APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2007, p. 7703–7710 Vol. 73, No. 23 0099-2240/07/$08.00ϩ0 doi:10.1128/AEM.01577-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Sequence Characterization and Comparative Analysis of Three Plasmids Isolated from Environmental Vibrio spp.ᰔ† Tracy H. Hazen,1 Dongying Wu,2,3 Jonathan A. Eisen,2,3 and Patricia A. Sobecky1* School of Biology, Georgia Institute of Technology, Atlanta, Georgia 303321; The Institute for Genomic Research, Rockville, Maryland 208502; and UC Davis Genome Center, University of California, Davis, Davis, California 956163 Received 11 July 2007/Accepted 26 September 2007 The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022 kb) had a predicted 99, 64, and 32 protein-coding sequences and G؉C contents of 49.2%, 44.7%, and 31.0) 42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and V. campbellii, respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii. Many predicted proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%). Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes among diverse Vibrio spp. The Vibrionaceae are gram-negative Gammaproteobacteria associated with well-characterized human and fish pathogens. that occur in temperate to tropical, coastal, and estuarine ma- Among these are plasmids isolated from V. anguillarum (20, rine systems (62). Vibrio spp. occupy a diverse range of eco- 67), V. cholerae (46, 47), V. vulnificus (14), V. parahaemolyticus logical niches, including sediments, the water column, and in (41), and V. salmonicida. The lack of plasmid sequence data, association with organisms either as symbionts (48) or patho- particularly of plasmids from Vibrio hosts isolated from coastal gens (26, 37). Phages contribute to Vibrio evolution and ecol- water and sediment, limits our understanding of Vibrio plasmid ogy by regulating host abundance (29) and transferring viru- evolution and diversity. lence genes, such as the cholera toxin encoded by ctxAB of the In the present study we provide a comparative assessment of CTX␾ phage of V. cholerae (64). Plasmids such as pJM1 of V. plasmids with diverse sizes and gene contents isolated from anguillarum (20) have also been shown to play a role in Vibrio vibrios. Similarities of replication initiation and hypothetical pathogenicity. In recent years, sequencing has revealed the vast proteins revealed relatedness of plasmids from vibrios occupy- diversity of phage genomes (10) and their globally significant ing diverse niches. In addition, these elements contained nu- contributions to horizontal gene transfer within marine envi- merous phage-like proteins, including proteins with consider- ronments (35). In contrast to the demonstrated genetic diver- able similarity and conserved gene order to enterobacteria sity of vibriophages (16, 66), much less is known of Vibrio phage P1. To our knowledge, this is the first report of P1-like plasmid diversity and the role of plasmids in gene transfer. A phage sequences isolated from a marine bacterium. A previous few studies have reported the occurrence of plasmids among study identified two P1-like genes as part of a marine viral Vibrio populations (19–21, 44, 63), and several have reported metagenome (10); however, no additional P1 genes or nearly complete sequences of Vibrio plasmids associated with patho- complete P1 genomes have been characterized from the ma- genic vibrios; however, the distribution and sequence diversity rine environment. of Vibrio plasmids has not been studied as extensively as vibrio- phages. MATERIALS AND METHODS As of September 2007, there are 16 plasmid and 20 phage Bacterial strains, media, and plasmid isolation. Vibrio sp. strains 0908, 23023, sequences in GenBank that were isolated from vibrios (12–14, and 09022 were isolated from salt marsh sediment of Charleston, SC, in Decem- 20, 21, 23, 25, 27, 28, 31, 38, 41, 43, 45–48, 51, 67). These ber 1998 (17). DNA for sequencing was obtained by purification of supercoiled sequences are biased toward small elements (i.e., nine plas- plasmid DNA by cesium chloride density gradient centrifugation as previously Ͻ Ͻ described (52). mids of 8 kb and 10 phages of 9 kb) and are primarily Plasmid sequencing and sequence analysis. Plasmids were sequenced using whole-genome shotgun sequencing and finishing methods (26). Initial open read- ing frame designations and annotation of select open reading frames was done * Corresponding author. Mailing address: 311 Ferst Drive, Atlanta, using an automated annotation system (26). Protein-coding sequences (CDSs) GA 30332-0230. Phone: (404) 894-5819. Fax: (404) 385-4440. E-mail: were confirmed by independent analysis using GeneMark software (7). Putative [email protected]. similarity to known proteins was determined by amino acid sequence comparison † Supplemental material for this article may be found at http://aem and identification of common motif and domain structure using a combination of .asm.org/. PSI-BLAST (3) from the National Center for Biotechnology Information, ᰔ Published ahead of print on 5 October 2007. SMART (50), COG (57), and Pfam (6) Web-based software. PSI-BLAST anal- 7703 Downloaded from https://journals.asm.org/journal/aem on 20 August 2021 by 73.2.105.168. 7704 HAZEN ET AL. APPL.ENVIRON.MICROBIOL. FIG. 1. A concatenation of 16S rRNA and rpoA nucleotide sequences of the plasmid hosts, Vibrio sp. strains 0908, 23023, and 09022, was used to determine relatedness of the hosts to other Vibrio spp. as examined in a previous study (60). The neighbor-joining method with the Jukes-Cantor model of distance estimation (30) was used to generate the tree with a concatenation of 16S rRNA (1,452 nucleotides) and rpoA (772 nucleotides) sequences. Bootstrap values represent 1,000 replications, and only those with values of Ն50 are shown. ysis was performed with the default threshold E-value of 0.005 and a maximum (11). To date, the only report of mobile genetic elements threshold of 1.0 over one to two iterations. ClustalW was used to generate all (MGEs) associated with any of these Vibrio species is an SXT- alignments (61). Phylogenetic analyses and sequence alignments. Host strains were identified like element of V. fluvialis with similarity to the multiple anti- by a concatenated phylogenetic analysis of 16S rRNA and rpoA nucleotide biotic resistance element SXT previously characterized from V. sequences as previously described (18). The neighbor-joining tree was generated cholerae (2). This previous study indicated there may be trans- using MEGA with the Jukes-Cantor (30) distance estimation model with 1,000 fer of MGEs among well-characterized pathogens such as V. replications for the nucleotide concatenation or the Poisson correction for the amino acid RecA tree (42). Percent identities of the nucleotide sequences to the cholerae and emerging marine pathogens such as V. fluvialis most related organism were determined using BLASTN (3) and BLAST2 (58) (11, 34, 55). sequences. Sequencing was performed by the University of Nevada, Reno, The nucleotide sequences of the Vibrio plasmids p0908, Genomics Center and the Core Genomics Facility at the Georgia Institute of p23023, and p09022 were 81,413 bp, 52,527 bp, and 31,036 bp Technology. ϩ Identification of phage-like proteins. Prophage Finder (9) was used with in length with overall G C contents of 49.2%, 44.7%, and BLAST analysis (3, 49) of a phage sequence database to identify prophages and 42.4%, respectively (see Table S1 in the supplemental mate- proteins with similarities to phage-associated proteins for all sequenced Vibrio rial). With the exception of p0908, the GϩC contents of the plasmids available in GenBank as of July 2007. An E value of 0.001 with 10 plasmids were within the range of percentages reported for hits/prophage and a hit spacing of 3,500 were used as parameters for all plasmids examined. Vibrio genomes (38 to 47%) (14, 26, 37, 48). The plasmids Nucleotide sequence accession numbers. The plasmid sequences have been p0908, p23023, and p09022 encoded 99, 64, and 32 predicted submitted to the GenBank database under accession numbers CP000755 to CDSs, respectively (see Table S1 in the supplemental mate- CP000757. All additional sequences have been submitted to the GenBank data- rial). The predicted proteins were assigned primarily to the base under accession numbers EU022567 to EU022572. following functional categories: replication, stable mainte- nance, partitioning, and recombination. Additional predicted RESULTS AND DISCUSSION proteins identified on one or more of the plasmids may be Host phylogeny and plasmid features. In this study we ex- involved in mobilization, restriction modification, or transcrip- amined the sequence diversity of plasmids previously isolated tional regulation (see Tables S1 to S3 in the supplemental from three Vibrio hosts (17). A concatenation of 16S rRNA material). The only genes common to at least two of the three sequences and rpoA nucleotide sequences was used for greater plasmids were the putative replication initiation and partition- resolution of related Vibrio spp. (60). The 16S rRNA and rpoA ing proteins. The predicted replication initiation protein of nucleotide sequences of Vibrio sp.