Characterization of a variant vlhA gene of Mycoplasma synoviae, strain WVU 1853, with a highly divergent haemagglutinin region.

BACKGROUND: In Mycoplasma synoviae, type strain WVU 1853, a single member of the haemaglutinin vlhA gene family has been previously shown to be expressed. Variants of vlhA are expressed from the same unique vlhA promoter by recruiting pseudogene sequences via site-specific recombination events, thus generating antigenic variability. Using a bacterial stock of M. synoviae WVU 1853 that had been colony purified thrice and maintained in our laboratory at low passage level, we previously identified a vlhA gene-related partial coding sequence, referred to as MS2/28.1. The E. coli-expressed product of this partial coding sequence was found to be immunodominant, suggesting that it might be expressed. RESULTS: Reverse transcription-PCR amplification (RT-PCR), using a sense primer located at the 5'-end region of the expected vlhA transcript and a reverse primer located at the 3' end of MS2/28.1 coding sequence, yielded a consistent amplification product showing that MS2/28.1 was indeed transcribed. Nucleotide sequence analysis of the RT-PCR product identified an 1815-nucleotide full-length open reading frame (ORF), immediately preceded by a nucleotide sequence identical to that previously reported for expressed vlhA genes. PCR amplifications using genomic DNA isolated from single colonies further confirmed that the full-length ORF of MS2/28.1 was located downstream of the unique vlhA promoter sequence. The deduced 604-amino acid (aa) sequence showed a perfect sequence identity to the previously reported vlhA expressed genes along the first 224 residues, then highly diverged with only 37.6% aa identity. Despite the fact that this M. synoviae clone expressed a highly divergent and considerably shorter C-terminal haemagglutinin product, it was found to be expressed at the surface of the bacterium and was able to haemagglutinate chicken erythrocytes. Importantly, the E. coli-expressed C-terminal highly divergent 60 residues of MS2/28.1 proved haemagglutination competent. CONCLUSIONS: In contrast to the previously characterized vlhA expressed variants, MS2/28.1 displayed a highly divergent sequence, while still able to haemagglutinate erythrocytes. Overall, the data provide an indication as to which extent the M. synoviae vlhA gene could vary its antigenic repertoire.

The Pta-AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis.

DegU is considered to be an orphan response regulator in Listeria monocytogenes since the gene encoding the cognate histidine kinase DegS is absent from the genome. We have previously shown that DegU is involved in motility, chemotaxis and biofilm formation and contributes to L. monocytogenes virulence. Here, we have investigated the role of DegU phosphorylation in Listeria and shown that DegS of Bacillus subtilis can phosphorylate DegU of L. monocytogenes in vitro. We introduced the B. subtilis degS gene into L. monocytogenes, and showed that this leads to highly increased expression of motility and chemotaxis genes, in a DegU-dependent fashion. We inactivated the predicted phosphorylation site of DegU by replacing aspartate residue 55 with asparagine and showed that this modified protein (DegU(D55N)) is no longer phosphorylated by DegS in vitro. We show that although the unphosphorylated form of DegU retains much of its activity in vivo, expression of motility and chemotaxis genes is lowered in the degU(D55N) mutant. We also show that the small-molecular-weight metabolite acetyl phosphate is an efficient phosphodonor for DegU in vitro and our evidence suggests this is also true in vivo. Indeed, a L. monocytogenesDeltaptaDeltaackA mutant that can no longer synthesize acetyl phosphate was found to be strongly affected in chemotaxis and motility gene expression and biofilm formation. Our findings suggest that phosphorylation by acetyl phosphate could play an important role in modulating DegU activity in vivo, linking its phosphorylation state to the metabolic status of L. monocytogenes.

The DegU orphan response regulator of Listeria monocytogenes autorepresses its own synthesis and is required for bacterial motility, virulence and biofilm formation.

The Gram-positive intracellular pathogen Listeria monocytogenes is endowed with 17 sets of genes encoding two-component systems. L. monocytogenes is closely related to the Gram-positive model bacterium Bacillus subtilis, in which we have shown previously that the DegS/DegU system plays a central role in controlling stationary phase adaptive responses, including degradative enzyme synthesis and competence. Although an orthologue of the DegU response regulator is present in L. monocytogenes, the gene encoding the cognate DegS kinase is conspicuously absent. We have inactivated the degU gene of L. monocytogenes and shown that DegU negatively regulates its own synthesis. Direct binding of L. monocytogenes DegU to its own promoter region was shown in vitro by gel mobility shift and DNase I footprinting experiments. DegU was also shown to bind upstream from the motB operon, which also encodes the GmaR anti-repressor of flagellar synthesis. In contrast to the situation in B. subtilis, DegU was shown to be essential for flagellar synthesis and bacterial motility in L. monocytogenes and is cotranscribed with the yviA gene located downstream. We also show that DegU is required for growth at high temperatures, adherence to plastic surfaces and the formation of efficient biofilms by L. monocytogenes. DegU plays a role in virulence of L. monocytogenes as well: in a murine intravenous infection model, an 11-fold increase in LD(50) was observed for the degU mutant. Taken together, our results indicate that despite the lack of the DegS kinase, DegU is fully functional as an orphan response regulator, and plays a central role in controlling several crucial adaptive responses in L. monocytogenes.