Conventional and normalized melt curve analysis of mixed strains using <i>pvpA</i>-PCR.

<p>Mixture of ts-11 and F strain (a and b), ts-11 and 6/85 strain (c and d), ts-11 and S6 strain (e and f), ts-11 and 86026 field isolate (g and h). Specimens A, B, C, D and E each contains 1 ng of ts-11 DNA and 1-10^-4 ng of contaminant MG DNA and specimens F, G, H and I each contains 1-10^-4 ng of ts-11 DNA and 1 ng of contaminant MG DNA, respectively.</p

<i>Mycoplasma gallisepticum</i> cultures used in this study and their origin.

<p><i>Mycoplasma gallisepticum</i> cultures used in this study and their origin.</p

Analysis of Obg expression in <i>M</i>. <i>synoviae</i> strains using SDS-PAGE and immunoblotting.

<p>(A) SDS-PAGE (7.5%) of whole cell proteins from <i>M</i>. <i>synoviae</i> MS-H transformed with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). Location of ~ 50 kDa protein in pKS-VOTL transformants is indicated with an arrow. (B) Immunostaining of untransformed 86079/7NS, MS-H, and transformed MS-H with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) using polyclonal rabbit serum against <i>M</i>. <i>synoviae</i> Obg_3 peptide. Location of Obg-MBP fusion protein and overexpressed Obg is indicated with arrows on the right. (C) SDS-PAGE (8.75%) of whole cell proteins from <i>M</i>. <i>synoviae</i> 86079/7NS transformed with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-T8 and 7NS-T4) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). (D) Immunostaining of untransformed 86079/7NS and transformed 86079/7NS with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-8 and 7NS-T4) using polyclonal rabbit serum against <i>M</i>. <i>synoviae</i> Obg_1 peptide. Location of overexpressed Obg is indicated with an arrow on the right. (E) For comparison of Obg expression in MS-H and 86079/7NS, approximately equal amounts (88 μg/lane) of whole cell proteins from MS-H, MS-H-C28, MS-H-T75, 86079/7NS, 7NS-C38 and 7NS-T30 were separated by SDS-PAGE (8.75%) and stained with Coomassie Brilliant Blue. (F) Immunostaining at two different concentrations of whole cell proteins (i.e. 1/3 and 2/3 of that loaded onto SDS-PAGE as shown in panel E) of each strain/transformant using polyclonal rabbit serum against <i>M</i>. <i>synoviae</i> Obg_1 peptide. MW, protein marker (Precision Plus Protein, Dual Color, Bio-Rad).</p

Temperature-sensitive phenotyping, growth curve analysis and loss of viability study of MS-H and 86079/7NS transformants.

<p>(A) Using <i>vlhA</i>-QPCR as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194528#pone.0194528.ref029" target="_blank">29</a>], quantification of <i>vlhA</i> gene copy number was performed at 33 and 39.5°C for untransformed MS-H (<i>ts</i>⁺ control) and 86079/7NS (<i>ts</i>^<i>−</i>control), transformed MS-H and 86079/7NS with pMAS-LoriC (clone MS-H-C28 and 7NS-C38, respectively) and pKS-VOTL (clone MS-H-T75 and 7NS-T30, respectively). Growth was lower at non-permissive temperature (39.5°C) than permissive temperature (33°C) for all strains/transformants except 86079/7NS. Data is expressed as mean ± SD of <i>vlhA</i> gene copy numbers (n = 3). The 33/39.5°C <i>vlhA</i> gene copy number ratios, as established by Shahid et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194528#pone.0194528.ref029" target="_blank">29</a>], indicated MS-H, MS-H-C28, MS-H-T75, 7NS-C38 and 7NS-T30 as <i>ts</i>⁺ and 86079/7NS as <i>ts</i>^<i>–</i>. (B) Growth curve analysis and loss of viability study of MS-H and 86079/7NS transformants. (a and b) For growth rate comparison, cultures of MS-H-C28 (Δ), MS-H-T75 (ϒ), 7NS-C38 (◊) and 7NS-T30 (○) transformants were grown at 33°C for 192 h. Aliquots were collected at 24 h intervals, and then titrated by incubation at 33°C for two weeks. (c and d) For loss of viability study, cultures of MS-H and 86079/7NS transformants, as indicated in panel a and b, were grown at 39.5°C for 72 h, aliquots collected at 12 h intervals, and then titrated by incubation at 33°C for two weeks. All cultures were grown in MB containing tetracycline (3 μg mL^–1). Bars indicate standard deviations of log₁₀-transformed CCU mL^–1 values from three independent cultures and "*" indicates significant differences (p < 0.05) using Student's <i>t</i>-test.</p

Southern blot hybridisation to localise pKS-VOTL plasmid in MS-H transformants.

<p>(A) Schematic presentation for the integration event of pKS-VOTL plasmid into genomic DNA of MS-H. A single putative homologous recombination event between the <i>oriC</i> copy carried by plasmid and chromosomal <i>oriC</i> region is represented by crossed lines. P indicates the promoter region of <i>vlhA</i> gene. (B, C, and D) <i>Bgl</i>II digested DNA of pKS-VOTL, untransformed MS-H and pKS-VOTL MS-H transformants MS-H-T75, MS-H-T78, MS-H-T90 (at passage 4^th and 8^th) was hybridised with DIG labeled <i>tetM</i> (B), <i>oriC</i> (C) and <i>vlhA</i>-<i>obg</i> probes (D).</p

<i>M</i>. <i>synoviae</i> strains and plasmids used in this study.

<p><i>M</i>. <i>synoviae</i> strains and plasmids used in this study.</p

Northern analysis of total RNA (~ 50 μg/lane) from <i>M</i>. <i>synoviae</i> 86079/7NS, MS-H, MS-H-C28, MS-H-T75, MS-H-T78, MS-H-T90, MS-H5 and 93198/1-24b.

<p>(A) Agarose gel of total RNA from untransformed (86079/7NS, MS-H, MS-H5 and 93198/1-24b) and transformed (pMAS-LoriC transformant MS-H-C28 and pKS-VOTL transformants MS-H-T75, MS-H-T78 and MS-H-T90) <i>M</i>. <i>synoviae</i> MS-H stained with GelRed. (B, C, and D) Northern blot of the above gel hybridised with DIG-labeled <i>vlhA</i>-<i>obg</i> probe (spanning the joining site of <i>vlhA</i> promoter with <i>obg</i> CDS), <i>obg</i> specific probe and <i>vlhA</i> coding sequence specific probe, respectively. Arrowheads indicate the location of the specific bands identified by probes described above. The location of bands for the DIG-labeled RNA molecular weight marker RNA I (Roche) is indicated on the left side of all northern blots. ImageJ (1.48v; NIH, USA) was used to quantify intensity of <i>obg</i> transcripts.</p

Melting points and genotype confidence percentages (C%) generated in obg-F3R3 HRM from different <i>M. synoviae</i> strains/isolates.

a<p>Melting points and C% values are from one HRM experiment for each strain using each sample DNA tested in triplicate.</p>b<p>These strains are rarely occurring <i>ts</i>^– MS-H reisolates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092215#pone.0092215-Shahid2" target="_blank">[18]</a> and can only be discriminated from MS-H using obg-F3R3 HRM.</p

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

<div><p>Temperature-sensitive (<i>ts</i>⁺) vaccine strain MS-H is the only live attenuated <i>M. synoviae</i> vaccine commercially available for use in poultry. With increasing use of this vaccine to control <i>M. synoviae</i> infections, differentiation of MS-H from field <i>M. synoviae</i> strains and from rarely occurring non-temperature-sensitive (<i>ts</i>^–) MS-H revertants has become important, especially in countries where local strains are indistinguishable from MS-H by sequence analysis of variable lipoprotein haemagglutinin (<i>vlhA</i>) gene. Single nucleotide polymorphisms (SNPs) in the <i>obg</i> of MS-H have been found to associate with <i>ts</i> phenotype. In this study, four PCRs followed by high-resolution melting (HRM)-curve analysis of the regions encompassing these SNPs were developed and evaluated for their potential to differentiate MS-H from 36 <i>M. synoviae</i> strains/isolates. The nested-obg PCR-HRM differentiated <i>ts</i>⁺ MS-H vaccine not only from field <i>M. synoviae</i> strains/isolates but also from <i>ts</i>^– MS-H revertants. The mean genotype confidence percentages, 96.9±3.4 and 8.8±11.2 for <i>ts</i>⁺ and <i>ts</i>^– strains, respectively, demonstrated high differentiating power of the nested-obg PCR-HRM. Using a combination of nested-obg and obg-F3R3 PCR-HRM, 97% of the isolates/strains were typed according to their <i>ts</i> phenotype with all MS-H isolates typed as MS-H. A set of respiratory swabs from MS-H vaccinated specific pathogen free chickens and <i>M. synoviae</i> infected commercial chicken flocks were tested using obg PCR-HRM system and results were consistent with those of <i>vlhA</i> genotyping. The PCR-HRM system developed in this study, proved to be a rapid and reliable tool using pure <i>M. synoviae</i> cultures as well as direct clinical specimens.</p></div

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

<div><p><i>Mycoplasma synoviae</i> strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (<i>ts</i>⁺) vaccine used for control of <i>M. synoviae</i> 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 (<i>ts</i>^–) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in <i>ts</i>^– MS-H reisolates. Nucleotide change from G to A at position 369 of the <i>obg</i> gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete <i>obg</i> 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 <i>obg</i> gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. <i>In silico</i> analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions <i>in vivo</i> 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.</p></div