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 H(2)O(2) 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 H(2)O(2). The ability of Mycoplasma sp. bovine group 7 to generate significant amounts of hydrogen peroxide may be important in pathogenesis.