Characterisation of a Transposon-induced Pleiotropic Mutant ofClostridium acetobutylicumP262

Abstract

Transposon-induced metronidazole resistance was used as a selection system for the isolation ofClostridium acetobutylicumP262 mutants with altered electron transport pathways. The metronidazole resistant transconjugant of interest, mutant 3R, displayed resistance to DNA damaging agents, UV and bleomycin, and harboured a single transposon insertion within a structural gene, designatedsum(susceptibility tometronidazole). Thesumgene encoded a 334 amino-acid protein, with 36% identity and 57–58% similarity at the amino acid level to two archaebacterial protein sequences which appear to represent a class of uncharacterised reductase enzymes. Physiological studies of mutant 3R revealed a number of pleiotropic characteristics which included enhanced autolysin activity, increased motility, impaired clostridial cell formation, and resistance to the toxic tripeptide analogue, bialaphos. The introduction of thesumgene in multiple copies on a plasmid vector into the related strainClostridium beijerinckiiNCIMB 8052, resulted in inhibition of cell division, motility and autolysin activity. Thesumgene appears to be a member of a new subgroup of activases with reducing activity, which may control a regulon affecting different stationary phase processes such as clostridial differentiation and sporulation inC. acetobutylicumP262. The metronidazole resistant phenotype of thesummutant can be attributed to an increased capacity for DNA repair.