Downstream from this region, a very high divergence was observed with 37,6%, 37,8%, and 38,7% aa identity, respectively. Likewise, in this region, MS2/28.1 shared only 39,8% and 38,8% identity, respectively, with the two vlhA1 expressed variants, vlhA4 and vlhA5, previously identified in M. synoviae strain WVU 1853 (Figure 2). Overall, the haemagglutinin region of MS2/28.1
was found to be considerably reduced in size (148 aa less than in vlhA1) and displayed high level of sequence divergence in comparison to the previously reported vlhA expressed genes, namely vlhA1, vlhA4 (GenBank accession no. AF181033), Selleckchem BIIB057 and vlhA5 (GenBank accession no. AF181034) . Figure 2 Comparison of the amino acid sequence predicted from M. synoviae MS2/28.1 gene with vlhAs 1 to 5. Alignment of the completed full-length MS2/28.1 deduced amino acid sequence with vlhAs 1 to 5 (GenBank accession numbers AF035624, AF085697, AF085698, AF181033, and AF181034, respectively). Identical aa regions are selleck kinase inhibitor shaded in black while similar aa residues are shaded in grey. Demonstration that MS2/28.1 sequence is preceded by the vlhA1 promoter To confirm that in our bacterial
stock MS2/28.1 was located downstream of the unique vlhA promoter sequence, we performed PCR amplifications on single colonies using oligonucleotide primers placed in the vlhA promoter sequence with either vlhA1- or MS2/28.1-specific reverse primers. As shown in Figure 3, amplicons migrating at the expected mobility were obtained solely with MS2/28.1-specific reverse primers. Sequence analysis selleck chemical further confirmed that the upstream sequence is identical to that of the vlhA1 promoter, a result consistent with the finding that MS2/28.1 is transcriptionally active and that, in its transcript, the region preceding its ATG initiation codon was identical to that reported for vlhA1. Figure 3 Confirmation
that MS2/28.1 is preceded by the unique vlhA1 promoter sequence. Primer EXpro, which anneals to the vlhA1 promoter, was combined with either vlhA1R (lanes b) or with ORF5.1R (lanes c). No amplification from genomic DNA extracted from the four colonies was obtained with the vlhA1-specific reverse primer (lanes b). Expected amplicon was obtained with primers EXpro/ORF5.1R (lanes c). PCR amplification of the full length MS2/28.1 was Vorinostat in vivo obtained with the primers pair EXproint and 2/28.1Rev (lanes d). As negative control, PCR was performed with no genomic M. synoviae DNA (lane a). Lane M; DNA size marker (1 kb). MS2/28.1 encoded full-length product is post-translationally cleaved with its C-terminal portion exposed at the bacterium’s surface To characterize MS2/28.1 encoded product and to examine whether it was processed similarly as the vlhA1 product, we generated antisera towards four bacterially expressed distinct regions of the coding sequence. The reactivity of these antisera is shown in Figure 4.