Molecular characterization of a trans-acting, positive effector (ipaR) of invasion plasmid antigen synthesis in Shigella flexneri serotype 5

Abstract A trans -acting, positive effector of invasion plasmid antigen (Ipa) synthesis has been identified and mapped on the pWR100 invasion plasmid of Shigella flexneri serotype 5 (strain M90T-W). Recombinant plasmids carrying this regulatory gene, designated ipaR , were found to restore full virulence to a non-invasive ipaR ::Tn 5 insertion mutant [M90T-W(pHS1042)] that had lost the ability to synthesize four Ipa antigens (IpaA, 70 kDa; IpaB, 62 kDa; IpaC, 42 kDa; and IpaD, 37 kDa). Genetic mapping of the ipaR gene positioned the locus on a 2.6 kb Pst I- Acc I fragment contained within a larger 8.0 kb Eco RI molecule that also encoded IpaD, IpaA, and two small proteins (27 kDa and 28 kDa). The trans regulatory effect of the ipaR product on ipaB , ipaC , ipaD , and ipaA expression was demonstrated by transforming compatible ipaBC , ipaDA , ipaR and ipaDAR plasmid recombinants, in various combinations, into M90T-A 3 , an isogenic invasion plasmid mutant of M90T-W that contained a deletion of the pWR100 ipaBCDA and ipaR loci; such transformants produced wild type levels of the IpaB, IpaC, IpaD and IpaA antigens only in the presence of IpaR + plasmids. DNA sequence analysis of the ipaR region established that the initiation codon for ipaR is 459 bp from the 3′-end of the ipaA gene and that ipaR encodes a 309 amino acid residue protein. An interesting feature of the IpaR polypeptide was its strong sequence homology with the bacteriophage P1 partition protein ParB, consisting of a 42.8% amino acid identity over a 278 residue section of the aligned proteins.