Introduction

The genus Bacillus comprises more than 222 species with validated names (http://www.bacterio.net). Therefore, classification and identification of Gram-positive, endospore-forming rods should be performed by using a polyphasic taxonomic approach that integrates phylogenetic analysis based on 16S rRNA gene sequences, genomic relatedness and an extensive range of phenotypic characteristics (Yoon et al. 1998; Tcherpakov et al. 1999). For a long time, most aerobic, endospore forming rods were assigned to the genus Bacillus (Claus and Berkeley 1986). Subsequently 16S rRNA gene sequence analyses revealed the presence of several phylogenetically distinct lineages within the genus Bacillus. Consequently, some phylogenetic groups have been established as new genera such as Alicyclobacillus (Wisotzkey et al. 1992), Paenibacillus (Ash et al. 1993), Aneurinibacillus (Shida et al. 1996), Brevibacillus (Shida et al. 1996), Virgibacillus (Heyndrickx et al. 1998), Salibacillus (Wainø et al. 1999), Ureibacillus (Fortina et al. 2001), Marinibacillus (Yoon et al. 2001a), Alkalibacillus (Jeon et al. 2005), Pullulanibacillus (Hatayama et al. 2006), Sporolactobacillus (Hatayama et al. 2006) and Viridibacillus (Albert et al. 2007). Moreover, several species of the genus Bacillus have been reclassified or transferred to the other genera such as Gracilibacillus (Wainø et al. 1999), Sporosarcina (Yoon et al. 2001b), Geobacillus (Nazina et al. 2001), Salimicrobium (Yoon et al. 2007), Lysinibacillus (Ahmed et al. 2007) and Rummeliibacillus (Vaishampayan et al. 2009). In addition to these, recently Glaeser et al. (2013) further proposed the genus Fictibacillus and reclassified Bacillus nanhaiensis, B. barbaricus, B. arsenicus, B. rigui, B. macauensis and B. gelatini as Fictibacillus nanhaiensis, Fictibacillus barbaricus, Fictibacillus arsenicus, Fictibacillus rigui, Fictibacillus macauensis and Fictibacillus gelatini comb. nov., respectively. In the course of our study on bacterial diversity, a Gram-stain positive, endospore-forming bacterial strain, designated strain NIO-1003T, was isolated from a marine sediment sample collected at Chorao Island, Goa, India and was the subject of a taxonomic investigation.

Materials and methods

Bacterial strains

Strain NIO-1003T, isolated on marine agar (MA) medium after about 2 weeks incubation at 30 °C, originates from a sediment sample collected from an intertidal region of mangroves at Chorao Island in Goa, India (GPS coordinates 15°32′34″N and 73°55′15″E). The strain was maintained on nutrient agar (NA) slants at 4 °C and as glycerol suspensions (20 %, v/v) at −80 °C. Biomass for chemical and molecular-systematic studies was obtained following growth in shake flasks (about 200 rpm) of Tryptic Soy broth, supplemented with the vitamin mixtures of the HV medium (Hayakawa and Nonomura, 1987) at 30 °C for 5 days.

Fictibacillus rigui KCTC 13278T and Fictibacillus solisalsi KCTC 13181T were obtained from Korean Type Culture Collection, Daejeon, South-Korea and cultured under comparable conditions as reference strains.

Phenotypic tests

Gram staining was carried out by using the standard Gram reaction and cell motility was confirmed by the development of turbidity throughout a tube containing semisolid medium (Leifson, 1960). Morphological features were determined on NA after 48 h at 30 °C. Strain NIO-1003T was observed with light microscopy (BH2; Olympus). For scanning electron microscopy examination, 1 ml samples were fixed overnight at 4 °C by adding formaldehyde to a final concentration of 7 %. Nine ml PBS (130 mM NaCl, 10 mM sodium phosphate, pH 7 ± 2) was added to the samples, which were then filtered through 0.2 μm Millipore filters and washed with PBS. The filters were then serially dehydrated in 25, 50, 70 and 100 % ethanol solutions (three times for 10 min at each stage), critical-point dried, mounted on scanning electron microscope stubs, sputter-coated with gold and viewed on a FEI Qunta 200 3D dual beam scanning electron microscope. Transmission electron microscopy was used to observe the flagella. Wet mount preparations were used: bacterial suspensions were settled onto specimen grids, stained with 1 % phosphotungstinate and viewed with a transmission electron microscope (JEOL 1200 EX).

For biochemical and physiological properties, cultures were incubated at 30 °C and properties were recorded for up to 4 days with 24 h intervals. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with NA that had been prepared anaerobically. Growth at various NaCl concentrations was investigated on NA broth without NaCl and NaCl concentrations (0–15 %) added separately (at intervals of 1 %, w/v). Growth at various temperatures was measured on NA at 4–45 °C, and pH (pH 5.0–12.0) at an intervals of 0.5 pH units using the following buffer systems: pH 4.0–5.0: 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0: 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0: 0.1 M NaHCO3/0.1 M Na2CO3.

The utilization of sugars and acid production from carbohydrates as carbon sources was determined using API 50CH kits (bioMérieux) according to the manufacturer’s instructions, with API 50CHB as inoculation medium. Fingerprints of enzymic activities were obtained using API ZYM test strips (bioMérieux) according to the manufacturer’s instructions. Nitrogen assimilation was assessed using NA broth. Catalase activity was determined by production of bubbles after the addition of a drop of 3 % H2O2. Oxidase activity was determined using the API oxidase reagent. Hydrolysis of urea was determined on peptone-glucose agar (l −1): peptone 1 g, glucose 1 g, NaCl 5 g, KH2PO4 2 g, containing 2 % (w/v) urea and 0.001 % (w/v) phenol red. Hydrolysis of starch was determined on peptone-beef extract agar containing 0.2 % (w/v) soluble starch by flooding of the plates with iodine solution. Hydrolysis of casein was tested on casein agar by observation of clear zones around the colonies. The incubation period for hydrolysis of urea, starch and casein was 24–48 h d at 30 °C. Gelatin hydrolysis was determined by incubation for one week at 30 °C on peptone-gelatin medium (l −1) (peptone 5 g, gelatin 120 g). Milk coagulation and peptonization was determined using 20 % (w/v) skimmed milk as medium incubation for 48–72 h at 30 °C.

Molecular analyses

Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene from strain NIO-1003T was performed as described by Li et al. (2007). The resulting 16S rRNA gene sequence was compared with available 16S rRNA gene sequences from GenBank using the BLAST program to determine the nearest neighbours of the strain NIO-1003T. Multiple alignments with sequences of the most closely related bacteria and calculations of levels of sequence similarity were carried out using CLUSTAL_X (Thompson et al. 1997). Phylogenetic analyses were performed using four tree-making algorithms: the neighbour-joining (Saitou and Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods. A phylogenetic tree was constructed using the neighbour-joining method of Saitou and Nei (1987) from Knuc values (Kimura, 1980) using MEGA version 5.0 (Tamura et al. 2011). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1,000 replicates.

To further study the genetic relatedness of NIO-1003T with the two closest type strains, F. rigui KCTC 113278T and F. solisalsi KCTC 13181T, the strains were analyzed by AP-PCR. In this method, arbitrarily selected primers are annealed to template DNA under low stringency conditions for the initial cycles of DNA amplifications, which allows interactions between the primers and target DNA in regions containing base mismatches. The AP-PCR fingerprinting were performed by using the M13F primer [(–20): GTAAAACGACGGCCAGT] and the following PCR program: two cycles of 94 °C for 5 min, 40 °C for 5 min, and 72 °C for 5 min; followed by 40 high-stringency cycles of 94 °C for 1 min, 60 °C for 1 min and 72° for 2 min. Amplified DNA product were resolved by electrophoresis n agarose 2 % w/v gels.

Chemotaxonomic analyses

Sugar analysis of the purified cell walls followed procedures described by Staneck and Roberts (1974). Polar lipids were extracted from freeze-dried cells and separated by two dimensional silica gel thin layer chromatography (Minnikin et al. 1984). The first direction was developed in chloroform/methanol/water (65:25:3.8, by vol.) and the second in chloroform/methanol/acetic acid/water (40:7.5:6:1.8, by vol.), first spraying with ninhydrin and developing at 120–160 °C in an incubator. Reaction sites were outlined with a soft pencil before executing a second spraying treatment with the molybdenum-Blue and drying in an incubator at 100 °C. Menaquinones were isolated according to Minnikin et al. (1984) and separated by reversed phase HPLC (Kroppenstedt 1982). For fatty acids analysis, strains NIO-1003T F. rigui KCTC 113278T and F. solisalsi KCTC 13181T were cultured on tryptic soy agar (TSA, Difco) at 30 °C for 48 h. Cellular fatty acids analysis was performed as described by Sasser (1990) according to the MIDI protocols by gas chromatography with flame ionization detector (GC-FID) and identified using the Microbial Identification Software (MIDI Sherlock aerobe method and TSBA library version Aerobic Bacteria Library TSBA6/RTSBA6 v6.10; Newark, DE, USA).

For the determination of G+C composition, genomic DNA was prepared according to the method of Marmur and Doty (1962). Genomic DNA was hydrolysed and the resultant nucleotides were analysed by reversed phase HPLC (Tamaoka and Komagata 1984).

DNA–DNA hybridization

For DNA–DNA hybridization experiments, genomic DNA was extracted and purified according to the method of Marmur (1961) with additional RNAse treatment. DNA–DNA hybridization was carried out based on principles and equations described by De Ley et al. (1970) under the consideration of modifications done by Huss et al. (1983) with optimized fluorimetric procedure evaluated by Loveland-Curtze et al. (2011), using a Step One Plus Real-Time PCR system (Applied Biosystems) fitted with 96 well thermal cycling block. DNA suspended in 2X SSC buffer was used for the analysis in triplicate. The reassociation was carried out at optimum renaturation temperature 70 °C (Gillis et al. 1970, Marmur and Doty 1962). The relatedness values are expressed by the means of the three values and the results DNA–DNA hybridizations were taken from the three means of relatedness values. The reassociation of DNA was carried out at optimum renaturation temperature 70 °C [Tor = 0.51 × (% G+C) + 47.0] according to De Ley et al. (1970).

Results and discussion

Colonies of strain NIO-1003T were observed to be cream coloured on nutrient agar and tryptone soy agar; no aerial mycelia were observed. No diffusible pigment was observed. Strain NIO-1003T was found to have morphological characteristics typical of the genus Bacillus, including endospore formation. Cells were observed to be Gram-positive short rods, 0.45–0.46 × 3.0–3.2 μm, and motile (Online Supplementary Fig. 1). Strain NIO-1003T was found to grow at 15–42 °C, pH 5.0–12.0 and with 0–12 % (w/v) NaCl, with optimum growth observed at 30 °C, pH 7.0–7.5 and 0–5 % (w/v) NaCl concentration. No growth was observed below 15 °C, pH 5.0 and above 12 % (w/v) NaCl. The detailed physiological and biochemical characteristics of strain NIO-1003T are given in the species description and Table 1.

Table 1 Phenotypic characteristics that differentiate strain NIO-1003 from its phylogenetic neighbours in the genus Fictibacillus
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Strain NIO-1003T was found to contain meso-diaminopimelic acid as the diagnostic amino-acid in the cell-wall peptidoglycan and ribose, glucose, galactose as cell-wall sugars. The predominant menaquinone was identified as unsaturated menaquinone with seven isoprene units (MK-7). The major polar lipids detected in strain NIO-1003T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified amino lipid (Supplementary Fig. 2). The genomic DNA G+C content of strain NIO-1003T was determined to be 42.6 mol%. The major fatty acids (>5 %) detected in strain NIO-1003T and the reference strains F. rigui KCTC 13278T and F. solisalsi KCTC 13181T were anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:0. The detailed comparative percent profile of fatty acid analysis is given in Table 2.

Table 2 Cellular fatty acid profiles (%) of strain NIO-1003T and its phylogenetically related species of the genus Fictibacillus
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The almost complete 16S rRNA gene sequence (1,419 bp) of strain NIO-1003T was obtained (GenBank/EMBL/DDBJ accession number JF893461). Strain NIO-1003T shares highest sequence similarity with F. rigui KCTC 13278T, F. solisalsi KCTC 13181T and F. barbaricus DSM 14730T with 98.2, 98.0, 97.2 % and 25, 28, 39 nucleotide differences respectively. A phylogenetic tree, based on 16S rRNA gene sequence data from strain NIO-1003T and corresponding sequences from the type strains of the genus Fictibacillus was constructed using the neighbour-joining (Fig. 1) and maximum parsimony (Suppl. Fig. 3) algorithms. The comparative analysis of 16S rRNA gene sequences and phylogenetic relationships showed that strain NIO-1003T forms a subclade in the tree with F. rigui KCTC 13278T supported by a high bootstrap value (Fig. 1), with which it shares the highest 16S rRNA gene sequence similarity. The affiliation of strain NIO-1003T and it closest neighbours was also supported by the maximum parsimony and maximum-likelihood algorithms with high bootstrap values. The determined DNA–DNA relatedness value between strain NIO-1003T and F. rigui KCTC 13278T and between strain NIO-1003T and F. solisalsi KCTC 13181T were 53.9 ± 1.2 and 54.8 ± 2.1 % respectively, which is well below the 70 % cutoff point accepted for the recognition of genomic species (Wayne et al. 1987). Depending on the investigated taxo-nomic group, the threshold value of DNA–DNA hybridizations is considered necessary has been increased to between 98.2- 99.0 % 16S rRNA sequence similarity (Stackebrandt and Ebers, 2006; Meier-Kolthoff et al. 2013). The 16S rRNA gene sequence similarity and DNA–DNA relatedness data therefore suggest that the strain NIO-1003T should be considered as a different genomic species of the genus Fictibacillus. Furthermore, AP-PCR amplicon fingerprint profiles showed marked differences in the banding patterns between strains NIO-1003T, F. rigui KCTC 13278T, and F. solisalsi KCTC 13181T (Supplementary Fig. 4), consistent with the assignment of these strains to separate species.

Fig. 1
figure 1

Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing relationships between strain NIO-1003T and related members of the genus Fictibacillus. Bootstrap values (>70 %; 1,000 resamplings) are given at branch points. Astrikes indicate that the corresponding nodes (groupings) are also recovered in Fitch–Margoliash, maximum-parsimony and maximum-likelihood trees. Bar, 0.01 nucleotide substitutions per position

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The phenotypic and chemotypic properties of strain NIO-1003T, and the 16S rRNA gene sequence comparison and DDH results, supports the proposal to classify the isolate NIO-1003T as a novel member of the genus Fictibacillus. The phenotypic, genotypic and phylogenetic data distinguish strain NIO-1003T from other validly named members of the genus Fictibacillus. Therefore, we propose that isolate NIO-1003T represents a novel species within the genus, for which the name Fictibacillus enclensis sp. nov. is proposed.

Description of Fictibacillus enclensis sp. nov.

Fictibacillus enclensis (en.clen’sis. N.L. masc. adj. enclensis arbitrary name formed from NCL, the acronym for the National Chemical Laboratory, India, where taxonomic studies on this species were performed).

Cells are aerobic, Gram stain-positive, motile rods 0.45–0.46 × 3.0–3.2 μm in size, which are endospore-forming and occur singly or in chains. Endospores are ellipsoidal. After 2 days incubation on nutrient agar, colonies are 0.5– 1.0 mm in diameter, cream in colour, opaque, circular, smooth and convex. Grows at 15–42 °C (optimum, 30 °C) and pH 5–12 (optimum, pH 7–7.5). Tolerates up to 12 % NaCl. Growth does not occur under anaerobic conditions. Growth occurs on nutrient agar, but not on Simmons’ citrate agar, cetrimide agar or MacConkey agar. Catalase- and oxidase-positive. Phenylalanine deaminase negative. In Hi25 (Hi-media, Mumbai) tests, positive for glucose, fructose, inositol, d-mannose, beta-galactosidase, lysine decarboxylase and Voges–Proskauer reaction. Negative for arginine dihydrolase, arginine decarboxylase, ornithine decarboxylase, indole and H2S production, urease and nitrate reduction. Starch, DNA, tyrosine, Tween 20, aesculin and casein are hydrolysed, but Tween 80, gelatin and carboxymethyl cellulose are not. Acid is produced from trehalose, salicin and d-fructose. Utilizes d-xylose, d-fructose, d-mannose, trehalose, d-mannitol, l-arabinose and salicin, but not d-lactose, sucrose, d-galactose, d-glucose, maltose, melibiose, turanose, cellobiose, d-ribose, melezitose, raffinose, l-rhamnose, l-sorbose, adonitol, l-arabitol, i-erythritol, xylitol, d-sorbitol, inositol, dextrin, glycerol, acetate, gluconate, inulin, amygdalin, N-acetyl-d-glucosamine, pyruvate or methyl alpha-glucoside. The diagnostic diamino acid of the cell-wall peptidoglycan is meso-diaminopimelic acid and MK-7 is the predominant quinone. The cellular fatty acid profile consists of significant amounts of anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:0. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The DNA G + C content of the type strain is 42.6 mol%.

The type strain, NIO-1003T (= NCIM 5458T = DSM 25142T), was isolated from a sediment sample taken from the Chorao Island in the Goa Province, India. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NIO-1003T is JF893461.