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 Grampositive 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.

SummaryDegU 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 degUD55N 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. monocytogenes Dpta DackA 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.

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.Mycoplasma gallisepticum and M. synoviae are considered important in commercial poultry industries. M. gallisepticum causes chronic respiratory disease in chickens and sinusitis in turkeys (17), and M. synoviae is commonly involved in respiratory tract infection, synovitis in chickens, and poor growth (12). The antigenic relatedness (1, 2, 7, 11) of these organisms make them difficult to identify with conventional serological tests (5, 6). Attempts to differentiate between these two major avian mycoplasmas by using molecular methods, such as PCR tests, were mainly based on the 16S rRNA gene (4, 10, 13). However, it often requires additional steps, such as restriction fragment length analysis (9) or hybridization with species-specific probes (3, 10). In addition, these analyses resulted in concomitant amplification of unrelated bacterial DNA, making this approach useless for the testing of clinical material.We show here that PCR amplification targeting speciesspecific structural genes provides an efficient tool for the simultaneous detection and differentiation of M. gallisepticum and M. synoviae.The Mycoplasma species and walled bacteria used in the present study are listed in Table 1. All of the mycoplasma strains were propagated in Frey's medium (8). Unrelated bacterial species were cultured in brain heart infusion broth (Difco Laboratories, Detroit, Mich.). Forty tracheal swabs were immediately processed for culture and used as clinical mycoplasma samples to test the applicability of the duplex PCR assay.The template DNA of M. synoviae and M. gallisepticum was prepared from 200 l of culture, to which an equal volume of nonionic detergent mix solution (0.45% Nonidet P-40, 0.45% Tween 20, and 100 g of proteinase K/ml) was added. The sample was incubated at 56°C for 1 h, boiled for 10 min, and then centrifuged at 14,000 ϫ g for 5 min. Then, 10 l of the resulting supernatant was directly used for PCR.All of the primers used in the present study are listed in Table 2. They targeted pMGA1.2 gene encoding a hemagglutinin protein (pMGA) from M. gallisepticum (14) and M. synoviae2/12 DNA fragment (accession no. M. synoviaeU66314) (3) of the M. synoviae hemagglutinin vlhA gene (15, 16). To confirm the specificity of the amplification reaction, a nested PCR was performed by using inner primers (Tab.2). Additional avian Mycoplasma spp. and other common bacteria (Table 1) were tested to determine the specificity of the duplex PCR assay.The amplification reaction was performed in a total volume of 50 l containing 5 l of 10ϫ PCR buffer (Amersham Biosciences), 250 M concentrations of each deoxynucleoside triphosphate (Pharmacia), 50 pmol of each external or internal primers, 2.25 mM MgCl 2 , 2 U of Taq D...

BackgroundIn 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.ResultsReverse 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.ConclusionsIn contrast to the previously characterized vlhA expressedvariants, 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.