Allobacillus halotolerans gen. nov., sp. nov. isolated from shrimp paste

During the characterization of micro-organisms present in shrimp paste, a white-transparent bacterial strain, designated B3A^T, was isolated and incubated on marine 2216 agar (MA; BD Difco) or in marine 2216 broth (MB; BD Difco) at 37 °C for 2 days. Subcultivation was done on MA at 37 °C for 24 h to 48 h. On this medium, strain B3A^T was able to grow at 20–45 °C but not at 15 or 50 °C. Halalkalibacillus halophilus BH2^T was obtained from the Japan Collection of Microorganisms (JCM) and used as a reference strain for phenotypic and genotypic tests.

The 16S rRNA gene sequence of strain B3A^T was determined and analysed as described previously (Chen et al., 2001). Analysis of the sequence data was performed with the software packages BioEdit (Hall, 1999) and mega version 3.1 (Kumar et al., 2004), after multiple alignments of the data by clustal x (Thompson et al., 1997).

The 16S rRNA gene sequence of strain B3A^T was a continuous stretch of 1431 bp. Phylogenetic analyses (distance options according to the Kimura-2 model; Kimura, 1983) were carried out using the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris, 1969) and maximum-likelihood (Felsenstein, 1981) methods. The neighbour-joining tree was reconstructed with a distance matrix calculated using the method described by Jukes & Cantor (1969). The stability of the tree topology was evaluated by bootstrap analysis (based on 1000 replications). 16S rRNA gene sequence analysis indicated that strain B3A^T belonged to the class Bacilli and was a member of the family Bacillaceae. Sequence similarity calculations using BioEdit software (over 1400 bp) indicated that strain B3A^T is distinct from any other genera in the family Bacillaceae and showed low levels of 16S rRNA gene sequence similarity (<94.0 %) with the members of the genera Alkalibacillus, Aquisalibacillus, Bacillus, Filobacillus, Halalkalibacillus, Piscibacillus and Tenuibacillus (Fig. 1 and Supplementary Fig. S1, available in IJSEM Online). The novel strain was closely related to H. halophilus BH2^T (Echigo et al., 2007) (93.8 % 16S rRNA gene sequence similarity). The overall topologies of the phylogenetic trees obtained either with neighbour-joining, maximum-likelihood or maximum-parsimony methods were similar. Since strain B3A^T and H. halophilus BH2^T formed a stable clade, the latter was used as a reference strain for subsequent phenotypic and genotypic analyses.

The morphology of bacterial cells was observed by phase-contrast microscopy (Leica DM 2000) and scanning electron microscopy (S-3500N; Hitachi) (Supplementary Fig. S2) using cells grown in MB at 37 °C for 6 h (lag growth phase), 18 h (exponential phase) and 36 h (stationary phase). Motility was tested by the hanging drop method. Flagellar staining was performed using the Spot Test flagella stain (BD Difco). The Gram stain set (BD Difco), the Ryu non-staining KOH method (Powers, 1995) and Bactident Aminopeptidase test strips (Merck) were used to ascertain Gram reaction. Presence of endospores was checked by the method of Wirtz-Conklin (Murray et al., 1999). Accumulation of poly-β-hydroxybutyrate granules was observed by light microscopy after staining cells with Sudan black. Colony morphology was examined using a stereoscopic microscope (SMZ 800; Nikon). Details of cell morphology are given in the species description.

The optimum pH range for growth was determined in MB using appropriate biological buffers such as glycine/HCl, citrate/Na₂HPO₄, phosphate buffer and glycine/NaOH for adjusting the pH to 3.0–4.0, 4.0–8.0, 6.0–8.0 and 9.0–11.0 (at 1.0 pH unit intervals), respectively. pH was adjusted prior to sterilization and post-sterilization controls revealed only minor changes. NaCl requirement was determined using nutrient broth (BD Difco) containing 0, 0.5 and 1.0–20.0 % (w/v) NaCl (at 1.0 % intervals). The temperature range for growth was examined at 4, 10, 15, 20, 25, 30, 35, 37, 40, 42, 45 and 50 °C in MB. Growth was determined by measuring the turbidity (OD₆₀₀) of cultures grown at various pH, NaCl concentrations and temperatures. Anaerobic growth was assessed using an Oxoid AnaeroGen system (Miller et al., 1995).

Catalase, oxidase and DNase activities and hydrolysis of starch, casein, gelatin and Tweens 20, 40, 60 and 80 were determined using standard methods (Gerhardt et al., 1994). The commercially available API 20NE, API ZYM (bioMérieux) and MicroPlate GP2 (Biolog) microtest systems were used, according to the manufacturers’ instructions, to determine biochemical properties, enzyme activities and carbohydrate oxidation pattern. The API ZYM strip was read after 4 h of incubation at 37 °C, whereas API 20NE and MicroPlate GP2 were read after 72 h at 37 °C. The three commercial systems were inoculated with a cell suspension in artificial seawater.

Polar lipids were analysed by two-dimensional TLC as described by Ventosa et al. (1993). The cellular polar lipids of strain B3A^T were diphosphatidylglycerol, phosphatidylglycerol, minor to trace amounts of two unknown phospholipids and one aminolipid (Supplementary Fig. S3).

Fatty acid methyl esters were prepared, separated and identified according to the instructions of the Microbial Identification System (Microbial ID; Sasser, 1990). The fatty acid constituents of strain B3A^T were 16 : 0 iso (28.8 %), 15 : 0 anteiso (28.0 %), 14 : 0 iso (14.6 %), 17 : 1 anteiso ω9c (6.8 %), 17 : 0 anteiso (5.6 %), 15 : 0 iso (5.3 %), 16 : 1 ω7c alcohol (2.5 %), 16 : 0 (2.0 %), 18 : 0 (1.9 %) and 18 : 1 iso H (1.7 %) (Supplementary Table S1).

Isoprenoid quinones of strain B3A^T were extracted and separated as described by Minnikin et al. (1984) and analysed by HPLC as described by Collins (1985). Strain B3A^T contained menaquinone MK-7 as the major respiratory quinone (at a peak area ratio of approximately 93 %) and MK-6 as the minor respiratory quinone (at approximately 7 %). The diagnostic cell-wall amino acid of strain B3A^T was determined by TLC (Staneck & Roberts, 1974). The isolate contained meso-diaminopimelic acid.

For guanine-plus-cytosine (G+C) content determination, DNA was prepared and degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was then separated by high-performance liquid chromatography. The DNA G+C content of strain B3A^T was 45.3±1.0 mol%.

Sensitivity of strain B3A^T to different antibiotics was analysed by the diffusion method on MA. The following antibiotic discs (Oxoid) were used: ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G (10 U), streptomycin (10 µg) and tetracycline (30 µg). The effect of antibiotics on cell growth was assessed after 2 days of incubation at 37 °C and susceptibility was scored based on the distance from the edge of the clear zone to the disc.

Detailed results of the physiological characterization of strain B3A^T are provided in the genus and species descriptions and in Table 1. Analysis of the 16S rRNA gene sequence showed that the isolate represented a novel lineage within the family Bacillaceae. Phenotypic properties, such as reduced NaCl range for growth, non-alkaliphilic nature, presence of C4 esterase, trypsin and α-chymotrypsin activities, and its ability to oxidize more carbohydrates in the Biolog GP2 test system (Table 1), distinguish strain B3A^T from H. halophilus BH2^T. The quantities of fatty acids 15 : 0 iso and 17 : 0 anteiso in strain B3A^T are much lower than those of H. halophilus BH2^T, while the quantity of fatty acid 14 : 0 iso is much higher (Table 1 and Supplementary Table S1). As well as these differences, strain B3A^T shows a very high DNA G+C content (45.3 mol%) compared to H. halophilus BH2^T (35.1 mol%).

In addition, several chemotaxonomic and physiological properties, such as Gram-staining, cell motility, alkaliphilic nature, optimal growth pH, ability to grow at pH 10, with 0.5 % NaCl or at 45 °C, presence of oxidase activity, ability to reduce nitrate to nitrite, cell-wall peptidoglycan type/variant and the distinctive major fatty acid, distinguish strain B3A^T from its closely related phylogenetic neighbours, Alkalibacillus, Aquisalibacillus, Filobacillus, Piscibacillus and Tenuibacillus (Supplementary Table S2). On the basis of these results, we suggest that strain B3A^T represents a novel genus and species in the family Bacillaceae, for which the name Allobacillus halotolerans gen. nov., sp. nov. is proposed.