Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics

Abstract

The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.