Abstract||Selected strains of Streptococcus equi subsp. zooepidemicus produce a bacteriocin-like inhibitory substance called zoocin A. Zoocin A has bacteriolytic activity against closely related species of streptococci, including the etiological agents of dental caries and Streptococcus pyogenes. Zoocin A functions to hydrolyse the peptidoglycan of bacterial cell walls. Producer-cell immunity to zoocin A is encoded by zif, the zoocin A immunity factor. The zooA-zif gene locus is chromosomally encoded and flanked by two transposon-like sequences with significant similarity to the lss-lif locus encoding lysostaphin in Staphylococcus simulans. This suggested the possibility that the zooA-zif locus was acquired by S. equi subsp. zooepidemicus through a horizontal gene transfer event. Studies conducted to examine this hypothesis were unable to establish the mechanism by which zooA-zif was acquired. Examination of 24 S. equi subsp. zooepidemicus strains using random amplified polymorphic DNA analysis and pulse field gel electrophoresis showed that the species was highly heterogeneous. Examination of the 24 strains using Southern hybridisation revealed only three S. equi subsp. zooepidemicus strains contained the zooA and zif genes; however the partial open reading frames (flaR and rgg-like) flanking zooA and zif appeared to be conserved in all tested strains. Long template PCR was used to examine this chromosomal area containing the zooA and zif genes in 24 S. equi subsp. zooepidemicus strains. These results, along with comparison of sequence data from two zoocin A producing strains, and analysis of the sequenced S. equi subsp. zooepidemicus H70 and S. equi subsp. equi 4047 genomes showed the area surrounding the zooA-zif locus to be one of considerable genetic variation substantiating the hypothesis that zooA-zif were acquired via horizontal gene transfer.
Studies on zoocin A production suggested that zooA expression was under carbon catabolite regulation, (CCR). In the Gram-positive bacteria CCR, is mediated by the carbon catabolite control protein (CcpA). CcpA is a DNA binding protein that can negatively regulate transcription of its target gene by binding to a catabolite-responsive element (CRE). This study reports the regulation of zif and zooA expression in response to cellular growth on different carbon sources, the presence of a ccpA-like gene in the genome of S. equi subsp. zooepidemicus, the presence of a CRE sequence in the promoter region of zooA, and the effect of a ccpA deletion on zooA expression. Zoocin A activity, zooA and zif mRNA transcript levels, and zooA and zif reporter gene activities were monitored during growth of S. equi subsp. zooepidemicus 4881 in a pH-controlled batch culture system. Zoocin A titres were maximal when the bacteria were grown on a starch-based medium and minimal when grown on a glucose-based medium. Northern hybridization showed that zif was constitutively expressed during growth regardless of the carbon source available. In contrast, zooA was transcribed at low levels in cells grown on a glucose-based medium but at much higher levels in cells grown on a starch-based medium. Use of a zooA promoter-chloramphenicol acetyl transferase (CAT) reporter demonstrated that zooA expression increased 17-fold in cells grown on a starch-based medium. Use of a zif promoter-CAT reporter showed that in cells grown on a starch-based medium zif was transcribed approximately 4 times as strongly as in cells grown on glucose. Transcription of zif was always much less than that of zooA. A ccpA deletion mutant appeared to exhibit complete de-repression of zooA under all growth conditions. Together these data showed that zoocin A expression was regulated in response to the carbon source on which the bacteria were grown.