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