew; Guirado, Evelyn; Schlesinger, Larry S.; Shuman, Howard; Amer, Amal O.

    2013-01-01

    Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant is the first L. pneumophila mutant to replicate in wild-type (WT) mice and their bone marrow derived macrophages (BMDMs). Less legA9 mutant- containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila -containing vacuole for autophagy uptake. PMID:23420491

  13. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ.

    PubMed

    Nepomuceno, Gabriella M; Chan, Katie M; Huynh, Valerie; Martin, Kevin S; Moore, Jared T; O'Brien, Terrence E; Pollo, Luiz A E; Sarabia, Francisco J; Tadeus, Clarissa; Yao, Zi; Anderson, David E; Ames, James B; Shaw, Jared T

    2015-03-12

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ's GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3.

  14. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ

    PubMed Central

    2015-01-01

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ’s GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3. PMID:25815151

  15. Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation.

    PubMed

    Baker, Jenny L; Çelik, Eda; DeLisa, Matthew P

    2013-05-01

    Glycosylation is the most prevalent post-translational modification found on proteins, occurring in all domains of life. Ever since the discovery of asparagine-linked (N-linked) protein glycosylation pathways in bacteria, major efforts have been made to harness these systems for the creation of novel therapeutics, vaccines, and diagnostics. Recent advances such as the ability to produce designer glycans in bacteria, some containing unnatural sugars, and techniques for evolving glycosylation enzymes have spawned an entirely new discipline known as bacterial glycoengineering. In addition to their biotechnological and therapeutic potential, bacteria equipped with recombinant N-linked glycosylation pathways are improving our understanding of the N-glycosylation process. This review discusses the key role played by microorganisms in glycosciences, particularly in the context of N-linked glycosylation.

  16. Flocculation behaviour of hematite-kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides.

    PubMed

    Poorni, S; Natarajan, K A

    2014-02-01

    Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells.

  17. Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci.

    PubMed

    Kästner, Julia; Saluz, Hans Peter; Hänel, Frank

    2015-05-01

    Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines.

  18. Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins.

    PubMed

    Groff, Dan; Armstrong, Stephanie; Rivers, Patrick J; Zhang, Juan; Yang, Junhao; Green, Evan; Rozzelle, James; Liang, Shengwen; Kittle, Joseph D; Steiner, Alexander R; Baliga, Ramesh; Thanos, Christopher D; Hallam, Trevor J; Sato, Aaron K; Yam, Alice Y

    2014-01-01

    Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

  19. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    PubMed

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  20. Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS.

    PubMed

    Ziegler, Dominik; Pothier, Joël F; Ardley, Julie; Fossou, Romain Kouakou; Pflüger, Valentin; de Meyer, Sofie; Vogel, Guido; Tonolla, Mauro; Howieson, John; Reeve, Wayne; Perret, Xavier

    2015-07-01

    Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.

  1. The bacterial cell cycle checkpoint protein Obg and its role in programmed cell death

    PubMed Central

    Dewachter, Liselot; Verstraeten, Natalie; Fauvart, Maarten; Michiels, Jan

    2016-01-01

    The phenomenon of programmed cell death (PCD), in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli). Importantly, the PCD pathway mediated by mutant Obg (Obg*) differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway.

  2. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    PubMed

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  3. The participation of outer membrane proteins in the bacterial sensitivity to nanosilver.

    PubMed

    Kędziora, Anna; Krzyżewska, Eva; Dudek, Bartłomiej; Bugla-Płoskońska, Gabriela

    2016-06-13

    The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

  4. Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

    PubMed

    Tomalty, Heather E; Walker, Virginia K

    2014-09-26

    Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.

  5. Ultrastructural and molecular characterization of a bacterial symbiosis in the ecologically important scale insect family Coelostomidiidae.

    PubMed

    Dhami, Manpreet K; Turner, Adrian P; Deines, Peter; Beggs, Jacqueline R; Taylor, Michael W

    2012-09-01

    Scale insects are important ecologically and as agricultural pests. The majority of scale insect taxa feed exclusively on plant phloem sap, which is carbon rich but deficient in essential amino acids. This suggests that, as seen in the related aphids and psyllids, scale insect nutrition might also depend upon bacterial symbionts, yet very little is known about scale insect-bacteria symbioses. We report here the first identification and molecular characterization of symbiotic bacteria associated with the New Zealand giant scale Coelostomidia wairoensis, using fluorescence in situ hybridization (FISH), transmission electron microscopy (TEM) and 16S rRNA gene-based analysis. Dissection and FISH confirmed the location of the bacteria in large, paired, multilobate organs in the abdominal region of the insect. TEM indicated that the dominant pleomorphic bacteria were confined to bacteriocytes in the sheath-enclosed bacteriome. Phylogenetic analysis revealed the presence of three distinct bacterial types, the bacteriome-associated B-symbiont (Bacteroidetes), an Erwinia-related symbiont (Gammaproteobacteria) and Wolbachia sp. (Alphaproteobacteria). This study extends the current knowledge of scale insect symbionts and is the first microbiological investigation of the ecologically important coelostomidiid scales.

  6. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  7. The CCN Family Proteins: Modulators of Bone Development and Novel Targets in Bone-Associated Tumors

    PubMed Central

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors. PMID:24551846

  8. The CCN family proteins: modulators of bone development and novel targets in bone-associated tumors.

    PubMed

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Lin, Chiao-Wen; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors.

  9. STUDIES ON THE BLOOD PROTEINS : I. THE SERUM GLOBULINS IN BACTERIAL INFECTION AND IMMUNITY.

    PubMed

    Hurwitz, S H; Meyer, K F

    1916-11-01

    The progress of an infection is usually associated with marked changes in the serum proteins. There may be an increase in the percentage of the total protein during some stage of the infection, and there is usually a change in the albumin-globulin ratio with an increase in the total globulins. This rise may antedate the development of any resistance by a considerable period of time. The non-protein constituents of the blood show fluctuations with a tendency to rise as the infection progresses. The process of immunization is in almost all instances associated with a definite increase in the globulins of the blood, and in some cases with a complete inversion of the normal albumin-globulin ratio. This may be produced both by living and dead organisms and by bacterial endotoxins. Massive doses usually result in an upset which shows no tendency to right itself during the period of observation. A rise in the globulins has been shown to occur long before the animal develops immune bodies in any appreciable concentration; and where the globulin curve and antibody curve appear to parallel one another, it can be shown by a careful analysis of both curves that there is a definite lack of correspondence at various periods of the experiment. Animals possessing a basic immunity show a more rapid rise in the globulin curve following inoculation. There is no parallelism between the leukocytic reaction and the globulin reaction. During periods of leukopenia the globulins may be as high as during the period of a leukocytosis. Bacterial endotoxins produce as striking an increase in the serum globulins as do living and killed bacteria. This would seem to indicate that a bacterial invasion of the organism is not absolutely essential for the globulin changes, and that the toxogenic factor in infection and immunity must play a part in the production of the changes noted. Inflammatory irritants injected intraperitoneally also result in a globulin increase. In this case the changes

  10. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    PubMed Central

    Branco, Luis M; Matschiner, Alex; Fair, Joseph N; Goba, Augustine; Sampey, Darryl B; Ferro, Philip J; Cashman, Kathleen A; Schoepp, Randal J; Tesh, Robert B; Bausch, Daniel G; Garry, Robert F; Guttieri, Mary C

    2008-01-01

    Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination. PMID:18538016

  11. The Sorcerer II Global Ocean Sampling Expedition: Expanding theUniverse of Protein Families

    SciTech Connect

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B.; Halpern,Aaron L.; Williamson, Shannon J.; Remington, Karin; Eisen, Jonathan A.; Heidelberg, Karla B.; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S.; Li, Huiying; Mashiyama, Susan T.; Joachimiak, Marcin P.; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A.; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael,Benjamin J.; Bafna, Vineet; Friedman, Robert; Brenner, Steven E.; Godzik,Adam; Eisenberg, David; Dixon, Jack E.; Taylor, Susan S.; Strausberg,Robert L.; Frazier, Marvin; Venter, J.Craig

    2006-03-23

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  12. The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families.

    PubMed

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J Craig

    2007-03-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  13. The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

    PubMed Central

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J. Craig

    2007-01-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature. PMID:17355171

  14. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

  15. The Staphylococcus aureus extracellular adherence protein promotes bacterial internalization by keratinocytes independent of fibronectin-binding proteins.

    PubMed

    Bur, Stephanie; Preissner, Klaus T; Herrmann, Mathias; Bischoff, Markus

    2013-08-01

    Staphylococcus aureus, the leading causal pathogen of skin infections, is strongly associated with skin atopy, and a number of bacterial adhesins allow the microbe to adhere to and invade eukaryotic cells. One of these adhesive molecules is the multifunctional extracellular adherence protein (Eap), which is overexpressed in situ in authentic human wounds and was shown to delay wound healing in experimental models. Yet, its role during invasion of keratinocytes is not clearly defined. By using a gentamicin/lysostaphin protection assay we demonstrate here that preincubation of HaCaT cells or primary keratinocytes with Eap results in a concentration-dependent significant increase in staphylococcal adhesion, followed by an even more pronounced internalization of bacteria by eukaryotic cells. Flow cytometric analysis revealed that Eap increased both the number of infected eukaryotic cells and the bacterial load per infected cell. Moreover, treatment of keratinocytes with Eap strongly enhanced the internalization of coagulase-negative staphylococci, as well as of E. coli, and markedly promoted staphylococcal invasion into extended-culture keratinocytes, displaying expression of keratin 10 and involucrin as differentiation markers. Thus, wound-related staphylococcal Eap may provide a major cellular invasin function, thereby enhancing the pathogen's ability to hide from the host immune system during acute and chronic skin infection.

  16. Nucleo-cytoplasmic functions of the PDZ-LIM protein family: new insights in organ development

    PubMed Central

    Krcmery, Jennifer; Camarata, Troy; Kulisz, Andre; Simon, Hans-Georg

    2010-01-01

    Summary Recent work on the PDZ-LIM protein family has revealed important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ-LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ-LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ-LIM proteins have wide-ranging and multi-compartmental cell functions during development and homeostasis while, in contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T-box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ-LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture and control gene transcription. PMID:20091751

  17. Discovery and Analysis of 4H-Pyridopyrimidines, a Class of Selective Bacterial Protein Synthesis Inhibitors▿

    PubMed Central

    Ribble, Wendy; Hill, Walter E.; Ochsner, Urs A.; Jarvis, Thale C.; Guiles, Joseph W.; Janjic, Nebojsa; Bullard, James M.

    2010-01-01

    Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyl-tRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC50s) ranging from 3 to 14 μM. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d]pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five- or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 μg/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition. PMID:20696870

  18. Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins.

    PubMed

    Zav'yalov, V P; Zav'yalova, G A; Denesyuk, A I; Gaestel, M; Korpela, T

    1995-07-01

    The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli. One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 x 10(-4). IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.

  19. Selective molecular transport through the protein shell of a bacterial microcompartment organelle

    DOE PAGES

    Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; ...

    2015-02-23

    Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. In this paper, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux ofmore » propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Finally and overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.« less

  20. Fluorescence quenching as a tool to investigate quinolone antibiotic interactions with bacterial protein OmpF.

    PubMed

    Neves, Patrícia; Sousa, Isabel; Winterhalter, Mathias; Gameiro, Paula

    2009-02-01

    The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp(214), Trp(61)), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp(61) and iodide Trp(214). Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp(214) and moxifloxacin near Trp(61). These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.

  1. The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

    PubMed Central

    Govindarajan, Sutharsan; Elisha, Yair; Nevo-Dinur, Keren; Amster-Choder, Orna

    2013-01-01

    ABSTRACT The bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near the Escherichia coli cell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shaped E. coli cells are comparable, implying that negatively curved “pole-like” sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein from Bacillus subtilis at strongly curved sites in both B. subtilis and E. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness. PMID:24129255

  2. Self-organized partitioning of dynamically localized proteins in bacterial cell division.

    PubMed

    Di Ventura, Barbara; Sourjik, Victor

    2011-01-04

    How cells manage to get equal distribution of their structures and molecules at cell division is a crucial issue in biology. In principle, a feedback mechanism could always ensure equality by measuring and correcting the distribution in the progeny. However, an elegant alternative could be a mechanism relying on self-organization, with the interplay between system properties and cell geometry leading to the emergence of equal partitioning. The problem is exemplified by the bacterial Min system that defines the division site by oscillating from pole to pole. Unequal partitioning of Min proteins at division could negatively impact system performance and cell growth because of loss of Min oscillations and imprecise mid-cell determination. In this study, we combine live cell and computational analyses to show that known properties of the Min system together with the gradual reduction of protein exchange through the constricting septum are sufficient to explain the observed highly precise spontaneous protein partitioning. Our findings reveal a novel and effective mechanism of protein partitioning in dividing cells and emphasize the importance of self-organization in basic cellular processes.

  3. Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter.

    PubMed

    Skillman, Kristen M; Barnard, Travis J; Peterson, Janine H; Ghirlando, Rodolfo; Bernstein, Harris D

    2005-11-01

    Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

  4. Selective molecular transport through the protein shell of a bacterial microcompartment organelle

    SciTech Connect

    Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; Sawaya, Michael R.; Sinha, Sharmistha; Yeates, Todd O.; Bobik, Thomas A.

    2015-02-23

    Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. In this paper, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Finally and overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.

  5. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  6. A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

    PubMed

    Amari, Khalid; Boutant, Emmanuel; Hofmann, Christina; Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L; Heinlein, Manfred; Mély, Yves; Maule, Andrew J; Ritzenthaler, Christophe

    2010-09-23

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

  7. Wnt family proteins are secreted and associated with the cell surface.

    PubMed Central

    Smolich, B D; McMahon, J A; McMahon, A P; Papkoff, J

    1993-01-01

    Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1. Images PMID:8167409

  8. ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

    PubMed Central

    East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

    2012-01-01

    The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

  9. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination.

    PubMed

    Suzuki, Toru; Kim, Minsoo; Kozuka-Hata, Hiroko; Watanabe, Masato; Oyama, Masaaki; Tsumoto, Kouhei; Yamamoto, Tadashi

    2011-05-27

    Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  10. Prokaryotic Virus Orthologous Groups (pVOGs): a resource for comparative genomics and protein family annotation

    PubMed Central

    Grazziotin, Ana Laura; Koonin, Eugene V.; Kristensen, David M.

    2017-01-01

    Viruses are the most abundant and diverse biological entities on earth, and while most of this diversity remains completely unexplored, advances in genome sequencing have provided unprecedented glimpses into the virosphere. The Prokaryotic Virus Orthologous Groups (pVOGs, formerly called Phage Orthologous Groups, POGs) resource has aided in this task over the past decade by using automated methods to keep pace with the rapid increase in genomic data. The uses of pVOGs include functional annotation of viral proteins, identification of genes and viruses in uncharacterized DNA samples, phylogenetic analysis, large-scale comparative genomics projects, and more. The pVOGs database represents a comprehensive set of orthologous gene families shared across multiple complete genomes of viruses that infect bacterial or archaeal hosts (viruses of eukaryotes will be added at a future date). The pVOGs are constructed within the Clusters of Orthologous Groups (COGs) framework that is widely used for orthology identification in prokaryotes. Since the previous release of the POGs, the size has tripled to nearly 3000 genomes and 300 000 proteins, and the number of conserved orthologous groups doubled to 9518. User-friendly webpages are available, including multiple sequence alignments and HMM profiles for each VOG. These changes provide major improvements to the pVOGs database, at a time of rapid advances in virus genomics. The pVOGs database is hosted jointly at the University of Iowa at http://dmk-brain.ecn.uiowa.edu/pVOGs and the NCBI at ftp://ftp.ncbi.nlm.nih.gov/pub/kristensen/pVOGs/home.html. PMID:27789703

  11. Overexpression of EVE1, a novel ubiquitin family protein, arrests inflorescence stem development in Arabidopsis.

    PubMed

    Hwang, Hyun-Ju; Kim, Hoyeun; Jeong, Young-Min; Choi, Monica Y; Lee, So-Young; Kim, Sang-Gu

    2011-08-01

    In Arabidopsis, inflorescence stem formation is a critical process in phase transition from the vegetative to the reproductive state. Although inflorescence stem development has been reported to depend on the expression of a variety of genes during floral induction and repression, little is known about the molecular mechanisms involved in the control of inflorescence stem formation. By activation T-DNA tagging mutagenesis of Arabidopsis, a dominant gain-of-function mutation, eve1-D (eternally vegetative phase1-Dominant), which has lost the ability to form an inflorescence stem, was isolated. The eve1-D mutation exhibited a dome-shaped primary shoot apical meristem (SAM) in the early vegetative stage, similar to that seen in the wild-type SAM. However, the SAM in the eve1-D mutation failed to transition into an inflorescence meristem (IM) and eventually reached senescence without ever leaving the vegetative phase. The eve1-D mutation also displayed pleiotropic phenotypes, including lobed and wavy rosette leaves, short petioles, and an increased number of rosette leaves. Genetic analysis indicated that the genomic location of the EVE1 gene in Arabidopsis thaliana corresponded to a bacterial artificial chromosome (BAC) F4C21 from chromosome IV at ∼17cM which encoded a novel ubiquitin family protein (At4g03350), consisting of a single exon. The EVE1 protein is composed of 263 amino acids, contains a 52 amino acid ubiquitin domain, and has no glycine residue related to ubiquitin activity at the C-terminus. The eve1-D mutation provides a way to study the regulatory mechanisms that control phase transition from the vegetative to the reproductive state.

  12. A simple model for DNA bridging proteins and bacterial or human genomes: bridging-induced attraction and genome compaction

    NASA Astrophysics Data System (ADS)

    Johnson, J.; Brackley, C. A.; Cook, P. R.; Marenduzzo, D.

    2015-02-01

    We present computer simulations of the phase behaviour of an ensemble of proteins interacting with a polymer, mimicking non-specific binding to a piece of bacterial DNA or eukaryotic chromatin. The proteins can simultaneously bind to the polymer in two or more places to create protein bridges. Despite the lack of any explicit interaction between the proteins or between DNA segments, our simulations confirm previous results showing that when the protein-polymer interaction is sufficiently strong, the proteins come together to form clusters. Furthermore, a sufficiently large concentration of bridging proteins leads to the compaction of the swollen polymer into a globular phase. Here we characterise both the formation of protein clusters and the polymer collapse as a function of protein concentration, protein-polymer affinity and fibre flexibility.

  13. A simple model for DNA bridging proteins and bacterial or human genomes: bridging-induced attraction and genome compaction.

    PubMed

    Johnson, J; Brackley, C A; Cook, P R; Marenduzzo, D

    2015-02-18

    We present computer simulations of the phase behaviour of an ensemble of proteins interacting with a polymer, mimicking non-specific binding to a piece of bacterial DNA or eukaryotic chromatin. The proteins can simultaneously bind to the polymer in two or more places to create protein bridges. Despite the lack of any explicit interaction between the proteins or between DNA segments, our simulations confirm previous results showing that when the protein-polymer interaction is sufficiently strong, the proteins come together to form clusters. Furthermore, a sufficiently large concentration of bridging proteins leads to the compaction of the swollen polymer into a globular phase. Here we characterise both the formation of protein clusters and the polymer collapse as a function of protein concentration, protein-polymer affinity and fibre flexibility.

  14. Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis.

    PubMed

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J F; Vollmer, Waldemar; Breukink, Eefjan; Terrak, Mohammed

    2017-02-24

    Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.

  15. Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis

    PubMed Central

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J. F.; Vollmer, Waldemar; Breukink, Eefjan; Terrak, Mohammed

    2017-01-01

    Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell’s need to ensure appropriate use of the limited pool of lipid II. PMID:28233869

  16. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer

    PubMed Central

    Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

    2016-01-01

    Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. DOI: http://dx.doi.org/10.7554/eLife.20352.001 PMID:27805565

  17. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer.

    PubMed

    Berger, Stephanie; Procko, Erik; Margineantu, Daciana; Lee, Erinna F; Shen, Betty W; Zelter, Alex; Silva, Daniel-Adriano; Chawla, Kusum; Herold, Marco J; Garnier, Jean-Marc; Johnson, Richard; MacCoss, Michael J; Lessene, Guillaume; Davis, Trisha N; Stayton, Patrick S; Stoddard, Barry L; Fairlie, W Douglas; Hockenbery, David M; Baker, David

    2016-11-02

    Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

  18. Making novel bio-interfaces through bacterial protein recrystallization on biocompatible polylactide derivative films

    NASA Astrophysics Data System (ADS)

    Lejardi, Ainhoa; López, Aitziber Eleta; Sarasua, José R.; Sleytr, U. B.; Toca-Herrera, José L.

    2013-09-01

    Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (Tg) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below Tg. The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 μm2), followed by PDLLA (0.034 μm2), and PLGA (0.039 μm2), and the largest size for PLCL (0.09 μm2). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (˜1800 ng cm-2) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest Tg). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

  19. Holo- and apo-bound structures of bacterial periplasmic heme-binding proteins.

    PubMed

    Ho, Winny W; Li, Huiying; Eakanunkul, Suntara; Tong, Yong; Wilks, Angela; Guo, Maolin; Poulos, Thomas L

    2007-12-07

    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(228) in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg(228), and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B(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(12), compared with ligands for FhuD, a peptide siderophore.

  20. PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence

    PubMed Central

    Singh, Parul; Rao, Rameshwaram Nagender; Reddy, Jala Ram Chandra; Prasad, RBN; Kotturu, Sandeep Kumar; Ghosh, Sudip; Mukhopadhyay, Sangita

    2016-01-01

    The role of the unique proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins in the pathophysiology and virulence of Mycobacterium tuberculosis is not clearly understood. One of the PE family proteins, PE11 (LipX or Rv1169c), specific to pathogenic mycobacteria is found to be over-expressed during infection of macrophages and in active TB patients. In this study, we report that M. smegmatis expressing PE11 (Msmeg-PE11) exhibited altered colony morphology and cell wall lipid composition leading to a marked increase in resistance against various environmental stressors and antibiotics. The cell envelope of Msmeg-PE11 also had greater amount of glycolipids and polar lipids. Msmeg-PE11 was found to have better survival rate in infected macrophages. Mice infected with Msmeg-PE11 had higher bacterial load, showed exacerbated organ pathology and mortality. The liver and lung of Msmeg-PE11-infected mice also had higher levels of IL-10, IL-4 and TNF-α cytokines, indicating a potential role of this protein in mycobacterial virulence. PMID:26902658

  1. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  2. Immunological characterization of a bacterial protein isolated from salmonid fish naturally infected with Piscirickettsia salmonis.

    PubMed

    Marshall, Sergio H; Conejeros, Pablo; Zahr, Marcela; Olivares, Jorge; Gómez, Fernando; Cataldo, Patricio; Henríquez, Vitalia

    2007-03-01

    The Salmon Rickettsia syndrome (SRS) remains a major infectious disease in the Chilean aquaculture. A limited number of Piscirickettsia salmonis proteins have been characterized so far for their use as potential candidates for vaccines studies. In this study, we identified and expressed a highly immunogenic protein of P. salmonis extracted by selective hydrophobicity from crude-cell macerates of naturally infected salmonid fish. One and two-D PAGE gels followed by Western blot analysis with a battery of polyclonal anti-P. salmonis antibodies have allowed the isolation of the target protein. Basic local alignment search (BLAST) done after partial sequencing of the pure protein identified it as a member of the heat-shock protein (HSP) family of prokaryotes. The protein, named ChaPs, was cloned as a single open reading frame encoding 545 amino acid residues with a predicted molecular mass of 57.3 kDa. The amplicon representing the entire novel gene was expressed in vitro in different heterologous systems: the PurePro Caulobacter crescentus expression system from where most of the characterization was attained, and also in the Escherichia coli BL-21 CodonPlus model for commercially potential purposes. The immunologic potential of ChaPs was determined with serum from naturally infected fish.

  3. Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

    PubMed

    Ibrahim, Susan A; Crack, Jason C; Rolfe, Matthew D; Borrero-de Acuña, José Manuel; Thomson, Andrew J; Le Brun, Nick E; Schobert, Max; Stapleton, Melanie R; Green, Jeffrey

    2015-07-03

    The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

  4. Platform for identification of Salmonella serovar differentiating bacterial proteins by top-down mass spectrometry: S. Typhimurium vs S. Heidelberg.

    PubMed

    McFarland, Melinda A; Andrzejewski, Denis; Musser, Steven M; Callahan, John H

    2014-07-15

    Intact protein expression profiling has proven to be a powerful tool for bacterial subspecies differentiation. To facilitate typing, epidemiology, and trace-back of Salmonella contamination in the food supply, a minimum of serovar level differentiation is required. Subsequent identification and validation of marker proteins is integral to rapid screening development and to determining which proteins are subject to environmental pressure. Bacterial sequencing efforts have expanded the number of sequenced genomes available for single-nucleotide polymorphism (SNP) analyses, but annotation is often missing, start site errors are not uncommon, and the likelihood of expression is not known. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed serovar specific nonsynonymous SNPs. Combinations of these marker proteins can be used in assays for rapid differentiation of bacteria. LC-MS generated intact protein expression profiles establish which bacterial protein masses differ across samples and can be determined without prior knowledge of the sample. Subsequent top-down LC-MS/MS is used to identify expressed proteins and their post-translational modifications (PTM), identify serovar specific markers, and validate genomic predicted orthologues as expressed biomarkers.

  5. Evolution of Bacterial Protein-Tyrosine Kinases and Their Relaxed Specificity Toward Substrates

    PubMed Central

    Shi, Lei; Kolar-Znika, Lorena; Boskovic, Ana; Jadeau, Fanny; Combet, Christophe; Grangeasse, Christophe; Franjevic, Damjan; Talla, Emmanuel; Mijakovic, Ivan

    2014-01-01

    It has often been speculated that bacterial protein-tyrosine kinases (BY-kinases) evolve rapidly and maintain relaxed substrate specificity to quickly adopt new substrates when evolutionary pressure in that direction arises. Here, we report a phylogenomic and biochemical analysis of BY-kinases, and their relationship to substrates aimed to validate this hypothesis. Our results suggest that BY-kinases are ubiquitously distributed in bacterial phyla and underwent a complex evolutionary history, affected considerably by gene duplications and horizontal gene transfer events. This is consistent with the fact that the BY-kinase sequences represent a high level of substitution saturation and have a higher evolutionary rate compared with other bacterial genes. On the basis of similarity networks, we could classify BY kinases into three main groups with 14 subgroups. Extensive sequence conservation was observed only around the three canonical Walker motifs, whereas unique signatures proposed the functional speciation and diversification within some subgroups. The relationship between BY-kinases and their substrates was analyzed using a ubiquitous substrate (Ugd) and some Firmicute-specific substrates (YvyG and YjoA) from Bacillus subtilis. No evidence of coevolution between kinases and substrates at the sequence level was found. Seven BY-kinases, including well-characterized and previously uncharacterized ones, were used for experimental studies. Most of the tested kinases were able to phosphorylate substrates from B. subtilis (Ugd, YvyG, and YjoA), despite originating from very distant bacteria. Our results are consistent with the hypothesis that BY-kinases have evolved relaxed substrate specificity and are probably maintained as rapidly evolving platforms for adopting new substrates. PMID:24728941

  6. Incorporation of radiolabeled leucine into protein to estimate bacterial production in plant litter, sediment, epiphytic biofilms, and water samples.

    PubMed

    Buesing, N; Gessner, M O

    2003-03-01

    The present study assessed the application of tritiated leucine incorporation into protein, as a measure of bacterial biomass production, within four benthic habitats of a littoral freshwater wetland dominated by emergent vegetation. Basic assumptions underlying the method, such as linearity of leucine incorporation, saturation level of incorporation rates, and specificity of incorporation for bacterial assemblages, were tested, and two procedures for extracting radiolabeled protein were compared. TCA precipitation followed by ultrasonication, and subsequent alkaline dissolution in 0.5 M NaOH, 25 mM EDTA, and 0.1% w/v SDS, gave best results in terms of both extraction efficiency and signal-to-noise ratio. Incorporation of leucine was linear for all habitats for up to 1 h. Saturation concentrations of leucine incorporation into protein were 150 nM for littoral surface waters, >960 nM for biofilms on plant surfaces, and 50 mM for aerobic sediment and submerged plant litter. An experiment with prokaryotic and eukaryotic inhibitors designed to examine specificity of leucine incorporation into bacterial protein showed no significant leucine incorporation into eukaryotes during short-term incubations. Calculations based on kinetic parameters of fungal leucine uptake suggest, nevertheless, that significant leucine incorporation cannot be ruled out in all situations. Thus, the leucine methodology can be used for estimating bacterial production in benthic aquatic habitats, provided that substrate saturation and isotope dilution are determined and that the active biomass of eukaryotes, such as fungi, does not greatly exceed bacterial biomass.

  7. Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco.

    PubMed

    Felix, Georg; Boller, Thomas

    2003-02-21

    To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

  8. Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins

    PubMed Central

    Dempwolff, Felix; Schmidt, Felix K.; Hervás, Ana B.; Stroh, Alex; Rösch, Thomas C.; Riese, Cornelius N.; Dersch, Simon; Heimerl, Thomas; Lucena, Daniella; Hülsbusch, Nikola; Stuermer, Claudia A. O.; Takeshita, Norio; Fischer, Reinhard; Graumann, Peter L.

    2016-01-01

    Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds. PMID:27362352

  9. Lactoferrin binding protein B – a bi-functional bacterial receptor protein

    PubMed Central

    Ostan, Nicholas K. H.; Yu, Rong-Hua; Ng, Dixon; Lai, Christine Chieh-Lin; Sarpe, Vladimir; Schriemer, David C.

    2017-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation. PMID:28257520

  10. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  11. Structure and evolutionary history of a large family of NLR proteins in the zebrafish

    PubMed Central

    Zielinski, Julia; Kondrashov, Fyodor

    2016-01-01

    Multicellular eukaryotes have evolved a range of mechanisms for immune recognition. A widespread family involved in innate immunity are the NACHT-domain and leucine-rich-repeat-containing (NLR) proteins. Mammals have small numbers of NLR proteins, whereas in some species, mostly those without adaptive immune systems, NLRs have expanded into very large families. We describe a family of nearly 400 NLR proteins encoded in the zebrafish genome. The proteins share a defining overall structure, which arose in fishes after a fusion of the core NLR domains with a B30.2 domain, but can be subdivided into four groups based on their NACHT domains. Gene conversion acting differentially on the NACHT and B30.2 domains has shaped the family and created the groups. Evidence of positive selection in the B30.2 domain indicates that this domain rather than the leucine-rich repeats acts as the pathogen recognition module. In an unusual chromosomal organization, the majority of the genes are located on one chromosome arm, interspersed with other large multigene families, including a new family encoding zinc-finger proteins. The NLR-B30.2 proteins represent a new family with diversity in the specific recognition module that is present in fishes in spite of the parallel existence of an adaptive immune system. PMID:27248802

  12. The MagA Protein of Magnetospirilla Is Not Involved in Bacterial Magnetite Biomineralization

    PubMed Central

    Uebe, René; Henn, Verena

    2012-01-01

    Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria. PMID:22194451

  13. The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of σ54-Dependent Transcription

    PubMed Central

    2012-01-01

    Summary: Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ54 for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ54-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ54 to the bacterial cell and its unique role in regulating transcription. PMID:22933558

  14. No human protein is exempt from bacterial motifs, not even one

    PubMed Central

    Trost, Brett; Lucchese, Guglielmo; Stufano, Angela; Bickis, Mik; Kusalik, Anthony

    2010-01-01

    The hypothesis that mimicry between a self and a microbial peptide antigen is strictly related to autoimmune pathology remains a debated concept in autoimmunity research. Clear evidence for a causal link between molecular mimicry and autoimmunity is still lacking. In recent studies we have demonstrated that viruses and bacteria share amino acid sequences with the human proteome at such a high extent that the molecular mimicry hypothesis becomes questionable as a causal factor in autoimmunity. Expanding upon our analysis, here we detail the bacterial peptide overlapping to the human proteome at the penta-, hexa-, hepta- and octapeptide levels by exact peptide matching analysis and demonstrate that there does not exist a single human protein that does not harbor a bacterial pentapeptide or hexapeptide motif. This finding suggests that molecular mimicry between a self and a microbial peptide antigen cannot be assumed as a basis for autoimmune pathologies. Moreover, the data are discussed in relation to the microbial immune escape phenomenon and the possible vaccine-related autoimmune effects. PMID:21487508

  15. Predicting Outcome of Childhood Bacterial Meningitis With a Single Measurement of C-Reactive Protein

    PubMed Central

    Peltola, Heikki; Roine, Irmeli; Cruzeiro, Manuel Leite; Bernardino, Luis

    2016-01-01

    Introduction: C-reactive protein (CRP), a marker of inflammation, shows high serum levels in invasive bacterial infections. We investigated the potential of a single CRP measurement at different phases of acute childhood bacterial meningitis to predict outcomes. Methods: Using whole-blood finger-prick samples with no centrifugation, CRP was measured quantitatively on arrival and on day 3 or 4 in children participating in 2 prospective, randomized, double-blind treatment studies conducted in Latin America or Angola. The results were compared with patient outcomes. Results: Although initial CRP values from 669 children gave useful prognostic information, the 3rd or 4th day measurements taken from 275 children associated significantly with seizures, slow recovery and low scores on the Glasgow Outcome Scale, with odds ratios for CRP values above the median (62 mg/L) ranging from 2 to 6, 2 to 5, and 3 to 5 (Latin America–Angola), respectively. Hearing impairment, although not full deafness, was 3 to 7 times more likely if CRP was above the median soon after hospitalization. Conclusions: Especially in resource-poor settings, clinicians have few simple-enough tools to identify the child with meningitis who requires maximum attention. CRP is a worthy addition. PMID:26986770

  16. A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development

    PubMed Central

    Hill, Jennifer Hampton; Franzosa, Eric A; Huttenhower, Curtis; Guillemin, Karen

    2016-01-01

    Resident microbes play important roles in the development of the gastrointestinal tract, but their influence on other digestive organs is less well explored. Using the gnotobiotic zebrafish, we discovered that the normal expansion of the pancreatic β cell population during early larval development requires the intestinal microbiota and that specific bacterial members can restore normal β cell numbers. These bacteria share a gene that encodes a previously undescribed protein, named herein BefA (β Cell Expansion Factor A), which is sufficient to induce β cell proliferation in developing zebrafish larvae. Homologs of BefA are present in several human-associated bacterial species, and we show that they have conserved capacity to stimulate β cell proliferation in larval zebrafish. Our findings highlight a role for the microbiota in early pancreatic β cell development and suggest a possible basis for the association between low diversity childhood fecal microbiota and increased diabetes risk. DOI: http://dx.doi.org/10.7554/eLife.20145.001 PMID:27960075

  17. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus.

    PubMed

    Matsui, Takashi; Yamane, Junji; Mogi, Nobuyuki; Yamaguchi, Hiroto; Takemoto, Hiroshi; Yao, Min; Tanaka, Isao

    2012-09-01

    FtsZ is a key molecule in bacterial cell division. In the presence of GTP, it polymerizes into tubulin-like protofilaments by head-to-tail association. Protofilaments of FtsZ seem to adopt a straight or a curved conformation in relation to the bound nucleotide. However, although several bacterial and archaeal FtsZ structures have been determined, all of the structures reported previously are considered to have a curved conformation. In this study, structures of FtsZ from Staphylococcus aureus (SaFtsZ) were determined in apo, GDP-bound and inhibitor-complex forms and it was found that SaFtsZ undergoes marked conformational changes. The accumulated evidence suggests that the GDP-bound structure has the features of the straight form. The structural change between the curved and straight forms shows intriguing similarity to the eukaryotic cytoskeletal protein tubulin. Furthermore, the structure of the apo form showed an unexpectedly large conformational change in the core region. FtsZ has also been recognized as a novel target for antibacterial drugs. The structure of the complex with the inhibitor PC190723, which has potent and selective antistaphylococcal activity, indicated that the inhibitor binds at the cleft between the two subdomains.

  18. PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport.

    PubMed

    Vasseur, Perrine; Soscia, Chantal; Voulhoux, Romé; Filloux, Alain

    2007-08-01

    Pseudomonas aeruginosa is a gram-negative bacterium, opportunistic pathogen, which causes severe acute or chronic infections, as is the case with cystic fibrosis patients. Chronic infections are frequently accompanied by the development of the bacterial population into a specialized community called biofilm. The pelA-G gene cluster of P. aeruginosa has been shown to be involved in pellicle production and biofilm formation. The pel genes have been proposed to contribute to the formation of the exopolysaccharide-containing pellicle. However, the function and the subcellular localization of the seven different Pel proteins are poorly understood. Based on bioinformatics analysis, we have previously considered that PelF is a putative glycosyltransferase (GT4 family), whereas PelG is a Wzx-like polysaccharide transporter from the PST family. In this study we have further characterized the PelC protein. We have shown that PelC is an outer membrane lipoprotein. The N-terminal signal peptide of the PelC lipoprotein is sufficient to target the protein into the membranes. However, by constructing various PelC hybrid proteins we also proposed that efficient and functional outer membrane insertion of PelC requires not only the signal peptide and the lipid modification, but also requires the C-terminal domain of PelC. Because the gene encoding the outer membrane lipoprotein PelC is part of a putative gene cluster involved in exopolysaccharide biogenesis, we suggest that PelC is a new member of the outer membrane auxiliary (OMA) family of lipoprotein whose Wza, involved in Escherichia coli capsular polysaccharide transport, is an archetype.

  19. Exploring the chemistry of uncultivated bacterial symbionts: antitumor polyketides of the pederin family.

    PubMed

    Piel, Jörn; Butzke, Daniel; Fusetani, Nobuhiro; Hui, Dequan; Platzer, Matthias; Wen, Gaiping; Matsunaga, Shigeki

    2005-03-01

    Symbiotic bacteria have long been proposed as being responsible for the production of numerous natural products isolated from invertebrate animals. However, systematic studies of invertebrate-symbiont associations are usually associated with serious technical challenges, such as the general resistance of symbionts to culturing attempts and the complexity of many microbial consortia. Herein an overview is provided on the culture-independent, metagenomic strategies recently employed by our group to contribute to a better understanding of natural product symbiosis. Using terrestrial Paederus spp. beetles and the marine sponge Theonella swinhoei as model animals, the putative genes responsible for the production of pederin-type antitumor polyketides have been isolated. In Paederus fuscipes, which uses pederin for chemical defense, these genes belong to an as-yet unculturable symbiont closely related to Pseudomonas aeruginosa. To study the extremely complex association of T. swinhoei and its multispecies bacterial consortium, we used a phylogenetic approach that allowed the isolation of onnamide/theopederin polyketide synthase genes from an uncultured sponge symbiont. Analysis of the biosynthesis genes provided unexpected insights into a possible evolution of pederin-type pathways. Besides revealing new facets of invertebrate chemical ecology, these first gene clusters from uncultivated symbiotic producers suggest possible biotechnological strategies to solve the supply problem associated with the development of most marine drug candidates.

  20. Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc{sub 1} complex

    SciTech Connect

    Martin, Daniel R.; Matyushov, Dmitry V.

    2015-04-28

    Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc{sub 1} bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

  1. Eos and pegasus, two members of the Ikaros family of proteins with distinct DNA binding activities.

    PubMed

    Perdomo, J; Holmes, M; Chong, B; Crossley, M

    2000-12-08

    Members of the Ikaros family of transcription factors, Ikaros, Aiolos, and Helios, are expressed in lymphocytes and have been implicated in controlling lymphoid development. These proteins contain two characteristic clusters of zinc fingers, an N-terminal domain important for DNA recognition, and a C-terminal domain that mediates homo- and heterotypic associations between family members. The conservation of these domains is such that all three proteins recognize related DNA sequences, and all are capable of dimerizing with other family members. Here we describe two additional Ikaros family proteins, Eos and Pegasus. Eos is most highly related to Helios and shares its DNA binding and protein association properties. Pegasus is related to other Ikaros proteins in its C-terminal dimerization domain but contains a divergent N-terminal zinc finger domain. Pegasus self-associates and binds to other family members but recognizes distinct DNA-binding sites. Eos and Pegasus repress the expression of reporter genes containing their recognition elements. Our results suggest that these proteins may associate with previously described Ikaros family proteins in lymphoid cells and play additional roles in other tissues.

  2. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    PubMed Central

    Wu, Fabai; van Schie, Bas G.C.; Keymer, Juan E.; Dekker, Cees

    2016-01-01

    The boundary of a cell defines the shape and scale for its subcellular organisation. However, the effects of the cell’s spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shape Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2x1x1 to 11x6x1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe, and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale in adaptation to the cell size within a characteristic length range of 3–6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling, and an adaptive range, when and only when facilitated by the three-dimensional confinement of cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood. PMID:26098227

  3. Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts.

    PubMed Central

    Piñeiro, M; García-Olmedo, F; Diaz, I

    1994-01-01

    Activity of neomycin phosphotransferase II (NPTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1' or 35S promoter was not similarly affected, thus excluding a redox modulation of transcription as the mechanism of NPTII activation by dithiothreitol. Western-blot analyses showed an increase in the amount of protein in response to dithiothreitol, whereas neither the steady-state level of NPTII mRNA nor the specific activity of the purified enzyme was affected. The same type of modulation was observed for transiently expressed beta-glucuronidase (nine cysteines) produced from a fusion with the 35S promoter, with or without a signal peptide. Limitation of cotranslational and/or early posttranslational steps by excessively oxidizing sulfhydryl/disulfide redox potentials is postulated to explain the low net accumulation of cysteine-rich proteins of bacterial origin (i.e., NPTII and beta-glucuronidase) when expressed in plant protoplasts, and the marked increase in such proteins in response to externally added dithiothreitol. Images PMID:8171004

  4. Prion-like characteristics of the bacterial protein Microcin E492

    PubMed Central

    Shahnawaz, Mohammad; Park, Kyung-Won; Mukherjee, Abhisek; Diaz-Espinoza, Rodrigo; Soto, Claudio

    2017-01-01

    Microcin E492 (Mcc) is a pore-forming bacteriotoxin. Mcc activity is inhibited at the stationary phase by formation of amyloid-like aggregates in the culture. Here we report that, in a similar manner as prions, Mcc naturally exists as two conformers: a β-sheet-rich, protease-resistant, aggregated, inactive form (Mccia), and a soluble, protease-sensitive, active form (Mcca). The exogenous addition of culture medium containing Mccia or purified in vitro-generated Mccia into the culture induces the rapid and efficient conversion of Mcca into Mccia, which is maintained indefinitely after passaging, changing the bacterial phenotype. Mccia prion-like activity is conformation-dependent and could be reduced by immunodepleting Mccia. Interestingly, an internal region of Mcc shares sequence similarity with the central domain of the prion protein, which is key to the formation of mammalian prions. A synthetic peptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducing the conversion of Mcca into Mccia in vivo, suggesting that this region corresponds to the prion domain of Mcc. Our findings suggest that Mcc is the first prokaryotic protein with prion properties which harnesses prion-like transmission to regulate protein function, suggesting that propagation of biological information using a prion-based conformational switch is an evolutionary conserved mechanism. PMID:28361921

  5. A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

    PubMed Central

    Tian, Tian; Salis, Howard M.

    2015-01-01

    Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

  6. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    NASA Astrophysics Data System (ADS)

    Wu, Fabai; van Schie, Bas G. C.; Keymer, Juan E.; Dekker, Cees

    2015-08-01

    The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

  7. Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.

    PubMed

    Ross, Caná L; Liang, Xiaowen; Liu, Qing; Murray, Barbara E; Höök, Magnus; Ganesh, Vannakambadi K

    2012-10-05

    The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N(1) and N(2) subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.

  8. Structure of the Bacterial Cytoskeleton Protein Bactofilin by NMR Chemical Shifts and Sequence Variation.

    PubMed

    Kassem, Maher M; Wang, Yong; Boomsma, Wouter; Lindorff-Larsen, Kresten

    2016-06-07

    Bactofilins constitute a recently discovered class of bacterial proteins that form cytoskeletal filaments. They share a highly conserved domain (DUF583) of which the structure remains unknown, in part due to the large size and noncrystalline nature of the filaments. Here, we describe the atomic structure of a bactofilin domain from Caulobacter crescentus. To determine the structure, we developed an approach that combines a biophysical model for proteins with recently obtained solid-state NMR spectroscopy data and amino acid contacts predicted from a detailed analysis of the evolutionary history of bactofilins. Our structure reveals a triangular β-helical (solenoid) conformation with conserved residues forming the tightly packed core and polar residues lining the surface. The repetitive structure explains the presence of internal repeats as well as strongly conserved positions, and is reminiscent of other fibrillar proteins. Our work provides a structural basis for future studies of bactofilin biology and for designing molecules that target them, as well as a starting point for determining the organization of the entire bactofilin filament. Finally, our approach presents new avenues for determining structures that are difficult to obtain by traditional means.

  9. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  10. Expanding the Imine Reductase Toolbox by Exploring the Bacterial Protein-Sequence Space.

    PubMed

    Wetzl, Dennis; Berrera, Marco; Sandon, Nicolas; Fishlock, Dan; Ebeling, Martin; Müller, Michael; Hanlon, Steven; Wirz, Beat; Iding, Hans

    2015-08-17

    Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates. Each of the identified enzymes was characterized against a set of nine cyclic imine model substrates. A refined clustering towards putative active-site residues was performed and was consistent both with our screening and previously reported results. Finally, preparative scale experiments on a 100 mg scale with two purified IREDs, IR_20 from Streptomyces tsukubaensis and IR_23 from Streptomyces vidiochromogenes, were carried out to provide (R)-2-methylpiperidine in 98% ee (71% yield) and (R)-1-methyl-1,2,3,4-tetrahydroisoquinoline in >98% ee (82% yield).

  11. Development of robust biocompatible silicone with high resistance to protein adsorption and bacterial adhesion.

    PubMed

    Lin, Weifeng; Zhang, Juan; Wang, Zhen; Chen, Shengfu

    2011-05-01

    A new biocompatible silicone comprising a carboxybetaine (CB) ester analogue, 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS) and an organic silicone macromer (bis-α,ω-(methacryloxypropyl) polydimethylsiloxane) has been developed using photo-polymerisation. Following interfacial hydrolysis of the CB ester, the resulting zwitterionic material became significantly more hydrophilic and exhibited high resistance to both non-specific protein adsorption and bacterial adhesion. Moreover, the stability of these non-fouling properties was dramatically improved by using a slow and controlled rate of ester hydrolysis of the original protective hydrophobic matrix. The subsequent ability to maintain the original optical and mechanical properties of the bare silicone following surface activation makes this material an ideal candidate for preparing contact lenses and other medical devices.

  12. Production of Green Fluorescent Protein Transgenic Embryonic Stem Cells Using the GENSAT Bacterial Artificial Chromosome Library

    PubMed Central

    Tomishima, Mark J.; Hadjantonakis, Anna-Katerina; Gong, Shiaoching; Studer, Lorenz

    2010-01-01

    Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology. PMID:16990587

  13. Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library.

    PubMed

    Tomishima, Mark J; Hadjantonakis, Anna-Katerina; Gong, Shiaoching; Studer, Lorenz

    2007-01-01

    Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology.

  14. Functional Display of Ice Nucleation Protein InaZ on the Surface of Bacterial Ghosts.

    PubMed

    Kassmannhuber, Johannes; Rauscher, Mascha; Schöner, Lea; Witte, Angela; Lubitz, Werner

    2017-01-25

    In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between -5.6 °C and -6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

  15. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  16. Genome-scale phylogenetic function annotation of large and diverse protein families.

    PubMed

    Engelhardt, Barbara E; Jordan, Michael I; Srouji, John R; Brenner, Steven E

    2011-11-01

    The Statistical Inference of Function Through Evolutionary Relationships (SIFTER) framework uses a statistical graphical model that applies phylogenetic principles to automate precise protein function prediction. Here we present a revised approach (SIFTER version 2.0) that enables annotations on a genomic scale. SIFTER 2.0 produces equivalently precise predictions compared to the earlier version on a carefully studied family and on a collection of 100 protein families. We have added an approximation method to SIFTER 2.0 and show a 500-fold improvement in speed with minimal impact on prediction results in the functionally diverse sulfotransferase protein family. On the Nudix protein family, previously inaccessible to the SIFTER framework because of the 66 possible molecular functions, SIFTER achieved 47.4% accuracy on experimental data (where BLAST achieved 34.0%). Finally, we used SIFTER to annotate all of the Schizosaccharomyces pombe proteins with experimental functional characterizations, based on annotations from proteins in 46 fungal genomes. SIFTER precisely predicted molecular function for 45.5% of the characterized proteins in this genome, as compared with four current function prediction methods that precisely predicted function for 62.6%, 30.6%, 6.0%, and 5.7% of these proteins. We use both precision-recall curves and ROC analyses to compare these genome-scale predictions across the different methods and to assess performance on different types of applications. SIFTER 2.0 is capable of predicting protein molecular function for large and functionally diverse protein families using an approximate statistical model, enabling phylogenetics-based protein function prediction for genome-wide analyses. The code for SIFTER and protein family data are available at http://sifter.berkeley.edu.

  17. Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.

    PubMed

    Jekabsons, Mika B; Echtay, Karim S; Brand, Martin D

    2002-09-01

    Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90% pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, N-methylanthraniloyl-tagged purine nucleoside di- and tri-phosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by least-squares non-linear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 3-5 microM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with [8-14C]ATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 2-5 microM affinity. Affinities for ADP and GDP were approx. 10-fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosyl-solubilized bacterial inclusion bodies by a two-step treatment with C12E9 detergent and hydroxyapatite; (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity; (c) binds GDP with the same affinity as GDP inhibits superoxide-stimulated uncoupling by kidney mitochondria; and (d) exhibits a different nucleotide preference than kidney mitochondria.

  18. Vaccinia Virus Protein A49 Is an Unexpected Member of the B-cell Lymphoma (Bcl)-2 Protein Family*

    PubMed Central

    Neidel, Sarah; Maluquer de Motes, Carlos; Mansur, Daniel S.; Strnadova, Pavla; Smith, Geoffrey L.; Graham, Stephen C.

    2015-01-01

    Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus. PMID:25605733

  19. Regulation and roles for claudin-family tight junction proteins

    PubMed Central

    Findley, Mary K.; Koval, Michael

    2009-01-01

    Transmembrane proteins known as claudins play a critical role in tight junctions by regulating paracellular barrier permeability. The control of claudin assembly into tight junctions requires a complex interplay between several classes of claudins, other transmembrane proteins and scaffold proteins. Claudins are also subject to regulation by post-translational modifications including phosphorylation and palmitoylation. Several human diseases have been linked to claudin mutations, underscoring the physiologic function of these proteins. Roles for claudins in regulating cell phenotype and growth control also are beginning to emerge, suggesting a multifaceted role for claudins in regulation of cells beyond serving as a simple structural element of tight junctions. PMID:19319969

  20. TRIM family proteins and their emerging roles in innate immunity.

    PubMed

    Ozato, Keiko; Shin, Dong-Mi; Chang, Tsung-Hsien; Morse, Herbert C

    2008-11-01

    The superfamily of tripartite motif-containing (TRIM) proteins is conserved throughout the metazoan kingdom and has expanded rapidly during vertebrate evolution; there are now more than 60 TRIM proteins known in humans and mice. Many TRIM proteins are induced by type I and type II interferons, which are crucial for many aspects of resistance to pathogens, and several are known to be required for the restriction of infection by lentiviruses. In this Review, we describe recent data that reveal broader antiviral and antimicrobial activities of TRIM proteins and discuss their involvement in the regulation of pathogen-recognition and transcriptional pathways in host defence.

  1. Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

    PubMed Central

    Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

    2005-01-01

    Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins. PMID:15775965

  2. Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

    PubMed Central

    Liu, Di; Sun, Wei; Yuan, Yaowu; Zhang, Ning; Hayward, Alice; Liu, Yongliang; Wang, Ying

    2014-01-01

    Background and Aims The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized. Methods Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato. Key Results Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family. Conclusions This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants. PMID:24812252

  3. Immune regulatory functions of DOCK family proteins in health and disease.

    PubMed

    Nishikimi, Akihiko; Kukimoto-Niino, Mutsuko; Yokoyama, Shigeyuki; Fukui, Yoshinori

    2013-09-10

    DOCK proteins constitute a family of evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho family of GTPases. Although DOCK family proteins do not contain the Dbl homology domain typically found in GEFs, they mediate the GTP-GDP exchange reaction through DHR-2 domain. Accumulating evidence indicates that the DOCK proteins act as major GEFs in varied biological settings. For example, DOCK2, which is predominantly expressed in hematopoietic cells, regulates migration and activation of leukocytes through Rac activation. On the other hand, it was recently reported that mutations of DOCK8, another member of the DOCK family proteins, cause a combined immunodeficiency syndrome in humans. This article reviews the structure, functions and signaling of DOCK2 and DOCK8, especially focusing on their roles in immune responses.

  4. Gut Commensal E. coli Proteins Activate Host Satiety Pathways following Nutrient-Induced Bacterial Growth.

    PubMed

    Breton, Jonathan; Tennoune, Naouel; Lucas, Nicolas; Francois, Marie; Legrand, Romain; Jacquemot, Justine; Goichon, Alexis; Guérin, Charlène; Peltier, Johann; Pestel-Caron, Martine; Chan, Philippe; Vaudry, David; do Rego, Jean-Claude; Liénard, Fabienne; Pénicaud, Luc; Fioramonti, Xavier; Ebenezer, Ivor S; Hökfelt, Tomas; Déchelotte, Pierre; Fetissov, Sergueï O

    2016-02-09

    The composition of gut microbiota has been associated with host metabolic phenotypes, but it is not known if gut bacteria may influence host appetite. Here we show that regular nutrient provision stabilizes exponential growth of E. coli, with the stationary phase occurring 20 min after nutrient supply accompanied by bacterial proteome changes, suggesting involvement of bacterial proteins in host satiety. Indeed, intestinal infusions of E. coli stationary phase proteins increased plasma PYY and their intraperitoneal injections suppressed acutely food intake and activated c-Fos in hypothalamic POMC neurons, while their repeated administrations reduced meal size. ClpB, a bacterial protein mimetic of α-MSH, was upregulated in the E. coli stationary phase, was detected in plasma proportional to ClpB DNA in feces, and stimulated firing rate of hypothalamic POMC neurons. Thus, these data show that bacterial proteins produced after nutrient-induced E. coli growth may signal meal termination. Furthermore, continuous exposure to E. coli proteins may influence long-term meal pattern.

  5. Coordinate synthesis and protein localization in a bacterial organelle by the action of a penicillin-binding-protein

    PubMed Central

    Hughes, H. Velocity; Lisher, John P.; Hardy, Gail G.; Kysela, David T.; Arnold, Randy J.; Giedroc, David P.; Brun, Yves V.

    2013-01-01

    SUMMARY Organelles with specialized form and function occur in diverse bacteria. Within the Alphaproteobacteria, several species extrude thin cellular appendages known as stalks, which function in nutrient uptake, buoyancy and reproduction. Consistent with their specialization, stalks maintain a unique molecular composition compared to the cell body, but how this is achieved remains to be fully elucidated. Here we dissect the mechanism of localization of StpX, a stalk-specific protein in Caulobacter crescentus. Using a forward genetics