- Volume 155, Issue 7, 2009

The Hrp type III secretion system (TTSS) is essential for the pathogenicity of Ralstonia solanacearum on host plants. Hrp TTSS is a specialized secretion system that injects virulence proteins, the so-called type III effector proteins, into plant cells. In R. solanacearum, the expression of Hrp TTSS-related genes is regulated by an AraC-type transcriptional activator, HrpB. We have identified 30 hrpB-regulated hpx ( hrpB-dependent expression) genes and three well-known hrpB-regulated genes, popA, popB and popC, as candidate effector genes in R. solanacearum strain RS1000. In this study, we newly cloned 11 additional candidate effector genes that share homology with known hpx genes from R. solanacearum RS1000. Using a Cya reporter system, we investigated the translocation of these 44 gene products into plant cells via the Hrp TTSS and identified 34 effector proteins. These include three effector families composed of more than four members, namely the Hpx4, Hpx30 and GALA families. The Hpx30 family effectors are 2200–2500 aa in size and appear to be the largest class of effector proteins among animal- and plant-pathogenic bacteria. Members of this family contain 12–18 tandem repeats of a novel 42 aa motif, designated SKWP repeats.

The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each of which has 8–13 ORFs. Although the function of the Mce protein family has not been clearly established, its members are believed to be membrane lipid transporters. Based on functional experiments, we found that the regulator of the mce3 locus, Mce3R, negatively regulates the expression of the Rv1933c–Rv1935c and Rv1936–Rv1941 transcriptional units. These operons are adjacent to one another and divergently transcribed. The predicted functions of most of these genes are related to either lipid metabolism or redox reactions. Bioinformatic analysis of the 5′ UTR sequences of the differentially expressed genes allowed us to define a putative Mce3R motif. Importantly, the Mce3R motif was present six and three times in the mce3R–yrbE3A and Rv1935c–Rv1936 intergenic regions, respectively. Two occurrences of this motif mapped within the two regions of the mce3 operon that were protected by Mce3R in a footprinting analysis, thus indicating that this motif is likely to serve as an operator site for the Mce3R regulator in the promoter. In addition, alterations in the lipid content of M. tuberculosis were detected in the absence of Mce3R. Taken together, these results suggest that Mce3R controls the expression of both the putative transport system encoded in the mce3 operon and the enzymes implicated in the modification of the Mce3-transported substrates.

A novel protective monoclonal antibody (mAb) that recognizes a lipopolysaccharide (LPS) epitope common between serotypes Ogawa and Inaba of the O1 serogroup of Vibrio cholerae was characterized and the potential to develop peptide mimics of this protective LPS epitope was investigated. mAb 72.1 recognizes both Ogawa and Inaba LPS and it is vibriocidal and protective in passive immunization against infection by strains of both serotypes. The cDNA-derived amino acid sequence of mAb 72.1 is closely related to the previously characterized mAb ZAC-3, which is thought to recognize an epitope in the lipid A core region of O1 LPS. In an attempt to develop a peptide mimic-based vaccine against V. cholerae, phage display libraries were screened with mAb 72.1 and 11 peptide mimics were identified. Remarkably, all of the peptide sequences identified from linear phage display libraries contained two cysteine residues, suggesting that mAb 72.1 preferentially binds to peptides constrained with a disulphide bond. One of the peptide mimics was immunologically characterized. Although immunization of mice with this peptide mimic conjugated to KLH elicited antibodies against the peptide itself, these antibodies did not cross-react with Ogawa or Inaba LPS. Effectiveness of a peptide mimic as a vaccine may depend on how well the peptide can mimic the carbohydrate interactions when binding to the anti-carbohydrate antibody. Thus, investigating how peptides and LPS bind to mAb 72.1 may be useful in improving current peptide mimics or designing more effective peptide mimics. Identification and characterization of novel protective anti-LPS antibodies may be useful in studying protective epitopes of LPS, which may help develop LPS-based therapeutics against V. cholerae.

Salmonella enterica serovar Choleraesuis (SC) is an important enteric pathogen that causes serious systemic infections in swine and humans. To identify the genes required for resistance to antimicrobial peptides, we constructed a bank of SC transposon mutants and screened them for hypersensitivity to the cationic peptide polymyxin B. Here we report one isolated polymyxin B-susceptible mutant that also exhibited increased sensitivity toward human neutrophil peptide alpha-defensin 1 (HNP-1) and hydrophobic antibiotics including erythromycin and novobiocin. The mutant had a mutation in an ORF identified as outer membrane β-barrel protein gene omb. The purified recombinant Omb protein was characterized as a ferrous iron-binding protein. The constructed omb isogenic mutant grew more slowly in iron-limiting conditions than the wild-type (WT) parent strain. In addition, compared with the WT strain, the omb mutant exhibited an increase in net negative charge upon the cell surface and was more easily killed by polymyxin B, HNP-1 and hydrophobic antibiotics. The omb gene was transcribed, regardless of the iron content within the growth medium, and the Omb protein appeared exclusively in the outer membrane fraction. Infection experiments demonstrated virulence attenuation when the mutant was administered orally or intraperitoneally to mice. This study indicates that Omb is a previously unrecognized ferrous iron-binding protein. In vivo, Omb may be involved in the acquisition of ferrous iron during the initial stages of SC infection and appears to be an important virulence factor for SC in mice.

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5 % of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway.

The laboratory diagnosis of pulmonary tuberculosis mainly relies on the detection of Mycobacterium tuberculosis complex (MTC) organisms in the sputum. In patients who do not give sputum, alternative respiratory tract specimens can be obtained only by invasive procedures. Based on the known survival of MTC organisms in the gastric fluid, we hypothesized that swallowed MTC organisms would be detectable in stool samples. We compared the presence of MTC organisms in respiratory tract specimens and stool specimens collected in parallel from the same patients. MTC was detected in cultures grown on egg-based medium after appropriate decontamination, by microscopic examination after Ziehl–Neelsen staining and by real-time PCR detection of IS6110 using internal controls. A case of pulmonary tuberculosis was defined by the presence of (i) clinical and radiological signs and symptoms suggestive of pulmonary tuberculosis, and (ii) culture of MTC organisms from at least one respiratory tract specimen or (iii) the presence of acid-fast bacilli in the sputum that were subsequently identified as MTC organisms by real-time PCR. The observation of 134 patients suspected to be suffering pulmonary tuberculosis led to the identification of 24 cases and 110 non-infected control patients. Cases and controls did not significantly differ with respect to sex but cases were significantly younger than controls. The sensitivity/specificity was 37.5 %/100 % for the microscopic examination of stools, 54.2 %/100 % for culturing and 100 %/97.3 % for real-time PCR. The positive predicted value was 100 %, 100 % and 88.9 %, respectively, and the negative predicted value was 88 %, 90.9 % and 100 %, respectively. In four patients, a stool specimen initially yielded the correct diagnosis of pulmonary tuberculosis before evaluation of the respiratory tract specimen confirmed the diagnosis. These data indicate that stools could be used in conjunction with sputum testing or as an alternative specimen upon which to base the diagnosis of pulmonary tuberculosis by molecular identification of acid-fast bacilli and culture. This non-invasive alternative procedure is of particular interest for patients who cannot expectorate.

We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of β-sheets with only a minor proportion of α-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.

Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPIases), the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that SlrA has PPIase activity, and that the enzyme plays a role in pneumococcal virulence. Here, we investigated the contribution of PpmA to pneumococcal pathogenesis. Pneumococcal mutants of D39 and TIGR4 lacking the gene encoding PpmA were less capable of persisting in the nasopharynx of mice, demonstrating the contribution of PpmA to pneumococcal colonization. This observation was partially confirmed in vitro, as the pneumococcal mutants NCTC10319ΔppmA and TIGR4ΔcpsΔppmA, but not D39ΔcpsΔppmA, were impaired in adherence to Detroit 562 pharyngeal cells. This suggests that the contribution of PpmA to pneumococcal colonization is not solely the result of its role in adherence to epithelial cells. Deficiency in PpmA did not result in reduced binding to various extracellular matrix and serum proteins. Similar to SlrA, we observed that PpmA was involved in immune evasion. Uptake of PpmA-deficient D39Δcps and NCTC10319 by human polymorphonuclear leukocytes was significantly enhanced compared to the isogenic wild-types. In addition, ingestion of D39ΔppmA, but not that of either NCTC10319ΔppmA or TIGR4ΔppmA, by murine macrophage cell line J774 was also enhanced, whereas intracellular killing remained unaffected. We conclude that PpmA contributes to the early stages of infection, i.e. colonization. The contribution of PpmA to virulence can be explained by its strain-specific role in adherence to epithelial cells and contribution to the evasion of phagocytosis.

Carnitine is a quaternary amine compound prevalent in animal tissues, and a potential carbon, nitrogen and energy source for pathogens during infection. Characterization of activities in Pseudomonas aeruginosa cell lysates has previously shown that carnitine is converted to 3-dehydrocarnitine (3-dhc) which is in turn metabolized to glycine betaine (GB), an intermediate metabolite in the catabolism of carnitine to glycine. However, the identities of the enzymes required for carnitine catabolism were not known. We used a genetic screen of the P. aeruginosa PA14 transposon mutant library to identify genes required for growth on carnitine. We identified two genomic regions and their adjacent transcriptional regulators that are required for carnitine catabolism. The PA5388–PA5384 region contains the predicted P. aeruginosa carnitine dehydrogenase homologue along with other genes required for growth on carnitine. The second region identified, PA1999–PA2000, encodes the α and β subunits of a predicted 3-ketoacid CoA-transferase, an enzymic activity hypothesized to be involved in the first step of deacetylation of 3-dhc. Furthermore, we confirmed that an intact GB catabolic pathway is required for growth on carnitine. The PA5389 and PA1998 transcription factors are required for growth on carnitine. PA5389 is required for induction of the PA5388–PA5384 transcripts in response to carnitine, and the PA1999–PA2000 transcripts are induced in a PA1998-dependent manner and induction appears to depend on a carnitine catabolite, possibly 3-dhc. These results provide important insight into elements required for carnitine catabolism in P. aeruginosa and probably in other bacteria.

The growth pattern of Pseudomonas putida KT2440 in the presence of glucose and phenylacetic acid (PAA), where the sugar is used in preference to the aromatic compound, suggests that there is carbon catabolite repression (CCR) of PAA metabolism by glucose or gluconate. Furthermore, CCR is regulated at the transcriptional level. However, this CCR phenomenon does not occur in PAA-amended minimal medium containing fructose, pyruvate or succinate. We previously identified 2-keto-3-deoxy-6-phosphogluconate (KDPG) as an inducer of glucose metabolism, and this has led to this investigation into the role of KDPG as a signal compound for CCR. Two mutant strains, the edd mutant (non-KDPG producer) and the eda mutant (KDPG overproducer), grew in the presence of PAA but not in the presence of glucose. The edd mutant utilized PAA even in the presence of glucose, indicating that CCR had been abolished. This observation has additional support from the finding that there is high phenylacetyl-CoA ligase activity in the edd mutant, even in the presence of glucose+PAA, but not in wild-type cells under the same conditions. Unlike the edd mutant, the eda mutant did not grow in the presence of glucose+PAA. Interestingly, there was no uptake and/or metabolism of PAA in the eda mutant cells under the same conditions. Targeted disruption of PaaX, a repressor of the PAA operon, had no effect on CCR of PAA metabolism in the presence of glucose, suggesting that there is another transcriptional repression system associated with the KDPG signal. This is the first study to demonstrate that KDPG is the true CCR signal of PAA metabolism in P. putida KT2440.

In order to survive in the host and initiate infection, Salmonella enterica needs to undergo a transition between aerobic and anaerobic growth by modulating its central metabolic pathways. In this study, a comparative analysis of the proteome of S. enterica serovar Typhimurium grown in the presence or absence of oxygen was performed. The most prominent changes in expression were measured in a semiquantitative manner using difference in-gel electrophoresis (DIGE) to reveal the main protein factors involved in the adaptive response to anaerobiosis. A total of 38 proteins were found to be induced anaerobically, while 42 were repressed. The proteins of interest were in-gel digested with trypsin and identified by MALDI TOF mass spectrometry using peptide mass fingerprinting. In the absence of oxygen, many fermentative enzymes catalysing reactions in the mixed-acid or arginine fermentations were overexpressed. In addition, the enzyme fumarate reductase, which is known to provide an alternative electron acceptor for the respiratory chains in the absence of oxygen, was shown to be induced. Increases in expression of several glycolytic and pentose phosphate pathway enzymes, as well as two malic enzymes, were detected, suggesting important roles for these in anaerobic metabolism. Substantial decreases in expression were observed for a large number of periplasmic transport proteins. The majority of these are involved in the uptake of amino acids and peptides, but permeases transporting iron, thiosulphate, glucose/galactose, glycerol 3-phosphate and dicarboxylic acids were also repressed. Decreases in expression were also observed for a superoxide dismutase, ATP synthase, inositol monophosphatase, and several chaperone and hypothetical proteins. The changes were monitored in two different isolates, and despite their very similar expression patterns, some variability in the adaptive response to anaerobiosis was also observed.

The multi-modular non-cellulosomal endo-1,3(4)-β-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191–426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca2+. The highest affinity to CBMX was shown for xylan (binding constant K=3.1×104 M−1), yeast cell wall glucan (K=1.4×105 M−1) and chitin (K=3.3.105 M−1 in the presence of Ca2+). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

In some antibiotic producers, p-aminobenzoic acid (PABA) or its immediate precursor, 4-amino-4-deoxychorismate (ADC), is involved in primary metabolism and antibiotic biosynthesis. In Streptomyces sp. FR-008, a gene pabC-1 putatively encoding a fold-type IV pyridoxal 5′-phosphate (PLP)-dependent enzyme was found within the antibiotic FR-008/candicidin biosynthetic gene cluster, whose inactivation significantly reduced the productivity of antibiotic FR-008 to about 20 % of the wild-type level. Its specific role in PABA formation was further demonstrated by the successful complementation of an Escherichia coli pabC mutant. Moreover, a free-standing gene pabC-2, probably encoding another fold-type IV PLP-dependent enzyme, was cloned from the same strain. Inactivation of pabC-2 reduced antibiotic FR-008 yield to about 57 % of the wild-type level in the mutant, and the complementation of the E. coli pabC mutant established its involvement in PABA biosynthesis. Furthermore, a pabC-1/pabC-2 double mutant only retained about 4 % of the wild-type antibiotic FR-008 productivity, clearly indicating that pabC-2 also contributed to biosynthesis of this antibiotic. Surprisingly, apparently retarded growth of the double mutant was observed on minimal medium, which suggested that both pabC-1 and pabC-2 are involved in PABA biosynthesis for primary metabolism. Finally, both PabC-1 and PabC-2 were shown to be functional ADC lyases by in vitro enzymic lysis with the release of pyruvate. pabC-1 and pabC-2 appear to represent the first two functional ADC lyase genes identified in actinomycetes. The involvement of these two ADC lyase genes in both cell growth and antibiotic FR-008 biosynthesis sets an example for the interplay between primary and secondary metabolisms in bacteria.