Extracytoplasmic Proteins Research Articles

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.

Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6778-8) contains supplementary material, which is available to authorized users.

Legionella pneumophila utilizes a single-player disulfide-bond oxidoreductase system to manage disulfide bond formation and isomerization.

Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein-disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S-S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared with E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant-negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single-player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.

Lactic Acid Bacteria as a Surface Display Platform for Campylobacter jejuni Antigens

Background: Food poisoning and diarrheal diseases continue to pose serious health care and socioeconomic problems worldwide. Campylobacter spp. is a very widespread cause of gastroenteritis. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles. They represent an attractive opportunity for vaccination in addition to vaccination with attenuated bacterial pathogens. Methods: We examined the binding ability of hybrid proteins to nontreated or trichloroacetic acid (TCA)-pretreated LAB cells by immunofluorescence and Western blot analysis. Results: In this study we evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of Lactobacillus salivarius as a binding platform for 2 conserved, immunodominant, extracytoplasmic Campylobacter jejuni proteins: CjaA and CjaD. We analyzed the binding ability of recombinant proteins that contain C. jejuni antigens (CjaA or CjaD) fused with the protein anchor (PA) of the L. lactis peptidoglycan hydrolase AcmA, which comprises 3 LysM motifs and determines noncovalent binding to the cell wall peptidoglycan. Both fused proteins, i.e. 6HisxCjaAx3LysM and 6HisxCjaDx3LysM, were able to bind to nontreated or TCA-pretreated L. salivarius cells. Conclusion: Our results documented that the LysM-mediated binding system allows us to construct GEM particles that present 2 C. jejuni antigens.

Promiscuous Nickel Import in Human Pathogens: Structure, Thermodynamics, and Evolution of Extracytoplasmic Nickel-Binding Proteins

In human pathogenic bacteria, nickel is required for the activation of two enzymes, urease and [NiFe]-hydrogenase, necessary for host infection. Acquisition of Ni(II) is mediated by either permeases or ABC-importers, the latter including a subclass that involves an extracytoplasmic nickel-binding protein, Ni-BP. This study reports on the structure of three Ni-BPs from a diversity of human pathogens and on the existence of three new nickel-binding motifs. These are different from that previously described for Escherichia coli Ni-BP NikA, known to bind nickel via a nickelophore, and indicate a variegated ligand selectivity for Ni-BPs. The structures are consistent with ligand affinities measured in solution by calorimetry and challenge the hypothesis of a general requirement of nickelophores for nickel uptake by canonical ABC importers. Phylogenetic analyses showed that Ni-BPs have different evolutionary origins and emerged independently from peptide-binding proteins, possibly explaining the promiscuous behavior of this class of Ni(II) carriers.

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli: STRUCTURAL AND FUNCTIONAL STUDIES

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA' (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.

Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans

In Streptomyces lividans an extracytoplasmic copper-binding Sco protein plays a role in two unlinked processes: (i) initiating a morphological development switch and (ii) facilitating the co-factoring of the CuA domain of CcO (cytochrome c oxidase). How Sco obtains copper once secreted to the extracytoplasmic environment is unknown. In the present paper we report on a protein possessing an HX₆MX₂₁HXM motif that binds a single cuprous ion with subfemtomolar affinity. High-resolution X-ray structures of this extracytoplasmic copper chaperone-like protein (ECuC) in the apo- and Cu(I)-bound states reveal that the latter possesses a surface-accessible cuprous-ion-binding site located in a dish-shaped region of β-sheet structure. A cuprous ion is transferred under a favourable thermodynamic gradient from ECuC to Sco with no back transfer occurring. The ionization properties of the cysteine residues in the Cys⁸⁶xxxCys⁹⁰ copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys⁸⁶ in initiating an inter-complex ligand-exchange reaction with Cu(I)-ECuC. Generation of the genetic knockouts, Δsco, Δecuc and Δsco/ecuc, and subsequent in vivo assays lend support to the existence of a branched extracytoplasmic copper-trafficking pathway in S. lividans. One branch requires both Sco and to a certain extent ECuC to cofactor the CuA domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.

A Gateway(®) -compatible bacterial adenylate cyclase-based two-hybrid system.

The bacterial adenylate cyclase two-hybrid (BACTH) system has been widely used to characterize protein-protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of adenylate cyclase activity in Escherichia coli by bringing together two complementary fragments of the catalytic domain of the adenylate cyclase toxin of Bordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway(®) (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW ) system using several models of protein-protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein-protein interactions.

Phylum‐wide general protein O‐glycosylation system of the Bacteroidetes

The human gut symbiont Bacteroides fragilis has a general protein O-glycosylation system in which numerous extracytoplasmic proteins are glycosylated at a three amino acid motif. In B. fragilis, protein glycosylation is a fundamental and essential property as mutants with protein glycosylation defects have impaired growth and are unable to competitively colonize the mammalian intestine. In this study, we analysed the phenotype of B. fragilis mutants with defective protein glycosylation and found that the glycan added to proteins is comprised of a core glycan and an outer glycan. The genetic region encoding proteins for the synthesis of the outer glycan is conserved within a Bacteroides species but divergent between species. Unlike the outer glycan, an antiserum raised to the core glycan reacted with all Bacteroidetes species tested, from all four classes of the phylum. We found that diverse Bacteroidetes species synthesize numerous glycoproteins and glycosylate proteins at the same three amino acid motif. The wide-spread conservation of this protein glycosylation system within the phylum suggests that this system of post-translational protein modification evolved early, before the divergence of the four classes of Bacteroidetes, and has been maintained due to its physiological importance to the diverse species of this phylum.

Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

Subcellular Localization of Extracytoplasmic Proteins in Monoderm Bacteria: Rational Secretomics-Based Strategy for Genomic and Proteomic Analyses

Genome-scale prediction of subcellular localization (SCL) is not only useful for inferring protein function but also for supporting proteomic data. In line with the secretome concept, a rational and original analytical strategy mimicking the secretion steps that determine ultimate SCL was developed for Gram-positive (monoderm) bacteria. Based on the biology of protein secretion, a flowchart and decision trees were designed considering (i) membrane targeting, (ii) protein secretion systems, (iii) membrane retention, and (iv) cell-wall retention by domains or post-translocational modifications, as well as (v) incorporation to cell-surface supramolecular structures. Using Listeria monocytogenes as a case study, results were compared with known data set from SCL predictors and experimental proteomics. While in good agreement with experimental extracytoplasmic fractions, the secretomics-based method outperforms other genomic analyses, which were simply not intended to be as inclusive. Compared to all other localization predictors, this method does not only supply a static snapshot of protein SCL but also offers the full picture of the secretion process dynamics: (i) the protein routing is detailed, (ii) the number of distinct SCL and protein categories is comprehensive, (iii) the description of protein type and topology is provided, (iv) the SCL is unambiguously differentiated from the protein category, and (v) the multiple SCL and protein category are fully considered. In that sense, the secretomics-based method is much more than a SCL predictor. Besides a major step forward in genomics and proteomics of protein secretion, the secretomics-based method appears as a strategy of choice to generate in silico hypotheses for experimental testing.

Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

BackgroundMany bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain.ResultsIn the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter.ConclusionsThe present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The dba expression is not only essential for the translation of the downstream dsbI gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI.

DsbA2 (27 kDa Com1‐like protein) of Legionella pneumophila catalyses extracytoplasmic disulphide‐bond formation in proteins including the Dot/Icm type IV secretion system

In Gram-negative bacteria, thiol oxidoreductases catalyse the formation of disulphide bonds (DSB) in extracytoplasmic proteins. In this study, we sought to identify DSB-forming proteins required for assembly of macromolecular structures in Legionella pneumophila. Here we describe two DSB-forming proteins, one annotated as dsbA1 and the other annotated as a 27 kDa outer membrane protein similar to Com1 of Coxiella burnetii, which we designate as dsbA2. Both proteins are predicted to be periplasmic, and while dsbA1 mutants were readily isolated and without phenotype, dsbA2 mutants were not obtained. To advance studies of DsbA2, a cis-proline residue at position 198 was replaced with threonine that enables formation of stable disulphide-bond complexes with substrate proteins. Expression of DsbA2 P198T mutant protein from an inducible promoter produced dominant-negative effects on DsbA2 function that resulted in loss of infectivity for amoeba and HeLa cells and loss of Dot/Icm T4SS-mediated contact haemolysis of erythrocytes. Analysis of captured DsbA2 P198T-substrate complexes from L. pneumophila by mass spectrometry identified periplasmic and outer membrane proteins that included components of the Dot/Icm T4SS. More broadly, our studies establish a DSB oxidoreductase function for the Com1 lineage of DsbA2-like proteins which appear to be conserved among those bacteria also expressing T4SS.

Theoretical and Experimental Characterization of the Scope of Protein O-Glycosylation in Bacteroides fragilis

Among bacterial species demonstrated to have protein O-glycosylation systems, that of Bacteroides fragilis and related species is unique in that extracytoplasmic proteins are glycosylated at serine or threonine residues within the specific three-amino acid motif D(S/T)(A/I/L/M/T/V). This feature allows for computational analysis of the proteome to identify candidate glycoproteins. With the criteria of a signal peptidase I or II cleavage site or a predicted transmembrane-spanning region and the presence of at least one glycosylation motif, we identified 1021 candidate glycoproteins of B. fragilis. In addition to the eight glycoproteins identified previously, we confirmed that another 12 candidate glycoproteins are in fact glycosylated. These included four glycoproteins that are predicted to localize to the inner membrane, a compartment not previously shown to include glycosylated proteins. In addition, we show that four proteins involved in cell division and chromosomal segregation, two of which are encoded by candidate essential genes, are glycosylated. To date, we have not identified any extracytoplasmic proteins containing a glycosylation motif that are not glycosylated. Therefore, based on the list of 1021 candidate glycoproteins, it is likely that hundreds of proteins, comprising more than half of the extracytoplasmic proteins of B. fragilis, are glycosylated. Site-directed mutagenesis of several glycoproteins demonstrated that all are glycosylated at the identified glycosylation motif. By engineering glycosylation motifs into a naturally unglycosylated protein, we are able to bring about site-specific glycosylation at the engineered sites, suggesting that this glycosylation system may have applications for glycoengineering.

Canonical and ECF-type ATP-binding cassette importers in prokaryotes: diversity in modular organization and cellular functions

Since their discovery in the 1960s as 'osmotic shock-sensitive' transporters, a plethora of so-called binding protein-dependent (canonical) ATP-binding cassette (ABC) importers has been identified in bacteria and archaea. Their cellular functions go far beyond the uptake of nutrients. Canonical ABC importers play important roles in the maintenance of cell integrity, responses to environmental stresses, cell-to-cell communication and cell differentiation and in pathogenicity. A new class of abundant micronutrient importers, the 'energy-coupling factor' (ECF) transporters, was originally identified by functional genomics. ABC ATPases are an integral part of both canonical ABC and ECF importers. Fundamental differences include the modular architecture and the independence of ECF systems of extracytoplasmic solute-binding proteins. This review describes the roles of both types of transporters in diverse physiological processes including pathogenesis, points to the differences in modular assembly and depicts their common traits.

A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, β-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation.

The cell envelope stress response mediated by the LiaFSR Lm three-component system of Listeria monocytogenes is controlled via the phosphatase activity of the bifunctional histidine kinase LiaS Lm

Most members of the phylum Firmicutes harbour a two-component system (TCS), LiaSR, which is involved in the response to cell envelope stress elicited most notably by inhibitors of the lipid II cycle. In all LiaSR systems studied in detail, LiaSR-mediated signal transduction has been shown to be negatively controlled by a membrane protein, LiaF, encoded upstream of liaSR. In this study we have analysed the LiaSR orthologue of Listeria monocytogenes (LiaSR(Lm)). Whole-genome transcriptional profiling indicated that activation of LiaSR(Lm) results in a remodelling of the cell envelope via the massive upregulation of membrane-associated and extracytoplasmic proteins in the presence of inducing stimuli. As shown for other LiaSR TCSs, LiaSR(Lm) is activated by cell wall-active antibiotics. We demonstrate that the level of phosphorylated LiaR(Lm), which is required for the induction of the LiaSR(Lm) regulon, is controlled by the interplay between the histidine kinase and phosphatase activities of the bifunctional sensor protein LiaS(Lm). Our data suggest that the phosphatase activity of LiaS(Lm) is stimulated by LiaF(Lm) in the absence of cell envelope stress.

Campylobacter protein oxidation influences epithelial cell invasion or intracellular survival as well as intestinal tract colonization in chickens

The Dsb family of redox proteins catalyzes disulfide bond formation and isomerization. Since mutations in dsb genes change the conformation and stability of many extracytoplasmic proteins, and since many virulence factors of pathogenic bacteria are extracytoplasmic, inactivation of dsb genes often results in pathogen attenuation. This study investigated the role of 2 membrane-bound oxidoreductases, DsbB and DsbI, in the Campylobacter jejuni oxidative Dsb pathway. Campylobacter mutants, lacking DsbB or DsbI or both, were constructed by allelic replacement and used in the human intestinal epithelial T84 cell line for the gentamicin protection assay (invasion assay) and chicken colonization experiments. In C. coli strain 23/1, the inactivation of the dsbB or dsbI gene separately did not significantly affect the colonization process. However, simultaneous disruption of both membrane-bound oxidoreductase genes significantly decreased the strain’s ability to colonize chicken intestines. Moreover, C. jejuni strain 81-176 with mutated dsbB or dsbI genes showed reduced invasion/intracellular survival abilities. No cells of the double mutants (dsbB⁻ dsbI⁻) of C. jejuni 81-176 were recovered from human cells after 3 h of invasion.

Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli

Two members of the widely conserved HtrA family of serine proteases, DegP and DegS, are key players in extracytoplasmic protein quality control. The underlying mechanisms of their main functions in stress sensing, regulation and protection during the unfolded protein response are discussed.

YtxR Acts as an Overriding Transcriptional Off Switch for the Yersinia enterocolitica Ysc-Yop Type 3 Secretion System

The Yersinia enterocolitica YtxR protein is a LysR-type transcriptional regulator that induces expression of the ytxAB locus, which encodes a putative ADP-ribosylating toxin. The ytxR and ytxAB genes are not closely linked in the Y. enterocolitica chromosome, and whereas ytxR is present in all sequenced Yersinia spp., the ytxAB locus is not. These observations suggested that there might be other YtxR-regulon members besides ytxAB and prompted us to investigate coregulated genes and gene products by using transcriptional and proteomic approaches. Microarray and reverse transcription-PCR analysis showed that YtxR strongly activates expression of the yts2 locus, which encodes a putative type 2 secretion system, as well as several uncharacterized genes predicted to encode extracytoplasmic proteins. Strikingly, we also discovered that under Ysc-Yop type 3 secretion system-inducing conditions, YtxR prevented the appearance of Yop proteins in the culture supernatant. Microarray and lacZ operon fusion analysis showed that this was due to specific repression of ysc-yop gene expression. YtxR was also able to repress VirF-dependent Phi(yopE-lacZ) and Phi(yopH-lacZ) expression in a strain lacking the virulence plasmid, which suggested a direct repression mechanism. This was supported by DNase I footprinting, which showed that YtxR interacted with the yopE and yopH control regions. Therefore, YtxR is a newly identified regulator of the ysc-yop genes that can act as an overriding off switch for this critical virulence system.