THE MODIFIED NUCLEOSIDE 2-METHYLTHIO-N-6-ISOPENTENYLADENOSINE IN TRANSFER-RNA OF SHIGELLA-FLEXNERI IS REQUIRED FOR EXPRESSION OF VIRULENCE GENES

The virulence of the human pathogen Shigella flexneri is dependent on both chromosome-and large-virulence-plasmid-encoded genes. A kanamycin resistance cassette mutation in the miaA gene (miaA::Km Sma), which e ncodes the tRNA N-6-isopentyladenosine (i(6)A37) synthetase and is inv olved in the first step of the synthesis of the modified nucleoside 2- methylthio-N-6-isopentenyladenosine (ms(2)i(6)A), was transferred to t he chromosome of S. flexneri 2a by phage P1 transduction. In the wild- type bacterium, ms(2)i(6)A37 is present in position 37 (next to and 3' of the anticodon) in a subset of tRNA species-reading codons starting with U (except tRNA(Ser) species SerI and SerV). The minA::Km Sma mut ant of S, flexneri accordingly lacked ms(2)i(6)A37 in its tRNA, In add ition, the mutant strains showed reduced expression of the virulence-r elated genes ipaB, ipaC, ipaD, virG, and virF, accounting for sixfold- reduced contact hemolytic activity and a delayed response in the focus plaque assay. A cloned sequence resulting from PCR amplification of t he wild-type Shigella chromosome and exhibiting 99% homology with the nucleotide sequence of the Escherichia coli miaA gene complemented the virulence-associated phenotypes as well as the level of the modified nucleoside ms(2)i(6)A in the tRNA of the miaA mutants, In the miaA mut ant, the level of the virulence-associated protein VirF was reduced 10 -fold compared with the wild type, However, the levels of virF mRNA we re identical in the mutant and in the wild type. These findings sugges t that a posttranscriptional mechanism influenced by the presence of t he modified nucleoside ms(2)i(6)A in the tRNA is involved in the expre ssion of the virF gene. The role of the miaA gene in the virulence of other Shigella species and in enteroinvasive E. coil was further gener alized.