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Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov.
Systematic and Applied Microbiology 33 (2010) 367–373
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Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov.夽 S. Krishnamurthi a,1 , A. Ruckmani a,1,2 , R. Pukall b , T. Chakrabarti a,∗ a b
Microbial Type Culture Collection & Gene Bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India Deutsche Sammlung vom Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7b, D-38124 Braunschweig, Germany
a r t i c l e
i n f o
Article history: Received 18 May 2010 Keywords: Bacillus rRNA group 2 16S rRNA gene FAME Polar lipids Ribotype
a b s t r a c t The taxonomic status of three Bacillus species, Bacillus insolitus, B. psychrodurans and B. psychrotolerans was reexamined using a polyphasic approach. In our analysis, these three Bacillus species formed a cluster separate from other members of Bacillus rRNA group 2 [5] and from Bacillus sensu stricto. These three species shared high 16S rRNA gene sequence similarities between them (97.8–99.7%) and showed closest sequence similarity (95.3–96.3%) to Paenisporosarcina quisquiliarum gen. nov., sp. nov. [18]. Sequence similarities with other related genera ranged between 90.9% and 94.5%. Phylogenetic coherence of the three species was supported by phenotypic characteristics, such as growth at low temperatures, negative oxidation and assimilation of many carbohydrates, MK8 as the major isoprenoid quinine and broadly similar polar lipid profiles. All three species had a similar peptidoglycan type of the variation A4 and similar genomic G + C contents (35.7–36.6 mol% [1]). Genomic relatedness among them was shown to be less than 70% and justified their separate species status [1]. These three species could be differentiated from each other and from related taxa on the basis of phenotypic, including chemotaxonomic, characteristics and ribotype patterns. On the basis of our analysis, we propose a new genus Psychrobacillus gen. nov. and to transfer B. insolitus, B. psychrodurans and B. psychrotolerans to the new genus as Psychrobacillus insolitus comb. nov. (type species of the genus; type strain W16BT = DSM 5T ), P. psychrodurans comb. nov. (type strain 68E3T = DSM 11713T ) and P. psychrotolerans comb. nov. (type strain 3H1T = DSM 11706T ). © 2010 Elsevier GmbH. All rights reserved.
On the basis of the 16S rRNA gene sequence analysis, heterogeneity in the genus Bacillus was first pointed out by Ash et al. [5] and they recognized five distinct groups. Members belonging to rRNA group 3 were placed in a new genus Paenibacillus [6]. In their analysis, Bacillus rRNA group 2 was comprised of six Bacillus species and the genus Sporosarcina. The genus Bacillus, at present, is heterogeneous and the need for reclassification of many of its members has been indicated by many authors [2,3,9,14,15,31] Subsequently, three species from the Bacillus rRNA group 2 of Ash et
夽 The GenBank accession number for the 16S rRNA sequence of Bacillus insolitus DSM 5T is AM980508. ∗ Corresponding author at: Microbial Culture Collection Project, National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India. Tel.: +91 20 20270840; fax: +91 20 25692259. E-mail address: [email protected] (T. Chakrabarti). 1 Both authors have contributed equally to the study. 2 Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India. 0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.06.003
al. [5] were transferred to the genus Sporosarcina [31] and two to Lysinibacillus [2]. Taxonomic status of the sole remaining member of the original six species, Bacillus insolitus, has not been reexamined so far. In spite of the uncertain taxonomic position, several new species were still being added to Bacillus rRNA group 2 (Bacillus silvestris [25]; Bacillus pycnus and B. neidei [22]; Bacillus psychrodurans and Bacillus psychrotolerans [1]; Bacillus odysseyi [19]; B. arvi and B. arenosi [12]; Bacillus massiliensis [11]; Bacillus decisifrondis [32]). Of these, B. arvi, B. arenosi and B. neidei were recently transferred to the genus Viridibacillus [3] and B. silvestris to a new genus Solibacillus [17]. In an attempt to bring homogeneity in the genus Bacillus, Kämpfer et al. [15] suggested a set of chemotaxonomic properties present in the type species of the genus, Bacillus subtilis, “should in future constitute the ‘core characteristics’ of the genus”. Unfortunately, many of these properties were not reported for most of the recently described species of Bacillus. In our study of prokaryotic diversity of a landfill, we isolated a few strains showing affiliation to Bacillus rRNA group 2. Polyphasic taxonomic characterization of one of these strains designated
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as SK 55T led to the proposal of a novel genus Paenisporosarcina and reclassification of Sporosarcina macmurdoensis as P. macmurdoensis [18]. Detailed study of these strains also prompted us to reexamine the taxonomic status of three psychrotolerant Bacillus species, Bacillus insolitus DSM 5T , Bacillus psychrotolerans DSM 11706T and Bacillus psychrodurans DSM 11713T , because of their phylogenetic closeness to the genus Paenisporosarcina [18]. In a previous study, Abd El Rahman et al. [1] investigated many strains of these three Bacillus species and, based on data from biochemical tests and DNA–DNA hybridization experiments, concluded separate species status for each group of strains. We examined several phenotypic and chemotaxonomic (fatty acids, menaquinones, polar lipids) properties of the type strains of these three species. In addition, we also investigated many biochemical properties (using the Biolog system and by API 50CH) and fatty acids of additional strains of the three species [strains 84E1 (DSM 22887) and 4H2 (DSM 11718) belonging to B. psychrotolerans, strains 67E1 (DSM 11712) and 61E1 (DSM 22889) belonging to B. psychrodurans, and strain T16B (DSM 2272) belonging to B. insolitus (Supplementary Tables S2, S3 and S4)]. This was carried out in order to take care of the intraspecies variations in the phenotypic properties of the bacterial species. Furthermore, riboprinting was also undertaken using an automated Riboprinter (Dupont) with EcoRI as the restriction enzyme [4] in order to analyze the diversity of the ribotype pattern within strains of the different species. On the basis of a polyphasic taxonomic analysis of the type strains, along with phenotypic characteristics of other strains (mentioned above), we show in this study that these three species are distinct from other members of Bacillus rRNA group 2 and they merit reclassification into a novel genus. We, therefore, propose creation of a new genus Psychrobacillus and to transfer these three Bacillus species to this genus. It is appropriate to mention here that a novel generic status of B. insolitus was speculated earlier in the extensive phylogenetic study of this group by Stackebrandt and Swiderski [28]. Eight strains of these three species were tested for acid production from, and oxidation of, various substrates using API 50CH strips (bioMèrieux, France) and Biolog GP2 plates (Biolog Inc., USA), respectively, according to the manufacturers’ instructions, except that incubation was carried out at 20–25 ◦ C depending on the growth behavior of strains. In addition, B. psychrotolerans DSM 11706T , B. psychrodurans DSM 11713T and B. insolitus DSM 5T were subjected to further biochemical characterization using API ZYM and API NE test kits (bioMèrieux, France), according to the manufacturer’s instructions except that incubation was carried out at 22 ◦ C. Motility was determined using the hanging drop method and by observing cells under a phase contrast microscope (Zeiss, Germany). For analysis of cellular fatty acids, all eight strains were grown on TSBA at 20–25 ◦ C according to their growth behavior. Fatty acid methyl esters were extracted and analyzed using the Microbial Identification System (MIDI), as described previously [23]. For analyses of isoprenoid quinones and polar lipids, B. psychrotolerans DSM 11706T and B. psychrodurans DSM 11713T were cultivated on TSB, and B. insolitus DSM 5T was grown on marine broth for 2 days in a rotary shaker (200 rpm) and harvested by centrifugation at 3000 × g. Isoprenoid quinones were extracted and purified as described previously [26]. The purified quinones were separated by reversed phase HPLC (SCL-10AVP, Shimadzu) using the solvent system of acetonitrile and isopropanol in a ratio of 65:35 with a flow rate of 1 mL min−1 monitored at a wavelength of 269 nm. Extraction of polar lipids was carried out based on the modified protocol of Bligh and Dyer [7]. Two-dimensional TLC was run for identification of polar lipids according to procedures described by Komagata and Suzuki [16]. Lipid spots were detected using the following spray reagents: molybdatophosphoric acid (5%; w/v) in absolute ethanol, molybdenum blue spray reagent (1.3%, Sigma),
ninhydrin (0.2%; w/v) in acetone and anisaldehyde reagent (Sigma) for detection of total lipids, phospholipids, aminolipids and glycolipids, respectively. Since the 16S rRNA gene sequence of B. insolitus DSM 5T available in the GenBank database (X60642) was found to be of poor quality, it was sequenced again and the sequence was deposited (AM980508). Partial sequences of the 16S rRNA gene of B. psychrodurans DSM 11713T and B. psychrotolerans DSM 11706T and other strains were also determined to check the authenticity of the strains. The genomic DNA of the strains was isolated according to the method described by Pitcher et al. [24]. The 16S rRNA gene was amplified by PCR using the universal primers 8-27F (5 -AGAGTTTGATC- CTGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ). The amplification reaction and purification of the product was carried out as mentioned earlier [23]. The amplified 16S rDNA was sequenced by the dideoxy chain terminator method using the BigDye Terminator kit followed by capillary electrophoresis on an ABI 310 genetic analyzer (Applied Biosystems, USA). The primers used for sequencing were 357F (5 -CTCCTACGGGAGGCAGCAG-3 ), 685R (5 -TCTACGCATTTCACCGCTAC-3 ), 926F (5 -AAACTCAAAGGAATTGACGG-3 ), and 907R (5 -CCGTCAATTCMTTTRAGTTT-3 ). The 16S rRNA gene sequences of B. psychrodurans DSM 11713T , B. psychrotolerans DSM 11706T and B. insolitus DSM 5T , along with sequences of other closely related taxa, were retrieved from the GenBank database and aligned using the Clustal X software [29], and the alignments were edited manually. The 16S rRNA gene sequence similarities were calculated from the alignment. Gaps at the 5 and 3 ends of the alignment were omitted from further analysis. Evolutionary distance matrices were calculated by using the algorithm of Jukes and Cantor [13] with the DNADIST program within the TREECON software package [30]. A phylogenetic tree was constructed using the neighbour-joining method [27] and bootstrap analysis was performed to assess the confidence limits of the branching. Trees based on the maximum parsimony and maximum likelihood methods were also constructed using the Phylip software package version 3.5c [10]. Results of biochemical analysis revealed that strains of the three species were non-reactive towards most of the substrates in oxidation and acid production tests (Supplementary Table S3 and S4). Out of the 95 substrates in the Biolog microplate most of the strains were unable to oxidize more than five substrates (except strain DSM 11712 which oxidized six substrates). Similarly, out of the 50 carbon sources tested in API 50 CH strips none of the strains could produce acid from more than three substrates (Supplementary Table S4). These observations were in agreement with previous and recent findings that species belonging to the so-called Bacillus rRNA group 2 are not reactive towards most of the substrates and these tests alone offer little diagnostic importance [2,3,12,21,22]. Analysis of cellular fatty acids of eight strains revealed that anteiso-C15:0 was the most predominant fatty acid (30.2–50.5%; Supplementary Table S2). The other major fatty acids present were C16:1 7c alcohol (7.5–16.5%; 4.71% in Bacillus psychrodurans strain 67E1), iso-C14:0 (6.2–21.1%) and isoC15:0 (10.5–21.9%; 4.7% in B. insolitus DSM 5T ). As compared to its closest relative Paenisporosarcina quisquiliarum, the three species contained relatively higher amounts of anteiso-C15:0 and iso-C14:0 , and lower amount of iso-C15:0 . Moreover, as compared to B. subtilis, the three species showed both quantitative and qualitative variations in the amount of a few fatty acids, such as C16:1 7c alcohol and iso-C14:0 (Supplementary Table S2). These three species were also characterized by the presence of A4 type peptidoglycan with ornithine as the diaminoacid at position 3 of the peptide subunit [1], whereas all other genera of Bacillus group 2 contained A4␣ type peptidoglycan with lysine as the diaminoacid (Table 1). This is one of the most important chemotaxonomic parameters which distin-
MK-7, MK-8 DPG, PG, PE, APL, UL i-C15:0 , ai-C15:0 , C16:1 7c alcohol MK-8, MK-7, MK-6, MK-9 DPG, PG, PE, 2PL, 4UL ai-C15:0 , C16:1 7c alcohol, iso-C15:0 , iso-C14:0 , Quinone system Polar lipids Major fatty acids
Data for Psychrobacillus are from this study, except for peptidoglycan type which was taken from [1]. * Data adopted from Krishnamurthi et al. [17]. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; APL, aminophospholipid; APGL, aminophosphoglycolipid; GBG, gentiobiosyldi-acylglycerol; UL, unknown lipid. Components listed in parentheses were present in major amounts in some members of the taxon.
MK-7 DPG, PG, PE, GBG, APL i-C15:0 , ai-C15:0 , ai-C17:1 MK-7 DPG, PG, PE, PS, UPL i-C15:0 , i-C16:1 MK-7 DPG, PE, PG i-C15:0 , ai-C15:0 MK-7 DPG, PG, APGL i-C15:0 , (ai-C15:0 , i-C16:0 , ai-C17:0 , C16:1 7c alcohol) MK-7, MK-8 DPG, PG, PE, APL, 2PL i-C15:0 , ai-C15:0
Rod + meso-DAP direct Rod + l-Lys-d-Glu Rod l-Lys-d-Asp Rod + l-Lys-d-Asp Rod + l-Lys-d-Asp/l-Lys-d-Glu
Rod/spherical + l-Lys-l-Ala-d-Asp/l-LysGly-d-Glu/l-Lys-d-Glu MK-7 * DPG, PG, PE, APL, 3 UL ai-C15:0 , (i-C15:0 ) Rod/spherical + l-Orn-d-Glu Cell shape Spore formation Peptidoglycan type
Rod/spherical + l-Lys-d-Asp
Lysinibacillus* Viridibacillus* Sporosarcina* Paenisporosarcina* Psychrobacillus Characteristics
Table 1 Characteristics that distinguish the genus Psychrobacillus from other closely related genera and the type species of the genus Bacillus, B. subtilis.
Kurthia*
Solibacillus*
B. subtilis*
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guished these three species from other members of the group and supported their placement as members of a new genus. Analysis of the menaquinone system of the type strains of these three species revealed that MK-8 was the major quinone, followed by MK-7 with MK-6 and MK-9 detected in minor amounts. This quinone system is unusual for bacilli which mainly contain MK-7 as the major menaquinone. However, presence of such a menaquinone pattern has recently been reported for the genus Viridibacillus [3] but MK-6 could not be detected, which until now has been reported only in the genus Caryophanon of Bacillus rRNA group 2 (Table 1). The menaquinone pattern of these three Bacillus species therefore appears to be unique and demarcates them from other genera of Bacillus rRNA group 2. Two dimensional polar lipid profiles of type strains of these three species showed the presence of some diagnostic lipids that could differentiate the three species from other closely related genera of the group (Table 1, Supplementary Fig. S1 and Supplementary Table S1). Polar lipids present in these three species were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two phospholipids (PL 1 and PL2) and four unknown lipids (UL 1, 2, 6, and 7). In addition to these lipids, each species also had its own characteristic lipids, such as the presence of unknown lipids UL 12 to UL 14 in B. psychrotolerans DSM 11706T and an aminolipid AL1 in B. insolitus (Supplementary Fig. S1). Overall, all three strains shared quite a number of common lipids (as mentioned above; Supplementary Fig. S1) that supported their placement within a single genus. It is to be noted that the presence of the aminophospholipid APL just behind PE (in B. psychrotolerans and B. psychrodurans; Supplementary Fig. S1) has been reported in the genus Viridibacillus [3] and was also seen in the lipid profile in Paenisporosarcina quisquiliarum [18]. The chemotaxonomic properties of the three species (menaquinone type, fatty acids, peptidoglycan type and polar lipids profile) were quite different from the type species of the genus Bacillus, B. subtilis (Supplementary Table S1), and, according to the criteria recommended by Kämpfer et al. [15], these three species should not belong to the genus Bacillus. Therefore, they merit reclassification. These three species were closely related at the 16S rRNA gene sequence level with binary similarity values in the range of 97.8–99.7%. The three species showed closest sequence similarity to Paenisporosarcina quisquiliarum (95.3–96.3%). The close relation of these three species with P. quisquiliarum was also evident in the phylogenetic trees constructed using the distance based, maximum parsimony and maximum likelihood methods. In the phylogenetic tree, the genus Paenisporosarcina was recovered as a sister clade of Bacillus insolitus, B. psychrotolerans and B. psychrodurans (Fig. 1). The 16S rRNA gene sequence similarities of these three species with other closely related genera were low (90.9–94.5%). As is evident in Fig. 1, most of the nodes were recovered in all three-tree construction methods. These three Bacillus species formed a coherent cluster separate from other genera supported by a high bootstrap value (Fig. 1). The phylogenetic data, in conjunction with other phenotypic characteristics, strengthen our proposal for the creation of a new genus Psychrobacillus enclosing these three species. The genus Psychrobacillus can be differentiated from its phylogenetically closest genus Paenisporosarcina and from the type species of the genus Bacillus, B. subtilis, in containing A4 type peptidoglycan, different diagnostic polar lipids, anteiso-C15:0 as the most predominant fatty acid (iso-C14:0 in B. insolitus) and the presence of menaquinone MK-6 (Table 1). Although the three Bacillus species of the newly proposed genus were closely related at the sequence level, they could be differentiated from each other by a number of biochemical and chemotaxonomic characteristics (Table 2). Extensive DNA–DNA hybridization between various strains of these three species revealed that the genomic relatedness among the strains
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Fig. 1. Phylogenetic tree based on the neighbor-joining method showing the relationship between members of the genus Psychrobacillus and other members of rRNA group 2 bacilli. Bootstrap values as a percentage of 1000 replications are shown at the branches. Filled circles represent nodes also recovered in maximum parsimony and maximum likelihood approaches. The sequence from Paenibacillus polymyxa IAM 13419T was used as an outgroup. Bar represents 0.02 substitutions per site.
within a species was between 86% and 100% but was lower than 70% among the three species, thus justifying separate species status for these three groups of strains [1]. Ribotype patterns of eight strains belonging to these three species placed them in three separate groups (Fig. 2). Strain 61E1 (DSM22889) was described as belonging to B. psychrodurans [1] but, according to ribotype data,
it was found to be embedded among the strains of B. psychrotolerans. Therefore, based on data from biochemical, chemotaxonomic and phylogenetic analyses we propose a novel genus Psychrobacillus gen. nov. and reclassify the former three Bacillus species, B. insolitus, B. psychrodurans and B. psychrotolerans as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrodurans comb. nov., and
Fig. 2. Diversity of normalized ribotype patterns found within the species Bacillus insolitus, Bacillus psychrodurans and Bacillus psychrotolerans. Cluster analysis was performed by the unweighted pair group method with arithmetic averages (UPGMA) based on the Pearson correlation coefficient, using an optimization of 1.20 (BioNumerics, Kortrijk, Belgium).
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Table 2 Phenotypic characteristics of (1) B. psychrotolerans 3H1T (DSM 11706T ), (2) B. psychrodurans 68E13T (DSM 11713T ), and (3) B. insolitus W16BT (DSM 5T ). Characteristics
1
2
3
Cell shape Spore shape Spore location Motility* Growth temperature range NaCl (%) tolerance Nitrate reduction* Production of* : alkaline phosphatase esterase Hydrolysis of: DNA starch esculin* PNPG* gelatin Acid production from* : d-glucose d-mannitol G + C content (mol%) Menaquinone type (In %)*
Rod R T + −2 ◦ C to 30 ◦ C Up to 3% −
Rod R T + −2 ◦ C to 30 ◦ C Up to 5% +
Rod/spherical R/C T + −2 ◦ C to 25 ◦ C Up to 2% −
+ +
+ −
− −
+ + + + −
+ + − − −
− − − − −
− − 36.5 MK-8 (60.9%), MK-7 (30.5%), MK-6 (7.9%), MK-9 (Tr)
+ w 36.5 MK-8 (64.5%), MK-7 (27.4%), MK-6 (8.0%), MK-9 (Tr)
− − 35.8 MK-8 (68.8%), MK-7 (12.9%), MK-9 (11.4%), MK-6 (6.7%)
Some data are from Abd El-Rahman et al. [1]; some data for B. insolitus are from Larkin and Stokes [20] and Claus and Berkeley [8]. These three species can grow at low temperatures (down to -2 ◦ C) and in the presence of up to 3% (w/v) NaCl but not at 7% (w/v). Type strains of these three species have the following properties in common (as determined in this study): positive for esterase-lipase, Naphthol-AS-BI-phosphohydrolase and oxidation of d-xylose. Negative for production of lipase, cysteine arylamidase, phosphatase acid, ␣-galactosidase, -galactosidase, -glucorunidase, ␣-glucosidase, -glucosidase, N-acetyl--glucosaminidase, ␣-mannosidase and ␣-fucosidase. Negative for indole production, glucose fermentation, arginine dihydrolase and urease. No assimilation of the following carbon sources: d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. R-round, C-cylindrical, T-terminal, w-weak reaction, Tr-Traces. * Data generated in the present study. Additional characteristics are given in the genus and species description, Supplementary Tables S1, S2, S3 and S4.
Psychrobacillus psychrotolerans comb. nov., respectively. The type species is Psychrobacillus insolitus W16BT (=DSM 5T ). Description of Psychrobacillus gen. nov. (Psy. chro. ba.cilˇılus. Gr. adj. psychros cold; L. masc. n. bacillus, rod; N. L. masc. n. Psychrobacillus cold loving bacillus/rod). Cells are Gram-positive, endospore forming motile rods (coccoid/spherical morphology may be seen on some media for B. insolitus [20]) and strictly aerobic. Spores terminal in position, may be round or cylindrical in swollen/or non-swollen sporangia [1,20]. Negative for citrate utilization, gelatin hydrolysis, indole production and urease [1,20]. MK-8 is the major menaquinone, followed by MK-7 and minor quantities of MK-6 and MK-9. The major polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and minor quantities of two phospholipids (PL 1 and PL 2), and four unknown lipids (UL 1, 2, 6, and 7). The cell wall peptidoglycan is of the A4 type with ornithine as the diaminoacid at position 3 of the peptide subunit [1]. Major fatty acids are anteiso-C15:0 , iso-C14:0 and C16:1 7c alcohol. The G + C content of the genomic DNA ranges from 35.7 to 36.6 mol% [1]. The type species is Psychrobacillus insolitus W16BT (=DSM 5T =ATCC 23299T ). Description of Psychrobacillus insolitus (Larkin and Stokes, 1967) comb. nov. (in.so’li.tus. L. masc. adj. insolitus, unaccustomed, unusual) Basonym: Bacillus insolitus Larkin and Stokes [20]. The description is based on two strains, W16BT (DSM5T ) and T16B (DSM 2272). In addition to the characteristics of the genus, the species has the following characteristics. Negative for oxidation of 95 substrates in the Biolog system and for acid production from 50 substrates in API 50CH strips [strain T16B (DSM 2272) is positive for oxidation of -hydroxybutyric acid, ␣-ketovaleric acid, pyruvic acid methyl ester, succinic acid monomethyl ester and pyruvic acid,
and for acid production from d-mannose (Supplementary Tables S3 and S4)]. The type strain is negative for production of alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, trypsin and ␣-chymotrypsin, esculin and PNPG hydrolysis. Negative for starch hydrolysis, citrate utilization and nitrate reduction [20]. Other characteristics are given in Table 2. The polar lipid profile of the type strain consists of the ones mentioned in the genus description, as well as an additional phospholipid (PL 3), one aminolipid (AL1) and seven unknown lipids (UL 3, 4, 5, 8, 9, 10 and 11). Major fatty acids are iso-C14:0 , anteiso-C15:0 and C16:1 7c alcohol. The species also contains C15:0 , C16:1 11c, and C18:1 9c in minor amounts. The genomic DNA G + C content is 35.8 mol% [1]. The type strain is W16BT (=DSM 5T = ATCC 23299T ) and was isolated from marshy and normal soil [20]. Description of Psychrobacillus psychrodurans (Abd El-Rahman et al., 2002) comb. nov. (psy.chro.dur’ans. Gr. adj. psychros cold; L. pres. part. durans enduring; N.L. part. adj. psychrodurans cold-enduring) Basonym: Bacillus psychrodurans Abd El-Rahman et al. (2002). The description is based on three strains, 68E3T (DSM 11713T ), 67E1 (DSM 11712) and 61E1 (DSM22889). In addition to the characteristics of the genus, the species has the following characteristics. Positive for oxidation of l-glutamic acid and ␣-ketovaleric acid (strain 61E1 is weakly positive), the type strain is negative for oxidation of other substrates of the Biolog system (strain 67E1 is positive for oxidation of acetic acid, l-alanine, l-alanyl glycine, 2,3 butanediol, weakly positive for inosine; strain 61E1 shows positive or weak oxidation of arbutin, D-fructose, d-mannitol, d-sorbitol, pyruvic acid methylester, succinic acid mono-methylester, pyruvic acid, l-alanyl glycine, glycyl-l-glutamic acid, 2-deoxyadenosine, inosin, thymidine, and uridine; Supplementary Table S3) The type strain is positive for alkaline phosphatase, weak production of leucine arylamidase, valine arylamidase, trypsin and ␣-chymotrypsin but negative for esterase production. Positive
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for catalase, oxidase, starch hydrolysis and nitrate reduction [1]. Negative for gelatin, esculin and PNPG hydrolysis and citrate utilization. Positive or weak acid production from d-glucose, dfructose, esculin, mannitol and cellobiose (strain 61E1 is negative for acid production). Other characteristics are given in Table 2, Supplementary Tables S3 and S4. In addition to the polar lipids mentioned in the genus description, the type strain also contains one phospholipid (PL 3), an aminophospholipid (APL) and three unknown lipids (UL 3, 4, 5). The major fatty acids are anteisoC15:0 and iso-C15:0 . In addition, the species also contains iso-C14:0 , iso-C16:0 and C16:1 7c alcohol in moderate amounts. The genomic DNA G + C content 35.8–36.5 mol% (36.5 mol% for the type strain). The type strain is 68E3T (=DSM 11713T = (NCIMB 13837T = ATCC BAA796T = CIP 107791T ) and was isolated from garden soil, Elkanater, Egypt [1]. Description of Psychrobacillus psychrotolerans (Abd El-Rahman et al., 2002) comb. nov. (psy.chro.to’le.rans. Gr. adj. psychros cold; L. pres. part. tolerans tolerating; N.L. part. adj. psychrotolerans cold-tolerating) Basonym: Bacillus psychrotolerans Abd El-Rahman et al. (2002). The description is based on three strains, 3H1T (DSM11706T ), 4H2 (DSM 11718) and 84E1 (DSM 22887). In addition to the characteristics described for the genus, the species showed the following characteristics. Positive for oxidation of ␣-ketoglutaric acid, esculin and PNPG hydrolysis. Positive for catalase, oxidase, hydrolysis of starch but negative for citrate utilization, nitrate reduction and gelatin hydrolysis [1]. The type strain is positive for alkaline phosphatase and esterase production but negative for production of leucine arylamidase, valine arylamidase, trypsin and ␣-chymotrypsin. Acid is produced from esculin but not from dglucose, d-fructose, d-mannitol, d-cellobiose and d-maltose, weak or negative from D-fructose. Other characteristics are given in Table 2, Supplementary Tables S2, S3 and S4. In addition to the polar lipids mentioned in the genus description, the type strain also contains an aminophospholipid (APL) and five unknown lipids (UL 10, 11, 12, 13, 14). The major fatty acids are anteiso-C15:0 , followed by iso-C15:0 , iso-C14:0 and C16:1 7c alcohol. The genomic DNA G + C content of the type strain is 36.5 mol% [1]. The type strain is DSM 11706T (=NCIMB 13838T = CIP 107792T ) and was isolated from field soil, Göttingen-Weende, Germany [1]. Acknowledgements We sincerely thank Prof. Euzeby (Ecole Nationale Veterinaire, France) for help in forming the specific epithet for Psychrobacillus insolitus. Financial assistance from DBT, Government of India and CSIR is duly acknowledged. SK is a recipient of a CSIR fellowship. AR is a recipient of a DBT post doctoral fellowship. We thank Mr. Deepak for his help in DNA sequencing. This is IMTECH communication number 48/2008.
[3]
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.syapm.2010.06.003.
[26]
References
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Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov.夽 S. Krishnamurthi a,1 , A. Ruckmani a,1,2 , R. Pukall b , T. Chakrabarti a,∗ a b
Microbial Type Culture Collection & Gene Bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India Deutsche Sammlung vom Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7b, D-38124 Braunschweig, Germany
a r t i c l e
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Article history: Received 18 May 2010 Keywords: Bacillus rRNA group 2 16S rRNA gene FAME Polar lipids Ribotype
a b s t r a c t The taxonomic status of three Bacillus species, Bacillus insolitus, B. psychrodurans and B. psychrotolerans was reexamined using a polyphasic approach. In our analysis, these three Bacillus species formed a cluster separate from other members of Bacillus rRNA group 2 [5] and from Bacillus sensu stricto. These three species shared high 16S rRNA gene sequence similarities between them (97.8–99.7%) and showed closest sequence similarity (95.3–96.3%) to Paenisporosarcina quisquiliarum gen. nov., sp. nov. [18]. Sequence similarities with other related genera ranged between 90.9% and 94.5%. Phylogenetic coherence of the three species was supported by phenotypic characteristics, such as growth at low temperatures, negative oxidation and assimilation of many carbohydrates, MK8 as the major isoprenoid quinine and broadly similar polar lipid profiles. All three species had a similar peptidoglycan type of the variation A4 and similar genomic G + C contents (35.7–36.6 mol% [1]). Genomic relatedness among them was shown to be less than 70% and justified their separate species status [1]. These three species could be differentiated from each other and from related taxa on the basis of phenotypic, including chemotaxonomic, characteristics and ribotype patterns. On the basis of our analysis, we propose a new genus Psychrobacillus gen. nov. and to transfer B. insolitus, B. psychrodurans and B. psychrotolerans to the new genus as Psychrobacillus insolitus comb. nov. (type species of the genus; type strain W16BT = DSM 5T ), P. psychrodurans comb. nov. (type strain 68E3T = DSM 11713T ) and P. psychrotolerans comb. nov. (type strain 3H1T = DSM 11706T ). © 2010 Elsevier GmbH. All rights reserved.
On the basis of the 16S rRNA gene sequence analysis, heterogeneity in the genus Bacillus was first pointed out by Ash et al. [5] and they recognized five distinct groups. Members belonging to rRNA group 3 were placed in a new genus Paenibacillus [6]. In their analysis, Bacillus rRNA group 2 was comprised of six Bacillus species and the genus Sporosarcina. The genus Bacillus, at present, is heterogeneous and the need for reclassification of many of its members has been indicated by many authors [2,3,9,14,15,31] Subsequently, three species from the Bacillus rRNA group 2 of Ash et
夽 The GenBank accession number for the 16S rRNA sequence of Bacillus insolitus DSM 5T is AM980508. ∗ Corresponding author at: Microbial Culture Collection Project, National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India. Tel.: +91 20 20270840; fax: +91 20 25692259. E-mail address: [email protected] (T. Chakrabarti). 1 Both authors have contributed equally to the study. 2 Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012, India. 0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.06.003
al. [5] were transferred to the genus Sporosarcina [31] and two to Lysinibacillus [2]. Taxonomic status of the sole remaining member of the original six species, Bacillus insolitus, has not been reexamined so far. In spite of the uncertain taxonomic position, several new species were still being added to Bacillus rRNA group 2 (Bacillus silvestris [25]; Bacillus pycnus and B. neidei [22]; Bacillus psychrodurans and Bacillus psychrotolerans [1]; Bacillus odysseyi [19]; B. arvi and B. arenosi [12]; Bacillus massiliensis [11]; Bacillus decisifrondis [32]). Of these, B. arvi, B. arenosi and B. neidei were recently transferred to the genus Viridibacillus [3] and B. silvestris to a new genus Solibacillus [17]. In an attempt to bring homogeneity in the genus Bacillus, Kämpfer et al. [15] suggested a set of chemotaxonomic properties present in the type species of the genus, Bacillus subtilis, “should in future constitute the ‘core characteristics’ of the genus”. Unfortunately, many of these properties were not reported for most of the recently described species of Bacillus. In our study of prokaryotic diversity of a landfill, we isolated a few strains showing affiliation to Bacillus rRNA group 2. Polyphasic taxonomic characterization of one of these strains designated
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as SK 55T led to the proposal of a novel genus Paenisporosarcina and reclassification of Sporosarcina macmurdoensis as P. macmurdoensis [18]. Detailed study of these strains also prompted us to reexamine the taxonomic status of three psychrotolerant Bacillus species, Bacillus insolitus DSM 5T , Bacillus psychrotolerans DSM 11706T and Bacillus psychrodurans DSM 11713T , because of their phylogenetic closeness to the genus Paenisporosarcina [18]. In a previous study, Abd El Rahman et al. [1] investigated many strains of these three Bacillus species and, based on data from biochemical tests and DNA–DNA hybridization experiments, concluded separate species status for each group of strains. We examined several phenotypic and chemotaxonomic (fatty acids, menaquinones, polar lipids) properties of the type strains of these three species. In addition, we also investigated many biochemical properties (using the Biolog system and by API 50CH) and fatty acids of additional strains of the three species [strains 84E1 (DSM 22887) and 4H2 (DSM 11718) belonging to B. psychrotolerans, strains 67E1 (DSM 11712) and 61E1 (DSM 22889) belonging to B. psychrodurans, and strain T16B (DSM 2272) belonging to B. insolitus (Supplementary Tables S2, S3 and S4)]. This was carried out in order to take care of the intraspecies variations in the phenotypic properties of the bacterial species. Furthermore, riboprinting was also undertaken using an automated Riboprinter (Dupont) with EcoRI as the restriction enzyme [4] in order to analyze the diversity of the ribotype pattern within strains of the different species. On the basis of a polyphasic taxonomic analysis of the type strains, along with phenotypic characteristics of other strains (mentioned above), we show in this study that these three species are distinct from other members of Bacillus rRNA group 2 and they merit reclassification into a novel genus. We, therefore, propose creation of a new genus Psychrobacillus and to transfer these three Bacillus species to this genus. It is appropriate to mention here that a novel generic status of B. insolitus was speculated earlier in the extensive phylogenetic study of this group by Stackebrandt and Swiderski [28]. Eight strains of these three species were tested for acid production from, and oxidation of, various substrates using API 50CH strips (bioMèrieux, France) and Biolog GP2 plates (Biolog Inc., USA), respectively, according to the manufacturers’ instructions, except that incubation was carried out at 20–25 ◦ C depending on the growth behavior of strains. In addition, B. psychrotolerans DSM 11706T , B. psychrodurans DSM 11713T and B. insolitus DSM 5T were subjected to further biochemical characterization using API ZYM and API NE test kits (bioMèrieux, France), according to the manufacturer’s instructions except that incubation was carried out at 22 ◦ C. Motility was determined using the hanging drop method and by observing cells under a phase contrast microscope (Zeiss, Germany). For analysis of cellular fatty acids, all eight strains were grown on TSBA at 20–25 ◦ C according to their growth behavior. Fatty acid methyl esters were extracted and analyzed using the Microbial Identification System (MIDI), as described previously [23]. For analyses of isoprenoid quinones and polar lipids, B. psychrotolerans DSM 11706T and B. psychrodurans DSM 11713T were cultivated on TSB, and B. insolitus DSM 5T was grown on marine broth for 2 days in a rotary shaker (200 rpm) and harvested by centrifugation at 3000 × g. Isoprenoid quinones were extracted and purified as described previously [26]. The purified quinones were separated by reversed phase HPLC (SCL-10AVP, Shimadzu) using the solvent system of acetonitrile and isopropanol in a ratio of 65:35 with a flow rate of 1 mL min−1 monitored at a wavelength of 269 nm. Extraction of polar lipids was carried out based on the modified protocol of Bligh and Dyer [7]. Two-dimensional TLC was run for identification of polar lipids according to procedures described by Komagata and Suzuki [16]. Lipid spots were detected using the following spray reagents: molybdatophosphoric acid (5%; w/v) in absolute ethanol, molybdenum blue spray reagent (1.3%, Sigma),
ninhydrin (0.2%; w/v) in acetone and anisaldehyde reagent (Sigma) for detection of total lipids, phospholipids, aminolipids and glycolipids, respectively. Since the 16S rRNA gene sequence of B. insolitus DSM 5T available in the GenBank database (X60642) was found to be of poor quality, it was sequenced again and the sequence was deposited (AM980508). Partial sequences of the 16S rRNA gene of B. psychrodurans DSM 11713T and B. psychrotolerans DSM 11706T and other strains were also determined to check the authenticity of the strains. The genomic DNA of the strains was isolated according to the method described by Pitcher et al. [24]. The 16S rRNA gene was amplified by PCR using the universal primers 8-27F (5 -AGAGTTTGATC- CTGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ). The amplification reaction and purification of the product was carried out as mentioned earlier [23]. The amplified 16S rDNA was sequenced by the dideoxy chain terminator method using the BigDye Terminator kit followed by capillary electrophoresis on an ABI 310 genetic analyzer (Applied Biosystems, USA). The primers used for sequencing were 357F (5 -CTCCTACGGGAGGCAGCAG-3 ), 685R (5 -TCTACGCATTTCACCGCTAC-3 ), 926F (5 -AAACTCAAAGGAATTGACGG-3 ), and 907R (5 -CCGTCAATTCMTTTRAGTTT-3 ). The 16S rRNA gene sequences of B. psychrodurans DSM 11713T , B. psychrotolerans DSM 11706T and B. insolitus DSM 5T , along with sequences of other closely related taxa, were retrieved from the GenBank database and aligned using the Clustal X software [29], and the alignments were edited manually. The 16S rRNA gene sequence similarities were calculated from the alignment. Gaps at the 5 and 3 ends of the alignment were omitted from further analysis. Evolutionary distance matrices were calculated by using the algorithm of Jukes and Cantor [13] with the DNADIST program within the TREECON software package [30]. A phylogenetic tree was constructed using the neighbour-joining method [27] and bootstrap analysis was performed to assess the confidence limits of the branching. Trees based on the maximum parsimony and maximum likelihood methods were also constructed using the Phylip software package version 3.5c [10]. Results of biochemical analysis revealed that strains of the three species were non-reactive towards most of the substrates in oxidation and acid production tests (Supplementary Table S3 and S4). Out of the 95 substrates in the Biolog microplate most of the strains were unable to oxidize more than five substrates (except strain DSM 11712 which oxidized six substrates). Similarly, out of the 50 carbon sources tested in API 50 CH strips none of the strains could produce acid from more than three substrates (Supplementary Table S4). These observations were in agreement with previous and recent findings that species belonging to the so-called Bacillus rRNA group 2 are not reactive towards most of the substrates and these tests alone offer little diagnostic importance [2,3,12,21,22]. Analysis of cellular fatty acids of eight strains revealed that anteiso-C15:0 was the most predominant fatty acid (30.2–50.5%; Supplementary Table S2). The other major fatty acids present were C16:1 7c alcohol (7.5–16.5%; 4.71% in Bacillus psychrodurans strain 67E1), iso-C14:0 (6.2–21.1%) and isoC15:0 (10.5–21.9%; 4.7% in B. insolitus DSM 5T ). As compared to its closest relative Paenisporosarcina quisquiliarum, the three species contained relatively higher amounts of anteiso-C15:0 and iso-C14:0 , and lower amount of iso-C15:0 . Moreover, as compared to B. subtilis, the three species showed both quantitative and qualitative variations in the amount of a few fatty acids, such as C16:1 7c alcohol and iso-C14:0 (Supplementary Table S2). These three species were also characterized by the presence of A4 type peptidoglycan with ornithine as the diaminoacid at position 3 of the peptide subunit [1], whereas all other genera of Bacillus group 2 contained A4␣ type peptidoglycan with lysine as the diaminoacid (Table 1). This is one of the most important chemotaxonomic parameters which distin-
MK-7, MK-8 DPG, PG, PE, APL, UL i-C15:0 , ai-C15:0 , C16:1 7c alcohol MK-8, MK-7, MK-6, MK-9 DPG, PG, PE, 2PL, 4UL ai-C15:0 , C16:1 7c alcohol, iso-C15:0 , iso-C14:0 , Quinone system Polar lipids Major fatty acids
Data for Psychrobacillus are from this study, except for peptidoglycan type which was taken from [1]. * Data adopted from Krishnamurthi et al. [17]. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; APL, aminophospholipid; APGL, aminophosphoglycolipid; GBG, gentiobiosyldi-acylglycerol; UL, unknown lipid. Components listed in parentheses were present in major amounts in some members of the taxon.
MK-7 DPG, PG, PE, GBG, APL i-C15:0 , ai-C15:0 , ai-C17:1 MK-7 DPG, PG, PE, PS, UPL i-C15:0 , i-C16:1 MK-7 DPG, PE, PG i-C15:0 , ai-C15:0 MK-7 DPG, PG, APGL i-C15:0 , (ai-C15:0 , i-C16:0 , ai-C17:0 , C16:1 7c alcohol) MK-7, MK-8 DPG, PG, PE, APL, 2PL i-C15:0 , ai-C15:0
Rod + meso-DAP direct Rod + l-Lys-d-Glu Rod l-Lys-d-Asp Rod + l-Lys-d-Asp Rod + l-Lys-d-Asp/l-Lys-d-Glu
Rod/spherical + l-Lys-l-Ala-d-Asp/l-LysGly-d-Glu/l-Lys-d-Glu MK-7 * DPG, PG, PE, APL, 3 UL ai-C15:0 , (i-C15:0 ) Rod/spherical + l-Orn-d-Glu Cell shape Spore formation Peptidoglycan type
Rod/spherical + l-Lys-d-Asp
Lysinibacillus* Viridibacillus* Sporosarcina* Paenisporosarcina* Psychrobacillus Characteristics
Table 1 Characteristics that distinguish the genus Psychrobacillus from other closely related genera and the type species of the genus Bacillus, B. subtilis.
Kurthia*
Solibacillus*
B. subtilis*
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guished these three species from other members of the group and supported their placement as members of a new genus. Analysis of the menaquinone system of the type strains of these three species revealed that MK-8 was the major quinone, followed by MK-7 with MK-6 and MK-9 detected in minor amounts. This quinone system is unusual for bacilli which mainly contain MK-7 as the major menaquinone. However, presence of such a menaquinone pattern has recently been reported for the genus Viridibacillus [3] but MK-6 could not be detected, which until now has been reported only in the genus Caryophanon of Bacillus rRNA group 2 (Table 1). The menaquinone pattern of these three Bacillus species therefore appears to be unique and demarcates them from other genera of Bacillus rRNA group 2. Two dimensional polar lipid profiles of type strains of these three species showed the presence of some diagnostic lipids that could differentiate the three species from other closely related genera of the group (Table 1, Supplementary Fig. S1 and Supplementary Table S1). Polar lipids present in these three species were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two phospholipids (PL 1 and PL2) and four unknown lipids (UL 1, 2, 6, and 7). In addition to these lipids, each species also had its own characteristic lipids, such as the presence of unknown lipids UL 12 to UL 14 in B. psychrotolerans DSM 11706T and an aminolipid AL1 in B. insolitus (Supplementary Fig. S1). Overall, all three strains shared quite a number of common lipids (as mentioned above; Supplementary Fig. S1) that supported their placement within a single genus. It is to be noted that the presence of the aminophospholipid APL just behind PE (in B. psychrotolerans and B. psychrodurans; Supplementary Fig. S1) has been reported in the genus Viridibacillus [3] and was also seen in the lipid profile in Paenisporosarcina quisquiliarum [18]. The chemotaxonomic properties of the three species (menaquinone type, fatty acids, peptidoglycan type and polar lipids profile) were quite different from the type species of the genus Bacillus, B. subtilis (Supplementary Table S1), and, according to the criteria recommended by Kämpfer et al. [15], these three species should not belong to the genus Bacillus. Therefore, they merit reclassification. These three species were closely related at the 16S rRNA gene sequence level with binary similarity values in the range of 97.8–99.7%. The three species showed closest sequence similarity to Paenisporosarcina quisquiliarum (95.3–96.3%). The close relation of these three species with P. quisquiliarum was also evident in the phylogenetic trees constructed using the distance based, maximum parsimony and maximum likelihood methods. In the phylogenetic tree, the genus Paenisporosarcina was recovered as a sister clade of Bacillus insolitus, B. psychrotolerans and B. psychrodurans (Fig. 1). The 16S rRNA gene sequence similarities of these three species with other closely related genera were low (90.9–94.5%). As is evident in Fig. 1, most of the nodes were recovered in all three-tree construction methods. These three Bacillus species formed a coherent cluster separate from other genera supported by a high bootstrap value (Fig. 1). The phylogenetic data, in conjunction with other phenotypic characteristics, strengthen our proposal for the creation of a new genus Psychrobacillus enclosing these three species. The genus Psychrobacillus can be differentiated from its phylogenetically closest genus Paenisporosarcina and from the type species of the genus Bacillus, B. subtilis, in containing A4 type peptidoglycan, different diagnostic polar lipids, anteiso-C15:0 as the most predominant fatty acid (iso-C14:0 in B. insolitus) and the presence of menaquinone MK-6 (Table 1). Although the three Bacillus species of the newly proposed genus were closely related at the sequence level, they could be differentiated from each other by a number of biochemical and chemotaxonomic characteristics (Table 2). Extensive DNA–DNA hybridization between various strains of these three species revealed that the genomic relatedness among the strains
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Fig. 1. Phylogenetic tree based on the neighbor-joining method showing the relationship between members of the genus Psychrobacillus and other members of rRNA group 2 bacilli. Bootstrap values as a percentage of 1000 replications are shown at the branches. Filled circles represent nodes also recovered in maximum parsimony and maximum likelihood approaches. The sequence from Paenibacillus polymyxa IAM 13419T was used as an outgroup. Bar represents 0.02 substitutions per site.
within a species was between 86% and 100% but was lower than 70% among the three species, thus justifying separate species status for these three groups of strains [1]. Ribotype patterns of eight strains belonging to these three species placed them in three separate groups (Fig. 2). Strain 61E1 (DSM22889) was described as belonging to B. psychrodurans [1] but, according to ribotype data,
it was found to be embedded among the strains of B. psychrotolerans. Therefore, based on data from biochemical, chemotaxonomic and phylogenetic analyses we propose a novel genus Psychrobacillus gen. nov. and reclassify the former three Bacillus species, B. insolitus, B. psychrodurans and B. psychrotolerans as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrodurans comb. nov., and
Fig. 2. Diversity of normalized ribotype patterns found within the species Bacillus insolitus, Bacillus psychrodurans and Bacillus psychrotolerans. Cluster analysis was performed by the unweighted pair group method with arithmetic averages (UPGMA) based on the Pearson correlation coefficient, using an optimization of 1.20 (BioNumerics, Kortrijk, Belgium).
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Table 2 Phenotypic characteristics of (1) B. psychrotolerans 3H1T (DSM 11706T ), (2) B. psychrodurans 68E13T (DSM 11713T ), and (3) B. insolitus W16BT (DSM 5T ). Characteristics
1
2
3
Cell shape Spore shape Spore location Motility* Growth temperature range NaCl (%) tolerance Nitrate reduction* Production of* : alkaline phosphatase esterase Hydrolysis of: DNA starch esculin* PNPG* gelatin Acid production from* : d-glucose d-mannitol G + C content (mol%) Menaquinone type (In %)*
Rod R T + −2 ◦ C to 30 ◦ C Up to 3% −
Rod R T + −2 ◦ C to 30 ◦ C Up to 5% +
Rod/spherical R/C T + −2 ◦ C to 25 ◦ C Up to 2% −
+ +
+ −
− −
+ + + + −
+ + − − −
− − − − −
− − 36.5 MK-8 (60.9%), MK-7 (30.5%), MK-6 (7.9%), MK-9 (Tr)
+ w 36.5 MK-8 (64.5%), MK-7 (27.4%), MK-6 (8.0%), MK-9 (Tr)
− − 35.8 MK-8 (68.8%), MK-7 (12.9%), MK-9 (11.4%), MK-6 (6.7%)
Some data are from Abd El-Rahman et al. [1]; some data for B. insolitus are from Larkin and Stokes [20] and Claus and Berkeley [8]. These three species can grow at low temperatures (down to -2 ◦ C) and in the presence of up to 3% (w/v) NaCl but not at 7% (w/v). Type strains of these three species have the following properties in common (as determined in this study): positive for esterase-lipase, Naphthol-AS-BI-phosphohydrolase and oxidation of d-xylose. Negative for production of lipase, cysteine arylamidase, phosphatase acid, ␣-galactosidase, -galactosidase, -glucorunidase, ␣-glucosidase, -glucosidase, N-acetyl--glucosaminidase, ␣-mannosidase and ␣-fucosidase. Negative for indole production, glucose fermentation, arginine dihydrolase and urease. No assimilation of the following carbon sources: d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. R-round, C-cylindrical, T-terminal, w-weak reaction, Tr-Traces. * Data generated in the present study. Additional characteristics are given in the genus and species description, Supplementary Tables S1, S2, S3 and S4.
Psychrobacillus psychrotolerans comb. nov., respectively. The type species is Psychrobacillus insolitus W16BT (=DSM 5T ). Description of Psychrobacillus gen. nov. (Psy. chro. ba.cilˇılus. Gr. adj. psychros cold; L. masc. n. bacillus, rod; N. L. masc. n. Psychrobacillus cold loving bacillus/rod). Cells are Gram-positive, endospore forming motile rods (coccoid/spherical morphology may be seen on some media for B. insolitus [20]) and strictly aerobic. Spores terminal in position, may be round or cylindrical in swollen/or non-swollen sporangia [1,20]. Negative for citrate utilization, gelatin hydrolysis, indole production and urease [1,20]. MK-8 is the major menaquinone, followed by MK-7 and minor quantities of MK-6 and MK-9. The major polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and minor quantities of two phospholipids (PL 1 and PL 2), and four unknown lipids (UL 1, 2, 6, and 7). The cell wall peptidoglycan is of the A4 type with ornithine as the diaminoacid at position 3 of the peptide subunit [1]. Major fatty acids are anteiso-C15:0 , iso-C14:0 and C16:1 7c alcohol. The G + C content of the genomic DNA ranges from 35.7 to 36.6 mol% [1]. The type species is Psychrobacillus insolitus W16BT (=DSM 5T =ATCC 23299T ). Description of Psychrobacillus insolitus (Larkin and Stokes, 1967) comb. nov. (in.so’li.tus. L. masc. adj. insolitus, unaccustomed, unusual) Basonym: Bacillus insolitus Larkin and Stokes [20]. The description is based on two strains, W16BT (DSM5T ) and T16B (DSM 2272). In addition to the characteristics of the genus, the species has the following characteristics. Negative for oxidation of 95 substrates in the Biolog system and for acid production from 50 substrates in API 50CH strips [strain T16B (DSM 2272) is positive for oxidation of -hydroxybutyric acid, ␣-ketovaleric acid, pyruvic acid methyl ester, succinic acid monomethyl ester and pyruvic acid,
and for acid production from d-mannose (Supplementary Tables S3 and S4)]. The type strain is negative for production of alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, trypsin and ␣-chymotrypsin, esculin and PNPG hydrolysis. Negative for starch hydrolysis, citrate utilization and nitrate reduction [20]. Other characteristics are given in Table 2. The polar lipid profile of the type strain consists of the ones mentioned in the genus description, as well as an additional phospholipid (PL 3), one aminolipid (AL1) and seven unknown lipids (UL 3, 4, 5, 8, 9, 10 and 11). Major fatty acids are iso-C14:0 , anteiso-C15:0 and C16:1 7c alcohol. The species also contains C15:0 , C16:1 11c, and C18:1 9c in minor amounts. The genomic DNA G + C content is 35.8 mol% [1]. The type strain is W16BT (=DSM 5T = ATCC 23299T ) and was isolated from marshy and normal soil [20]. Description of Psychrobacillus psychrodurans (Abd El-Rahman et al., 2002) comb. nov. (psy.chro.dur’ans. Gr. adj. psychros cold; L. pres. part. durans enduring; N.L. part. adj. psychrodurans cold-enduring) Basonym: Bacillus psychrodurans Abd El-Rahman et al. (2002). The description is based on three strains, 68E3T (DSM 11713T ), 67E1 (DSM 11712) and 61E1 (DSM22889). In addition to the characteristics of the genus, the species has the following characteristics. Positive for oxidation of l-glutamic acid and ␣-ketovaleric acid (strain 61E1 is weakly positive), the type strain is negative for oxidation of other substrates of the Biolog system (strain 67E1 is positive for oxidation of acetic acid, l-alanine, l-alanyl glycine, 2,3 butanediol, weakly positive for inosine; strain 61E1 shows positive or weak oxidation of arbutin, D-fructose, d-mannitol, d-sorbitol, pyruvic acid methylester, succinic acid mono-methylester, pyruvic acid, l-alanyl glycine, glycyl-l-glutamic acid, 2-deoxyadenosine, inosin, thymidine, and uridine; Supplementary Table S3) The type strain is positive for alkaline phosphatase, weak production of leucine arylamidase, valine arylamidase, trypsin and ␣-chymotrypsin but negative for esterase production. Positive
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for catalase, oxidase, starch hydrolysis and nitrate reduction [1]. Negative for gelatin, esculin and PNPG hydrolysis and citrate utilization. Positive or weak acid production from d-glucose, dfructose, esculin, mannitol and cellobiose (strain 61E1 is negative for acid production). Other characteristics are given in Table 2, Supplementary Tables S3 and S4. In addition to the polar lipids mentioned in the genus description, the type strain also contains one phospholipid (PL 3), an aminophospholipid (APL) and three unknown lipids (UL 3, 4, 5). The major fatty acids are anteisoC15:0 and iso-C15:0 . In addition, the species also contains iso-C14:0 , iso-C16:0 and C16:1 7c alcohol in moderate amounts. The genomic DNA G + C content 35.8–36.5 mol% (36.5 mol% for the type strain). The type strain is 68E3T (=DSM 11713T = (NCIMB 13837T = ATCC BAA796T = CIP 107791T ) and was isolated from garden soil, Elkanater, Egypt [1]. Description of Psychrobacillus psychrotolerans (Abd El-Rahman et al., 2002) comb. nov. (psy.chro.to’le.rans. Gr. adj. psychros cold; L. pres. part. tolerans tolerating; N.L. part. adj. psychrotolerans cold-tolerating) Basonym: Bacillus psychrotolerans Abd El-Rahman et al. (2002). The description is based on three strains, 3H1T (DSM11706T ), 4H2 (DSM 11718) and 84E1 (DSM 22887). In addition to the characteristics described for the genus, the species showed the following characteristics. Positive for oxidation of ␣-ketoglutaric acid, esculin and PNPG hydrolysis. Positive for catalase, oxidase, hydrolysis of starch but negative for citrate utilization, nitrate reduction and gelatin hydrolysis [1]. The type strain is positive for alkaline phosphatase and esterase production but negative for production of leucine arylamidase, valine arylamidase, trypsin and ␣-chymotrypsin. Acid is produced from esculin but not from dglucose, d-fructose, d-mannitol, d-cellobiose and d-maltose, weak or negative from D-fructose. Other characteristics are given in Table 2, Supplementary Tables S2, S3 and S4. In addition to the polar lipids mentioned in the genus description, the type strain also contains an aminophospholipid (APL) and five unknown lipids (UL 10, 11, 12, 13, 14). The major fatty acids are anteiso-C15:0 , followed by iso-C15:0 , iso-C14:0 and C16:1 7c alcohol. The genomic DNA G + C content of the type strain is 36.5 mol% [1]. The type strain is DSM 11706T (=NCIMB 13838T = CIP 107792T ) and was isolated from field soil, Göttingen-Weende, Germany [1]. Acknowledgements We sincerely thank Prof. Euzeby (Ecole Nationale Veterinaire, France) for help in forming the specific epithet for Psychrobacillus insolitus. Financial assistance from DBT, Government of India and CSIR is duly acknowledged. SK is a recipient of a CSIR fellowship. AR is a recipient of a DBT post doctoral fellowship. We thank Mr. Deepak for his help in DNA sequencing. This is IMTECH communication number 48/2008.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.syapm.2010.06.003.
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