Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes

Abstract

Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes The central clade of the genus Vibrio, also called the Vibrio core group, comprises six species that are tightly related (DNA-DNA reassociation values are very close to 70 % for most species pairs). Identification of novel strains to the species level within this group is troublesome and results are quite often dependent on the methodology employed. Therefore, this group represents an excellent framework to test the robustness of multilocus sequence analysis (MLSA) not only for inferring phylogeny but also as an identification tool without the need for DNA-DNA hybridization assays. The genes selected, 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR, were amplified by direct PCR from 44 Vibrio core-group strains. Subsequent analysis allowed us to recognize toxR and rpoD as the most resolving individual genes and showed that concatenated sequences of rpoD, rctB and toxR were more useful than concatenated sequences of all seven genes. To validate our conclusions, MLSA similarities have been correlated with DNA-DNA relatedness values obtained in this study and values taken from the literature. Although the seven concatenated genes gave the best correlation, the concatenated sequences of rpoD, rctB and toxR have the practical advantage of showing a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity recorded, meaning that they can be used quite conveniently for species identification of vibrios. INTRODUCTIONMLSA (multilocus sequence analysis) is a recently developed technique derived from multilocus sequence typing that has been used successfully in epidemiology studies. The use of MLSA as an alternative method for species delineation in bacteriology is currently being evaluated, following a recommendation of the ad hoc committee for the re-evaluation of the species definition in bacteriology (Stackebrandt et al., 2002). In MLSA studies, several housekeeping genes (more than five) are analysed and relationships between taxa are established. One important advantage of this kind of approach is the availability of genomic sequences from any laboratory, avoiding the problems of lack of comparability that are associated with DNA-DNA reassociation data. However, the usefulness of MLSA for describing bacterial species has to be demonstrated for each of the taxa or group of species studied and for the selected housekeeping genes. Authors should prove that there is a sufficient degree of congruence between the alternative technique (i.e. MLSA) and DNA-DNA reassociation data. To date, the technique has been applied to discrete bacterial taxa, mainly genera of lactic acid bacteria (Naser et al. et al., 2007, 2008) and the Vibrionaceae (Goarant et al., 2006;Urbanczyk et al., 2007;Thompson et al., 2008;Rameshkumar et al., 2008).In the family Vibrionaceae, sequence similarities for the 16S rRNA gene are ¢97.6 % among members of the so-called Vib...

Photobacterium aphoticum sp. nov., isolated from coastal water

“…Cell pellets of each strain were collected and DNA was extracted using a microbial DNA isolation kit (Ultra Clean; MO BIO Laboratories). Amplification and partial sequencing of 16S rRNA, recA and gyrB genes were performed as described previously (Lucena et al, 2010;Pascual et al, 2010). The new sequences were compared with those available in gene databases using the NCBI BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).…”

Fermentative Production of Pyranone Derivate I from Marine Vibrio sp. SKMARSP9: Isolation, Characterization and Bioactivity Evaluation

“…In this regard our study is in sync with previous studies. Recently, the use of concatenated multi-gene analyses has led to considerable advances unravelling the phylogeny of Vibrio's [19,24,25]. The 16S rRNA sequence of this strain has been deposited at EMBL under the accession number HE798514.…”

Biochemical and Molecular Characteristics of Vibrio anguillarum Isolated from Alaska Pollock Theragra chalcogramma Seedlings

“…(Chang et al, 2008;Fabbro et al 2010). Pascual et al (2010) 16S rDNA sequences , Vibrio core group 6 (V. alginolyticus, V. campbellii, V. harveyi, V. parahaemolyticus, V. natriegens, V. rotifierianus) 16S rDNA sequences 97.6% 16S rDNA . Thompson et al (2005) pyrH recA gene PCR sequencing (Genbank accession no.…”