- Volume 150, Issue 11, 2004

An oligonucleotide probe microarray for investigation of the evolution of epidemic Staphylococcus aureus strains has been constructed. The array comprises 383 probes based on virulence-associated genes present in four key strains. Twelve strains including seven for which the complete chromosomal nucleotide sequence was available were tested on the array. Twenty-six per cent of the probes were able to differentiate between strains to give a minimum of two gene differences between pairs. A gene difference distance tree based on the array data had approximately the same topology as one prepared using concatenated MLST sequences. Differences in the topologies of these trees were found to indicate that large-scale recombination events had occurred during the evolution of the species. One such occurrence appears to have been a key event in the genesis of the EMRSA-15 clone (ST22) that currently represents the most prevalent methicillin-resistant S. aureus (MRSA) in the UK.

The gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for CamR transformants. The authors have shown that several phenotypic properties, resistance to antibiotics, dyes and detergent as well as efficiency of transformation, depend on the Dam methylation state of the DNA. Although the major role of the methyl-directed mismatch repair (MMR) system is to repair postreplicative errors, it seems that in H. influenzae its effect is more apparent in repairing DNA damage caused by oxidative compounds. In the dam mutant treated with hydrogen peroxide, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted into single- and double-strand DNA breaks. This is shown by the increased peroxide sensitivity of the dam mutant and the finding that the sensitivity can be suppressed by a mutH mutation inactivating MMR. In the dam mutant treated with nitrofurazone the resulting damage is not converted into DNA breaks but the high sensitivity is also suppressed by a mutH mutation.

In recent years, the recognition sequence of the SOS repressor LexA protein has been identified for several bacterial clades, such as the Gram-positive, green non-sulfur bacteria and Cyanobacteria phyla, or the ‘Alphaproteobacteria’, ‘Deltaproteobacteria’ and ‘Gammaproteobacteria’ classes. Nevertheless, the evolutionary relationship among these sequences and the proteins that recognize them has not been analysed. Fibrobacter succinogenes is an anaerobic Gram-negative bacterium that branched from a common bacterial ancestor immediately before the Proteobacteria phylum. Taking advantage of its intermediate position in the phylogenetic tree, and in an effort to reconstruct the evolutionary history of LexA-binding sequences, the F. succinogenes lexA gene has been isolated and its product purified to identify its DNA recognition motif through electrophoretic mobility assays and footprinting experiments. After comparing the available LexA DNA-binding sequences with the F. succinogenes one, reported here, directed mutagenesis of the F. succinogenes LexA-binding sequence and phylogenetic analyses of LexA proteins have revealed the existence of two independent evolutionary lanes for the LexA recognition motif that emerged from the Gram-positive box: one generating the Cyanobacteria and ‘Alphaproteobacteria’ LexA-binding sequences, and the other giving rise to the F. succinogenes and Myxococcus xanthus ones, in a transitional step towards the current ‘Gammaproteobacteria’ LexA box. The contrast between the results reported here and the phylogenetic data available in the literature suggests that, some time after its emergence as a distinct bacterial class, the ‘Alphaproteobacteria’ lost its vertically received lexA gene, but received later through lateral gene transfer a new lexA gene belonging to either a cyanobacterium or a bacterial species closely related to this phylum. This constitutes the first report based on experimental evidence of lateral gene transfer in the evolution of a gene governing such a complex regulatory network as the bacterial SOS system.

Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT, PAOΔmvaT, was constructed and characterized. The lysate of PAOΔmvaT failed to bind to the 150 bp probe. The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAOΔmvaT than in PAO1, but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.

Bacterial pathogens such as Staphylococcus aureus undergo major physiological changes when they infect their hosts, requiring the coordinated regulation of gene expression in response to the stresses encountered. Several environmental factors modify the expression of S. aureus virulence genes. This report shows that the expression of spa (virulence gene encoding the cell-wall-associated protein A) is down-regulated by high osmolarity (1 M NaCl, 1 M KCl or 1 M sucrose) in the wild-type strain and upregulated by novobiocin (a DNA gyrase inhibitor that relaxes DNA). A gyrB142 allele corresponding to a double mutation in the B subunit of DNA gyrase relaxed DNA and consequently induced spa expression, confirming that spa expression is regulated by DNA topology. Furthermore, in the presence of novobiocin plus 1 M NaCl, a good correlation was observed between DNA supercoiling and spa expression. The ArlS–ArlR two-component system is involved in the expression of virulence genes such as spa. Presence of an arlRS deletion decreased the effect of DNA supercoiling modulators on spa expression, suggesting that active Arl proteins are necessary for the full effect of DNA gyrase inhibitors and high osmolarity on spa expression. Indeed, evidence is provided for a relationship between the arlRS deletion and topological changes in plasmid DNA.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the α-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), β-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

The fbl gene of Staphylococcus lugdunensis encodes a protein Fbl that is 58 % identical to the clumping factor A (ClfA) of Staphylococcus aureus. The fbl gene was present in eight clinical isolates of S. lugdunensis. When Fbl was expressed on the surface of Lactococcus lactis it promoted adherence to immobilized fibrinogen and cell clumping in a fibrinogen solution. Purified recombinant Fbl region A bound to immobilized fibrinogen in a dose-dependent manner and inhibited the adherence of both Fbl-expressing and ClfA-expressing strains of L. lactis to fibrinogen. Adherence of S. lugdunensis and L. lactis Fbl+ to immobilized fibrinogen was also inhibited by rabbit anti-Fbl region A antibodies and rabbit anti-ClfA region A antibodies, as well as by human immunoglobulin with a high level of anti-ClfA antibodies. Alignment of the A domains of CflA and Fbl revealed that all of the ClfA residues implicated in binding to the γ-chain of fibrinogen are conserved in Fbl. Nevertheless Fbl had a tenfold lower affinity for fibrinogen, suggesting that sequence differences that occur elsewhere in the protein, possibly in β-strand E of domain N2, affect ligand binding.

Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor σ B, encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0·3 M NaCl) or acid (pH 4·5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes ΔsigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that σ B is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that σ B contributes to the ability of L. monocytogenes to cause foodborne infections.

Release of colicin A was studied in Escherichia coli cells that differed in expressing the colicin A lysis protein (Cal). Pools of released and unreleased colicin A were harvested throughout colicin A induction. The amount of colicin A in each pool varied with the time of induction, allowing the definition of two sequential steps in colicin A release, one of which was dependent on Cal. Each step of colicin A release was differently affected in cells containing Cal mutants in which the N-terminal cysteine residue was substituted by either proline or threonine, preventing them from being acylated and matured. These Cal mutants were only observed in degP cells, indicating that the DegP protease cleaved the unacylated precursor of Cal. Cal was found in the insoluble fraction of the pools of released and unreleased colicin A together with the hetero-oligomers of colicin A and porins (colicins Au). The biogenesis of colicins Au was studied in temperature-sensitive secA and secY strains and found to be Sec-independent, indicating that they are formed by newly synthesized colicin A binding to mature porins already incorporated in the outer membrane. Cal is a lipoprotein similar to VirB7, a constituent of the type IV secretion system. It would interact with colicins Au to constitute the colicin A export machinery.

The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpRc) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpRA162T was as active as the PrpRc protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the PprpBCDE promoter.