Identification of Shigella sonnei form I plasmid genes necessary for cell invasion and their conservation among Shigella species and enteroinvasive Escherichia coli

Abstract

A series of Tn1 insertions in pSS120, the 120-megadalton form I plasmid of Shigella sonnei, were constructed by a Tn1-mediated conduction system previously described (H. Watanabe and A. Nakamura, Infect. Immun. 48:260-262, 1985, and screened for cell invasion in a tissue culture assay. The analysis of Tn1 insertion sites of seven noninvasive mutants suggested that four separate HindIII fragments were necessary for cell invasion. HindIII fragments including Tn1 of mutant plasmids were cloned into a vector plasmid, pACYC184. The DNA was used as a DNA probe to identify the corresponding, parental HindIII fragments. We identified one contiguous molecule of 2.6- and 4.1-kilobase pair (kb) HindIII fragments as being responsible for restoring cell invasiveness to the three mutant plasmids, pHW505, pHW510, and pHW511. Polypeptide analysis in minicells demonstrated that the contiguous HindIII fragments of 2.6 and 4.1 kb coded for at least four polypeptides, of 38, 41, 47, and 80 kilodaltons (kDa). A comparison of polypeptides synthesized by parental and mutant plasmids strongly suggested that the 38-kDa protein was essential for cell invasion. The 4.1-kb DNA which encoded the 38-kDa protein was conserved among plasmids of Shigella species and enteroinvasive Escherichia coli.