- Volume 151, Issue 12, 2005

Salmonella gallinarum (SG) is a non-motile host-adapted salmonella that causes fowl typhoid, a severe systemic disease responsible for significant economic losses to the poultry industry worldwide. This study describes the application of a PCR-based signature-tagged mutagenesis system to identify in vivo-essential genes of SG. Ninety-six pools representing 1152 SG mutants were screened in a natural-host chicken infection model. Twenty presumptive attenuated mutants were identified and examined further. The identity of the disrupted gene in each mutant was determined by cloning of the DNA sequences adjacent to the transposon, followed by sequencing and comparison with the bacterial genome database. In vitro and in vivo competition indices were determined for each identified mutant and a total of 18 unique, attenuating gene disruptions were identified. These mutations represented six broad genomic classes: Salmonella pathogenicity island-1 (SPI-1), SPI-2, SPI-10, SPI-13, SPI-14 and non-SPI-encoded virulence genes. SPI-13 and SPI-14 are newly identified and designated in this study. Most of the genes identified in this study were not previously believed or known to play a role in the pathogenesis of SG infection in chickens. Each STM identified mutant showed competitiveness and/or virulence defects, confirmed by in vitro and in vivo assays, and challenge tests. This study should contribute to a better understanding of the pathogenic mechanisms involved in progression of disease caused by SG, and identification of novel live vaccine candidates and new potential antibiotic targets.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis patients. P. aeruginosa colonizes the lungs by forming biofilm microcolonies throughout the lung. Quorum sensing (QS) renders the biofilm bacteria highly tolerant to otherwise lethal doses of antibiotics, and protects against the bactericidal activity of polymorphonuclear leukocytes (PMNs). It has been previously demonstrated that QS is inhibited by garlic extract. In this study, the synergistic effects of garlic and tobramycin, and PMNs activities have been evaluated. P. aeruginosa was grown in vitro in continuous-culture once-through flow chambers with and without garlic extract. The garlic-treated biofilms were susceptible to both tobramycin and PMN grazing. Furthermore, the PMNs showed an increase in respiratory burst activation, when incubated with the garlic-treated biofilm. Garlic extract was administered as treatment for a mouse pulmonary infection model. Mice were treated with garlic extract or placebo for 7 days, with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology, mortality, histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation, and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin, respiratory burst and phagocytosis by PMNs, as well as leading to an improved outcome of pulmonary infections.

In enteric bacteria, the contribution of endogenous energy sources to survival both inside and outside the host is poorly understood. The contribution of glycogen production to the virulence, colonization and environmental survival of different Salmonella enterica serotypes was assessed. Of 19 serotypes (339 strains) tested for glycogen production, 17 (256 strains) were positive. The avian-specific serovars S. Gallinarum (62 strains) and S. Pullorum (21 strains) did not produce glycogen. The sequence of glgC in three S. Gallinarum strains tested revealed an identical deletion of 11 consecutive bases, which was not present in S. Pullorum, and a CCC insertion after position 597. Transduction of S. Gallinarum and S. Pullorum to a glycogen-positive phenotype did not change the ability to colonize the intestine or affect virulence in the chicken. Mortality rates in chickens following oral infection with a S. Typhimurium glycogen mutant (glgC : : km) were not significantly reduced, although colonization of the intestine was reduced over the first 4 weeks of the trial. Growth and yield of the glgC : : km mutant were comparable to the parent. The glgC mutant survived less well in faeces and in water at 4 °C when the strain was grown in LB broth containing 0·5 % glucose, and in saline it died off more rapidly after 7 days. The data suggest that glycogen has a complex but comparatively minor role in virulence and colonization, but a more significant role in survival.

Helicobacter hepaticus is a pathogen of rodents, which causes diverse enteric and hepatic inflammatory diseases and malignancies. The urease enzyme is an important colonization factor of gastric Helicobacter species like Helicobacter pylori, but little is known about the role and regulation of urease in enterohepatic Helicobacter species. Here it is reported that urease activity of H. hepaticus does not contribute to acid resistance, and that it is nickel-responsive at the post-translational level. H. hepaticus strain ATCC 51449 did not grow or survive at pH 3·0, and supplementation with urea or NiCl2 did not abrogate this acid sensitivity. Furthermore, urease enzyme activity of H. hepaticus was acid-independent, which contrasts with the acid-induced urease system of H. pylori. Nickel supplementation of Brucella medium resulted in a tenfold increase in urease activity in both H. hepaticus and H. pylori, but the maximum level of urease activity in H. hepaticus was still three- to fivefold lower when compared to H. pylori in the same conditions. The increase in urease activity of H. hepaticus was not associated with elevation of urease mRNA or protein levels. Inhibition of protein synthesis by chloramphenicol did not affect nickel-responsive induction of urease activity in H. hepaticus, and confirmed that nickel induction occurs at the post-translational level, probably by activation of preformed apo-enzyme. In conclusion, both the role of the urease enzyme and the regulation of urease activity differ between the enterohepatic pathogen H. hepaticus and the gastric pathogen H. pylori.

PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to H2O2 challenge (P<0·05). Expression of eight genes with potential roles in the oxidative-stress response was determined in the wild-type and perR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.

CodY, a pleiotropic transcriptional regulator conserved in low G+C species of Gram-positive bacteria, was previously described to be the central regulator of proteolysis in Lactococcus lactis. In this study, over 100 potential CodY targets were identified by DNA-microarray analysis. Complementary transcriptional analysis experiments were carried out to validate the newly defined CodY regulon. Moreover, the direct role of CodY in the regulation of several target genes was demonstrated by gel retardation experiments. Interestingly, 45 % of CodY-dependent genes encode enzymes involved in amino acid biosynthesis pathways, while most of the other genes are involved in functions related to nitrogen supply. CodY of L. lactis represents the first example of a regulator in Gram-positive bacteria that globally controls amino acid biosynthesis. This global control leads to growth inhibition in several amino-acid-limited media containing an excess of isoleucine. A conserved 15 nt palindromic sequence (AATTTTCNGAAAATT), the so-called CodY-box, located in the vicinity of the −35 box of target promoter regions was identified. Relevance of the CodY-box as an operator for CodY was demonstrated by base substitutions in gel retardation experiments. This motif is also frequently found in the promoter region of genes potentially regulated by CodY in other Gram-positive bacteria.

Experiments were done to define the nature of the xylan-derived induction signal for xylanase activity, and evaluate which xylanase genes among the three known ones (xynA, xynB and xynC) are induced by the presence of xylan in Prevotella bryantii B14. During the later stages of exponential growth on glucose, addition of 0·05 % water-soluble xylan (WS-X) stimulated xylanase formation within 30 min. Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, arabinose and glucuronic acid all failed to induce the xylanase activity. An acid-ethanol-soluble fraction of WS-X (approximate degree of polymerization 30) enhanced the activity significantly, whereas the acid-ethanol-insoluble fraction had no effect, unless first digested by the cloned P. bryantii XynC xylanase. These results indicate that medium- to large-sized xylo-oligosaccharides are responsible for induction. The transcription of all three known xylanase genes from P. bryantii was upregulated coordinately by addition of WS-X. There have been relatively few investigations into the regulation of xylanase activity in bacteria, and it appears to be unique that medium- to large-sized xylo-oligosaccharides are responsible for induction.

To investigate the metabolic biochemistry of iron-oxidizing extreme acidophiles, a proteomic analysis of chemomixotrophic and chemo-organotrophic growth, as well as protein expression in the absence of organic carbon, was carried out in Ferroplasma species. Electron transport chain inhibitor studies, spectrophotometric analysis and proteomic results suggest that oxidation of ferrous iron may be mediated by the blue copper-haem protein sulfocyanin and the derived electron passes to a cbb 3 terminal electron acceptor. Despite previous suggestions of a putative carbon dioxide fixation pathway, no up-regulation of proteins typically associated with carbon dioxide fixation was evident during incubation in the absence of organic carbon. Although a lack of known carbon dioxide fixation proteins does not constitute proof, the results suggest that these strains are not autotrophic. Proteins putatively involved in central metabolic pathways, a probable sugar permease and flavoproteins were up-regulated during chemo-organotrophic growth in comparison to the protein complement during chemomixotrophic growth. These results reflect a higher energy demand to be derived from the organic carbon during chemo-organotrophic growth. Proteins with suggested function as central metabolic enzymes were expressed at higher levels during chemomixotrophic growth by Ferroplasma acidiphilum YT compared to ‘Ferroplasma acidarmanus’ Fer1. This study addresses some of the biochemical and bioenergetic questions fundamental for survival of these organisms in extreme acid-leaching environments.