By random transposon TnS insertions, we previously identified six virulence-associated SaIl fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigellaflexneri 2a. In this study, we analyzed the sites of 134 independent TnS insertions on four contiguous Sall fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichuz coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for fuDl expression of the virulence phenotype of shigeliae.Shigellae are enteroinvasive bacteria that cause bacillary dysentery in humans and monkeys. These organisms invade colonic epithelial cells, multiply intracellularly, and spread to adjacent cells (9). The genetic determinants required for these abilities are located on at least three separate sites of the chromosome (3-5, 18) and on a 100-to 140-megadalton (MDa) plasmid. Commonly, plasmids of shigellae and enteroinvasive Escherichia coli (EIEC) contain genetic regions that are required in the early steps of the invasion process (18, 19); loss of the plasmid or deletion of an essential region consistently leads to loss of virulence (13,21).Expression of virulence in shigellae is dependent on temperature (12). Shigellae grown at 37°C are fully virulent, whereas bacteria grown at 30°C neither invade epithelial cells (12) nor provoke keratoconjunctivitis in guinea pigs (24). Making use of this property, Hale et al. (6) identified at least seven plasmid-coded, virulence-associated peptides produced by Shigella flexneri 2a and 5 and EIEC strain 0143. By Western blot (immunoblot) analysis of extracts of whole cells, four peptides of 78, 62, 43, and 38 kDa were recognized by convalescent-phase monkey antisera. These workers proposed that these proteins function as components of the invasion phenotype and are expressed on the bacterial surface. Oaks et al. (15) identified an additional plasmid-encoded surface peptide of 140 kDa which was also specifically recognized by convalescent-phase human or monkey sera. To identify the genetic regions associated with invasion, Maurelli et al. (14) shotgun-cloned Sau3A digests of the plasmid DNA into a cosmid vector, which was subsequently introduced into plasmid-free S. flexneri 5. A clone containing a 37-kilobase (kb) minimum sequence necessary for invasion was isolated. This recombinant clone also produced the four virulence-associated peptides described by Hale et al. (6). A DNA fragment coding for three antigenic proteins of 57, 43, and 39 kDa was cloned into a A expression vector by Buysse et al. (1). T...
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 Pcrcell 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 Sall digests of 39 deletion derivatives plus 1 formed by insertion of an IS1-like element in independently isolated, noninvasive Pcrmutants. 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 Sail-generated partial digests of the wild-type plasmid cloned into pBR322. Three copies of IS1-like elements were found on three different Sall fragments by Southern hybridization. Segments required for the Pcr+ phenotype seemed to occur at several different locations in the plasmid. Each of 28 representative Pcrmutants were negative by the Sereny test. Hence, many, or possibly all, Pcr determinants were required for full virulence.
A DNA sequence of about 1.0 kilobase (kb) derived from a 230-kb (140-megadalton) plasmid in a fully virulent Shigellaflexneri 2a strain, YSH6000, was cloned into Escherichia coli K-12 by selecting for the ability to bind Congo red (Pcr+ phenotype). It was mapped and localized within the Sall restriction fragment F on the plasmid. This clone converted an S. flexneri strain which is avirulent as a result of a small deletion in the
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