- Volume 155, Issue 2, 2009

The physiological role and transcriptional expression of Rhizobium etli sigma factors rpoH1 and rpoH2 are reported in this work. Both rpoH1 and rpoH2 were able to complement the temperature-sensitive phenotype of an Escherichia coli rpoH mutant. The R. etli rpoH1 mutant was sensitive to heat shock, sodium hypochlorite and hydrogen peroxide, whereas the rpoH2 mutant was sensitive to NaCl and sucrose. The rpoH2 rpoH1 double mutant had increased sensitivity to heat shock and oxidative stress when compared with the rpoH1 single mutant. This suggests that in R. etli, RpoH1 is the main heat-shock sigma factor, but a more complete protective response could be achieved with the participation of RpoH2. Conversely, RpoH2 is involved in osmotic tolerance. In symbiosis with bean plants, the R. etli rpoH1 and rpoH2 rpoH1 mutants still elicited nodule formation, but exhibited reduced nitrogenase activity and bacterial viability in early and late symbiosis compared with nodules produced by rpoH2 mutants and wild-type strains. In addition, nodules formed by R. etli rpoH1 and rpoH2 rpoH1 mutants showed premature senescence. It was also determined that fixNf and fixKf expression was affected in rpoH1 mutants. Both rpoH genes were induced under microaerobic conditions and in the stationary growth phase, but not in response to heat shock. Analysis of the upstream region of rpoH1 revealed a σ 70 and a probable σ E promoter, whereas in rpoH2, one probable σ E-dependent promoter was detected. In conclusion, the two RpoH proteins operate under different stress conditions, RpoH1 in heat-shock and oxidative responses, and RpoH2 in osmotic tolerance.

Highly branched dendritic swarming of B. subtilis on synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded by phrC) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genes sfp (activation of surfactin synthases), comA, abrB and codY (global regulators), hag (flagellin), mecA and yvzB (hag-like), and swrB (motility), to the different swarming stages. Surprisingly, mutations in genes comPX, comQ, comS, rapC and oppD, which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for driving srfABCD transcription. We showed that the phrC mutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated with phrC sfp+ and phrC+ sfp (GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormal phrC type, these were predominantly populated by sfp cells, which migrated faster than the phrC cells. This and other results indicated a specific migration defect in the phrC mutant that could not be trans-complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.

Surface exposure of antigens on bacterial cells can be critical for eliciting an effective antibody response. Therefore, we investigated the cellular localization of the fimbrial F17a-G receptor-binding domain, fused to the translocator domain of the AIDA-I autotransporter. Synthesis of the fusion protein, under the control of the l-arabinose-inducible PBAD promoter, was shown to permeabilize Escherichia coli K-12 and Salmonella enterica serovar Typhimurium cells. The presence of permeable cells interfered with several methods that are typically used to determine surface exposure of proteins, such as protease treatment and whole-cell ELISA. Double immunofluorescence microscopy, using a second antibody directed against β-galactosidase, a bacterial protein expressed in the cytoplasm, allowed the simultaneous detection of antigen expression and permeability in individual cells.

Yersinia pestis cells were grown in vitro at 26 and 37 °C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37 °C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small β-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37 °C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26 °C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26 °C, but not at 37 °C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658–y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a M r range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.

Streptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibiotic mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high-phosphate brain heart infusion agar medium that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones for which we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups, including central metabolism, surface binding and sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when the gene is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the −10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.

Molecular aspects of the circadian clock in the cyanobacterium Synechococcus elongatus have been described in great detail. Three-dimensional structures have been determined for the three proteins, KaiA, KaiB and KaiC, that constitute a central oscillator of the clock. Moreover, a temperature-compensated circadian rhythm of KaiC phosphorylation can be reconstituted in vitro with the addition of KaiA, KaiB and ATP. These data suggest a relatively simple circadian system in which a single oscillator provides temporal information for all downstream processes. However, in vivo the situation is more complex, and additional components contribute to the maintenance of a normal period, the resetting of relative phases of circadian oscillations, and the control of rhythms of gene expression. We show here that two well-studied promoters in the S. elongatus genome report different circadian periods of expression under a given set of conditions in wild-type as well as mutant genetic backgrounds. Moreover, the period differs between these promoters with respect to modulation by light intensity, growth phase, and the presence or absence of a promoter-recognition subunit of RNA polymerase. These data contrast sharply with the current clock model in which a single Kai-based oscillator governs circadian period. Overall, these findings suggest that complex interactions among the circadian oscillator, perhaps other oscillators, and other cellular machinery result in a clock that is plastic and sensitive to the environment and to the physiological state of the cell.

Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8 % of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65 % of these strains also had a putative conjugative plasmid with a molecular size of 52–56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48–56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon–Wiener (J′) indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D=0.83; J′=0.88) and Aphanizomenon flos-aquae the highest ones (D=J′=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.

Amoeba-resistant bacteria (ARB), such as Legionella spp., are currently regarded as potential human pathogens that live in the natural environment, and thus their habitat is regarded as a reservoir of human pathogens. To detect ARB in human and environmental samples, co-culture with amoebae has been demonstrated to be an efficient tool. However, to date, only water samples from cooling towers and hospital water supplies have been investigated as possible reservoirs of ARB using this procedure. In the present study, we studied the ARB population of 11 diverse soil and sand sources in proximity to human environments; these sources included the university, the station, hospitals, the square, parks and public beaches in the city of Marseilles, France. As a result, a total of 33 different species of ARB were identified. The ability to grow within and/or lyse amoebae was demonstrated, for what is believed to be the first time, for several species; moreover, 20 of the isolates (61 %), including Streptococcus pneumoniae, have been described as human pathogens. However, Legionella spp. were not isolated. Four isolates are likely to be the members of new or uncharacterized genera or species, and their capability to be human pathogens needs to be determined. This preliminary work demonstrates that soils and sands in the vicinity of humans are reservoirs of human pathogenic ARB.

Colibacillosis is a common systemic disease of worldwide economic importance in poultry, caused by Escherichia coli. E. coli are normally found in the intestines of poultry, but some strains are able to cause extraintestinal disease. Plasmid pVM01 is essential for virulence in avian pathogenic Escherichia coli (APEC) strain E3 in chickens after aerosol exposure and contains the virulence-associated genes iucA, iss and tsh in distinct regions. The determination of the complete sequence of this plasmid identified many ORFs that were highly similar to genes found in the APEC O1 plasmid, as well as many hypothetical ORFs. Truncated versions of pVM01 were constructed and introduced into avirulent APEC strain E3/2.4 and the pathogenicity of these strains was assessed by aerosol exposure. The function of the region of pVM01 that contains the genes for conjugation was confirmed. Strains carrying the truncated plasmids appeared to be of intermediate virulence compared to the wild-type APEC strain E3. The conserved portion of the putative virulence region was found to contribute to the colonization of and generation of lesions in the air sacs. Both the conserved and variable portions of the putative virulence region were shown to contribute to the colonization of the trachea, but the variable portion of the putative virulence region was not required for the strain to confer a virulent phenotype. These results reveal that deletion of the conserved portion of the putative virulence region, but not the variable portion of the putative virulence region, is associated with a decrease in virulence of APEC.

We have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (CmR). Using a different antibiotic resistance marker, kanamycin (KmR), did not impair invasiveness. Despite the effect of plasmid-encoded CmR, the strain containing chromosomally encoded GFP, also carrying a CmR gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH : : gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating CmR or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying CmR , should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.

Salmonella enterica serovar Typhi causes a human-restricted systemic infection called typhoid fever. We have identified a Typhi genomic region encoding two ORFs, STY1498 and STY1499, that are expressed during infection of human macrophages and organized in an operon. STY1498 corresponds to clyA, which encodes a pore-forming cytolysin, and STY1499 encodes a 27 kDa protein, without any attributed function, which we have named TaiA (Typhi-associated invasin A). In order to evaluate the roles of these genes in Typhi pathogenesis, isogenic Typhi strains harbouring a non-polar mutation of either clyA or taiA were constructed. In macrophages, taiA was involved in increasing phagocytosis, as taiA deletion reduced bacterial uptake, whereas clyA reduced or controlled bacterial growth, as clyA deletion enhanced Typhi survival within macrophages without affecting cytotoxicity. In epithelial cells, deletion of taiA had no effect on invasion, whereas deletion of clyA enhanced the Typhi invasion rate, and reduced cytotoxicity. Overexpression of taiA in Typhi or in Escherichia coli resulted in a higher invasion rate of epithelial cells. We have demonstrated that TaiA is secreted independently of both the Salmonella pathogenicity island (SPI)-1 and the SPI-2 type three secretion systems. We have shown that this operon is regulated by the virulence-associated regulator PhoP. Moreover, our results revealed that products of this operon might be involved in promoting the use of macrophages as a sheltered reservoir for Typhi and allowing long-term persistence inside the host.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes and proliferates at the mucosal surface, inducing severe diarrhoea. Short-chain fatty acids (SCFAs) are abundant in the intestine owing to the metabolic activity of microflora, and are important for colonic health. We found that, although a high concentration of SCFAs inhibited the growth of EHEC, at low concentrations, the SCFAs markedly enhanced the expression of the virulence genes required for cell adherence and the induction of attaching and effacing (A/E) lesions. Of the SCFAs tested, butyrate markedly enhanced the expression of these virulence-associated genes, even at the low concentration of 1.25 mM, but acetate and propionate showed only a small effect at concentrations higher than 40 mM. Butyrate enhanced the promoter activity of the LEE1 operon, which encodes a global regulator of the LEE genes, Ler. This enhancement was dependent on a regulator, PchA. Butyrate sensing was completely abrogated by the deletion of lrp, the gene for the leucine-responsive regulatory protein, Lrp. Expression of a constitutively active mutant of Lrp enhanced the expression of the LEE genes in the absence of butyrate, and a response-defective Lrp derivative reduced the response to butyrate. Thus, upon entering the distal ileum, EHEC may respond to the higher butyrate level via Lrp by increasing its virulence expression, leading to efficient colonization of the target niche.

Enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 inhibits interferon (IFN)-γ-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-1 in epithelial cells. We determined the effects of probiotics on EHEC-mediated disruption of IFN-γ-stimulated STAT-1 activation in epithelial cell lines. Confluent Intestine 407, HEp-2 and Caco-2 epithelial cells were pre-treated (3 h) with either probiotics or surface-layer proteins derived from Lactobacillus helveticus R0052 prior to infection with EHEC O157 : H7 strain CL56 (m.o.i. 100 : 1, 6 h, 37 °C in 5 % CO2). Subsequently, cells were washed and stimulated with human recombinant IFN-γ (50 ng ml−1, 0.5 h, 37 °C) followed by whole-cell protein extraction and immunoblotting for tyrosine-phosphorylated STAT-1. Relative to uninfected cells, STAT-1-activation was reduced after EHEC O157 : H7 infection. Pre-incubation with the probiotic L. helveticus R0052 followed by EHEC infection abrogated pathogen-mediated disruption of IFN-γ–STAT-1 signalling. As determined using Transwell inserts, probiotic-mediated protection was independent of epithelial cell contact. In contrast, pre-incubation with boiled L. helveticus R0052, an equal concentration of viable Lactobacillus rhamnosus R0011, or surface-layer proteins (0.14 mg ml−1) did not restore STAT-1 signalling in EHEC-infected cells. The viable probiotic agent L. helveticus R0052 prevented EHEC O157 : H7-mediated subversion of epithelial cell signal transduction responses.