Salirhabdus euzebyi gen. nov., sp. nov., a Gram-positive, halotolerant bacterium isolated from a sea salt evaporation pond

Gram-positive rod-shaped, aerobic or facultatively anaerobic, spore-forming bacteria that grow optimally in media containing NaCl are currently assigned to 19 genera with similar characteristics and close phylogenetic relationships (Nunes et al., 2006). During a survey of bacterial diversity present in sea salt evaporation pond samples collected on the island of Sal in the Cape Verde Archipelago, several slightly halophilic Gram-positive bacteria were isolated and characterized further. One of these isolates shared several physiological and biochemical characteristics with other species of halophilic bacilli and was phylogenetically most closely related to species of the genus Salinibacillus (Ren & Zhou, 2005). However, this organism had a distinctly lower NaCl requirement for optimal growth and a distinctive fatty acid content. Furthermore, 16S rRNA gene sequence analysis indicated that this organism represents a novel phylogenetic lineage within the radiation of the genus Bacillus and related genera. Based on morphological, physiological, chemotaxonomic and phylogenetic characteristics, we describe a novel species in a new genus to accommodate strain CVS-14^T.

Strain CVS-14^T was isolated from a sea salt evaporation pond on solid R3A-V medium (Tiago et al., 2006), pH 7.0, containing 2 % NaCl (w/v). Soil samples (3 g) were resuspended on sterile blenders with sterile 2 % NaCl. One drop of the homogenate was spread on R3A-V agar plates. These preparations were incubated at 37 °C for up 5 days. The R3A-V medium contained the following components (l⁻¹): 1.0 g yeast extract (Difco), 1.0 g proteose peptone (Difco no. 3), 1.0 g Casamino acids, 1.0 g glucose, 0.6 g K₂HPO₄, 0.1 g MgSO₄.7H₂O, 0.05 g Na pyruvate, 15.0 g agar (Difco), 50 ml of a 10-fold concentrated macronutrients solution, 5 ml of a 100-fold concentrated trace elements solution and 100 ml of 1 M phosphate buffer (pH 7.0); macronutrients, trace elements and the phosphate buffer were autoclaved separately and added to the cooled R3A agar. The 10-fold concentrated macronutrients solution contained (l⁻¹): 1.0 g nitrilotriacetic acid, 0.6 g CaSO₄.2H₂O, 1.0 g MgSO₄.7H₂O, 0.8 g NaCl, 1.03 g KNO₃, 6.89 g NaNO₃ and 1.11 g NaHPO₄. The 100-fold concentrated trace elements solution contained (l⁻¹): 0.22 g MnSO₄.H₂O, 0.05 g ZnSO₄.7H₂O, 0.05 g H₃BO₃, 0.0025 g CuSO₄.5H₂O, 0.0025 g Na₂MoO₄.2H₂O and 0.0046 g CoCl₂.6H₂O.

Despite repeated attempts to isolate additional strains, only one isolate was obtained. Cultures were purified by subculturing on the same medium and maintained at −70 °C in Degryse medium 162 containing 3 % NaCl and 15 % (w/v) glycerol (Degryse et al., 1978). The organism was also routinely cultured in Degryse medium 162 containing 5 % NaCl at 37 °C for up to 5 days, unless stated otherwise (Albuquerque et al., 2005). Paucisalibacillus globulus B22^T was used for comparative purposes; the type strains of Salinibacillus kushneri and Salinibacillus aidingensis were not available from the Japanese Collection of Microorganisms (JCM).

The growth temperature range of strain CVS-14^T was examined in liquid medium in a reciprocal water bath shaker between 10 and 55 °C; the pH range for growth was determined at 37 °C in the same medium buffered at pH values between 5.5 and 10.5 by using 20 mM MES, Tris and CAPSO (Sigma). The NaCl range for growth was determined in liquid medium without additional NaCl and with up to 20 % NaCl. Enzymic activities were determined by using the API ZYM system (bioMérieux) at 37 °C. Anaerobic growth was assessed in solidified Degryse medium with 5.0 % NaCl and 1.0 g KNO₃ l⁻¹ in anaerobic chambers (GENbox anaer; bioMérieux). Single carbon source assimilation tests were performed in a defined medium composed of Degryse basal salts containing 5.0 % NaCl, 0.1 g yeast extract l⁻¹, 0.5 g ammonium sulfate l⁻¹, 2.0 g single carbon sources l⁻¹ and 2 % deionized water-washed agar (Oxoid). Cultures (24 h) from plates were resuspended in the basal salts medium (turbidity equivalent to a McFarland no. 1 standard) and a single drop of the suspension was placed on plates, each of which contained a single carbon source. Growth was examined visually for up to 5 days. Negative control plates did not include a carbon source. Carbon source assimilation profiles were also investigated in liquid media with the same composition indicated above, but this proved unsuccessful owing to the absence of growth or clumping of cells. Acid production from carbohydrates was determined by using the API 50 CH system (bioMérieux) according to the manufacturer's instructions, with API 50 CHB/E medium containing 5.0 % NaCl. Results were recorded after 24 h, 48 h and 5 days incubation at 37 °C.

Peptidoglycan analysis was performed according to the method of Schleifer & Kandler (1972) and Schleifer (1985); respiratory quinone analysis was performed according to the method of Tindall (1989). Cultures for fatty acid analyses were grown on solidified Degryse medium 162 containing 5 % NaCl incubated in sealed plastic bags submerged in a water-bath at 37 °C for 48 h. Fatty acid methyl esters were extracted as described by Moreira et al. (2000). Identification and quantification of fatty acid methyl esters, as well as numerical analysis of the fatty acid profiles, were performed by using the standard MIS Library Generation Software (Microbial ID). Polar lipid analyses were performed as described by Prado et al. (1988).

The G+C content of the DNA was determined by HPLC as described by Mesbah et al. (1989). The 16S rRNA gene was amplified by PCR and sequenced as described by Rainey et al. (1996). Phylogenetic analyses were performed using the arb software package (Ludwig et al., 2004) and a phylogenetic tree was constructed by using the neighbour-joining (Saitou & Nei, 1987) algorithm. Tree topologies were evaluated by performing bootstrap analysis (Felsenstein, 1985) of 1000 resamplings of the data set.

Comparative analyses of 1479 nt positions of the 16S rRNA gene sequence of strain CVS-14^T with those of other lineages within the low-G+C Gram-positive bacteria showed that the new isolate clustered with other moderately halophilic bacilli. Strain CVS-14^T showed highest 16S rRNA gene sequence similarities to species of the genus Salinibacillus, 95.7 % to S. aidingensis JCM 12389^T and 95.14 % to S. kushneri JCM 12390^T. The next closest relative was P. globulus B22^T with 94.9 % 16S rRNA gene sequence similarity. Similarity values in the range 90.3–95.2 % were found to members of the genera Lentibacillus, Oceanobacillus and Virgibacillus. Strain CVS-14^T grouped with species of the genus Salinibacillus but the branching point was not fully supported, with a bootstrap value of 69 % (Fig. 1⇓). The G+C content of the DNA of strain CVS-14^T was 37.0 mol%.

Strain CVS-14^T formed non-pigmented colonies and Gram-positive, motile, long rod-shaped cells (0.3–0.4 μm in width by 2.9–8.6 μm in length) with oval endospores at a terminal position within a swollen sporangium, giving the cells the appearance of spermatozoids (Fig. 2⇓). The optimum NaCl concentration for growth was 4–6 %, but good growth occurred both in the absence of additional NaCl and in medium containing up to 16 % NaCl (see Supplementary Fig. S1 in IJSEM Online). Species belonging to the genus Salinibacillus, in contrast with strain CVS-14^T, require 1 % NaCl for growth and have higher optimum and maximum NaCl ranges for growth (Table 1⇓).

Strain CVS-14^T was oxidase-, catalase- and DNase-positive and hydrolysed gelatin, hippurate and Tweens 20, 40, 60 and 80. Aesculin, arbutin, casein, elastin, starch and xylan were not hydrolysed. Yeast extract was required for growth on single carbon sources. Strain CVS-14^T assimilated carbohydrates, polyols, organic acids and amino acids. Acid was produced from several carbohydrates. Nitrate and nitrite were reduced. Anaerobic growth in the presence of nitrate was not observed.

Strain CVS-14^T had a cell-wall type A1γ peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid, which is common in the majority of endospore-forming Gram-positive rods. The polar lipid pattern on TLC revealed the presence of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol (see Supplementary Fig. S2 in IJSEM Online). The major respiratory lipoquinone was menaquinone 7 (MK-7). The fatty acid profile of strain CVS-14^T, as well as those of species of the genus Salinibacillus, was dominated by branched fatty acids, namely iso-15 : 0, anteiso-15 : 0, iso-17 : 0 and anteiso-17 : 0 (Table 2⇓). However, strain CVS-14^T and species of the genus Salinibacillus could be distinguished based on the relative amounts of these fatty acids. Moreover, strain CVS-14^T possessed acyl compounds such as 16 : 1ω7c alcohol, iso-17 : 1ω10c and summed feature 4 (iso-17 : 1 I and/or anteiso-17 : 1 B) that are not detected in the type strains of the two other recognized species of the genus Salinibacilus (Ren & Zhou, 2005) or in the type strain of P. globulus. Strain CVS-14^T could also be distinguished from other slightly halophilic endospore-forming bacilli (Tables 1 and 2⇑⇓).

Despite similar characteristics and phylogenetic relatedness to several moderately halophilic bacilli, strain CVS-14^T could be clearly distinguished from the type strains of species of the related genera Paucisalibacillus and Salinibacillus, namely based on NaCl requirements for optimal growth, fatty acid content as well as other phenotypic characteristics and, for the genus Paucisalibacillus, a different cell-wall type.

On the basis of these findings, strain CVS-14^T is considered to represent a novel species in a new genus, for which the name Salirhabdus euzebyi gen. nov., sp. nov. is proposed.