The genus Salinivibrio constitutes a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and includes moderate halophilic bacteria. This genus was created to accommodate the species Vibrio costicola based on the significant phenotypic and genotypic differences between this and other species of the genus Vibrio (Mellado et al. 1996). Currently, the genus Salinivibrio comprises four species, one of them with three subspecies: S. costicola subsp. costicola (Smith 1938; Mellado et al., 1996; Huang et al., 2000), S. costicola subsp. vallismortis (Huang et al., 2000), S. costicola subsp. alcaliphilus (Romano et al., 2005), Salinivibrio proteolyticus (Amoozegar et al., 2008), Salinivibrio siamensis (Chamroensaksri et al., 2009) and Salinivibrio sharmensis (Romano et al., 2011).
This genus comprises Gram-negative, obligatory halophilic bacteria, most of them growing between 5 and 20 % NaCl, 10 and 45 ºC and pH 5.0 and 9.0. They are facultative anaerobes. Moreover, S. costicola is considered as a model microorganism for studying osmoregulation and other physiological mechanisms in moderate halophiles (Oren 2003; Amoozegar et al., 2008). Therefore, members of this genus have developed cellular mechanisms to thrive in extremophilic conditions, such as hypersalinity (Garcia et al., 1987, Huang et al., 2000). Also, these microorganisms are facultative anaerobes and positive for catalase and oxidase tests. The DNA G+C content ranges from 49.0 to 51.0 mol% (Mellado et al., 1996; Huang et al., 2000; Romano et al., 2005; 2011; Amoozegar et al., 2008; Chamroensaksri et al., 2009). Besides, members of the genus Salinivibrio have been isolated from a wide variety of habitats, such as marine and hypersaline water, lagoons, lake, saline soil, salterns, salted food products, animals such as Artemia spp., etc.
The phylogeny of the family Vibrionaceae based on a 16S rRNA gene approach is confusing since closely related species within the family cannot always be distinguished reliably due to the high levels of sequence conservation. Several studies have shown that the 16S rRNA gene does not provide sufficient resolution for differentiation between closely related bacterial species (Thompson et al., 2005; 2008; Staley, 2006; Pascual et al., 2010; Gao et al., 2016). The classification of the genus Salinivibrio needs reappraisal since the three subspecies of Salinivibrio costicola do not form a monophyletic group; this conclusion is supported by antecedent studies carried out in the genus Salinivibrio that have shown one of the subspecies, S. costicola subsp. vallismortis, is not related to the other two, but forms a monophyletic group with another species of the genus, S. proteolyticus (Chamroensaksri et al., 2009; Amoozegar et al., 2008; Romano et al., 2011; Gorriti et al., 2014).
So as to resolve these problems, it has been demonstrated in a number of studies that concatenating the sequences of several protein-encoding gene fragments, i.e., a multilocus sequence analysis (MLSA), provides a more robust tree topology and an improved understanding of speciation events compared to a tree based solely on 16S rRNA gene sequences (Thompson et al., 2005; 2007; Sawabe et al., 2013; Dubert et al., 2016; González-Castillo et al., 2016).
The purpose of this Doctoral Thesis has been to study in detail the phylogenetic relationships of the species of the genus Salinivibrio to clarify the current classification of this genus by using several molecular approaches such as MLSA studies, DNA-DNA hybridization, phenotypic characteristics and the use of genomic data.
To begin with a total of two hundred eighty-eight strains were isolated of several solar salterns of different areas of the Spanish geographic (Alicante, Huelva, Granada, Mallorca) and of Puerto Rico. After the analysis of 16S rRNA a collection of seventy strains belonging to the genus Salinivibrio was obtained. Besides, type strains of this genus were used in this study too. With the objective to clarify the current classification of this genus, a MLSA approach was used basing of gyrB, recA, rpoA and rpoD sequences. These genes were selected based on previous studies in the family Vibrionaceae (Thompson et al., 2005; 2008; Pascual et al., 2010).
Phylogenetic trees based on the concatenation of these four housekeeping genes show that these seventy-six strains constituted four different phylogroups, with only one strain belonging to the species Salinivibrio sharmensis (with a high bootstrap value of 100%), which cannot be includedwithin any phylogroup since we could not isolate any strain belonging to this species and, therefore, S. sharmensis constitutes a phylotype. In the case of the phylogroup 1, this is composed for a group of strains of differents places of isolation but not from any species, therefore, this phylogroup can be possible to constitute a new species of the genus Salinivibrio. On the basis of this study, we have carried out a complete polyphasic investigation that has led us to propose the description of new taxa. On the other hand, the phylogroup 4 was constituted by five strains isolated from Huelva and Puerto Rico and by species S. proteolyticus DSM 11403T and subspecies S. costicola subsp. vallismortis DSM 8285T. We performed an exhaustive study to the aim to clarify the phylogenetic position of these species.
To validate MLSA study, DNA-DNA hybridization (DDH) was carried out in thirty-three strains, including the type strains. The DDH percentage values for strains within the same phylogroup were always above 70 %, a value established as cut-off for species delineation (Wayne et al., 1987; Stackebrandt and Goebel, 1994), confirming that they belong to the same species. DDH analyses among phylogroups always showed values lower than 70 %, indicating that each phylogroup constitutes a different species. After confirm that there was a correlation between both studies (MLSA and DDH), a value of 97 % was proposed as a cut-off for the genus Salinivibrio for MLSA study.
Besides, in this investigation genome fingerprinting analysis was performed to observe if these strains constituted clones. This study showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation.
After the MLSA analysis, an in-depth study of phylogroup 1 was carried out to the aim to verify these strains constituted a new species of the genus Salinivibrio. For that a polyphasic study was performed; including DNA-DNA hybridization, phenotypic characteristics and genomic data of the strains which genomes were sequenced. Afterward these studies, it was concluded that these strains constituted a new species of this genus, which name S. kushneri sp. nov. was proposed. As it mentioned above, the phylogroup 4 was constituted for the subspecies S. costicola subsp. vallismortis DSM 8285T, species S. proteolyticus DSM 11403T and the strains IB574, IB872, PR5, PR919 and PR932. Following the polyphasic studies and the genomic data; it concluded that both, species and subspecies, they were a single species, which was emended as S. vallismortis com. nov., ascending to range of species the subspecies S. costicola subsp. vallismortis DSM8285T; so the species S. proteolyticus constitute later heterotypic synonym of S. costicola subsp. vallismortis.
On the other side, a total of thirty-two strains were selected to sequence their genomes. The study of these genomes was performed, together with the type strain of the genus Salinivibrio, S. costicola subsp. costicola ATCC 33508, which were available from the GenBank database.
Based on different index (ANIb, ANIm, OrthoANI) and DDH in silico, these strains were classified as five different species belonging to the genus Salinivibrio. Each species correlated with each phylogroup from the MLSA study. Besides, phylogenomic study based on the core-genome showed a phylogenetic tree in which it was possible differentiates four phylogroup, the same as in the MLSA study.
Among the most represented subsystems of the members of the genus Salinivibrio are the genes involved in the synthesis and regulation of the flagellum, this is due to the polar flagellum that these strains have at one end and that allows them mobility.
In addition, this study of the genomes has permitted us to determine, using recruitments obtained from metagenomic databases of environments with intermediate salinities, that these strains are not abundant in these habitats, despite growing fast in the laboratory and being easily isolated from hypersaline environments
