Evidence for the Role of Horizontal Transfer in Generating pVT1, a Large Mosaic Conjugative Plasmid from the Clam Pathogen, Vibrio tapetis

Gaël Erauso,Patrick Le Chevalier,Fatma Lakhal,Christine Paillard,Adeline Bidault-Toffin,Annick Jacq,Philippe Bouloc,Michael Hensel

doi:10.1371/journal.pone.0016759

Abstract

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae.

Highlights

Vibrios are ubiquitous bacterial species in marine ecosystems
Cells were eluted from the filters in 1 ml LBS and 100 ml were spread on LBS +2 mg/ml chloramphenicol (V. harveyi) or LB+20 mg/ml chloramphenicol (E. coli) and incubated at 37uC
85 kb in size, as determined after pulse-field gel electrophoresis. pVT1 digested by NotI was religated between the two NotI sites of pBeloBac11 [33], giving pBac17. pBac17 DNA as well as cesium chloride preparation of pVT1 DNA from V. tapetis were used for sequence determination. pVT1 is 82,266 bp in size and contains 88 predicted ORFs of more than 50 amino acids (Table S1 and Fig. 1)

Summary

Introduction

Vibrios are ubiquitous bacterial species in marine ecosystems They constitute a genetically complex group, associated with a variety of ecological niches, as free-living organisms or in association with various hosts, the interactions ranging from symbiosis to pathogenicity. The majority of Vibrio species are symbionts, commensals or pathogens of fish and shellfish and among bacterial diseases, vibrioses are arguably the most important infections in mollusks (Cf. review by Paillard [4]). Cells were eluted from the filters in 1 ml LBS and 100 ml were spread on LBS +2 mg/ml chloramphenicol (V. harveyi) or LB+20 mg/ml chloramphenicol (E. coli) and incubated at 37uC This temperature was effective to counter-select V. tapetis since this species does not grow above 25uC. Putative exconjugants were purified twice on the same medium, before genomic and plasmid DNA extraction for further analysis

Methods

Results

Conclusion