- Volume 157, Issue 12, 2011

Human salmonellosis infections are usually acquired via the food chain as a result of the ability of Salmonella serovars to colonize and persist within the gastrointestinal tract of their hosts. In addition, after food ingestion and in order to cause foodborne disease in humans, Salmonella must be able to resist several deleterious stress conditions which are part of the host defence against infections. This review gives an overview of the main defensive mechanisms involved in the Salmonella response to the extreme acid conditions of the stomach, and the elevated concentrations of bile salts, osmolytes and commensal bacterial metabolites, and the low oxygen tension conditions of the mammalian and avian gastrointestinal tracts.

Bacterial pathogens use a range of protein secretion systems to colonize their host. One recent addition to this arsenal is the type VI secretion system (T6SS), which is found in many Gram-negative bacteria. The T6SS involves 12–15 components, including a ClpV-like AAA+ ATPase. Moreover, the VgrG and Hcp components have been proposed to form a puncturing device, based on structural similarity to the tail spike components gp5/gp27 and the tail tube component gp19 of the T4 bacteriophage, respectively. Another T6SS component shows similarity to a T4 phage protein, namely gp25. The gp25 protein has been proposed to have lysozyme activity. Other T6SS components do not exhibit obvious similarity to characterized T4 phage components. The genome of Pseudomonas aeruginosa contains three T6SS gene clusters. In each cluster a gene encoding a putative member of the gp25-like protein family was identified, which we called HsiF. We confirmed this similarity by analysing the structure of the P. aeruginosa HsiF proteins using secondary and tertiary structure prediction tools. We demonstrated that HsiF1 is crucial for the T6SS-dependent secretion of Hcp and VgrG. Importantly, lysozyme activity of HsiF proteins was not detectable, and we related this observation to the demonstration that HsiF1 localizes to the cytoplasm of P. aeruginosa. Finally, our data showed that a conserved glutamate, predicted to be required for proper HsiF folding, is essential for its function. In conclusion, our data confirm the central role of HsiF in the T6SS mechanism, provide information on the predicted HsiF structure, and call for reconsideration of the function of gp25-like proteins.

Metal homeostasis is important in all living cells in order to provide sufficient amounts of metal ions for biological processes but to prevent toxic effects by excess amounts. Here we show that the gene product of RSP_2889 of the facultatively photosynthetic bacterium Rhodobacter sphaeroides is homologous to CueR, a regulator of copper metabolism in Escherichia coli and other bacteria. CueR binds to the promoter regions of genes for a copper-translocating ATPase and for a copper chaperone and is responsible for their high expression when cells are exposed to elevated levels of copper ions. While deletion of RSP_2889 has no significant effect on copper resistance, expression from a low-copy-number plasmid mediates increased sensitivity to copper.

A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter Pxyl/tet. Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR–Pxyl/tet regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed Pxyl/tet-like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54 and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

The phytopathogenic bacterium Pectobacterium atrosepticum (Pba) strain SCRI1043 does not exhibit appreciable biofilm formation under standard laboratory conditions. Here we show that a biofilm-forming phenotype in this strain could be activated from a cryptic state by increasing intracellular levels of c-di-GMP, through overexpression of a constitutively active diguanylate cyclase (PleD*) from Caulobacter crescentus. Randomly obtained Pba transposon mutants defective in the pga operon, involved in synthesis and translocation of poly-β-1,6-N-acetyl-d-glucosamine (PGA), were all impaired in this biofilm formation. The presence of the PGA-degrading enzyme dispersin B in the growth media prevented biofilm formation by Pba overexpressing PleD*, further supporting the importance of PGA for biofilm formation by Pba. Importantly, a pga mutant exhibited a reduction in root binding to the host plant under conditions of high intracellular c-di-GMP levels. A modest but consistent increase in pga transcript levels was associated with high intracellular levels of c-di-GMP. Our results indicate tight control of PGA-dependent biofilm formation by c-di-GMP in Pba.

Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (GmR or Lac+) were obtained at frequencies ranging from 101 to 106 c.f.u. (µg DNA)−1. Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter PC were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

We have identified a methanol- and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P. pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucose-ammonium (Bio−) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC). Deletion of ROP results in a strain (ΔROP) that can grow under biotin-deficient conditions due to derepression of a biotin- and PC-independent pathway of anaplerotic synthesis of oxaloacetate. Northern analysis as well as microarray expression profiling of RNA isolated from WT and ΔROP strains cultured in Bio− medium indicate that expression of the phosphoenolpyruvate carboxykinase gene (PEPCK) is induced in ΔROP during biotin- or PC-deficiency even under glucose-abundant conditions. There is an excellent correlation between PEPCK expression and growth of ΔROP in Bio− medium, suggesting that ROP-mediated regulation of PEPCK may have a crucial role in the biotin- and PC-independent growth of the ΔROP strain. To our knowledge, ROP is the first example of a zinc finger transcription factor involved in the catabolite repression of PEPCK in yeast cells cultured under biotin- or PC-deficient and glucose-abundant conditions.

The soil bacterium Bacillus subtilis can utilize exogenous proline as a sole nitrogen or carbon source. The proline-inducible putBCP (formerly ycgMNO) operon encodes proteins responsible for proline uptake and two-step oxidation of proline to glutamate. We now report that a gene (formerly ycgP, now designated prcR) located downstream of the putBCP operon is essential for B. subtilis cells to utilize proline as a sole nitrogen or carbon source. Disruption of the prcR gene also abolished proline induction of putB transcription. prcR expression is not subject to autoregulation and proline induction. The PrcR protein shows no significant amino acid sequence similarity to the known transcriptional activators for proline utilization genes of other bacteria, but it does show partial amino acid sequence similarity to the transcriptional regulator PucR for the purine degradation genes of B. subtilis. PrcR orthologues of unknown function are present in some other Bacillus species. Primer-extension analysis suggests that both putB and prcR are transcribed by a σA-dependent promoter. Deletion and mutation analysis revealed that an inverted repeat (5′-TTGTGG-N5-CCACAA-3′) centred at position −76 relative to the transcriptional initiation site of putB is essential for putB expression. Electrophoretic mobility shift assays showed that the purified His-tagged PrcR was capable of binding specifically to this inverted repeat. Altogether, these results suggest that PrcR is a PucR-type transcriptional activator that mediates expression of the B. subtilis putBCP operon in response to proline availability.

Quorum sensing is a cell density-dependent gene expression mechanism found in many Gram-negative bacteria which involves the production of signal molecules such as N-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in Halomonas anticariensis FP35T, which is composed of luxR/luxI homologues: hanR (the putative transcriptional regulator gene) and hanI (the autoinducer synthase gene). To understand how the hanR/hanI system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the hanR and hanI genes are on the same transcript and that their transcription is growth phase-dependent. HanI seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of hanI resulted in the complete loss of its AHLs. We also found that the hanI gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35hanR mutant showed AHL-producing activity and hanI expression similar to that of the wild-type strain, the latter being measured by RT-PCR. Moreover, hanR is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by HanI.

The carbazole-degradative plasmid pCAR1 carries the class II transposon Tn4676, which contains the car and ant genes, essential for conversion of carbazole into anthranilate, and anthranilate into catechol, respectively. In our previous study, DNA rearrangements in pCAR1 were frequently detected in the host Pseudomonas fluorescens Pf0-1 in the presence of carbazole, resulting in the improvement of host survivability. Several Pf0-1 mutants harbouring pCAR1 were isolated, and deletion of DNA in the plasmid ant gene was found. Here, we compared genome sequences of the parent strain Pf0-1L(pCAR1 : : rfp) and one of its mutants, 5EP83, to assess whether other DNA rearrangements occurred in either the plasmid or the host chromosome. We found transposition of Tn4676 into the 5EP83 chromosome. In addition, ISPre1 had transposed into the car gene intergenic region on the pCAR1-derivative plasmid of 5EP83, which inhibited car transcription. As a result of these transpositions, 5EP83 was able to metabolize carbazole due to the Tn4676 on its chromosome, although the car genes on its plasmid were non-functional. We also found that one copy of duplicate carAa genes had been deleted, and that ISPre4 had transposed into both the host chromosome and the plasmid. Our findings suggest that Pf0-1 harbouring pCAR1 is subjected to DNA rearrangements not only on the plasmid but also on its chromosome in the presence of carbazole.

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Enterococci, especially Enterococcus faecium, are well-known pathogens of hospitalized patients and are frequently linked with resistance against multiple antibiotics, which compromises effective therapy. Rabbit immune serum raised against heat-killed E. faecium E155, a HiRECC clone, was used in an opsonophagocytic assay, an inhibition assay and a mouse bacteraemia model to identify targets of opsonic and protective antibodies. Serum against whole heat-killed bacteria was opsonic and recognized a protein of about 72 kDa that was abundantly secreted. This protein, identified as SagA by LC-ES-MS/MS, was expressed in Escherichia coli and purified. Rabbit serum raised against the purified protein showed opsonic killing activity that was inhibited by almost 100 % using 100 µg purified protein ml−1. In a mouse bacteraemia model, a statistically significant reduction of the colony counts in blood was shown with immune rabbit serum compared with preimmune serum using the homologous and a heterologous vancomycin-resistant enterococci (VRE) strain. These results indicate that SagA could be used as a promising vaccine target to treat and/or prevent VRE bacteraemia.

The naturally occurring reactive electrophilic species 12-oxo-phytodienoic acid (12-oxo-PDA) is a potent antifungal agent, whereas the plant growth regulator jasmonic acid, which is synthesized from 12-oxo-PDA, is ineffective. To address what structural features of the molecule endow it with antifungal activity, we synthesized a series of molecular mimics of 12-oxo-PDA varying in the length of the alkyl chain at its C-4 ring position. The octyl analogue (4-octyl cyclopentenone) was the most effective at suppressing spore germination and subsequent mycelial growth of a range of fungal pathogens and was particularly effective against Cladosporium herbarum and Botrytis cinerea, with minimum fungicidal concentrations in the range 100–200 µM. Introduction of a carboxyl group to the end of the chain, mimicking natural fatty acids, markedly reduced antifungal efficacy. Electrolyte leakage, indicative of membrane perturbation, was evident in both C. herbarum and B. cinerea exposed to 4-octyl cyclopentenone. Lipid composition analysis of the fungal spores revealed that those species with a high oil content, namely Fusarium oxysporum and Alternaria brassicicola, were less sensitive to 4-octyl cyclopentenone. The comparable hydrophobicity of 4-octyl cyclopentenone and 12-oxo-PDA accounts for the similar spore suppression activity of these two compounds. The relative ease of synthesis of 4-octyl cyclopentenone makes it an attractive compound for potential use as an antifungal agent.

Klebsiella pneumoniae community-acquired pyogenic liver abscess (PLA) is an emerging infectious disease. The rmpA gene (for regulator of mucoid phenotype A) has been reported to be associated with PLA in prevalence studies. NTUH-K2044, a K1 PLA isolate, carries three rmpA/A2 genes: two large-plasmid-carried genes (p-rmpA and p-rmpA2) and one chromosomal gene (c-rmpA). In this study, we re-examined the role of rmpA/A2 in PLA pathogenesis to clarify the relationship of rmpA/A2 and capsular serotype to virulence. Using isogenic gene deletion strains and complemented strains of NTUH-K2044, we demonstrated that only p-rmpA enhanced expression of capsular polysaccharide synthesis (cps) genes and capsule production. Nevertheless, the lethal dose and in vivo competitive index indicated that p-rmpA does not promote virulence in mice. The prevalence of these three rmpA/A2 and capsular types in 206 strains was investigated. This revealed a correlation of rmpA/A2 with six PLA-related capsular types (K1, K2, K5, K54, K57 and KN1). However, the correlation of rmpA/A2 with K1 strains from the West was less obvious than with the strains from Asia (17/22 vs 39/39, P = 0.0019). Among the three rmpA/A2 genes, p-rmpA was the most prevalent. Due to the strong correlation with PLA-related capsular types, p-rmpA could serve as a surrogate marker for PLA. We found an association of p-rmpA with three widely spaced loci in a large plasmid (30/32). Therefore, rmpA could be co-inherited together with virulence genes carried by this plasmid.

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR–LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

The Trichomonas vaginalis 120 kDa protein adhesin (AP120) is induced under iron-rich conditions and has sequence homology with pyruvate : ferredoxin oxidoreductase A (PFO A), a hydrogenosomal enzyme that is absent in humans. This homology raises the possibility that, like AP120, PFO might be localized to the parasite surface and participate in cytoadherence. Here, the cellular localization and function of PFO that was expressed under various iron concentrations was investigated using a polyclonal antibody generated against the 50 kDa recombinant C-terminal region of PFO A (anti-PFO50). In Western blot assays, this antibody recognized a 120 kDa protein band in total protein extracts, and proteins with affinity to the surface of HeLa cells from parasites grown under iron-rich conditions. In addition to localization that is typical of hydrogenosomal proteins, PFOs that were expressed under iron-rich conditions were found to localize at the surface. This localization was demonstrated using immunofluorescence and co-localization assays, as well as immunogold transmission electron microscopy. In addition to describing its enzyme activity, we describe a novel function in trichomonal host interaction for the PFO localized on the parasite surface. The anti-PFO50 antibody reduced the levels of T. vaginalis adherence to HeLa cell monolayers in a concentration-dependent manner. Thus, T. vaginalis PFO is an example of a surface-associated cell-binding protein that lacks enzyme activity and that is involved in cytoadherence. Additionally, PFO behaves like AP120 in parasites grown under iron-rich conditions. Therefore, these data suggest that AP120 and PFO A are encoded by the same gene, namely pfo a.

The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Malassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 °C and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.