IDENTIFICATION OF SIGMA(S)-DEPENDENT GENES ASSOCIATED WITH THE STATIONARY-PHASE ACID-RESISTANCE PHENOTYPE OF SHIGELLA-FLEXNERI

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribu te to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor sig ma(s). Using random TnphoA and TnlacZ mutagenesis we isolated five aci d-sensitive mutants of S. flexneri, which had lost their ability to su rvive at pH 2.5 for 2 h in vitro. Each transposon insertion with flank ing S. flexneri DNA was cloned and sequenced. Database searches indica ted that two TnlacZ mutants had an insertion within the hdeA gene, whi ch is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon . Further sequence analysis from the remaining TnlacZ and two TnphoA m utants demonstrated that they all had insertions within a previously u nidentified open reading frame (ORF), which is directly downstream fro m the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polyme rase chain reaction (PCR) product containing this gene was cloned, whi ch was able to restore acid resistance in each mutant. These fusions w ere induced during entry into late exponential phase and were positive ly regulated by RpoS. We confirmed that the expression of the acid-res istance phenotype in acidified minimal media was dependent upon the su pplementation of glutamic acid and that this glutamate-dependent syste m was RpoS regulated. Southern hybridization revealed that both the ga dC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS(-) derivative was extremel y acid sensitive. Two-dimensional gel electrophoresis of this mutant r evealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the exp ression of acid resistance in S. flexneri may be multifactorial and in volve proteins located at different subcellular locations.