- Volume 156, Issue 6, 2010
Volume 156, Issue 6, 2010
- Microbial Pathogenicity
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Regional variations in the population structure of Pseudomonas syringae pathovar phaseolicola from Spain are revealed by typing with PmeI pulsed-field gel electrophoresis, plasmid profiling and virulence gene complement
More LessOne hundred and twenty pathogenic isolates of Pseudomonas syringae pv. phaseolicola recovered in Spain were subjected to biochemical and genomic typing, and investigated for virulence gene complement. Fifty-six were recovered from common beans (Phaseolus vulgaris) of the type Granja Asturiana, grown in a northern Spanish region (Asturias), and 64 from other common beans cultured in the neighbouring region of Castilla y León. Typing by PmeI digestion followed by pulsed-field gel electrophoresis revealed 27 profiles, with only three being common to both regions. Relationships between profiles distributed the isolates into two clusters: A (subdivided into subclusters A1 and A2) and B. Cluster A included all isolates from Granja Asturiana and about a quarter of the isolates from Castilla y León. Isolates from cluster A were negative for mannitol utilization and hybridized to probes for the argK–tox region responsible for phaseolotoxin production. Isolates that grouped in cluster B, which were only found in Castilla y León, were able to utilize mannitol but did not hybridize to probes for the argK–tox region. Separation of the isolates into three genomic groups, subsequently termed PphA1, PphA2 and PphB, was also supported by effector gene complement and location. In PphB, all effector genes tested (hopX1, hopF1, avrB2 and avrD1) mapped on chromosomal fragments, but faint hybridization of avrB2 with plasmids of about 40 kb was also observed. In PphA hopX1 mapped on the chromosome; in PphA1 avrB2 and avrD1 were carried on virulence plasmids (most of approx. 125 kb) and hopF1 was not detected, while in PphA2 the three genes were located on plasmids (approx. 75–160 kb). These results can be used as a framework to investigate the basis of regional variation in population structure, and for further epidemiological surveillance of P. syringae pv. phaseolicola.
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Salmonella pathogenicity island 1 (SPI-1) type III secretion of SopD involves N- and C-terminal signals and direct binding to the InvC ATPase
More LessSalmonella enterica serovar Typhimurium (S. Typhimurium) is an important pathogen and a causative agent of gastroenteritis. During infection, S. Typhimurium assembles molecular-needle complexes termed type III secretion (T3S) systems to translocate effector proteins from the bacterial cytoplasm directly into the host cell. The T3S signals that direct the secretion of effectors still remain enigmatic. SopD is a key T3S effector contributing to the systemic virulence of S. Typhimurium and the development of gastroenteritis. We have scrutinized the distribution of the SopD T3S signals using in silico analysis and a targeted deletion approach. We show that amino acid residues 6–10 act as the N-terminal secretion signal for Salmonella pathogenicity island 1 (SPI-1) T3S. Furthermore, we show that two putative C-terminal helical regions of SopD are essential for its secretion and also help prevent erroneous secretion through the flagellar T3S machinery. In addition, using protein–protein interaction assays, we have identified an association between SopD and the SPI-1 T3S system ATPase, InvC. These findings demonstrate that T3S of SopD involves multiple signals and protein interactions, providing important mechanistic insights into effector protein secretion.
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The enteropathogenic Escherichia coli effector NleH inhibits apoptosis induced by Clostridium difficile toxin B
Clostridium difficile is a leading cause of nosocomial infections, causing a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis triggered by a range of virulence factors including C. difficile toxins A (TcdA) and B (TcdB). TcdA and TcdB are monoglucosyltransferases that irreversibly glycosylate small Rho GTPases, inhibiting their ability to interact with their effectors, guanine nucleotide exchange factors, and membrane partners, leading to disruption of downstream signalling pathways and cell death. In addition, TcdB targets the mitochondria, inducing the intrinsic apoptotic pathway resulting in TcdB-mediated apoptosis. Modulation of apoptosis is a common strategy used by infectious agents. Recently, we have shown that the enteropathogenic Escherichia coli (EPEC) type III secretion system effector NleH has a broad-range anti-apoptotic activity. In this study we examined the effects of NleH on cells challenged with TcdB. During infection with wild-type EPEC, NleH inhibited TcdB-induced apoptosis at both low and high toxin concentrations. Transfected nleH1 alone was sufficient to block TcdB-induced cell rounding, nuclear condensation, mitochondrial swelling and lysis, and activation of caspase-3. These results show that NleH acts via a global anti-apoptotic pathway.
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Characterization of the haem-uptake system of the equine pathogen Streptococcus equi subsp. equi
More LessStreptococcus equi possesses a haem-uptake system homologous to that of Streptococcus pyogenes and Streptococcus zooepidemicus. The system consists of two ligand-binding proteins (Shr and Shp) and proteins (HtsA–C) with homology to an ABC transporter. The haem-uptake system of S. equi differs from that of S. pyogenes and S. zooepidemicus in that Shr is truncated by two-thirds. This study focused on the SeShr, SeShp and SeHtsA proteins of S. equi. Analysis of shr, shp and shphtsA knockout mutants showed that all three proteins were expressed in vitro and that expression was upregulated under conditions of iron limitation. SeShr possesses no membrane-/cell wall-spanning sequences and was shown to be secreted. Both SeShp and SeHtsA were confirmed to be envelope-associated. Recombinant SeShp and SeHtsA proteins have been previously shown to bind haem and SeHtsA could capture haem from SeShp. This report extends these studies and shows that both SeShp and SeHtsA can sequester haem from haemoglobin but not from haemoglobin–haptoglobin complexes. Like full-length Shr, SeShr possesses haemoglobin and haemoglobin–haptoglobin binding ability but unlike full-length Shr, it lacks haem- or fibronectin-binding capabilities. Analysis of SeShr truncates showed that residues within and upstream of the near transporter (NEAT) domain are required for this ligand binding. Structural predictions suggest that truncation of NEAT1 in SeShr accounts for its impaired ability to bind haem. Haem and haemoglobin restored to almost normal the impaired growth rates of wild-type S. equi cultured under iron-limiting conditions. However, no difference in the growth rates of wild-type and mutants could be detected under the in vitro growth conditions tested.
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Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes
Immune adult horses have CD8+ cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-γ) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-γ, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-γ transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.
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- Physiology And Biochemistry
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Amino acid identity at one position within the α1 helix of both the histidine kinase and the response regulator of the WalRK and PhoPR two-component systems plays a crucial role in the specificity of phosphotransfer
More LessTwo-component systems usually function as cognate pairs, thereby ensuring an appropriate response to the detected signal. The ability to exclusively phosphorylate a partner protein, often in the presence of many competing homologous substrates, demonstrates a high level of specificity that must derive from the interacting surfaces of the two-component system. Here, we identify positions within the histidine kinases and response regulators of the WalRK and PhoPR two-component systems of Bacillus subtilis that make a major contribution to the specificity of phosphotransfer. Changing the identity of the amino acid at position 11 within the α1 helix of WalK and at position 17 within the α1 helix of PhoP altered discrimination and allowed phosphotransfer to occur with the non-cognate partner. Changing amino acids at additional positions of the WalK kinase increased phosphotransfer, while changes at additional positions in PhoP only had an effect in the presence of the change at position 17. The importance of amino acid identity at these two positions is supported by the fact that the amino acid combinations of Ile and Ser in WalRK, and Leu and Gly in PhoPR, are very highly conserved among orthologues, while modelling indicates that these amino acid pairs are juxtaposed in the WalRK and PhoPR complexes.
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Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate
More LessThe fermentative metabolism of d-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh−) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh− were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD+ ratio supported the proposed role of PDH. Moreover, the NADH : NAD+ ratio in a strain deficient in both PDH and the ox-PPP (Pdh−Zwf−) was even lower than that observed for Pdh−. Strain Pdh−Zwf− also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh−. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh− mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD+ ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.
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A new small regulatory protein, HmuP, modulates haemin acquisition in Sinorhizobium meliloti
More LessSinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted β-sheets are well conserved. Our data indicate that at least one of the β-sheets is important for HmuP activity.
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Structure analysis of the two-peptide bacteriocin lactococcin G by introducing d-amino acid residues
More LessThe importance of 3D structuring in the N- and C-terminal ends of the two peptides (39-mer LcnG-α and 35-mer LcnG-β) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding d-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-α with four d-residues in its C-terminal region and LcnG-β with four d-residues in either its N- or its C-terminal region were relatively active (two- to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-α and the C- and N-terminal regions in LcnG-β is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-α may be more important, as LcnG-α with four N-terminal d-residues was the least active of these four peptides (10- to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-α and -β form a transmembrane parallel helix–helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-α and at about residue 13 in LcnG-β. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these d-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the d-residue-containing peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N- and C-terminal residues with d-residues.
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Characterization and inactivation of the membrane-bound polyol dehydrogenase in Gluconobacter oxydans DSM 7145 reveals a role in meso-erythritol oxidation
More LessThe growth of Gluconobacter oxydans DSM 7145 on meso-erythritol is characterized by two stages: in the first stage, meso-erythritol is oxidized almost stoichiometrically to l-erythrulose according to the Bertrand–Hudson rule. The second phase is distinguished from the first phase by a global metabolic change from membrane-bound meso-erythritol oxidation to l-erythrulose assimilation with concomitant accumulation of acetic acid. The membrane-associated erythritol-oxidizing enzyme was found to be encoded by a gene homologous to sldA known from other species of acetic acid bacteria. Disruption of this gene in the genome of G. oxydans DSM 7145 revealed that the membrane-bound polyol dehydrogenase not only oxidizes meso-erythritol but also has a broader substrate spectrum which includes C3–C6 polyols and d-gluconate and supports growth on these substrates. Cultivation of G. oxydans DSM 7145 on different substrates indicated that expression of the polyol dehydrogenase was not regulated, implying that the production of biomass of G. oxydans to be used as whole-cell biocatalysts in the biotechnological conversion of meso-erythritol to l-erythrulose, which is used as a tanning agent in the cosmetics industry, can be conveniently carried out with glucose as the growth substrate.
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The dddP gene of Roseovarius nubinhibens encodes a novel lyase that cleaves dimethylsulfoniopropionate into acrylate plus dimethyl sulfide
More LessThe cloned dddP gene of the marine bacterium Roseovarius nubinhibens allows Escherichia coli to form the volatile dimethyl sulfide (DMS) from dimethylsulfoniopropionate (DMSP), an abundant anti-stress compatible solute made by many marine plankton and macroalgae. Using purified DddP, we show here that this enzyme is a DMSP lyase that cleaves DMSP to DMS plus acrylate. DddP forms a functional homodimeric enzyme, has a pH optimum of 6.0 and was a K m of ∼14 mM for the DMSP substrate. DddP belongs to the M24B family of peptidases, some members of which have metal cofactors. However, the metal chelators EDTA and bipyridyl did not affect DddP activity in vitro and the as-isolated enzyme did not contain metal ions. Thus, DddP resembles those members of the M24B family, such as creatinase, which also act on a non-peptide substrate and have no metal cofactor. Site-directed mutagenesis of the active-site region of DddP completely abolished its activity. Another enzyme, termed DddL, which occurs in other alphaproteobacteria, had also been shown to generate DMS plus acrylate from DMSP. However, DddL and DddP have no sequence similarity to each other, so DddP represents a second, wholly different class of DMSP lyase.
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