Characterization of a chromosomal region of Mycoplasma sp. bovine group 7 strain PG50 encoding a glycerol transport locus (gtsABC)

Steven P. Djordjevic1, Edy M. Vilei2 and Joachim Frey2

1 NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Private Mail Bag 8, Camden, NSW, Australia 2570
2 Institute for Veterinary Bacteriology, University of Berne, CH-3012 Berne, Switzerland

Correspondence
Steven P. Djordjevic
steve.djordjevic{at}agric.nsw.gov.au


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycoplasma species bovine group 7, represented by the type strain PG50, is one of six members of the Mycoplasma mycoides cluster and has been implicated in sporadic and outbreak cases of polyarthritis and mastitis in Australian dairy cattle. This study describes cloning and sequencing a 7·9 kb region of the PG50 chromosome and identification of genes involved in glycerol transport (gtsA, gtsB and gtsC) that are followed by a putative lipoprotein gene lppB and a genomic locus containing two ORFs encoding putative membrane proteins. Long range PCR using primers spanning gtsABC and downstream flanking genes, and Southern hybridization analyses using a suite of probes derived from M. mycoides subsp. mycoides small colony type (SC) strain Afadé for gtsA, gtsB and gtsC, lppB and the two downstream genes confirmed that these genes were conserved among Mycoplasma sp. bovine group 7 isolates and mycoplasmas belonging to the M. mycoides subcluster [M. mycoides subsp. mycoides SC, M. mycoides subsp. mycoides large colony type (LC) and M. mycoides subsp. capri] but were absent in mycoplasmas belonging to the Mycoplasma capricolum subcluster (M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae). M. capricolum subsp. capricolum type strain California kid did not hybridize with the probe for gtsA and gave only weak or no hybridization signals with probes derived from the loci downstream of gtsABC, suggesting that this region has diverged in mycoplasmas belonging to subspecies of M. capricolum. It is shown that PG50, after the addition of a physiological concentration of glycerol to the growth medium, generates H2O2 at levels comparable with strain Afadé, implying that the glycerol transport system is functional in Mycoplasma sp. bovine group 7. This suggests that in PG50, as in M. mycoides subsp. mycoides SC, glycerol uptake is followed by phosphorylation to glycerol 3-phosphate and then conversion to dihydroxyacetone phosphate, catalysed by L-{alpha}-glycerophosphate oxidase, resulting in the production of H2O2. The ability of Mycoplasma sp. bovine group 7 to generate significant amounts of hydrogen peroxide may be important in pathogenesis.

Abbreviations: LC, large colony type; SC, small colony type

The GenBank/EMBL accession number for the 7902 bp region of the PG50 chromosome encoding GtsA, GtsB, GtsC, LppB, ORF5 and ORF6 is AJ419906.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycoplasma sp. bovine group 7 has been associated with polyarthritis (Simmons & Johnston, 1963; Shiel et al., 1982), mastitis (Connole et al., 1967) and pneumonia (Alexander et al., 1985) in Australian cattle. Recently, Mycoplasma sp. bovine group 7 was isolated during a severe outbreak of polyarthritis, mastitis and abortion affecting about 120 cattle in a large dairy operation in New South Wales, Australia (Hum et al., 2000). Twenty-four isolates of Mycoplasma sp. bovine group 7 were recovered from multiple joint fluids, pericardial fluid and a lymph node of affected calves, from individual and bulk milk samples, and from the internal organs (liver, spleen, lung and stomach contents) of two aborted foetuses (Hum et al., 2000). Fingerprinting studies showed that all 24 isolates were indistinguishable from one another but could be readily differentiated from a collection of epidemiologically unrelated Australian and international isolates of this species (Djordjevic et al., 2001). These studies suggest that Mycoplasma sp. bovine group 7 can be an invasive pathogen capable of causing systemic infection with significant economic losses to dairy operations. However, its virulence mechanisms are unknown, and its phylogenetic and taxonomic position is unclear.

Mycoplasma sp. bovine group 7, Mycoplasma mycoides subsp. mycoides small colony type (SC), M. mycoides subsp. mycoides large colony type (LC), M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae represent the six recognized members of the M. mycoides cluster, and species identification within this cluster has been problematic. Electrophoretic, immunoblotting and DNA hybridization studies confirm the close phylogenetic relationship between members of the cluster (Bonnet et al., 1993; Christiansen & Ernø, 1990; Costas et al., 1987; Olsson et al., 1990; Rodwell, 1982; Rodwell & Rodwell, 1978) and serological cross-reactions between Mycoplasma sp. bovine group 7 and M. capricolum subsp. capripneumoniae (Guerin et al., 1993; Kibe et al., 1985; Thiaucourt et al., 1994), and M. capricolum subsp. capricolum (Bölske et al., 1988) have been reported. Genotyping studies using the insertion elements IS1296 and IS1634, and serological studies using antisera raised against a major surface lipoprotein (LppB) revealed the presence of two distinct clonal lineages within M. mycoides subsp. mycoides SC; a highly virulent African/Australian cluster and the moderately pathogenic cluster of strains from the re-emerging European outbreaks of contagious bovine pleuropneumonia (Cheng et al., 1995; Frey et al., 1995; Vilei et al., 1999, 2000). Detailed genetic studies comparing isolates within each of these clusters has provided evidence that the less pathogenic European isolates arose by deletion of an 8·84 kb chromosomal region, present only among isolates belonging to the African/Australian cluster. This region carries a copy of IS1634, encodes proteins for glycerol transport (GtsA, GtsB and GtsC) and a major surface lipoprotein (LppB), and also contains two ORFs encoding a putative surface lipoprotein (ORF6) and a proline-rich membrane protein (ORF5) (Vilei et al., 2000). African/Australian strains of M. mycoides subsp. mycoides SC are capable of importing and phosphorylating glycerol (Vilei & Frey, 2001). Glycerol 3-phosphate is presumably oxidized to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate with the concomitant production of H2O2, a potent haemolysing metabolite. Deletion of gtsC and part of gtsB (two of three genes in the putative operon involved in glycerol transport) in the moderately virulent European isolate L2 is correlated with a reduced ability to produce H2O2 and haemolyse sheep erythrocytes compared to the highly virulent Afadé strain, representative of the African/Australian cluster (Houshaymi et al., 1997; Rice et al., 2000; Vilei & Frey, 2001). Collectively, genes located within this region are important for virulence and have also been targets in studies to differentiate between members of the M. mycoides cluster.

It has been reported that isolates of Mycoplasma sp. bovine group 7 representative of the clonal cluster recovered from multiple animals and different tissue sites during an outbreak lysed sheep red blood cells when cultured on blood agar medium (Hum et al., 2000). The aim of this study was to identify the putative genes for glycerol transport (gtsABC), lppB, and the flanking segment containing ORF5 and ORF6 on the chromosome of PG50, and compare them with homologues in M. mycoides subsp. mycoides SC. The capacity of PG50 to transport glycerol and produce H2O2 when cultured in the presence of glycerol was also investigated.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, growth conditions and DNA extraction.
Mycoplasma strains used in this study are listed in Table 1. Mycoplasmas were cultured using standard procedures (Bannerman & Nicolet, 1971). Mycoplasma sp. bovine group 7 strains were cultured as described previously (Djordjevic et al., 2001) and were harvested when the indicator in the medium changed from a red to yellow (density of 108–109 cells ml-1). DNA from isolates of Mycoplasma sp. bovine group 7 was extracted as described previously (Djordjevic et al., 2001). DNA from the Afadé strain of M. mycoides subsp. mycoides SC and from M. capricolum subsp. capricolum strain California kid was isolated as described previously (Cheng et al., 1995).


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Table 1. Mycoplasma strains tested by long range PCR using primers 7500bp1L and 3480bp

 
Long range PCR.
This was based on the Expand Long Template PCR System (Roche Diagnostics) and used primers 7500bp1L (5'-GTTGGTTTTGGATCAACTGG-3') and 3480bp-R (5'-TCTGATTTAGTTGGATTGAGTTCA-3'). Reactions of 50 µl containing 50 mM Tris/HCl (pH 9·2), 350 µM each dNTP, 15 pmol primers 7500bp1L and 3480bp-R, 0·75 µl Taq/Pwo polymerases (Roche Diagnostics), 10–50 ng template DNA, 1·75 mM MgCl2 and sterile milli Q water were added to a 200 µl PCR tube. PCR amplification was carried out in a Perkin Elmer 9000 thermal cycler. After an initial denaturation step of 2 min at 94 °C, the samples were subjected to 30 cycles of denaturation (94 °C, 30 s), annealing (48 °C, 30 s) and extension (68 °C, 6 min). Amplification products were maintained at 8 °C overnight and stored at -20 °C.

Cloning and DNA sequence analyses.
A 5·9 kb fragment amplified from template DNA of Mycoplasma sp. bovine group 7 type strain PG50 with primers 7500bp1L and 3480bp-R was ligated into the pGEM-T vector (Promega). Ligation products were used to transform competent Escherichia coli XL-1 Blue cells and transformants were selected on LB agar containing ampicillin (100 µg ml-1), X-Gal and IPTG (30 mg l-1). Recombinant plasmids recovered from white colonies were screened for the presence of a 5·9 kb insert by digestion with NcoI and NotI. All manipulations were performed according to standard procedures (Sambrook et al., 1989). One construct, pSDW12, was isolated from a bacterial pellet using a Qiagen maxiprep column and used as template for sequencing. Sequencing was performed with a DNA sequenator AB310 and the Taq DyeDeoxy Terminator Cycle Kit (both from Applied Biosystems) using primers complementary to the SP6 and T7 promoters of the vector and primers derived from sequenced segments. Sequence coverage was extended to 7902 bp by primer walking on genomic DNA of strain PG50. The DNA and deduced amino acid sequences were analysed with the ScanProsite software (http://ca.expasy.org/tools/scanprosite/), SignalP (http://www.cbs.dtu.dk/services/SignalP-2·0/) and the TMpred software (http://www.isrec.isb-sib.ch/ftp-server/tmpred/www/TMPRED_form.html). Sequence comparisons with sequences in the GenBank and EMBL databases were made using the BLAST programs BLASTN, BLASTX and BLASTP (Altschul et al., 1990).

Southern hybridization.
Southern blots were performed essentially as described previously (Vilei & Frey, 2001). Genomic DNA was digested with HindIII, separated electrophoretically on a 0·7 % agarose gel and transferred to positively charged nylon membrane (Roche Diagnostics). The specific DNA probes were prepared by PCR amplification of genomic DNA, adding 0·5–1 µl digoxigenin-11-dUTP (Roche Diagnostics) to the reaction mix.

Immunoblotting.
Bovine serum collected from cattle experimentally infected with M. mycoides subsp. mycoides SC was prepared as described previously (Abdo et al., 1998). Cell lysates of type strains representative each of the six species in the M. mycoides cluster and Mycoplasma putrefaciens type strain KS1 were separated by SDS-PAGE and blotted onto PVDF membrane. The conditions and reagents used to detect the immunoreactive proteins on the membrane have been previously described (Abdo et al., 1998).

Production of hydrogen peroxide.
Measurements of time-dependent H2O2 production in Mycoplasma sp. bovine group 7 (PG50) and M. mycoides subsp. mycoides SC (Afadé) were carried out using the Merckoquant peroxidase test (Merck KgaA) as described previously (Vilei & Frey, 2001). Briefly, 30 ml mycoplasma cultures of PG50 and Afadé, grown to a density of approximately 108 cells ml-1, were centrifuged at 12 000 g for 10 min and the cell pellets were washed and resuspended in 10 ml isotonic HEPES buffer containing 7 mM MgCl2. For each strain, two cell suspensions each of 1·0 ml were adjusted to OD550 1·0. After starvation for 1 h at 37 °C, the isotonic buffer of one sample was adjusted to 100 µM glycerol while the buffer of the second sample was deprived of glycerol. At time intervals ranging from 5 s to 20 min the test strips were dipped for 1 s and subsequently read as suggested by the manufacturer.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence comparison between glycerol uptake loci of PG50 and the Afadé strain of M. mycoides subsp. mycoides SC
Primers 7500bp1L and 3480bp-R, which amplify a 9·3 kb fragment of the M. mycoides subsp. mycoides SC chromosome containing genes for gtsB, gtsC, lppB, IS1634 and ORF6 (Vilei et al., 2000; Table 1) also amplified a 5·9 kb fragment of the chromosome of Mycoplasma sp. bovine group 7 type strain PG50. DNA sequence analysis confirmed that gtsB, gtsC, lppB and ORF6 were encompassed within this fragment. Primer walking was used to extend the sequence coverage in both directions to 7·9 kb (Fig. 1), to include the sequences for gtsA and ORF5.



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Fig. 1. Maps of chromosomal regions of M. mycoides subsp. mycoides SC (Afadé) and Mycoplasma sp. bovine group 7 (PG50) encoding the glycerol transport operon (gtsA, gtsB and gtsC), lipoprotein B (lppB), ORF5, ORF6, IS1296 and IS1634. Both IS elements and nt 7834–8998 encoding 299 aa of the N terminus of ORF6 are absent in the PG50 genome. The remaining 205 aa of ORF6 in PG50 showed some homology with the N-terminal part of ORF5 in strain Afadé. Compared to ORF5 in strain Afadé the PG50 homologue was missing the N-terminal 199 aa. Southern hybridization probes are shown below the Afadé sequence.

 
DNA sequence analysis of a 3227 bp region within the 7·9 kb fragment of the PG50 chromosome region identified three ORFs, gtsA (nt 149–1369), gtsB (nt 1347–2375) and gtsC (nt 2353–3156), which were partially overlapping. These ORFs were preceded by a consensus sequence for a -10 signal box (nt 62–67) of a prokaryotic transcriptional promoter and were followed by a sequence capable of forming a hairpin structure (nt 3207–3227) with a {Delta}G of -9·1 kcal mol-1, representing a potential rho-independent transcription termination signal. No clear -35 box was evident as with many other mycoplasmal genes. gtsB overlapped gtsA and gtsC overlapped gtsB, each by 23 bp. The A+T content of the 3·23 kb segment spanning the putative gts operon was 80·5 mol%, a value consistent with mycoplasma genomes. The A+T content within the intergenic regions was consistently higher compared with that within ORFs. A+T were 85·1 mol% of the nucleotides in the first 148 bases preceding gtsA, 83 mol% of those in the region (nt 3157–3297) between gtsC and lppB and 83 mol% of those between lppB and ORF6 (nt 5203–5520).

gtsA encoded a peptide of 406 aa with a predicted molecular mass of 47·4 kDa and was almost identical to the sequence for GtsA of M. mycoides subsp. mycoides SC strain Afadé (Vilei & Frey, 2001); only 15 of 406 (96·3 % identity) aa were different. It was preceded by a canonical ribosome binding sequence 16 bp upstream (nt 127–133) of the ATG start codon and contained three mycoplasma-specific TGATrp codons. Although TMpred software failed to identify a significant (score more than 500) transmembrane domain, ScanProsite software identified an ATP/GTP binding site motif A (P-loop) between aa 48 and 55, and an ABC transporter family signature with an ATP binding protein motif between aa 213 and 227. GtsA sequences from PG50 and strain Afadé showed that the P-loop sequence GPSGSGKT and the ABC transporter signature LSGGQKQRVAFAKGI were identical in both species. A BLASTP analysis using GtsA from Mycoplasma sp. bovine group 7 identified, in addition to the high identity with GtsA from M. mycoides subsp. mycoides SC, 33·3 % identity and 52·6 % similar amino acids with an ABC transporter ATP-binding protein from Mycoplasma pulmonis (MyPu_4970).

gtsB encoded a 342 aa peptide with a predicted molecular mass of 39·9 kDa and varied by 13 aa (96·2 % identity) from the GtsB in M. mycoides subsp. mycoides SC strain Afadé. gtsC encoded a 267 aa peptide with a predicted molecular mass of 31·5 kDa and displayed 89·2 % identity (29 amino acid variations and two deletions) with GtsC in M. mycoides subsp. mycoides SC strain Afadé. Although database searches failed to identify ABC transporter peptide signatures, TMpred software identified six transmembrane domains in both GtsB and GtsC. BLASTP searches also demonstrated that GtsB had 33·2 % identical and 56·6 % similar amino acids to an ABC transporter permease protein in M. pulmonis (MyPu_4980). GtsC has 32·5 % identical and 54·8 % similar amino acids to another hypothetical ABC transporter permease protein in M. pulmonis (MyPu_4990).

Sequence comparison between lppB, ORF5 and ORF6 genes downstream of the glycerol uptake locus from PG50 and the Afadé strain of M. mycoides subsp. mycoides SC
An ORF (nt 3298–5202) encoding a peptide of 634 aa (predicted molecular mass of 71·0 kDa) showing 66·4 % aa identity (80·3 % similarity) with LppB of M. mycoides subsp. mycoides SC was identified immediately downstream of the glycerol uptake operon (Fig. 1). LppB sequences from M. mycoides subsp. mycoides SC and M. mycoides subsp. mycoides LC had 90·1 % identity (Vilei et al., 2000). The lppB homologue was preceded by a consensus sequence for a -10 box (nt 3252–3258) and was followed by a sequence (nt 5224–5250) that could form a hairpin structure with a {Delta}G of -13·0 kcal mol-1, representing a potential transcriptional termination signal. A canonical ribosomal binding sequence preceded the lppB gene by 8 nt (nt 3283–3290) and the gene contained eight potential TGATrp codons. SignalP identified a prokaryotic signal peptide cleavage site spanning aa 23–24 of LppB.

One hundred and eighty one nucleotides downstream of lppB was a sequence spanning 2309 nt (nt 5383–7691) containing two ORFs, termed ORF6 and ORF5 (Fig. 1). ORF6 (nt 5521–6789) was preceded by a consensus sequence for a -10 box (nt 5383–5388). ORF5 spanned nt 6762–7652 and overlapped ORF6 by 28 bp. No -10 box was identified for ORF5. A sequence that could form a hairpin structure with a {Delta}G of -16·4 kcal mol-1 spanned nt 7660–7691. ORF6 encoded a putative membrane protein of 422 aa (predicted molecular mass of 49·2 kDa) and was preceded by a canonical ribosomal binding sequence spanning nt 5506–5510. The first 217 aa of ORF6 showed 88·5 % sequence identity (90·3 % sequence similarity) with the carboxy-terminal 217 aa portion of ORF6 of M. mycoides subsp. mycoides SC, but 299 aa were missing from the N terminus compared with ORF6 of M. mycoides subsp. mycoides SC. ORF5 encoded a proline-rich peptide of 296 aa (predicted molecular mass of 32·9 kDa) which showed significant similarity to ORF5 in M. mycoides subsp. mycoides SC (86·3 % sequence identity and 88·6 % similarity). The Mycoplasma sp. bovine group 7 homologue was missing the N-terminal 199 aa. Proline constituted 14·5 % of the Mycoplasma sp. bovine group 7 ORF5 sequence.

PCR analysis of the gtsABC and lppB loci within members of the M. mycoides cluster
Primers 7500bp1L and 3480bp-R amplified different fragment sizes from genomic DNA of different members of the M. mycoides cluster (Table 1). A 5·9 kb fragment was amplified from Mycoplasma sp. bovine group 7 isolates recovered from geographically diverse regions of the world. A 9·3 kb fragment was amplified from M. mycoides subsp. mycoides SC strains derived from the African/Australian cluster and a 0·45 kb fragment from isolates derived from the less virulent European cluster, as expected (Vilei et al., 2000). An amplification product of 6·9 kb was observed when DNA from type strain Y-goat and field isolates of M. mycoides subsp. mycoides LC was used. Furthermore, a 7·5 kb fragment was amplified from PG3 and field isolates of M. mycoides subsp. capri (Table 1). However, no amplification products were observed using DNA from type strains representative of the phylogenetically related species M. capricolum subsp. capripneumoniae (F38) and M. capricolum subsp. capricolum (California kid) or from the serogroup L isolate (B144P). No amplification products were obtained using DNA from phylogenetically more distantly related Mycoplasma spp., including Mycoplasma bovis, Mycoplasma agalactiae and M. putrefaciens (Table 1).

Genetic analysis of the gtsABC and lppB loci
Variation in amplicon size demonstrated by long range PCR suggested that the chromosomal region spanning the glycerol uptake locus, lppB and the flanking segment including ORF5 and ORF6 may be prone to modification by insertion and/or deletion of gene sequences or by accumulation of sequence polymorphisms in PCR primer sites. Southern blot hybridization experiments using a suite of probes spanning regions within gtsABC, lppB, ORF5 and ORF6 derived from M. mycoides subsp. mycoides SC strain Afadé were used to determine the genetic composition of fragments amplified by long range PCR. Southern blots of HindIII-digested PG50 and field isolate 99/0361/4 of Mycoplasma sp. bovine group 7 identified fragments of 1·9 and 1·5 kb using a probe spanning a portion of gtsA. A minor RFLP (fragment sizes of 1·9 and 1·4 kb) was observed in strain PG50(54) and field isolate 99/8407/10 (Fig. 2); Afadé showed the characteristic fragments of 1·8 and 1·5 kb, whereas the European M. mycoides subsp. mycoides SC strain L2 showed bands of 3·4 kb and 1·5 kb. A probe spanning gtsB and gtsC hybridized to three fragments of 5·0, 2·5 and 1·9 kb on blots containing HindIII digests of Mycoplasma sp. bovine group 7 DNA from all four strains (Fig. 2); fragments of 3·8, 1·8 and 1·0 kb were observed on blots containing HindIII digests of Afadé DNA, whereas L2 DNA presented a band at 3·8 kb. The presence of the 3·8 kb band in M. mycoides subsp. mycoides SC and of the 2·5 kb band in Mycoplasma sp. bovine group 7 cannot be explained by the sequence of this genetic locus. However, since the probe for gtsBC spanned most of gtsB and all of gtsC (Fig. 1) it is conceivable that a portion(s) of this probe may share homology with domains characteristic of other ABC transporter genes known to be common in mycoplasma genomes (Chambaud et al., 2001; Fraser et al., 1995; Himmelreich et al., 1996) and could explain the presence of the unpredicted hybridization signal. An lppB gene probe identified a single 5·0 kb HindIII fragment on blots of genomic DNA from all four Mycoplasma sp. bovine group 7 strains (Fig. 2); fragments of 3·9 and 1·0 kb were evident on blots containing HindIII-digested Afadé DNA. A single 5·0 kb fragment of DNA from all four Mycoplasma sp. bovine group 7 strains hybridized with separate probes spanning regions of ORF5 and ORF6 (Fig. 2); a single fragment of 4·4 kb hybridized with these probes in HindIII-digested Afadé DNA (Fig. 2). As expected, probes for lppB, ORF5 and ORF6 did not react with the L2 DNA. These data showed that the genomic region encompassing genes for the glycerol uptake operon, lppB, ORF5 and ORF6 was quite stable among geographically diverse strains of Mycoplasma sp. bovine group 7.



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Fig. 2. Southern blots of HindIII digests of chromosomal DNA isolated from two strains of M. mycoides subsp. mycoides SC and four strains of Mycoplasma sp. bovine group 7 hybridized with probes spanning regions within gtsABC, lppB, ORF6 and ORF5. Molecular size markers (M) were digoxigenin-labelled {lambda} DNA digested with HindIII (23·1, 9·4, 6·6, 4·4, 2·3, 2·0 and 0·6 kb). Lanes: 1 and 2, M. mycoides subsp. mycoides SC strains L2 (Europe) and Afadé (Africa), respectively; 3–6, Mycoplasma sp. bovine group 7 strains PG50, PG(54), 99/0361/4 and 99/8407/10, respectively.

 
Southern blots of HindIII-digested chromosomal DNA from M. capricolum subsp. capricolum (California kid) and M. mycoides subsp. mycoides SC strain Afadé were used to determine the degree of similarity between these two species. Under low stringency conditions hybridization signals were observed using probes spanning gtsB and gtsC (fragment sizes of 2·1, 1·2 and 1·0 kb) and lppB (fragment size of 1·4 kb) in HindIII digests of California kid DNA (Fig. 3). We interpret the presence of additional, faint gtsBC signals to be due to the several ABC transporter genes that might be present in the California kid genome. The faint bands obtained with the lppB probe under low stringency conditions could be due to the presence of highly conserved signal sequence domains of other lipoprotein genes in California kid. No signals were observed for gtsA or ORF5, and only a weak signal was detected with the ORF6 probe (fragment size of 5 kb) (Fig. 3). These data suggested that an incomplete glycerol uptake locus resided in California kid and that parts of the 7·9 kb region found in Mycoplasma sp. bovine group 7 were poorly conserved in M. capricolum subsp. capricolum (California kid).



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Fig. 3. Southern blots of HindIII digests of chromosomal DNA isolated from M. mycoides subsp. mycoides SC strain Afadé (A) and M. capricolum subsp. capricolum strain California kid (C). Molecular size markers (M) were digoxigenin-labelled {lambda} DNA digested with HindIII.

 
Antigenic analysis
To investigate the degree of antigenic cross-reactivity between members of the M. mycoides cluster, cell lysates representative of mycoplasma type strains were reacted with antiserum from a cow infected with M. mycoides subsp. mycoides SC strain Afadé. The characteristic pattern of immunoreactive antigens with molecular masses between 38 and 110 kDa (Gonçalves et al., 1998) were seen in lanes containing African and European strains of M. mycoides subsp. mycoides SC (Fig. 4). A particularly strong antigenic reaction with a band of the same molecular mass as LppB was seen in Mycoplasma sp. bovine group 7 strain PG50 (Fig. 4). A band of this size was also present in M. mycoides subsp. mycoides SC strains PG1 and Afadé, but not in strain L2, as expected (Vilei et al., 2000). Several other cross-reactive antigens were observed with all other members of the M. mycoides cluster and with M. putrefaciens strain KS1. However, the pattern of cross-reactive antigens in Mycoplasma sp. bovine group 7 type strain PG50 was most similar to that observed with M. mycoides subsp. mycoides SC strains PG1, Afadé and L2. Complex patterns of cross-reactive antigens were also observed in lanes containing lysates of M. mycoides subsp. mycoides LC strain Y-goat, M. capricolum subsp. capricolum strain California kid, M. capricolum subsp. capripneumoniae strain F38 and M. mycoides subsp. capri strain PG3.



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Fig. 4. Immunoblot of cell lysates (~10 µg) of strains representative of the M. mycoides cluster reacted with antiserum obtained from a cow experimentally infected with M. mycoides subsp. mycoides SC strain Afadé. Strain designations are shown above each lane: M. mycoides subsp. mycoides SC strains PG1, Afadé and L2; M. mycoides subsp. mycoides LC strain Y-goat; Mycoplasma sp. bovine group 7 strain PG50; M. mycoides subsp. capri strain PG3; M. capricolum subsp. capricolum strain California kid; M. capricolum subsp. capripneumoniae strain F38; and M. putrefaciens strain KS1. Molecular mass markers are indicated in kDa. Arrow and arrowheads indicate the position of LppB and bands of a similar size in Y-goat and PG50 on the immunoblot.

 
Production of hydrogen peroxide
Previous studies have demonstrated that African/Australian isolates of M. mycoides subsp. mycoides SC are capable of transporting [U-14C]glycerol (but not L-{alpha}-glycerophosphate) in a time-dependent manner with the concomitant production of hydrogen peroxide (Vilei & Frey, 2001). We compared the capacity of Mycoplasma sp. bovine group 7 strain PG50 to produce hydrogen peroxide in the presence of glycerol with M. mycoides subsp. mycoides SC strain Afadé. PG50 was capable of producing up to 9·0 µg hydrogen peroxide ml-1 within 10 min of the addition of 100 µM glycerol, whereas strain Afadé only reached a maximum of 5·0 µg H2O2 ml-1 over the same time period (Fig. 5). After 20 min incubation, no further increase in the production of H2O2 was observed with strain PG50, whereas strain Afadé slightly increased H2O2 production to 6·25 µg ml-1 (Fig. 5). The European strain L2 of M. mycoides subsp. mycoides SC gradually produced hydrogen peroxide (0–2·7 µg ml-1) over the 20 min period, which probably reflects the passive diffusion of glycerol across the membrane. A similar level of hydrogen peroxide was previously reported to be produced by strain L2 in identical experiments (Vilei & Frey, 2001) (Fig. 5). When strain PG50 was incubated with 1 mM glycerol at 37 °C, the pattern of H2O2 production was similar to that observed with 100 µM glycerol, whereas strain Afadé reached a maximum of almost 10 µg hydrogen peroxide ml-1 after 20 min incubation (data not shown). In the absence of glycerol, H2O2 production by all strains was at a background level (<1·0 µg ml-1) (Fig. 5).



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Fig. 5. Production of H2O2 by M. mycoides subsp. mycoides SC strain Afadé, M. mycoides subsp. mycoides SC strain L2 and Mycoplasma sp. bovine group 7 strain PG50 in HEPES buffer containing 7 mM MgCl2 and 100 µM glycerol at 37 °C. For each mycoplasma strain, 1x109 cells were used to measure H2O2 production. {square}, Afadé strain; {triangleup}, strain PG50 and {circ}, strain L2 incubated in the absence of glycerol. {blacksquare}, Afadé strain; {blacktriangleup}, strain PG50 and {bullet}, strain L2 incubated with 100 µM glycerol. The data are the means of three independent measurements. Error bars, standard errors.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this report, we demonstrate that Mycoplasma sp. bovine group 7 strain PG50 and other isolates of this group possess a chromosomal gene locus (gtsA, gtsB and gtsC) for the transport of glycerol. The polypeptides encoded by gtsABC of PG50 had predicted amino acid sequences that had more than 89 % identity with three homologues in M. mycoides subsp. mycoides SC strain Afadé. Furthermore, we show that Mycoplasma sp. bovine group 7 reference strain PG50 was capable of generating a significantly higher level of H2O2 upon addition of a physiological concentration of glycerol (100 µM) to the growth medium than M. mycoides subsp. mycoides SC strain Afadé. The high level of sequence identity of glycerol uptake genes between these two species and their abilities to generate physiologically significant levels of H2O2 when cultured in the presence of glycerol suggest that Mycoplasma sp. bovine group 7 strain PG50 has a functional glycerol uptake mechanism. Moreover, the ability to amplify a 5·9 kb fragment among the geographically diverse collection of Mycoplasma sp. bovine group 7 isolates suggests that these genes are widespread in this species. Although the role of H2O2 in the pathogenesis of Mycoplasma sp. bovine group 7 infections is unclear, field strains isolated during a recent outbreak of polyarthritis, mastitis and abortion were able to haemolyse red blood cells when cultured on sheep blood agar (Hum et al., 2000). These observations suggest that the release of H2O2 by Mycoplasma sp. bovine group 7 strains contributes to their virulence as it does in M. mycoides subsp. mycoides SC (Vilei & Frey, 2001).

Although the gtsABC locus of M. sp. bovine group 7 displays strong sequence identity with homologous genes in Mycoplasma mycoides subsp. mycoides SC strain Afadé, the regions immediately downstream of these loci differ significantly. In PG50, IS1634 and IS1296 are missing and there are truncations of large portions of the 5'-terminal halves of both ORF5 (proline-rich protein) and ORF6 (putative surface located membrane protein) compared with homologous genes in strain Afadé. However, both PG50 and strain Afadé possess the lipoprotein gene lppB. PCR and Southern blot analyses of other members of the M. mycoides cluster, including M. mycoides subsp. mycoides LC, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae revealed the latter two to be most different from Mycoplasma sp. bovine group 7. Hybridization of PG50 DNA with probes spanning gtsABC, ORF5, ORF6 and lppB derived from M. mycoides subsp. mycoides SC showed that these genes were present in Mycoplasma sp. bovine group 7, while M. capricolum subsp. capricolum (California kid) DNA only yielded positive signals with probes spanning gtsB, gtsC and lppB; no signal was observed with probes spanning gtsA and ORF5, and only a very weak signal was observed with ORF6. Furthermore, immunoblotting data showed that PG50 possessed a panel of cross-reactive antigens that most closely resembled the pattern generated with lysates of M. mycoides subsp. mycoides SC strains when reacted with anti-strain Afadé serum raised during an experimental infection. The least similar patterns of cross-reactive antigens were observed with type strains California kid and KS1 of M. putrefaciens.

Salih and others examined 24 mycoplasma strains representing all six members of the M. mycoides cluster for the presence of 35 enzymes by horizontal starch gel electrophoresis. These authors concluded that Mycoplasma sp. bovine group 7 constituted a new species and was most similar to the M. mycoides subsp. mycoides SC type strain PG1 (Salih et al., 1983). Recent evidence that Mycoplasma sp. bovine group 7 is closely related to M. mycoides subsp. mycoides SC has also emerged from studies of surface accessible lipoproteins present among members of the M. mycoides cluster. The lppA gene has been characterized by DNA sequence analysis in M. mycoides subsp. mycoides SC and LC, M. mycoides subsp. capri and Mycoplasma sp. bovine group 7 (Monnerat et al., 1999) and unlike lppB, is located at a site unrelated to the glycerol uptake locus. Comparisons of the predicted amino acid sequences of LppA from type strains of Mycoplasma sp. bovine group 7 and M. mycoides subsp. mycoides SC showed 91 % identity. However, comparisons of predicted LppA sequences of PG50 (Mycoplasma sp. bovine group 7) and California kid (M. capricolum subsp. capricolum) showed only 53 % identity (Frey et al., 1998; Monnerat et al., 1999). Moreover, antisera raised against recombinant LppA from M. capricolum subsp. capricolum (California kid) did not cross-react with LppA homologues in any other member of the M. mycoides cluster (Monnerat et al., 1999). LppB sequences from M. mycoides subsp. mycoides SC and M. mycoides subsp. mycoides LC had 90·1 % identity (Vilei et al., 2000), while LppB proteins of M. mycoides subsp. mycoides SC and Mycoplasma sp. bovine group 7 had 66·4 % sequence identity. Antisera raised against recombinant LppB from strain Afadé reacted strongly with LppB in PG50, PG1 (M. mycoides subsp. mycoides SC type strain) and Y-goat (M. mycoides subsp. mycoides LC type strain) but only weakly in Western blots of whole cell lysates of California kid, F38 (M. capricolum subsp. capripneumoniae type strain) and PG3 (M. mycoides subsp. capri type strain) (Vilei et al., 2000).

In conclusion, our data suggest that PG50 and other strains of Mycoplasma sp. bovine group 7 possess a functional glycerol transport locus. Predicted amino acid sequences of GtsA, B and C, LppB, ORF6 and ORF5 from Mycoplasma sp. bovine group 7 showed strong sequence identity with M. mycoides subsp. mycoides SC but IS1634 and IS1296 were absent from this region in the PG50 chromosome. Southern hybridization and long range PCR studies indicated that this chromosomal region has diverged in mycoplasmas belonging to subspecies of M. capricolum.


   ACKNOWLEDGEMENTS
 
We thank Y. Schlatter (Institute for Veterinary Bacteriology, Berne, Switzerland) for her practical help. S. P. D. performed all of this study at the Institute for Veterinary Bacteriology, University of Berne, Switzerland via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems. This study was supported by grant no. C96.0073 of the Swiss Ministry of Education and Science and by the Swiss Federal Veterinary Office.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 17 May 2002; revised 8 October 2002; accepted 9 October 2002.



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