Ann Microbiol (2016) 66:895–905 DOI 10.1007/s13213-015-1174-2

ORIGINAL ARTICLE

Bacillus pseudoflexus sp. nov., a moderately halophilic bacterium isolated from compost

Piyush Chandna1 & Shanmugam Mayilraj3 & Ramesh Chander Kuhad1,2

Received: 28 May 2015 /Accepted: 20 October 2015 /Published online: 14 November 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract A Gram-stain-positive, motile, rod-shaped, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyleth- endospore-forming moderately halophilic bacterium (RC1T) anolamine, and five unidentified phospholipids, as well as one was isolated from compost. The strain, designated RC1T,was unidentified aminophospholipid) supported the affiliation of grown at 2–11 % (w v−1)NaCl[optimum5%(wv−1)], 15– RC1T to the genus Bacillus. The polyphasic taxonomic investi- 42 °C (optimum 30 °C), and pH of 5.0–11.0 (optimal pH 7.0). gation (phenotypic and chemotaxonomic characteristics, phylo- The G+C content of DNAwas 40.4±0.2 mol %. The 16S rRNA genetic data, and genomic distinctiveness) indicated that RC1T gene sequence of RC1T showed 97 % similarity with Bacillus represents a novel species within the genus Bacillus,forwhich paraflexus MTCC 9831T and99%similaritywithB. flexus the name B. pseudoflexus sp. nov. is now proposed. The type DSM 1320T. Phylogenetic analysis based on 16S ribosomal strain B. pseudoflexus is RC1T (= MTCC 9830T=KCTC RNA gene sequences showed that RC1T was a member of the 13723T=CCM 7753T). genus Bacillus and most closely related to B. paraflexus MTCC 9831T and B. flexus DSM 1320T. Further, DNA-DNA hybridi- Keywords Bacillus pseudoflexus sp. nov. . zation revealed that the relatedness between RC1T and phyloge- Gram-stain-positive . Moderately halophilic . Mesophilic netically closely related species, B. paraflexus MTCC 9831T and bacteria . Compost B. flexus DSM 1320T, was 41 and 58 %, respectively. Chemo- taxonomic data (peptidoglycan type, meso-diaminopimelic acid; predominant isoprenoid quinone, MK-7; predominant fatty Introduction acids, anteiso-C15:0 (28.9 %), iso-C15:0 (15.9 %), iso-C17:1E (7.8 %), and iso-C18:1H (5.4 %); major polar lipids, Composting is a complex biological process, involving many species of bacteria and fungi that convert low-value materials The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene into high-value products. A large number of strains, belonging to sequence of RC1T is FN999944. The type strain Bacillus pseudoflexus is the genera Bacillus, Micrococcus, Clostridium, Staphylococcus, T RC1 has been deposited in the Microbial Type Culture Collection and Citrobacter, Serratia, Klebsiella, Pseudomonas, Enterobacter, Gene Bank (MTCC 9830T), Korean Collection for Type Cultures (KCTC 13723T), and Czech Collection of Microorganisms (CCM 7753T). and Escherichia, were isolated from compost (Herrmann and Shann 1997; Ryckeboer et al. 2003a, b;Ashrafetal.2007; * Ramesh Chander Kuhad Yamamoto et al. 2009;Dangetal.2011;Sharmaetal.2013; [email protected] Jurado et al. 2014; Guo et al. 2015). Amongst the aerobic pro- karyotes isolated from compost, the majority belong to the genus 1 Lignocellulose Biotechnology Laboratory, Department of Bacillus. The genus Bacillus was first established by Ferdinand Microbiology, University of Delhi South Campus, New Delhi 110 Cohn and included three species of rod-shaped bacteria, 021, India B. subtilis, B. anthracis,andB. ulna (Cohn 1872). Presently, 2 Department of Microbiology, Central University of Haryana, the genus Bacillus comprises more than 295 species, including Mahendergarh District, Pali, Haryana 123 029, India seven subspecies (http://www.bacterio.net/). Members of this 3 Microbial Type Culture Collection (MTCC), Institute of Microbial genus are able to form endospore which resists adverse Technology (IMTECH), Chandigarh 160 036, India conditions. These were isolated from various sources, 896 Ann Microbiol (2016) 66:895–905 including volcanic ash (Logan et al. 2004), fresh and marine motility was observed using both light microscopy (Cayol water (Yoon et al. 2004;Baiketal.2010), hypersaline sites et al. 1994) and in motility agar medium (Farmer 1999). The (Hong et al. 2012), and different kinds of soils (Madhaiyan presence of flagella was determined by negative staining. et al. 2010;Chenetal.2011). Halophilic and/or halotolerant Growth at different temperatures (4.0, 15.0, 25.0, 30.0, 37.0, bacilli have been isolated from the Dead Sea (Arahal et al. 40.0, 42.0, 45.0, 50.0, 55.0, 60.0, and 65.0 °C) was 1999), sea water (Yoon et al. 2003), deep sea sediments (Bae ascertained in the trypticase soya medium agar (TSA) slants et al. 2005), solar saltern (Lim et al. 2006), and salt lake (Xue for 48 h. Growth under anaerobic conditions was also evalu- et al. 2008). Moderately halophilic/halotolerant bacteria that ated after incubating in an anaerobic chamber (BBL, USA) on grow optimally in media containing 3–15 % (w v−1)NaClare 0.1× TSA supplemented with 0.5 % (w v−1) glucose or 0.1 % widely distributed among many hypersaline habitats (Ventosa (w v−1) potassium nitrate. Growth at different pH 4.0, 5.0, 6.0, et al. 1989). Taxonomically, the genus Bacillus represents phys- 7.0, 8.0, 9.0, 10.0, and 11.0 was assessed in trypticase soya iologically a very heterogeneous group of Gram-stain-positive, (TS) broth at 30 °C; the pH was adjusted to acidic, neutral, and aerobic, or facultative anaerobic endospores-forming, rod- alkaline pH by sodium acetate, Tris–HCl, and sodium hydrox- shaped bacteria widely distributed in nature. Very few reports ide (NaOH), respectively. Growth at different NaCl concen- are available on the isolation of moderate halophilic Gram-stain- trations (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 %, positive bacterium from compost. The present article describes wv−1) was examined in nutrient broth medium at pH 7.3±0.1. the isolation of an aerobic, Gram-stain-positive, mesophilic, Growth was monitored by measuring the turbidity at optical moderately halophilic, and endospore-forming rod-shaped bac- density (OD)600 by using a spectroscopic method terium, RC1T, from compost. (SPECORD, Analytik Jena, Germany). These physiological The aim of this study was to determine the taxonomic sta- tests were carried out according to the methods described pre- tus of an isolate RC1T by using the polyphasic approach. The viously in the literature (Gordon et al. 1973;Barrowand minimal standards required for characterization of an isolate Feltham 1993; Logan et al. 2009). Other physiological char- were performed as recommended by Logan et al. (2009). acteristics, including methyl red tests and Voges–Proskauer, resistance to lysozyme, and utilization of citrate, were exam- ined as described by Smibert and Krieg (1994). Biochemical Materials and methods characteristics, such as: activity of catalase, oxidase, decar- boxylation of ornithine and lysine, nitrate reduction, hydroly- During the process of composting, various mesophilic and sis of starch, gelatin, casein, aesculin, ortho-Nitrophenyl-β- thermophilic bacteria were isolated and characterized, as de- galactoside (ONPG), and Tween 20, 40, 60, 80, and 100; scribed by Chandna et al. (2013a). A bacterium designated as assimilation of carbon source, production of hydrogen sulfide T RC1 isolated from the compost sample was grown on nutri- (H2S) and indole; susceptibility to different antibiotics ent agar (NA) (HiMedia M 012, India), pH 7.3±0.1 and tem- employing the disk diffusion method using commercially perature 30±2 °C, and was screened for various enzymes like available disks (HiMedia, India), were tested according to xylanase, cellulase, amylase, and protease. The pure culture the methods of Smibert and Krieg (1994). The urease test was maintained in 20 % (v v−1) glycerol suspension at −20 °C. was performed according to Kim et al. (2006). For comparison, a reference (type) strain was obtained from Utilization of various carbon sources, enzyme activity, and the Deutsche Sammlung von Mikroorganismen und acid production from different substrates were tested with the Zellkulturen, Braunschweig, Germany (DSMZ) [B. flexus commercially available Biolog (GEN III, USA) and 1320T (ABO21185)]. Unless otherwise indicated, morpholog- bioMérieux systems (Hazelwood, USA), as per the manufac- ical, physiological, phenotypic, chemotaxonomic, and molec- turers’ instructions. ular studies were performed with the cells grown on NA (pH 7.3±0.1) at 30±2 °C. Chemotaxonomic characteristics

Morphology, physiological, and biochemical For quantitative analysis of cellular fatty acid, the isolated characteristics RC1T and reference strains {B. flexus DSM 1320T and B. paraflexus MTCC 9831T) were grown at 30 °C for 24 h The colony morphology of an 18-h-old culture of RC1T was (late logarithmic phase) on TSA plates. The cells of both examined, employing light microscopy (Olympus DP70, Ja- strains were harvested, washed thrice with distilled water to pan), scanning electron microscopy (SEM; LEO435VP, remove traces of media, and freeze-dried. The fatty acid meth- USA), and transmission electron microscopy (TEM; yl esters (FAME) were extracted, saponified, and methylated Morgagni 268D, USA), as described previously (Chandna according to the protocol of the MIDI Sherlock Microbial et al. 2013b). Reaction to Gram stain was examined using Identification System (MIS) system (MIDI; Microbial ID, light microscopy according to Gerhardt et al. (1981). Cell USA). The fatty acids were analyzed by gas chromatography Ann Microbiol (2016) 66:895–905 897

(Agilent, 6850 Series II, USA) and identified by the MIS DNA-DNA hybridization software package (MIS, MIDI Technical Note 101, USA). To determine the polar lipid profile, total cellular polar lipids DNA-DNA hybridization of RC1T was performed following were extracted according to Minnikin et al. (1984). It was the membrane filter method (Tourova and Antonov 1987) analyzed by two-dimensional thin-layer chromatography with the reference strains B. flexus DSM 1320T and (2D-TLC), followed by spraying with the appropriate detec- B. paraflexus MTCC 9831T, the closest strains based on 16S tion reagent (Komagata and Suzuki 1987). Isoprenoid qui- rRNA gene sequencing. The probe DNA was labeled with nones were extracted with chloroform/methanol (2:1; v v−1) α32-P (ATP), whose mean specific activity obtained was from dried cells of RC1T and purified via TLC, as described 3000 Ci (m mol) from the Board of Radiation and Isotope by Collins and Jones (1981). The preparation of the cell wall Technology (BRIT), Hyderabad, India. The membrane filter and composition analysis of peptidoglycan were performed as method essentially had three steps: described by Schleifer and Kandler (1972) with cellulose sheets (Art 5577, DC-Plastikfolien cellulose; ohne I. Immobilization of the DNA fluoreszenzindikator, 25 folien 20×20 cm, Merck, Germany). II. Radioactive labeling of DNA to be used as a probe III. Hybridization between the immobilized DNA and the Phylogenetic analysis, DNA-DNA hybridization, and DNA radioactive DNA base composition

The 16S ribosomal RNA gene of RC1T was amplified by Immobilization of the DNA polymerase chain reaction (PCR) using the universal primer pairs 8f, 536r, 342f, 1115r, and 1542r. The PCR product was About 10 μg of DNA in a sterilized microfuge to which 20× purified with a GenElute™ PCR Clean-Up kit (Sigma- saline sodium citrate (SSC) was appended to a final concen- Aldrich, USA) and sequenced using a DNA sequencer (Ap- tration of 6× and the contents were boiled for 10 min. The plied Biosystems, USA). The 16S rRNA gene sequence was tubes were cooled instantly on ice and DNA was immobilized compared initially with reference sequences in GenBank onto a Hybond N++ membrane using a dot blot apparatus. The (http://www.ncbi.nlm.nih.gov) by using the BLAST program wells were rinsed with 100 μL of 0.5 N NaOH after, which the (Altschul et al. 1997)andEzTaxon(http://eztaxon-e. filter was removed, dried, and baked in a oven at 80 °C for 2 h ezbiocloud.net;Chunetal.2007), and multiple alignments under vacuum. were performed using the CLUSTAL_X program (Thompson et al. 1997). The flanking ambiguous Radioactive labeling of DNA to be used as a probe nucleotides present in all the sequences were omitted for further analysis. Evolutionary distances were calculated DNA labeling based on nick translation was achieved for a according to the Kimura two-parameter distance and unrooted microfuge in a 100-μL reaction mixture. In a microfuge tube, trees were constructed using the neighbor-joining (Saitou and these reagents were added in the following order: sterile H2O Nei 1987), maximum-likelihood (Guindon et al. 2005), and (64 μL), 10×nick translation buffer (5 μL), template DNA unweighted pair-group method with arithmetic mean (200 ng in 5 μL), dTTP (7 μL), dGTP (7 μL), dCTP (7 μL), (UPGMA) algorithms using MEGA version 6.0 (Tamura and α32-P-d ATP (50 μCi in 5 μL). The solution was thor- et al. 2013). The tree topologies were assessed by performing oughly mixed and kept in incubation for 2 h at 15 °C. After 2 h a bootstrap analysis with 1500 replications. of incubation at 15 °C, the reaction was stopped with 8 μLof

Fig. 1 Scanning electron microphotograph showing vegetative cells of RC1T. a Bar, 12 nm and M.P., 7.31 KX. b Bar, 12 nm and M.P., 3.80 KX 898 Ann Microbiol (2016) 66:895–905

Fig. 2 a Phase-contrast photomicrograph of cells of RC1T showing spore-forming rods. b A photomicrograph of sporangia and vegetative cells of RC1T showing ellipsoidal endospores that are located centrally. Bar, 500 nm

0.25 M EDTA; subsequently, 56 μLof5Mammoniumace- Hybridization between the immobilized DNA tate, 50 μL of carrier DNA, and 500 μL of chilled alcohol and the radioactive DNA were added. The contents were mixed in a tube and incubated at −20 °C overnight, and subsequently centrifuged at 4 °C for The baked filter was immersed in prehybridization buffer 20 min at 17,968×g. The pellet was washed twice with 70 % [0.5 M phosphate buffer, pH 7.2, 7 % sodium dodecyl sulfate −1 (v v ) alcohol, briefly dried under vacuum, dissolved in 6× (SDS), freshly prepared] for 1 h at 20 °C less than the Tm of SSC solution, boiled at 100 °C for 10 min, followed by im- DNA being used for hybridization. The prehybridization buff- mediate chilling on ice. The chilled sample was further used er was discarded and the probe was dissolved in the hybridi- for hybridization. zation buffer. Freshly prepared buffer was added and kept for

Fig. 3 Transmission electron microphotograph of RC1T a showing an endospore. a Bar, 200 nm. b Bar, 500 nm

Inner membrane Germ cell wall Core Cortex Coat Outer membrane

b Ann Microbiol (2016) 66:895–905 899

Table 1 Phenotypic characteristics that differentiate RC1T from its closest phylogenetic neighbor in the genus Bacillus

Characteristics RC1T B. flexus DSM 1320T B. paraflexus MTCC 9831T

Colony morphology Opacity Opaque Translucent Translucent Cell shape Short rods Long rods Short rods Endospore position Central Terminal Central Growth at 11.0 (% w v−1)NaCl + −− Optimum NaCl concentration for growth (%, w v−1)2–5.0 2–5.0 2–4.0 Optimum temperature for growth (°C) 30.0 35.0 30.0 pH for growth Range 5–11.0 5–8.0 5–11.0 Optimum 7.0 7.5 7.0 Biochemical tests Oxidase − + − Catalase − ++ Methyl red + − + Hydrolysis of Casein + − + Gelatin −− + Starch −− w Tween 80 + −− Tween 100 −− + Utilization of Citrate −− − Nitrate reduction − + − Acid production from Sucrose + − + Dextrose + + + Galactose + + − Lactose + −− Cellobiose − + − Sorbose − + − Lysine + − + Ornithine + − + Myo−inositol − + − Raffinose + − + Salicin − ++ Trehalose + − + Inulin + −− Glycerol + −− Glucosamine + −− Antibiotic susceptibility Chloramphenicol S R R Cefradine S R S Fusidic acid S R S

RC1T (this study), B. flexus DSM 1320T and B. paraflexus MTCC 9831T (Chandna et al. 2013b) Strains display the following properties. Gram-stain-positive, rod-shaped, motile, aerobic, regular margin with convex elevation, dull in appearance, and endospore-former with swollen sporangia, and the Voges–Proskauer (VP) test was negative. Growth in 2–10.0 % (w v−1 ) NaCl and at 15–42 °C. Positive for acid production from glucose, fructose, ONPG, urease, mannitol, and melibiose. Strains were negative for fluorescence (UV), lysozyme (0.001 %), hydrolysis of Tween 20, 40, and 60, urea, aesculin, production of indole and H2S, utilization of citrate, and acid production from mannose, malonate, rhamnose, melezitose, α-methyl-D-mannoside, α-methyl-D-glucoside, xylitol, xylose, D-arabinose, adonitol, sorbitol, dulcitol, ribose, phenylalanine deamination, and β-glucuronidase. None of the strains grew on MacConkey agar. They are susceptible to streptomycin, tetracycline, clindamycin, lincomycin, erythromycin, vancomycin, levofloxacin, cefotaxime, kanamycin, and resistant to novobiocin (μg). B+^,positive;B−^, negative; Bw^, weak; BS^ susceptible; BR^, resistant 900 Ann Microbiol (2016) 66:895–905

16 h for hybridization at the same temperature as stated above. susceptibilities and resistances of RC1T to different antibiotics The filter was washed with 0.5×SSC containing 0.1 % (w v−1) are also given in Table 1. The carbon substrate profile of SDS for 10 min at room temperature (RT) and with 0.1×SSC RC1T, as measured by the Biolog system, indicated an identi- containing 0.5 % (w v−1) SDS for 20 min at 50 °C. Subse- fication match for Bacillus. Phenotypically, as measured by quently, the filter was dried, exposed to an X-ray film for 24 h, the bioMérieux system, RC1T showed resemblance to the ge- and developed using Kodak X-ray film. The autoradiogram nus Bacillus (Table 2). was scanned and quantified using a phosphor imager. Simul- On the basis of morphological, physiological, and bio- taneously, the filter was processed for the determination of chemical characteristics, RC1T could be differentiated from radioactive counts in the scintillation counter (1450 LSC Lu- B. paraflexus MTCC 9831T and B. flexus DSM 1320T.The minescence Counter Wallac MicroBeta TriLux, Perkin Elmer, biochemical and physiological properties of RC1T are present- USA). The percent hybridization was calculated as follows: ed in Tables 1 and 2 in the species description. All tests used for biochemical characterization were carried out in triplicate. % hybridization Chemotaxonomic distinctiveness ¼ Counts obtained from heterologous hybridization Counts obtained from homologous hybridization The cellular fatty acid profile of RC1T is shown in Table 3, Â 100 together with B. flexus DSM 1320T and B. paraflexus MTCC 9831T (close relative reference strains), analyzed previously Reciprocal reactions (A × B and B × A) were performed for (Chandna et al. 2013 b). RC1T had large amounts of straight- each DNA pair and their variation was within the limits of this chain saturated and terminal branched-chain fatty acids, the method (Goris et al. 1998, 2007). Genomic DNA of RC1T was major compounds, and fatty acids iso-C , iso-C , and extracted using a standard method (Marmur 1961) and the G + 15:0 16:0 anteiso-C , which were characteristics of numerous taxa C content was determined by using the thermal denaturation 15:0 within bacilli (Kämpfer 1994) that still support in allotting method (Marmur and Doty 1962). RC1T to the genus Bacillus. In order to complete the chemotaxonomic characterization of RC1T, the analysis of polar lipids, quinone, and peptidogly- Results and discussion can of the cell wall was carried out. The major polar lipids detected in RC1T were diphosphatidylglycerol (DPG), Phenotypic distinctiveness phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and five unidentified phospholipids, as well as one unidenti- Colonies of RC1T that formed on NA after 18 h at 30 °C were fied amino-phospholipid, as shown in Fig. 4. The presence of circular, opaque, non-pigmented, with regular margin, convex DPG, PE, and PG in RC1T has also been reported for Bacillus elevation, and were dull in appearance. Cells of RC1T were (Minnikin and Goodfellow 1981). However, unknown phos- aerobic, motile spore-former, Gram-stain-positive, rod-shaped pholipids, amino-phospholipids, and polar lipids were also (∼1.0 μmwideand∼2.0 μm long), and occurred either sin- present in B. thaonhiensis NHI-38 (Van Pham and Kim gularly or in chains (Fig. 1a, b). Cells of RC1T produced 2014a, b). endospores, which were ellipsoidal and located centrally in Menaquinone-7 (MK-7) was the predominant isoprenoid position (Fig. 2a, b and 3a), ∼904 nm in length and quinone in RC1T, which supports the affiliation of RC1T to ∼507nmindiameter(Fig.3b), and usually cause the the genus Bacillus (Collins and Jones 1981). The diagnostic sporangia to swell. The spore coat was loosely associated with diamino acid in the cell-wall peptidoglycan was meso- the cortex. The nucleus might have been lost during sample diaminopimelic acid. RC1T contained meso-diaminopimelic preparation. Flagella were clearly seen on negatively stained acid as the diagnostic diamino acid in the cell-wall peptido- preparation in RC1T. It grew between 15 and 42 °C and in the glycan. Analysis of the cell-wall peptidoglycan indicated that pH range 5.0–11.0, with optimum growth at 30 °C and it was of A1‘γ’ type, i.e., meso-diaminopimelic acid as diag- pH 7.0. The NaCl concentration range for growth of RC1T nostic diamino acid, in common with the great majority of was 2–11 % (w v−1), with an optimal concentration of 5 % (w Gram-stain-positive, endospore-forming, and rod-shaped bac- v−1). RC1T was positive for methyl red, tetrazolium tests, and teria (Minnikin et al. 1984). The peptidoglycan and quinone of urease, but negative for catalase, oxidase, and Voges– RC1T are in agreement with those reported in various species Proskauer tests. Detailed results of the phenotypic characteri- of the genus Bacillus, including the type species, B. subtilis zation of RC1T are described in Table 1. The cells of RC1T (Claus and Berkeley 1986). were positive for methyl-α-d-glucopyranoside acidification The significant differences in cellular fatty acid profile can and negative for L-lactate alkalization, putrescine assimila- be used to distinguish RC1T from B. paraflexus MTCC 9831T tion, and phosphatidylinositol phospholipase C. The and B. flexus DSM 1320T. Ann Microbiol (2016) 66:895–905 901

Table 2 Phenotypic traits of RC1T, its closest phylogenetic neighbor in Table 3 FattyacidprofilesofRC1T, B. flexus DSM 1320T,and the genus Bacillus using the bioMérieux system, and those of related B. paraflexus MTCC 9831T species retrieved from Chandna et al. 2013b Fatty acid 1* 2 3 S. no. Test 1* 2 3

iso-C11:0 0.3 1.6 – 1 β-Xylosidase + −− iso-C12:0 ––0.2 3 L-Aspartate arylamidase − + − iso-C13:0 0.5 2.2 – 5 Phenylalanine arylamidase + − + C14:0 1.6 4.9 2.5 7 β-Galactosidase + − + iso-C14:0 – 7.4 – 8 L-Pyrrolidonyl arylamidase − + − iso-C15: 0 15.9 9.6 36.8 9 α-Galactosidase + − + anteiso-C15:0 28.8 33.7 49.1 10 Alanine arylamidase + −− iso-C15: 0 3-OH ––0.1 12 β-n-Acetyl-glucosaminidase − + − C16:0 2.9 14.9 5.1 13 Ala-phe-pro arylamidase −−+ iso-C16:0 – 8.6 – 15 D-Galactose + −− iso-C16:1 E 0.5 – 17 Myo-inositol + −− iso-C17:0 3.2 – 1.4 18 Methyl-α-d-glucopyranoside acidification + −− iso-C17:1 E 7.7 – 19 Ellman’s reagent + −− anteiso-C17:0 ––3.2 21 α-Mannosidase + −− iso-C17:1 w5c ––0.4 22 Maltotriose − ++ C18:0 0.8 7.0 0.2 23 Glycine arylamidase + −− iso-C18:1 H 5.3 –– 24 D-Mannose + + − C18:0 9 C 1.6 –– 26 N-Acetyl-d-glucosamine − + − iso-C18:1 w9c ––0.2 29 β-Glucosidase + −− C20:0 ––0.1 32 Pyruvate + −− T T 33 α-Glucosidase − ++ *Strains 1, RC1 (this study); strain 2, B. flexus DSM 1320 and strain 3, B. paraflexus MTCC 9831T (Chandna et al. 2013b) 34 D-Tagatose + −− Values are percentages of total fatty acid. B–^,notdetected 38 Esculin hydrolysis + + − 39 Tetrazolium red + + − 40 Polymixin B resistance − + − 47 β-Galactopyranosidase + − + 56 Sorbitol − ++ 64 Optochin resistance + − +

*Strain1,RC1T (this study); strain 2, B. flexus DSM 1320T and strain 3, B. paraflexus MTCC 9831T (Chandna et al. 2013b) PL1 Strains display the following properties. Strains were negative for L-ly- PE PL2 sine-arylamidase, L-proline arylamidase, cyclodextrin, methyl-d- DPG β PL3 xyloside, Palatinose, -mannosidase, putrescine assimilation, d-amygda- PG lin, phosphatase, phosphatidylinositol phospholipase C, arginine PL4 dihydrolase 1, β-glucuronidase, L-lactate alkalization, N-acetyl-D-glu- AL cosamine, arginine dihydrolase 2, glycogen, D-melezitose, L-rhamnose, and phosphoryl choline. They were all positive for leucine arylamidase, PL5 tyrosine arylamidase, α-glucuronidase, pullulan, and salicin. All strains were resistance to kanamycin, oleandomycin, novobiocin, and bacitracin. B+^,positive;B−B,negative

Phylogenetic and genotypic distinctiveness

The complete 16S ribosomal RNA gene sequence of RC1T

(1456 bps) was deposited in GenBank under accession num- T ber FN999944. The 16S rRNA gene sequence was subjected Fig. 4 2D-TLC of total polar lipids of RC1 . Abbreviations: AL unidentified aminophospholipid; DPG diphosphatidylglycerol; PE to similarity search by using the sequence matching tool of the phosphatidylethanolamine; PG phosphatidylglycerol; PL1–PL5 BLASTn program and EzTaxon-e. RC1T showed a close unidentified phospholipids 902 Ann Microbiol (2016) 66:895–905 relationship with the two Bacillus species. Comparative 16S independent cluster with a bootstrap value of 99 % (Fig. 5a). rRNA gene sequence analysis indicated that RC1T had the Similar tree topology was also seen in the phylogenetic trees highest sequence similarity to B. paraflexus MTCC 9831T made by the neighbor-joining method (Fig. 5b). The UPGMA (97 %) and B. flexus DSM 1320T (99 %). In the phylogenetic algorithm also gave a similar result, as illustrated in Fig. 5c. tree based on the maximum-likelihood algorithm, RC1T with The phylogenetic trees constructed using the maximum-like- B. paraflexus MTCC 9831T, B. flexus DSM 1320T formed an lihood, neighbor-joining, and UPGMA methods clearly

Fig. 5 a Maximum-likelihood phylogenetic tree based on 16S rRNA family Bacillaceae. Bootstrap values based on 1500 replicates are gene sequences, showing the relationship between B. pseudoflexus shown at the branch nodes. Alicyclobacillus acidocaldarius AJ496806 RC1T and representatives of the family Bacillaceae. Bootstrap values was used as an outgroup. Bar, 0.02 substitutions per nucleotide position. c based on 1500 replicates are shown at the branch nodes. UPGMA phylogenetic tree based on 16S rRNA gene sequences, showing Alicyclobacillus acidocaldarius AJ496806 was used as an outgroup. the relationship between B. pseudoflexus RC1T and representatives of the Bar, 0.02 substitutions per nucleotide position. b Neighbor-joining family Bacillaceae. Bootstrap values based on 1500 replicates are shown phylogenetic tree based on 16S rRNA gene sequences, showing the at the branch nodes. Alicyclobacillus acidocaldarius AJ496806 was used relationship between B. pseudoflexus RC1T and representatives of the as an outgroup. Bar, 0.02 substitutions per nucleotide position Ann Microbiol (2016) 66:895–905 903 revealed that RC1T is a member of the genus Bacillus and it singly or in chains. The endospores of RC1T were oval, locat- formed a distinct clustering with a small subgroup of species ed centrally, and made the swollen sporangia. The optimal (embracing B. paraflexus MTCC 9831T and B. flexus DSM temperature for growth, pH, and NaCl concentration were 1320T). Moreover, bootstrap resampling analysis demonstrated 30 °C, 7.0, and 5 % (w v−1), respectively. RC1T showed pos- a strong association between RC1T and the aforementioned itive tests for methyl red, tetrazolium red, methyl-α-d- species. The branching position of RC1T with respect to its glucopyranoside acidification, and urease, while oxidase, cat- closest phylogenetic relatives, B. paraflexus MTCC 9831T alase, Voges–Proskauer test, L-lactate alkalization, putrescine and B. flexus DSM 1320T, was relatively stable, according to assimilation, and phosphatidylinositol phospholipase C were the three algorithms used for phylogenetic analysis in this study. negative. Casein and Tween 80 were hydrolyzed, but Tween The topologies of phylogenetic trees built according to the 20, 40, 60, 100, gelatin, starch, urea, and aesculin were not maximum-likelihood and UPGMA algorithms also supported hydrolyzed. It showed acid production from glucose, sucrose, the results of the neighbor-joining algorithm (Fig. 5a–c). fructose, lactose, dextrose, galactose, D-mannose, ONPG, ly- To elucidate the interrelatedness, DNA-DNA hybridization sine, ornithine, mannitol, raffinose, trehalose, melibiose, inu- was performed with type strains of the genus Bacillus.The lin, glycerol, glucosamine, ellman, salicin, D-tagatose, and DNA-DNA hybridization results supported differentiation of pullulan, but not from malonate, rhamnose, cellobiose, RC1T from other closely related Bacillus species. RC1T exhib- melezitose, α-methyl-D-mannoside, α-methyl-D-glucoside, ited DNA-DNA hybridization of 41 % to B. paraflexus MTCC xylitol, D-arabinose, xylose, sorbose, adonitol, sorbitol, 9831T and58%toB. flexus DSM 1320T. The percentage relat- myo-inositol, phenylalanine deamination, dulcitol, ribose, edness between RC1T, B. paraflexus MTCC 9831T,andB. flexus glycogen, methyl-d-xyloside, maltotriose, D-melezitose, N- DSM 1320T were calculated on the basis of the data obtained acetyl-D-glucosamine, Palatinose, L-rhamnose, phosphoryl (mean of four replicates). The pooled standard deviations of all choline, D-amygdalin, and cyclodextrin. Nitrate was not re- T the hybridization experiments varied by up to 1 %. duced to nitrite and H2S and indole were not produced. RC1 DNA-DNA hybridization values were below the threshold showed resistance to novobiocin and displayed susceptibility value of 70 % that is recommended for the delineation of to streptomycin, chloramphenicol, tetracycline, clindamycin, bacterial species (Wayne et al. 1987; Stackebrandt and Goebel lincomycin, cefradine, erythromycin, vancomycin, fusidic ac- 1994), clearly indicating that RC1T represents a species of id, cefotaxime, and levofloxacin. The major amounts of fatty T Bacillus. The reciprocal values of DNA-DNA hybridization acid of RC1 were anteiso-C15:0 (28.9 %), iso-C15:0 (15.9 %), T T between RC1 , the type strain of B. paraflexus MTCC 9831 , iso-C17:1 E (7.8 %), and iso-C18:1 H (5.4 %). Apart from the and the reference strain of B. flexus DSM 1320T were not major polar lipids, RC1T contained five unidentified phospho- higher than 50 %. The reciprocal values of DNA-DNA hy- lipids and one unidentified aminophospholipid. The DNA G+ bridization of RC1T against the closest relatives were 33 % C content of RC1T was 40.4±0.2 mol %. The type strain RC1T and 46 % with B. paraflexus MTCC 9831T and B. flexus DSM has been successfully isolated and characterized from com- 1320T, respectively. Four replicates were used for each of the post with an experiment conducted at the University of Delhi two reciprocal pairings. The G+C content of the DNA of South Campus, New Delhi, India. RC1T was 40.4±0.2 mol %. The DNA G+C content of the genus Bacillus ranged from 32 to 66 mol % (Logan and De Vo s 2009). Thus, our results support the placement of RC1T Conclusion within a separate and previously unrecognized species. On the basis of the taxonomic data described above, RC1T The genotypic and phenotypic data indicated that RC1T merits represents a species of the genus Bacillus for which the name recognition as a distinct species of the genus Bacillus.Itis, B. pseudoflexus sp. is now proposed. therefore, proposed that RC1T be classified in the genus Bacillus as B. pseudoflexus sp.nov.(=MTCC9830T=KCTC Description of Bacillus pseudoflexus RC1T sp. nov. 13723T=CCM 7753T).

Pseudoflexus: pseu.do.fle’xus. Gr. adj. pseudês, false; L. Acknowledgments We are grateful to Prof. Rup Lal, Department of masc. adj. flexus, flexible and also a bacterial epithet [B. flexus Zoology, University of Delhi, Delhi, India for allowing us to make use (ex Batchelor 1919); Priest et al. 1988]; N.L. masc. adj. of the dot blot facility. The authors are also thankful to Dr. J.P. Euzeby for providing the etymological advice during the preparation of the manu- pseudoflexus, the false [Bacillus] flexus,ofcompost. script. The authors also gratefully acknowledge the timely help from Dr. Colonies of RC1T that grew at 30 °C on nutrient agar were Jung-Sook Lee, Curator, KCTC, Korea, Dr. Ana Luisa Toribio, European non-pigmented, opaque, dull in appearance, and convex in Nucleotide Archive, EMBL-EBI, Cambridge, UK, and Dr. Arvind elevation with regular margins, and did not show UV fluores- Kumar, Post-Doctoral fellow, University of Delhi South Campus, New T Delhi. The authors wish to express their gratitude to Ms. Urvashi Kuhad, cence. Cells of RC1 were aerobic, motile, straight, short Department of Modern Indian Languages and Literary Studies, Univer- Gram-stain-positive rods with rounded ends, and occurring sity of Delhi, Delhi, for editing the manuscript. The authors are extremely 904 Ann Microbiol (2016) 66:895–905 thankful to Prof. J.P. Khurana, Department of Plant Molecular Biology community diversity during composting. Huan Jing Ke Xue 32: and Biotechnology, University of Delhi South Campus, New Delhi, India 2689–2695 for his valuable suggestions and input to improve the manuscript. Farmer JJ III (1999) Enterobacteriaceae: introduction and identification. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds) Manual of clinical microbiology, 7th edn. ASM Press, Washington, Conflict of interest The authors declare that they have no competing pp 442–458 interests. 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