Fictibacillus Phosphorivorans Gen. Nov., Sp. Nov. and Proposal to Reclassify

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2934–2944 DOI 10.1099/ijs.0.049171-0

Fictibacillus phosphorivorans gen. nov., sp. nov. and proposal to reclassify Bacillus arsenicus, Bacillus barbaricus, Bacillus macauensis, Bacillus nanhaiensis, Bacillus rigui, Bacillus solisalsi and Bacillus gelatini in the genus Fictibacillus

Stefanie P. Glaeser,1 Wolfgang Dott,2 Hans-Ju¨rgen Busse3 and Peter Ka¨mpfer1

Correspondence 1Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Germany Peter Ka¨mpfer 2Institut fu¨r Hygiene und Umweltmedizin, RWTH Aachen, Germany peter.kaempfer 3Institut fu¨r Bakteriologie, Mykologie und Hygiene, Veterina¨rmedizinische Universita¨t, A-1210 Wien, @umwelt.uni-giessen.de Austria

A Gram-positive-staining, aerobic, endospore-forming bacterium (Ca7T) was isolated from a bioreactor showing extensive phosphorus removal. Based on 16S rRNA gene sequence similarity comparisons, strain Ca7T was grouped in the genus Bacillus, most closely related to Bacillus nanhaiensis JSM 082006T (100 %), Bacillus barbaricus V2-BIII-A2T (99.2 %) and Bacillus arsenicus Con a/3T (97.7 %). Moderate 16S rRNA gene sequence similarities were found to the type strains of the species Bacillus gelatini and Bacillus rigui (96.4 %), Bacillus macauensis (95.1 %) and Bacillus solisalsi (96.1 %). All these species were grouped into a monophyletic cluster and showed very low sequence similarities (,94 %) to the type species of the genus Bacillus, Bacillus subtilis.Thequinonesystemof strain Ca7T consists predominantly of menaquinone MK-7. The polar lipid profile exhibited the major compounds diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. In addition, minor compounds of an unidentified phospholipid and an aminophospholipid were detected. No glycolipids were found in strain Ca7T, which was consistent with the lipid profiles of B. nanhaiensis, B. barbaricus, B. arsenicus, B. rigui, B. solisalsi, B. macauensis and B. gelatini, but in contrast to B. subtilis. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid and the polyamine pattern contained predominantly spermidine and spermine. The major fatty acids, which were T iso-C15 : 0,anteiso-C15 : 0 and C16 : 0, supported the grouping of strain Ca7 in the family Bacillaceae. The strain showed DNA–DNA similarities of 48 % (reciprocal 47 %) to B. nanhaiensis DSM 23009T, 31 % (reciprocal 36 %) to B. barbaricus V2-BIII-A2T and 29 % (reciprocal 39 %) to B. arsenicus DSM 15822T, respectively. These results clearly demonstrate that strain Ca7T is a representative of a novel species, which can be differentiated from its closest relatives by physiological and biochemical tests. Because of the low sequence similarity of strain Ca7T to B. subtilis, which was shared by B. nanhaiensis, B. barbaricus, B. arsenicus, B. gelatini, B. rigui, B. solisalsi and B. macauensis, and their unique lipid patterns, we propose that strain Ca7T represents a novel species in a novel genus for which the name Fictibacillus phosphorivorans gen. nov., sp. nov. is proposed. The type strain is Ca7T (5CCM 8426T5LMG 27063T). In addition we propose the reclassification of B. nanhaiensis, B. barbaricus, B. arsenicus, B. rigui, B. macauensis, B. solisalsi and B. gelatini as Fictibacillus nanhaiensis comb. nov., Fictibacillus barbaricus comb. nov., Fictibacillus arsenicus comb. nov., Fictibacillus rigui comb. nov., Fictibacillus macauensis comb. nov., Fictibacillus solisalsi comb. nov. and Fictibacillus gelatini comb. nov., respectively [type strains JSM 082006T (5DSM 23009T5KCTC 13712T), V2-BIII-A2T (5CCM 4982T5DSM 14730T), Con a/3T (5MTCC 4380T5DSM 15822T5JCM 12167T), WPCB074T (5KCTC 13278T5JCM 16348T), ZFHKF-1T (5JCM 13285T5DSM 17262T), YC1T (5KCTC 13181T5CGMCC 1.6854T) and LMG 21881T (5DSM 15866T), respectively].

Abbreviations: APL, aminophospholipid; bGG, b-gentiobiosyldiacylglycerol; DPG, diphosphatidylglycerol; LPDG, lysylphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, phospholipid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Fictibacillus phosphorivorans Ca7T is JX258924. Three supplementary figures are available with the online version of this paper.

2934 049171 G 2013 IUMS Printed in Great Britain Fictibacillus phosphorivorans gen. nov., sp. nov.

The genus Bacillus comprises, at the time of writing, more (v1.2.9) according to the SILVA seed alignment [http:// than 150 species with validly published names (www. www.arb-silva.de; Pruesse et al. (2007)] and implemented bacterio.org) isolated from various sources, among them in the ARB database. The alignment was checked manually are halophilic, halotolerant, alkaliphilic and/or alkalitoler- based on secondary structure information. Pairwise ant species (Ash et al., 1991; Nielsen et al., 1994; Ventosa sequence similarities were calculated in ARB without the et al., 1998; Arahal & Ventosa, 2002; Romano et al., 2005; use of an evolutionary substitution model. Phylogenetic Lim et al., 2006 a, b; Carrasco et al., 2007; Yumoto, 2007; trees were reconstructed with the maximum-likelihood Aino et al., 2008; Chen et al., 2009, 2011; Liu et al., 2009). method using RAxML v7.04 (Stamatakis, 2006) with GTR- It has been already pointed out by Chen et al. (2011) that GAMMA and rapid bootstrap analysis, the neighbour- some of these bacteria may reveal some interesting joining method with the Jukes–Cantor correction (Jukes & properties, e.g. the potential use of their enzymes in Cantor, 1969) and the maximum-parsimony method using biotechnological applications (Horikoshi, 1999; Margesin DNAPARS v. 3.6 (Felsenstein, 2005). Phylogenetic trees were & Schinner, 2001; Nogi et al., 2005; Krulwich et al., 2007). calculated with 100 resamplings (Bootstrap analysis; Another interesting biotechnological application is the Felsenstein, 1985) and based on 16S rRNA gene sequences recovery of phosphorus from sewage sludge incineration between positions 96 and 1395 according to Escherichia coli ash. This was investigated by using the process of numbering, Brosius et al. (1978). phosphorus release by bioleaching bacteria belonging to The sequenced 16S rRNA gene fragment of strain Ca7T was species of the genus Acidithiobacillus and bioaccumulation a continuous stretch of 1444 unambiguous nucleotides of released phosphorus by polyphosphate accumulating (positions 46–1468, E. coli numbering). Strain Ca7T shared organisms [Acidithiobacillus enriched digested sludge 100 % similarity with B. nanhaiensis JSM 082006T, 99.2 % (AEDS)] (Zimmermann & Dott, 2009 a, b). During these with B. barbaricus V2-BIII-A2T and 97.7 % with B. studies, strain Ca7T was isolated from a wastewater arsenicus Con a/3T. Moderate 16S rRNA gene sequence treatment bioreactor showing extensive phosphorus similarities were found to the type strains of the species removal on nutrient agar (NA; Oxoid) at 30 uC. The Bacillus gelatini and Bacillus rigui (96.4 %), Bacillus organism was maintained on NA after incubation at 30 uC macauensis (95.1 %) and Bacillus solisalsi (96.1 %). The for 48 h. reconstruction of phylogenetic trees based on different Cell morphology, abundance and localization of endo- treeing methods showed that all these species were grouped spores and motility were observed using a Zeiss light into a monophylogenetic cluster, distinct from all the other microscope at a magnification of 61000, using cells that species of the genus Bacillus (Fig. 1 and Figs S1 and S2 had been grown for 3 days at 25 uC on tryptic soy agar available in IJSEM Online). The respective type strains (TSA; Oxoid). Gram-staining was performed by the furthermore showed very low sequence similarities (,94 %) modified Hucker method according to the protocol of to the type species of the genus Bacillus, B. subtilis. Gerhardt et al. (1994). The physiological characterization Biomass subjected to analyses of the diagnostic peptido- was done according to the methods described by Ka¨mpfer glycan diamino acid, polyamines, the quinone system and et al. (1991) and Ka¨mpfer (1990). In addition, the presence polar lipids was grown on PYE broth (0.3 % peptone from of urease was tested on urea agar (Merck) supplemented casein, 0.3 % yeast extract, pH 7.2) at 28 uC. with 2 % urea according to the method of Christensen (1946). Indole and sulphide production was tested in SIM Quinones and polar lipids were extracted and analysed by agar according to the instructions of the manufacturer applying the integrated procedure reported by Tindall (Merck). Bacillus nanhaiensis DSM 23009T, Bacillus (1990 a, b) and Altenburger et al. (1996). Polyamines were barbaricus V2-BIII-A2T, Bacillus arsenicus DSM 15822T extracted and analysed from cells harvested at the late and Bacillus gelatini LMG 21880T as well as Bacillus subtilis exponential growth phase as reported by Busse & Auling DSM 10T were tested in parallel. The results are listed in (1988) and Altenburger et al. (1997). HPLC analysis was the species description (below) and differentiating features carried out using the equipment described by Stolz et al. from the most closely related species and from B. subtilis (2007). The diagnostic diamino acid of the peptidoglycan DSM 10T are listed in Table 1. was determined according to the method of Schleifer (1985). Fatty acids were extracted and analysed as DNA isolation for phylogenetic analysis was performed described by Ka¨mpfer & Kroppenstedt (1996). Strains with a commercial DNA extraction kit (GenElute Plant were grown under identical conditions and the cells for Genomic DNA kit; Sigma). Universal primers 27F (59- extractions were taken from colonies of the same size. Fatty GAGTTTGATCMTGGCTCAG-39) and 1492R (59-TACC- acids were identified with Sherlock version 2.11, TSBA40 TTGTTACGACTT-39) (Lane, 1991) were used for PCR Rev. 4.1. amplification and sequencing of the 16S rRNA gene with the dideoxnucleotide method. Phylogenetic analyses were The polyamine pattern of strain Ca7T consisted predomi- 21 performed in ARB release 5.2 (Ludwig et al., 2004) using the nantly of spermidine [11.4 mmol (g dry weight) ] and ‘All-Species Living Tree’ Project (LTP; Yarza et al., 2008) spermine [7.3 mmol (g dry weight)21] and traces of putres- database release LTPs106 (August 2011). Sequences not cine [,0.1 mmol (g dry weight)21]. This polyamine pattern T included in the LTP database were aligned with SINA clearly distinguished strain Ca7 from representatives of http://ijs.sgmjournals.org 2935 S. P. Glaeser and others

Table 1. Characteristics used to distinguish strain Ca7T from the type strains of phylogenetically related species and the type species of the genus Bacillus, B. subtilis.

Strains: 1, Ca7T,2,F. nanhainensis DSM 23009T,3,F. barbaricus V2-BIII-A2T,4,F. arsenicus DSM 15822T,5,F. gelatini LMG 21880T,6,B. subtilis T DSM 10 . All data are from this study. All strains are negative for nitrate and nitrite reduction, H2S and indole production, methyl red and Voges– Proskauer tests and hydrolysis of cellulose, DNA, Tweens 20, 40 and 60, L-proline-p-nitroanilide, p-nitrophenyl-b-D-glucuronide and urea, acid production from a-D-glucose, a-lactose, dulcitol, D-salicin, D-adonitol, D-sorbitol, a-raffinose, maltose, trehalose, sucrose, 1-O-methyl-D- glucopyranoside, i-erythritol, a-melibiose and D-arabitol, assimilation of (a-)L-rhamnose, myo-inositol, adonitol, putrescine, adipate, itaconate, suberate, 3-hydroxybenzoate and (DL-3-)phenylacetate. All strains are positive for the hydrolysis of starch and aesculin. +, Positive; 2, negative; (+), weakly positive.

Characteristic 1 23456

Acid production from: Sucrose 22222(+) D-Mannitol 22222(+) i-Inositol 22222(+) L-Arabinose 2222(+) 2 a-L-Rhamnose 2222(+) 2 D-Xylose 2222(+) 2 Assimilation of: N-Acetyl-D-galactosamine 22+ 2 + 2 N-Acetyl-D-glucosamine + 22++2 L-Arabinose 22222+ p-Arbutin 2222(+) + Cellobiose 222+++ D-Fructose 22++++ D-Galactose 2222(+) 2 D-Gluconate 22+ 2 ++ a-D-glucose (+) 2 ++++ Maltose + 2 ++++ D-Mannose + 2 ++++ a-Melibiose 2222(+) + D-Ribose + 2 + 2 ++ Sucrose + 22+++ D-Salicin + 22+ 2 + Trehalose + 2 +++2 D-Xylose 2222+ 2 Maltitol + 22++2 D-Mannitol 2222++ D-Sorbitol 2222++ Acetate + 2 +++2 Propionate + 22+ 22 cis-Aconitate 222+++ trans-Aconitate 22222+ 4-Aminobutyrate 2222+ 2 Azelate + 22222 Citrate 2222++ Fumarate ++2 + 2 + Glutarate 2222+ 2 DL-3-Hydroxybutyrate + 2 +++2 DL-Lactate + 222++ L-Malate ++++++ Mesaconate 222+ 22 2-Oxoglutarate 2 + 2 ++2 Pyruvate + 2 ++2 + L-Alanine + 2 ++++ b-Alanine 2222+ 2 L-Aspartate 2222++ L-Histidine 22++++

2936 International Journal of Systematic and Evolutionary Microbiology 63 Fictibacillus phosphorivorans gen. nov., sp. nov.

Table 1. cont.

Characteristic 123456

L-Leucine 22+ 222 L-Ornithine 2 +++++ L-Phenylalanine 22+++2 L-Proline 22++++ L-Serine + 2 ++++ L-Tryptophan 222+ 22 4-Hydroxybenzoate 222+ 22 Hydrolysis of: o-Nitrophenyl-b-D-galactopyranoside 22222+ p-Nitrophenyl-a-D-glucopyranoside ++(+) +++ p-Nitrophenyl-b-D-glucopyranoside + 2 (+) +++ p-Nitrophenyl-b-D-xylopyranoside 22+ 22+ Bis-p-nitrophenylphosphate ++2 +++ p-Nitrophenyl-phenylphosphonate + 22+++ p-Nitrophenyl-phosphorylcholine + 2 (+) + 22 2-Deoxythymidine-5-p-nitrophenyl-phosphate ++(+) +++ L-Alanine-p-nitroanilide 22(+) 222 L-Glutamate-c-carboxy-p-nitroanilide 2222++

Fictibacillus arsenicus Con a/3T (AJ606700) 0.10 98* Fictibacillus barbaricus V2-BIII-A2T (AJ422145) Fictibacillus nanhaiensis JSM 082006T (GU477780) 100** 100** Fictibacillus phosphorivorans Ca7T (JX258924) 99* Fictibacillus rigui WPCB074T (EU939689) Fictibacillus macauensis ZFHKF-1T (AY373018) Fictibacillus solisalsi YC1T (EU046268) 71 Fictibacillus gelatini LMG 21880T (AJ551329) 99* Bacillus funiculus NAF001T (AB049195) Bacillus panaciterrae Gsoil 1517T (AB245380) 100** Bacillus licheniformis ATCC 14580T (CP000002) 86* Bacillus subtilis subsp. subtilis DSM10T (AJ276351) Bacillus azotoformans NBRC 15712T (AB363732) 100** Bacillus cohnii DSM 6307T (X76437) Bacillus halmapalus DSM 8723T (X76447) Bacillus niabensis 4T19T (AY998119) Bacillus asahii MA001T (AB109209) 90** Bacillus novalis LMG 21837T (AJ542512) Bacillus vireti LMG 21834T (AJ542509) Bacillus soli LMG 21838T (AJ542513) Bacillus drentensis LMG 21831T (AJ542506) 96** Bacillus niacini IFO 15566T (AB021194) 88* Bacillus bataviensis LMG 21833T (AJ542508) 93** Bacillus ginsengisoli DCY53T (HQ224517) Bacillus pocheonensis Gsoil 420T (AB245377) 86** Bacillus purgationiresistans DS22T (FR666703) Bacillus eiseniae A1-2T (HM035089) 95* Bacillus kochii WCC 4582T (FN995265) 98* Bacillus flexus IFO 15715T (AB021185) Bacillus megaterium IAM 13418T (D16273) Bacillus koreensis BR030T (AY667496) Listeria monocytogenes NCTC 10357T (X56153)

Fig. 1. Maximum-likelihood tree showing the phylogenetic position of strain Ca7T and all species of the proposed genus Fictibacillus relative to the most closely related species of the genus Bacillus. The tree was generated in ARB using RAxML (GTR-GAMMA, Rapid Bootstrap analysis, 100 bootstraps) and based on 16S rRNA gene sequences between positions 96 and 1395, E. coli numbering, Brosius et al. (1978). GenBank accession numbers are given in parentheses. Numbers at branch nodes refer to bootstrap values .70 % (100 replicates). Bootstrap values marked with one asterisk are supported by high bootstrap values by one of the other treeing methods used for analysis and two asterisks indicate support by the neighbour- joining and maximum-parsimony methods. Listeria monocytogenes NCTC 10357T was used as an outgroup. Bar, 0.10 substitutions per site. http://ijs.sgmjournals.org 2937 S. P. Glaeser and others the species of the genus Bacillus, including B. subtilis, Table 2. Fatty acid composition of strain Ca7T and the type Bacillus cereus and Bacillus megaterium which had been strains of phylogenetically related species of the genus shown (Hamana et al., 1989) to contain a polyamine Bacillus pattern with the single major compound spermidine and Strains: 1, Ca7T ;2,F. nanhaiensis DSM 23009T;3,F. barbaricus V2- only minor amounts of spermine (at least ninefold lower BIII-A2T;4,F. arsenicus DSM 15822T;5,B. subtilis DSM 10T. Data are than the spermidine content). The diagnostic diamino acid percentages of the total fatty acid content; fatty acids representing of the peptidoglycan was meso-diaminopimelic acid, which ,0.5 % have been omitted. 2, Not detected. All data are from this is also present in B. subtilis. Menaquinone MK-7 (86 %) study. Data in parentheses are from Chen et al. (2011), the respective was the major quinone and in addition moderate amounts analysis was performed using a different medium and different of MK-6 (14 %) are present, as well. This quinone system is growth conditions. * Data taken from Ka¨mpfer (1994), for B. subtilis also in agreement with the characteristics of species of T DSM 10 ; 8 % of total fatty acids are represented by other fatty acids, the genus Bacillus. A similar quinone system with MK-7 not specified in more detail. (99 %), MK-8 (1 %) and traces of MK-6 (0.4 %) was T detected in B. gelatini LMG 21880 . However, menaqui- Fatty acid 1 2 3 4 5* none MK-7 as the predominant quinone is widespread in bacilli including B. subtilis. The polar lipid profile of strain Saturated T Ca7 was composed of the major lipids diphosphatidylgly- C14 : 0 2.1 2 (2.8) 0.8 22 cerol (DPG), phosphatidylglycerol (PG) and phosphatidy- C16 : 0 7.8 4.4 (14.0) 3.5 5.6 1 lethanolamine (PE) and minor amounts of an unidentified C16 : 0 N alcohol 1.3 1.0 (1.8) 0.6 4.5 2 aminophospholipid APL1, a phospholipid (PL1) and a C17 : 0 2 0.5 (0.6) 222 C 2220.9 2 polar lipid (L1) not stainable with the specific reagents 18 : 0 Unsaturated used to detect phosphate-, amino- and sugar-groups, C v7c alcohol 2.0 1.6 (0.6) 1.0 2.6 2 respectively (Fig. S3a). B. gelatini LMG 21880T exhibited 16 : 1 C16 : 1v7c 22(2) 6.5 22 a polar lipid profile (Fig. S3b) that contained all lipids C16 : 1v11c 5.1 3.4 (1.5) 7.1 6.0 2 (DPG, PG, PE, APL1 and PL1) detected in strain Ca7T C18 : 1v9c 22(0.8) 222 except lipid L1. Furthermore, a major phospholipid (PL2) Branched and minor amounts of two aminophospholipids (APL2 iso-C14 : 0 3.7 2.6 (7.2) 5.2 1.5 1 T and APL3) were detected in B. gelatini LMG 21880 . This iso-C15 : 0 39.6 43.7 (17.8) 18.1 39.4 27 T polar lipid profile clearly distinguishes strain Ca7 and B. anteiso-C15 : 0 23.6 29.8 (40.7) 46.2 13.7 39 T gelatini LMG 21880 from B. subtilis, which has been iso-C16 : 0 4.5 3.8 (4.6) 2.3 3.2 1 reported to contain in addition to DPG, PG and PE iso-C16 : 1 1 significant amounts of one or two glycolipids and an iso-C17 : 0 I 2.5 2.8 (1.1) 1.6 9.9 8 aminophospholipid differing in its chromatographic mot- anteiso-C17 : 0 3.1 3.2 (2.5) 4.5 4.6 10 T ility from APL1 of strain Ca7 (Ka¨mpfer et al., 2006). On iso-C17 : 1 22223 the other hand, the polar lipid profile is most similar to anteiso-C17 : 1 222210 those of the closest phylogenetic relatives of strain Ca7T iso-C17 : 0v7c 2223.6 2 2 2 (Fig. 1), the species B. barbaricus (Ta¨ubel et al., 2003), B. Summed feature 3* 3.0 4.6 (1.3) 1.4 Summed feature 4* 1.7 2 (0.5) 1.6 2.9 2 nanhaiensis and B. arsenicus (Chen et al., 2011), which all share the major characteristics in the polar lipid profile and *Summed features represent groups of two or three fatty acids that differ only in the presence of some minor lipids and are cannot by separated by GLC with the MIDI system. Summed feature 3 also lacking any glycolipids. These results suggest that the comprises C v7c and/or C v6c. Summed feature 4 comprises phylogenetic cluster comprising strain Ca7T and the species 16 : 1 16 : 1 iso-C17 : 1 I and/or anteiso-C17 : 1 B. B. nanhaiensis, B. barbaricus and B. arsenicus together could be reclassified in a novel genus. Since B. solisalsi, B. macauensis (Liu et al., 2009) and B. rigui (Baik et al., 2010), nanhaiensis, B. barbaricus and B. arsenicus were studied in which are placed in the most closely related clade (Fig. 1), comparison. also share these major lipid characteristics, they can be placed in the same novel genus. The fact that all these DNA–DNA hybridization was performed according to the species show identical major lipid characteristics (DPG, method of Ziemke et al. (1998). Despite the high 16S rRNA gene sequence similarities within the B. barbaricus group, PG, PE and no glycolipids) strongly supports the T distinction of those species from B. subtilis. strain Ca7 showed DNA–DNA similarities of only 48 % (reciprocal 47 %) to B. nanhaiensis DSM 23009T,31% The fatty acids comprised mainly iso- and anteiso- (reciprocal 36 %) to B. barbaricus V2-BIII-A2T and 29 % branched fatty acids with the same predominant fatty (reciprocal 39 %) to B. arsenicus DSM 15822T, respectively. acids as the most closely related species of the genus These relatively low DNA–DNA hybridization values along Bacillus. The detailed fatty acid profile obtained from cells with high 16S rRNA gene sequence similarities had been grown on tryptic soy (TS)-medium after 72 h of incuba- reported previously by Chen et al. (2011). The DNA G+C tion at 28 uC is shown in Table 2. The type strains of B. mol% content of strain Ca7T was determined as described

2938 International Journal of Systematic and Evolutionary Microbiology 63 Fictibacillus phosphorivorans gen. nov., sp. nov. by Urdiain et al. (2008) and resulted in a value of PG and PE. A lipid profile with an aminophospholipid and 40.3 mol% (±0.2 mol%). a glycolipid showing the chromatographic motilities of LPG and bGG, respectively, has been reported for Bacillus In the pioneering study of Ash et al. (1991), the siamensis which is phylogenetically closely related to B. phylogenetic heterogeneity of the genus Bacillus was subtilis, as well (Sumpavapol et al., 2010). Different polar demonstrated and distribution of its species over five lipid profiles have been reported for several other species. major rRNA groups was shown. A sixth rRNA group was For instance, Bacillus herbersteinensis is characterized by the established by Nielsen et al. (1994). A much greater major lipids DPG and PG, the two glycolipids of B. subtilis, phylogenetic heterogeneity among species of the genus but no PE or LPG (Wieser et al., 2005). Bacillus iranensis Bacillus has been demonstrated recently, indicating that the shows the major lipids DPG, PG, two unidentified genus is not monophyletic (Munoz et al., 2011). However, glycolipids (whose chromatographic motilities are clearly the type species of the genus, B. subtilis, was placed in different from those of B. subtilis) and a phospholipid absent rRNA group 1 (Ash et al., 1991). Meanwhile, the majority in B. subtilis but no PE or LPG (Bagheri et al., 2012). Despite of species of the genus Bacillus of the remaining rRNA the phylogenetic distance B. megaterium, Bacillus flexus, groups have been reclassified in novel genera. But species Bacillus cohnii (Ta¨ubel et al., 2003), Bacillus sporother- of rRNA group 1 have also been reclassified in novel modurans, Bacillus oleronius (Heyndrickx et al., 2012) and genera such as Virgibacillus (Heyndrickx et al., 1998), Bacillus purgationiresistans (Vaz-Moreira et al., 2012) share Gracilibacillus (Wainø et al., 1999), Alkalibacillus (Jeon et rather similar polar lipid profiles with strain Ca7T and its close al., 2005), Pullulanibacillus (Hatayama et al., 2006) and phylogenetic relatives (Fig. S3). The presence of the major Salimicrobium (Yoon et al., 2007). Species of the genus lipids DPG, PG, PE and absence of any glycolipids distin- Bacillus of rRNA group 1 share with B. subtilis between guish these species from B. subtilis, as well. Based on these approximately 93 % and 99.9 % 16S rRNA gene sequence considerations, the core of a redefined genus Bacillus, similarity. Since in many other genera the borderline for consisting of B. subtilis, B. licheniformis and B. pumilus,is assignment of novel species to a genus is approximately characterized by a polar lipid profile that is composed of 95 % similarity, the question arises as to whether species DPG, PG, PE and bGG. Strain Ca7T and its close relatives can sharing less than 95 % similarity should be reclassified in be clearly distinguished from B. subtilis, B. licheniformis and B. novel genera. The borderline for genus separation might be pumilus based on the absence of any glycolipid. even higher, as for members of rRNA group 2 where the genera Viridibacillus and Rummeliibacillus are separated at In order to create a stable taxonomy from our point of approximately 97 % 16S rRNA gene similarity (Albert et al., view the only reference for assignment of novel species to 2007; Vaishampayan et al., 2009). The proposal of novel the genus Bacillus should be B. subtilis and its close genera among rRNA group 1 is hampered by rather high phylogenetic relatives that share at least 95 % sequence homogeneity in chemotaxonomic traits such as fatty acids, similarity with the type species. In conclusion, strain Ca7T quinones and diagnostic diamino acid of the peptidoglycan should not be described as a novel species of the genus and peptidoglycan structure as well (A1c; Schleifer & Bacillus but classified in a novel genus and its close relatives Kandler, 1972). Like B. subtilis these species contain fatty should be assigned to this novel genus as well. Hence, based acid profiles with predominantly iso-C15 : 0 and anteiso- on low 16S rRNA gene sequence similarities with B. subtilis, C15 : 0, a quinone system with the major compound phylogenetic distinctness, polar lipid profiles and pheno- menaquinone MK-7 and the diagnostic peptidoglycan typic traits (for an overview see Table 3) we here propose diamino acid meso-diaminopimelic acid. Even though the novel genus Fictibacillus gen. nov. with the type species polar lipid profiles have been included more often in Fictibacillus barbaricus comb. nov. and the reclassification recent years in species descriptions, information on this of B. arsenicus, B. barbaricus, B. macauensis, B. nanhaiensis, chemotaxonomic trait in species of the genus Bacillus is B. rigui, B. solisalsi and B. gelatini as Fictibacillus arsenicus still rather limited. However, available data indicate some comb. nov., Fictibacillus barbaricus comb. nov., Fictibacillus significant variability between species of the genus Bacillus macauensis comb. nov., Fictibacillus nanhaiensis comb. with respect to presence of major lipids, glycolipids and nov., Fictibacillus rigui comb. nov., Fictibacillus solisalsi aminophospholipids that may to some degree also reflect comb. nov. and Fictibacillus gelatini comb. nov., respect- the phylogenetic diversity. B. subtilis has been reported ively. Within the proposed genus we propose a novel (Ka¨mpfer et al., 2006) to contain the major lipids DPG, species, Fictibacillus phosphorivorans with strain Ca7T as the PG, PE, b-gentiobiosyldiacylglycerol (bGG), perhaps type strain. another glycolipid (GL3) and an aminophospholipid (PN2) that, considering the studies of Op den Kamp et al. Description of Fictibacillus gen. nov. (1969) and Gidden et al. (2009), should be lysylpho- Fictibacillus (Fic.ti.ba.cil9lus. L. adj. fictus false, L. masc. n. sphatidylglycerol (LPG). Also strains of Bacillus bacillus a rod and also a bacterial generic name; N.L. masc. licheniformis and Bacillus pumilus, both species being n. Fictibacillus false bacillus). closely related to B. subtilis (Reva et al., 2002), have been reported to contain bGG (Iwasaki et al., 1986) and the type Cells are Gram-stain-positive or Gram-stain-variable rods strain of B. licheniformis has been reported to contain DPG, (0.5–1.062–5 mm), which often form chains. They bear http://ijs.sgmjournals.org 2939 2940 others and Glaeser P. S.

Table 3. Overview of phenotypic and chemotaxonomic characteristics of strain Ca7T and the seven species of the genus Bacillus forming a distinct phylogenetic cluster (reclassified as members of the genus Fictibacillus)

Taxa: 1, Ca7T;2,F. barbaricus V2-BIII-A2T (Ta¨ubel et al., 2003); 3, F. macauensis ZFHKF-1T (Zhang et al., 2006); 4, F. solisalsi YC1T (Liu et al., 2009); 5, F. nanhaiensis JSM 082006T (Chen et al., 2011); 6, F. arsenicus Con a/3T (Shivaji et al., 2005); 7, F. rigui WPCB074T (Baik et al., 2010); F. gelatini LMG 21881T (De Clerck et al., 2004). Data for strain Ca7T are from this study. Data for all other species were taken from the literature except for polar lipid and quinone data of the type strain of B. gelatini, +, Positive; 2, negative; ND, no data available; opt, optimum.

Characteristic 1 2 3 4 5 6 7 8

Spores Oval, central, in Oval, central, in Endospore-forming Ellipsoidal, Oval, in subterminal Subterminal Ellipsoidal Oval, slightly swollen swollen sporangia subterminal in unswollen endospores in non- endospores, Paracentrally sporangia swollen sporangia sporangia swelling sporangia subterminal and position, cause subterminally in sporangia to swell non-swelling sporangia NaCl (w/v) 0–1 %, not at 2 % 5 or 10 % Up to 2 %, Up to 15 % 0–18 % Up to 1 % 0–9 % (opt. 1–2 %) opt. 15 % (weakly at 2 %) not at 5–10 % (opt. 0.4–4 %) pH growth range 6.5–10.5 7.2–9.5 (weakly at 6.0) 6.0–10.0 (opt 8.5) 5.0–13.0 (opt. 7–10) 6.0–10.5 (opt. 8.0) 5.5–8.0 6–9 (opt. 7–8) 5–8 (opt. 4–5) (weakly at 11.5) nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International Oxidase/ Catalase 2/22/+ ND/++/+ 2/+ ND/++/+ 2/+ Temperature 20–36, weakly at 18–37 uC, not at 20–40 uC 15–53 uC 15–45 uC 20–40 uC, not at 45 uC 10–45 uC Opt. 40–50 uC range 15 uC, not at 10 and 4 and 47 uC (opt. 30uC) (opt. 35–42 uC) (opt. 30 uC) (opt. 20–37 uC) (max. 58–60 uC) 45 uC DNA G+C 40.3 40.2 40.8 41.8 40.2 35.0 41.9 41.5 content (mol%) Major fatty acids i-C15 : 0 ai-C15 : 0 ai-C15 : 0 i-C15 : 0 i-C14 : 0 ai-C15 : 0 i-C15 : 0 i-C15 : 0 ai-C15 : 0 ai-C15 : 0 i-C15 : 0 i-C15 : 0 ai-C15 : 0 i-C15 : 0 ai-C15 : 0 i-C15 : 0 C16 : 0 (¢10 %) ai-C17 : 0 C16 : 0 i-C16 : 0, i-C16 : 1 v7c i-C17 : 0 Polar lipids DPG, PG, PE, PE, PG, DPG, APL, DPG, PE, PG, DPG, PG, PE, DPG, PG, PE PG, DPG, PE AL1, 2, DPG, PG, PE AL, DPG, PG, PE, APL1, PL, L AL, L; No GLs ALP, AL APL, AL APL, PL, L APL APL, PL APL1, 2, 3, PL1 Menaquinones MK-7 MK-7 MK-7 MK-7 MK-7 MK-7 MK-7 MK-7 (MK) Minor MK MK-6 ND ND ND MK-8, MK-6 ND ND MK-8, MK-6 (.10 %) Cell wall Meso-diaminopimelic Diaminopimelic acid Meso- Meso-diaminopimelic Meso- Diaminopimelic acid Meso- ND diamino acid acid diaminopimelic acid diaminopimelic diaminopimelic acid acid acid 63 Fictibacillus phosphorivorans gen. nov., sp. nov. spherical to ellipsoidal endospores, which are located either Basonym: Bacillus nanhaiensis Chen et al. (2011). centrally or terminally, sometimes subterminally. Spore The description is that of Chen et al. (2011). formation can be very sparse. Colonies are circular and slightly irregular, smooth, glossy or sometimes matt, flat, The type strain is JSM 082006T (5DSM 23009T5KCTC butyrous, creamy to yellowish white and almost opaque on 13712T). nutrient-rich media. Members of the genus are facultatively anaerobic and catalase-negative. The polyamine pattern of Description of Fictibacillus arsenicus comb. nov. the type species consists predominantly of spermidine and spermine. The quinone system consists predominantly of Fictibacillus arsenicus (ar.sen.i9cus. N.L. masc. adj. arsenicus menaquinone MK-7. The polar lipid profile is composed of pertaining to arsenic). the major compounds DPG, PG and PE. Glycolipids are Basonym: Bacillus arsenicus Shivaji et al. (2005). absent. The fatty acid profile comprises mainly iso- and anteiso-branched fatty acids. The diagnostic diamino acid The description is that of Shivaji et al. (2005). of the peptidoglycan is meso-diaminopimelic acid. The The type strain is Con a/3T (5MTCC 4380T5DSM + DNA G C content ranges from 35.0 mol% to 42 mol%. 15822T5JCM 12167T). The type species is Fictibacillus barbaricus.

Description of Fictibacillus rigui comb. nov. Description of Fictibacillus barbaricus comb. nov. Fictibacillus rigui (ri9gu.i. L. gen. n. rigui of a well-watered Fictibacillus barbaricus (bar.ba9ri.cus. L. adj. barbaricus place). strange, foreign, referring to the strange behaviour towards growth at different pH). Basonym: Bacillus rigui Baik et al. (2010). Basonym: Bacillus barbaricus Taübel et al. (2003). The description is that of Baik et al. (2010). T T The description is that of Taübel et al. (2003). The type strain is WPCB074 (5KCTC 13278 5JCM 16348T). The type strain is V2-BIII-A2T (5CCM 4982T5DSM 14730T). Emended description of Fictibacillus gelatini comb. nov. Description of Fictibacillus macauensis comb. nov. Fictibacillus gelatini (ge.la.ti9ni. N.L. neut. gen. n. gelatini from gelatin). Fictibacillus macauensis (ma.cau.en9sis. N.L. masc. adj. macauensis pertaining to Macau, the city where the type Basonym: Bacillus gelatini De Clerck et al. (2004). strain was isolated). The description is that of De Clerck et al. (2004) with the Basonym: Bacillus macauensis Zhang et al. (2006). following amendments. The quinone system consists predominantly of menaquinone MK-7. Minor amount of MK-8 and The description is that of Zhang et al. (2006). MK-6 are present. The polar lipid profile is composed of the The type strain is ZFHKF-1T (5JCM 13285T5DSM 17262T). major compounds DPG, PG and PE and in minor amounts an unknown aminophospholipid and a phospholipid. T T Description of comb. nov. The type strain is LMG 21881 (5DSM 15866 ). Fictibacillus solisalsi (so.li.sal9si. L. neut. n. solum -i soil; L. adj. salsus -a -um salty; N.L. gen. n. solisalsi of a salty soil). Description of Fictibacillus phosphorivorans sp. nov. Basonym: Bacillus solisalsi Liu et al. (2009). Fictibacillus phosphorivorans (phos.pho.ri.vo9rans. N.L. n. The description is that of Liu et al. (2009). phosphorus (from L. n. phosphorus, morning-star, the The type strain is YC1T (5KCTC 13181T5CGMCC light-bringer), phosphorus; L. part. adj. vorans devouring; 1.6854T). N.L. part. adj. phosphorivorans phosphorus-devouring). Cells (with rounded ends) are Gram-stain-variable to Description of Fictibacillus nanhaiensis comb. Gram-stain-positive. No chains or filaments can be observed after growth on TSA at 28 uC for 48 h. Cells are nov. 2.0–3.0 mm in length and 0.8–1.0 mm in width and show Fictibacillus nanhaiensis (nan.ai.en9sis. N.L. masc. adj. no motility. Centrally located endospores are present in nanhaiensis pertaining to Nanhai, the Chinese name for slightly swollen sporangia. No other cell inclusions can be the South China Sea, the source of the sample from which found. Colonies grown on TSA after 48 h of incubation are the type strain was isolated). circular, convex and beige with a shiny appearance with an http://ijs.sgmjournals.org 2941 S. P. Glaeser and others average diameter of 2–3 mm. No growth is observed on G+C analysis of strain Ca7T and Dr Jean Euze´by for his MacConkey agar. Optimum temperature for growth is 28– nomenclatural advice. 30 uC; growth occurs at 15–36 uC but not at 10 uC and 45 uC. Optimal pH for growth is pH 7–8; growth occurs at REFERENCES pH 6.5–11.5. Growth occurs in the presence of 1 % NaCl (but not at 2 % or above) tested in tryptic soy broth. Aino, K., Hirota, K., Matsuno, T., Morita, N., Nodasaka, Y., Fujiwara, Facultatively anaerobic. Tests for catalase and oxidase T., Matsuyama, H., Yoshimune, K. & Yumoto, I. (2008). Bacillus activities are negative. Tests for production of indole and polygoni sp. nov., a moderately halophilic, non-motile obligate sulphide, gelatinase, b-galactosidase, arginine dihydrolase, alkaliphile isolated from indigo balls. 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