Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri

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Abstract
A plasmid of about 140 megadaltons has been associated with the invasiveness of Shigella flexneri. Upon subculturing in liquid media of fully virulent isolates of Shigella flexneri 2a YSH6000, which contains only a 230-kilobase-pair (kbp) plasmid in addition to 3.3- and 4.2-kbp cryptic plasmids characteristic to all S. flexneri strains, loss of invasiveness, loss of Congo red binding activity (Pcr), and complete loss of, or a deletion, or even a single-site IS insertion in the plasmid occurred simultaneously. This was ascribed to the fact that, once a noninvasive Pcr- cell has emerged, it overgrows the wild type as a consequence of its selective advantage in artificial media. A deletion map of the 230-kbp plasmid was made by analyzing SalI digests of 39 deletion derivatives plus 1 formed by insertion of an IS1-like element in independently isolated, noninvasive Pcr- mutants. Of 39 deletion derivatives, 16 belonged to a single type, and 6 belonged to another, suggesting deletion hot spots. The deletion map was confirmed and extended by analyzing 359 SalI-generated partial digests of the wild-type plasmid cloned into pBR322. Three copies of IS1-like elements were found on three different SalI fragments by Southern hybridization. Segments required for the Pcr+ phenotype seemed to occur at several different locations in the plasmid. Each of 28 representative Pcr- mutants were negative by the Sereny test. Hence, many, or possibly all, Pcr determinants were required for full virulence.