Mutations in GTP Binding Protein Obg of Mycoplasma synoviae Vaccine Strain MS-H: Implications in Temperature-Sensitivity Phenotype

Abstract

Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts +) vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts –) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts – MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg) as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition

“…Despite the close structural similarity of these various PG II -helical structures, their function in proteins other than S16 LTFs remains unclear. The 6-PG II domain of Obg putatively plays a role in protein interaction for GTP/GDP binding, with current studies revealing that mutation of individual glycine residues can produce temperature-sensitive B. subtilis phenotypes (Buglino et al, 2002;Shahid et al, 2013). Similarly unrelated, the putative function of sfAfp is inhibition of ice growth by binding water molecules on the surface of ice crystals (Lin et al, 2007;Pentelute et al, 2008).…”

Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates

“… a and rare 94036-2-1a genotype ts + MS-H re-isolates [ 7 , 13 ]. b and MS-H 4 like genotype ts - MS-H re-isolates [ 7 , 13 ]. …”

“…Shahid and co-workers [ 7 , 13 ] discovered two specific SNPs on the obg gene and developed HRM-curve analysis based assays to differentiate the ts + MS-H vaccine strain, ts - MS-H re-isolates and wild-type M . synoviae strains.…”

“…Shahid and co-workers [ 13 ] discovered two single nucleotide polymorphisms (SNP) on the obg gene sequence, namely the A-G substitution at nt367 and C-T substitution at nt629 which are able to differentiate the ts + MS-H vaccine strain, its rare non-temperature sensitive (ts - ) MS-H re-isolates and wild-type M . synoviae isolates.…”

High-Resolution Melting-Curve Analysis of obg Gene to Differentiate the Temperature-Sensitive Mycoplasma synoviae Vaccine Strain MS-H from Non-Temperature-Sensitive Strains

“…A selection of MS-H strain specific SNPs was confirmed by Sanger sequencing. SNPs in obg associated with change in temperature sensitivity [18] were primarily targeted to enable differentiation of MS-H strain from ts – MS-H revertants as well as from ts – field strains. Initially a 335-bp obg-F1R3 PCR-HRM was developed but melt curves generated did not provide clear differentiation of MS-H from other strains/isolates.…”