Isolation and characterization of a topA mutant of Shigella flexneri

Abstract

Shigella flexneri was shown to possess a homologue of the Escherichia coli K-12 topA gene which encodes DNA topoisomerase I. The S. flexneri topA gene was replaced by a copy of the E. coli K-12 topA gene which has been insertionally inactivated by transposon Tn10. The topoisomer distribution of reporter plasmids showed that the presence of this topA lesion in S. flexneri correlated with an increase in the level of negative DNA supercoiling in the mutant, indicating that topoisomerase I is required to relax DNA in S. flexneri as it is in E. coli and Salmonella typhimurium. The introduction of the topA mutation also resulted in repression of transcription of a thermally regulated invasion gene located on the 230 kb virulence plasmid. In addition, the topA mutant was hypersensitive to growth medium osmolarity, was unable to grow on MacConkey indicator plates and exhibited an increased doubling time under all growth conditions tested. All of these phenotypes were fully complemented in trans by a cloned copy of the E. coli topA gene carried on a recombinant plasmid. Unlike E. coli topA mutants which acquire compensatory mutations at a high frequency, such compensatory mutations were not detected in the S. flexneri topA::Tn10 mutant.