OVEREXPRESSION OF THE SHIGELLA-FLEXNERI GENES-CODING FOR DNA TOPOISOMERASE-IV COMPENSATES FOR LOSS OF DNA TOPOISOMERASE-I - EFFECT ON VIRULENCE GENE-EXPRESSION

Introducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E, coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression, E. coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase I by amplifying the genes coding for topoismerase IV. Unlike the E. coli topA mutants, derivatives of S. flexneri harbouring this topA allele did not appear to acquire any compensatory mutations, We investigated the possibility that this was due in part to an inability of the S, flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed. The S. flexneri genes encoding the alpha- and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids, These plasmids complemented well-characterized Salmonella typhimurium temperature-sensitive topoisomerase IV mutations, showing that the S. flexneri and S, typhimurium proteins are capable of combining to form active complexes. When the S, flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S. flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling. The same plasmid also partially restored S, flexneri virulence gene transcription. These data show that overexpression of the S. flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression, The fact that S. flexneri does not exploit topoisomerase IV gene amplification as E, coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.