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Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

ABSTRACT: All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneri serotype 6 other than insertion sequence or related sequences and compared it with the pINV B form. More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions. This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative. There are also genes absent in the S. flexneri serotype 6 plasmid, including the sepA gene encoding serine protease, the major secreted protein of S. flexneri serotype 2a, and the stbAB genes, which encode one of the two partition systems found in S. flexneri serotype 5. The incompatibility of the two pINV forms appears to be due to either small differences in the mvpAT postsegregational killing system or the presence of an unknown system in pINVA.

SUBMITTER: Lan R

PROVIDER: S-EPMC219609 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

Lan Ruiting R Stevenson Gordon G Reeves Peter R PR

Infection and immunity 20031101 11

All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneri serotype 6 other th ...[more]

PMID: 14573649

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Project description:VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.ImportanceThis study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

Project description:BACKGROUND: Terpenoids constitute the largest class of secondary metabolites made by plants and display vast chemical diversity among and within species. Terpene synthases (TPSs) are the pivotal enzymes for terpenoid biosynthesis that create the basic carbon skeletons of this class. Functional divergence of paralogous and orthologous TPS genes is a major mechanism for the diversification of terpenoid biosynthesis. However, little is known about the evolutionary forces that have shaped the evolution of plant TPS genes leading to terpenoid diversity. RESULTS: The orthologs of Oryza Terpene Synthase 1 (OryzaTPS1), a rice terpene synthase gene involved in indirect defense against insects in Oryza sativa, were cloned from six additional Oryza species. In vitro biochemical analysis showed that the enzymes encoded by these OryzaTPS1 genes functioned either as (E)-?-caryophyllene synthases (ECS), or (E)-?-caryophyllene & germacrene A synthases (EGS), or germacrene D & germacrene A synthases (DAS). Because the orthologs of OryzaTPS1 in maize and sorghum function as ECS, the ECS activity was inferred to be ancestral. Molecular evolutionary detected the signature of positive Darwinian selection in five codon substitutions in the evolution from ECS to DAS. Homology-based structure modeling and the biochemical analysis of laboratory-generated protein variants validated the contribution of the five positively selected sites to functional divergence of OryzaTPS1. The changes in the in vitro product spectra of OryzaTPS1 proteins also correlated closely to the changes in in vivo blends of volatile terpenes released from insect-damaged rice plants. CONCLUSIONS: In this study, we found that positive Darwinian selection is a driving force for the functional divergence of OryzaTPS1. This finding suggests that the diverged sesquiterpene blend produced by the Oryza species containing DAS may be adaptive, likely in the attraction of the natural enemies of insect herbivores.

Dataset Information

Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

Publications

Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence.

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