|Houlihan, Adam - CORNELL UNIVERSITY|
|Mantovani, Hilario - CORNELL UNIVERSITY|
Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2004
Publication Date: May 23, 2004
Citation: Houlihan, A., Mantovani, H., Russell, J.B. 2004. Transposition of pgh9:iss1 in streptococcus bovis hc5 to identify genes responsible for bovicin hc5 production. American Society for Microbiology Annual Meeting. p. 363. Technical Abstract: Background: Streptococcus bovis is a rapidly growing lactic acid bacterium that normally inhabits the rumen and lower GI tract of mammals. S. bovis HC5 produces a pore-forming bacteriocin (bovicin HC5) that has a broad spectrum of activity and is most active at low pH. The N-terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unidentified amino acids (represented by X) had approximately the same position as dehydrated residues found in some lantibiotics. Bovicin HC5 shared similarity with a lantibiotic produced by S. pyogenes SF370 (55% identity). Methods: S. bovis HC5 and JB1 were grown anaerobically on M17 agar plates or aerobically in M17 broth. S. bovis HC5 cells grown in M17 broth were washed and resuspended in 0.5 M sucrose with glycerol (10% w/v). The cells were electroporated with pGh9:ISS1, serially diluted in M17 broth and spread on M17 agar plates containing erythromycin (5 ug/ml). The plates were incubated at 28° C for 3 to 5 days. Transformants were inoculated into M17 broth with erythromycin, and the tubes were incubated at 39° C for 24 h. The cultures were spread onto M17 agar plates containing erythromycin and incubated at 39° C for 24 h. Colonies were then picked and transferred to M17 plates that lacked erythromycin and incubated at 39° C for 24 h. These plates were overlaid with molten M17 agar inoculated with S. bovis JB1 (a susceptible strain). The agar overlays were incubated at 39° C for 24 h, and bacteriocin activity was assessed from zones of clearing. Results: S. bovis HC5 was transformed with the plasmid pGh9:ISS1 via electroporation. This plasmid has a temperature-sensitive origin of replication. By increasing the temperature to 39° C, it was possible to select erythromycin-resistant cells that had pGhost9:ISS1 integrated into the chromosome. Agar overlays with S. bovis JB1, a sensitive strain, indicated that four mutants no longer produced active bovicin HC5. Conclusions: The plasmid pGh9:ISS1 was used to create S. bovis HC5 mutants that no longer produced bovicin HC5. By using primers homologous to the insertion sequence, we will be able to locate genes responsible for bovicin HC5 production.