Spontaneous insertion of an IS1-like element into the virF gene is responsible for avirulence in opaque colonial variants of Shigella flexneri 2a

Abstract

Colonial variation of Shigella flexneri serotype 2a from the translucent (2457T) to the opaque form (2457O) occurs spontaneously once in 10(4) cell divisions, with concomitant loss of ipa gene expression and virulence. The appearance of 2457O was associated with the insertional inactivation of virF, an invasion plasmid-encoded positive regulator of ipa gene expression. Plasmid pWR110, a Tn5-tagged invasion plasmid that restores the invasive phenotype to plasmid-cured Shigella derivatives, was conjugally transferred into 2457O. Synthesis of the invasion-associated IpaB and IpaC polypeptides, normally present on the surface of virulent shigellae, and the invasive phenotype were restored in 2457O(pWR110) transconjugants. Plasmid DNA restriction endonuclease patterns of 2457T and 2457O, along with hybridization analysis, showed that a SalI fragment carrying the virF gene in 2457O had increased in size relative to its counterpart in 2457T. Analysis of virF DNA sequences amplified by the polymerase chain reaction revealed that the virF sequence from 2457O was 780 bp larger than that amplified from 2457T. Moreover, the virF sequence amplified from 2457O hybridized to an IS1 DNA probe whereas the amplified 2457T virF sequence did not. DNA sequence analysis mapped the insertion element, designated IS1SFO, within an A.T-rich region of the virF open reading frame and identified a 9-bp virF target sequence that was duplicated at the insertion site of IS1SFO. The DNA sequence of IS1SFO was greater than 99% homologous to IS1F. Plasmid pWR600, carrying a 1,260-bp HpaII fragment encoding a wild-type virF gene, was able to restore the virulent phenotype and translucent colonial morphology to nine independently isolated 2457O hosts.