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The plasmid R64 thin pilus identified as a type IV pilus.

ABSTRACT: The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.

SUBMITTER: Kim SR

PROVIDER: S-EPMC179153 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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The plasmid R64 thin pilus identified as a type IV pilus.

Kim S R SR Komano T T

Journal of bacteriology 19970601 11

The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in ty ...[more]

PMID: 9171405

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Project description:Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

Project description:Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer. Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.

Project description:Type IV pili (T4P) are common bacterial surface appendages involved in different biological processes such as adherence, motility, competence, pathogenesis, and conjugation. In this work, we describe the T4P of TP114, an IncI2 enterobacterial conjugative plasmid recently shown to disseminate at high rates in the mouse intestinal tract. This pilus is composed of the major PilS and minor PilV pilins that are both important for conjugation in broth and in the gut microbiota but not on a solid support. The PilV-coding sequence is part of a shufflon and can bear different C-terminal domains. The shufflon is a multiple DNA inversion system containing many DNA cassettes flanked by recombination sites that are recognized by a shufflon-specific tyrosine recombinase (shufflase) promoting the recombination between DNA segments. The different PilV variants act as adhesins that can modify the affinity for different recipient bacteria. Eight PilV variants were identified in TP114, including one that has not been described in other shufflons. All PilV variants allowed conjugative transfer with different recipient Escherichia coli strains. We conclude that the T4P carried by TP114 plays a major role in mating pair stabilization in broth as well as in the gut microbiota and that the shufflon acts as a biological switch modifying the conjugative host range specificity. IMPORTANCE Conjugative plasmids are involved in horizontal gene transfer in the gut microbiota, which constitutes an important antibiotic resistance gene reservoir. However, the molecular mechanisms used by conjugative plasmids to select recipient bacteria and transfer at high rates in the mouse gut microbiota remain poorly characterized. We studied the type IV pilus carried by TP114 and demonstrated that the minor pilin PilV acts as an adhesin that can efficiently select target cells for conjugative transfer. Moreover, the pilV gene can be rapidly modified by a shufflon, hence modulating the nature of the recipient bacteria during conjugation. Our study highlights the role of mating pair stabilization for conjugation in broth as well as in the gut microbiome and explains how the host spectrum of a plasmid can be expanded simply by remodeling the PilV adhesin.

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The plasmid R64 thin pilus identified as a type IV pilus.

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The plasmid R64 thin pilus identified as a type IV pilus.

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