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Bacillus Subtilis, Isolated from Soil in Death Valley, California

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Bacillus Subtilis, Isolated from Soil in Death Valley, California 7557 INTERNATIONAL JOURNAL OF SYSTEl\lATIC BACTERIOLOGY. Apr. 1996. p. 470-475 Vol. 46. NO.2 0020-7713/96/504.00+0 Copyright ;g 1996. International Union of Microbiological Societies Bacillus vallisnloltis sp. nov., a Close Relative of Bacillus subtilis, Isolated from Soil in Death Valley, California I 2 3 MICHAEL S. ROBERTS. ,; L. K. NAKAMURA. AND FREDERICK M. COHAN 1 Center for Microbial Ecology. Michigan State University. East Lansing. Michigan 48824-1101 ; Microbial Properties Research. National Center for Agriculwral Utilization Research. Agricultural Research Sen'ice, U.S. Department ofAgriculture, 2 3 Peoria, Illinois 61604 ; and Department 0.( Biology, Wesleyan University, iHiddletown, Connecticut 06459-0170 Five Bacillus strains isolated from Death Valley soil were shown to belong to a previously unidentified species, for which we propose the name Bacillus vallismortis. The type strain is strain DVI-F-3 (= NRRL B-14890). On the basis of previously published restriction digestion data, B. vallismortis is most closely related to Bacillus subtilis. At this time B. vallismortis can be distinguished from B. subtilis only by differences in whole-cell fatty acid compositions, DNA sequences, and levels of reassociation of genomic DNA. The species most closely related to Bacillus subtilis are easily eludes the prefix B-. which identifies organisms that were obtained directly from distinguished by differences in their DNA sequences. B. subti­ individuals or were isolated at the National Center for Agricultural Utilization Research. or the prefix NRS-_ which identifies strains obt';ined from the N. R. lis, Bacillus mojavensis, Bacillus atrophaeus, Bacillus amyloliq­ Smith Bacilllls collection.) uefaciens, and Bacillus licheniformis can be distinguished by Selection for spontaneous rifampin-resistant mutants. We obtained rifampin­ genomic hybridization data (11, 12, 16): these species form resistant (Ril": encoded by IpoB) mutants of each strain (which were used as distinct DNA sequence clusters for shared genes (15, 16), and donors in transformation experiments) by using previously described methods (16). as a result of sequence divergence, levels of transformation Purification of genomic DNA. For DNA preparation. the type strains of B. among these species are low (14, 16, 21). However, the se­ popilliae and B. lemimorbus were cultured in J medium by using the protocol of quence differences among these species are accompanied by Gordon et al. (6): all other strains were cultured in brain hean infusion medium very few phenotypic differences. B. subtilis can be distinguished (Difco). Genomic DNA for use in transformation experiments was obtained from a spontaneous RW mutant of each donor strain. as described previously (3). phenotypically from B. mojavensis only by its fatty acid com­ The procedure used to prepare highly purified DNA for reassociation experi­ position (16) and from B. atrophaeus only by its pigmentation ments has been described previously (13). (12). B. subtilis can be distinguished from B. amyloliquefaciens Transformation. Using the protocol of Cohan et al. (3). we induced strains to by only three phenotypic traits (9, 11) and from the more become competent for transformation in liquid cultures. Each strain was then transformed to rifampin resistance with genomic DNA from its own RiF mutant distantly related organism B. licheniforl71is by only five pheno­ (homogamic transformation) and with DNAs from RW mutants of other strains typic traits (9). The dearth of diagnostic phenotypic character­ (hetcrogamic transformation). \Ve calculated transformation frequencies by de­ istics in this group suggests there may be species which are termining the fraction of cells from each recipient culture that were transformed closeIv related to B. subtilis that have not been discovered yet to rifampin resistance. after we corrected for mutation. Sexual isolation (i.e.. atropha~lIs resistance to transformation) between a recipient and a lest donor was quantified (16). Indeed, two close relatives of B. subtilis (B. by determining the ratio of the frequency of homogamic transformation to the and B. l71ojavensis) have been discovered in only the last 6 years frequency of heterogamic transformation (14. 21). (12, 16). DNA-DNA reassociation and G+C content determinations. The renaturation A recent survey of genetic variation among soil isolates rates of genomic fragments from pairs of strains were determined spectropho­ tometrically with a model 250 UV spectrophotometer (Gilford Systems. CIBA­ having B. sllbtilis-like ph'enot:ypes revealed an un'expected clus­ Corning Diagnostics Corp., Oberlin. Ohio) equipped with a model 2527 ther­ ter of strains very closely related to B. subtilis (15). On the basis moprogrammer (13). The equation of De Ley et al. (4) was used to calculate the of the results of a restriction digestion analysis of the gyrA, extent of DNA-DNA reassociation. polc' and 'poB genes, a cluster of five strains obtained from The G + C content was estimated by the thermal melting procedure described bv Mandel and Marmur (lO). Escherichia coli DNA with a G+C content of 51 Death Valley formed a monophyletic group that was most ~olSc was used for comparison. closely related to B. subtilis (Fig. 1). In this paper we present Phenotypic characterization. Physiological. morphological. and metabolic evidence that these Death Valley group strains are members of characteristics (except fatty acid composition) were determined as described a previously unidentified Bacillus species. previously (6. 13). The whole-cell fatty acid composition was determined by using the MIDI system of Sasser (18) and organisms that had been grown for 24 h at 28'C on Trypticase soy agar. We determined the fatty acid compositions of the MATERl!\LS AND METHODS live members of the Death Valley group and compared these compositions with data for related species obtained previously (16). B. lelllimorbus and B. popilliae Bacterial strains. The five strains previously identified as members of Ihe were not characterized because their phenotypes were known to be very ditTerent Death Valley group (IS) are listed in Table I. These strains were isolated from from the phenotypes of B. Sliblilis-like organisms (2). soil obtained at four collection sites in Death Valley (15). A prototrophic. rifampin-susceptible derivative of B. sllblilis 168. designated strain lA2 (= NRRL B-148191, was obtained from the Bacilllls Genetic Stock Center. The RESULTS Agricultural Research Service Culture Collection at the National Center for Sexual isolation between the Death Valley group and B. Agricultural Utilization Research provided the following type strains: B. sllbtilis. Each Death Valley group strain failed to show any amyloliqllefaciells NRRL B-14393 (= ATCC 23350). B. alroplwells NRRL NRS­ 213. Bacilllls lelllimorblis NRRL B-2522 (= ATCC 147(7). B. IichCllilormis evidence of transformation toward rifampin resistance. NRRL NRS-1264 (= ATCC 145S0). B. moja\'cllsis NRRL B-14698. Bacilllls whether the donor DNA was from the recipient's own RiF popilliae NRRL B-2309 (= ATCC 147(6). and B. sllblilis NRRL NRS-744 (= mutant or from a RiF mutant of B. subtilis lA2: that is. the ATCC 6(51). (The prefix NRRL indicates that a strain is maintained in the frequency of appearance of rifampin-resistant colonies when Agricultural Research Service Culture Collection: each NRRL designation in- donor DNA was added was about the same as the frequency of appearance under mutation alone (data not shown). Because '" Corresponding author. Phone: (517) 432-3771. Fax: (517) 432­ of this apparent lack of competence in the Death Valley group 3770. Electronic mail address: robert62<f~pilot.msu.edu. strains, sexual isolation between the Death Valley group and 470 VOL. 46. 1996 BACILLUS k:4LLISMORTlS SP. NOV. 471 TABLE 2. Levels of sexual isolation when B. sl/blilis IA2 was the DV1-A-1 DV1-F-3 DV8-1.7-4= =J Death recipient and Death Valley group strains were the donors Valley Group Sexual Transformation DV4-D-3 DV7-C-1 Donor strain isolation frequenc),a b ,----B.slIb/ilis 1A2 value '-----B. slIb/ilis 2AZ Death Valley group strains DVI-A-l -2.39 = 0.12 0.22 =0.13 ,--__ B. mojavensis RQ..H-1 T DVI-F-3T -2.54 = 0.10 0.36 = 0.15 1 ,-----8.amy/oliqllefaciens ATCC 23350 DV4-D-3 -2.36 = 0.09 0.18 = 0.16 DV7-C-l -2.34 ::: 0.10 0.16=0.17 '-------B. a/rophaells N'R.RL NR5-Z13 T DV8-1.7-4 -2.24 = 0.06 0.06 = 0.15 '-------------- B. lichenifomlis ATCC 14580T Mean'" -2.37::: 0.05 0.20 = 0.05" FIG. 1. Phylogeny based on restriction digestion of the 'poB. &~·iA. and pole a Transformation frequencies were loglo transformed as described by Cohan ct genes of 84 wild isolates and 2 laboratorv strains of B. slib/ifis. 26 wild isolates of al. (3). The values are the mean standard error for each strain. based on five S. 1Il0jarensis. 14 wild isolates of B. U;·heniforlllis. and the type strains of B. experimental trials. alllyloliqll~I{,cieIl.L B. a/wphaell.L and B. lichenifomlis (phylogeny abridged from " Factor by which the recipient's transformation frequency' was reduced in the study of Roberts and Cohan [15]). The wild isolates were collected from the heterogamic transformation compared with the frequency at which the recipient Gobi Desert. the Sahara Desert. and three deserts in southwestern North Amer­ was transformed by its own Rif mutant's DNA. Sexual isolation values were ica. The Death Valley group strains were found only in Death Valley in Cali­ loglU transformed. The values are the mean = standard error for each strain, fornia. The hypothesis of monophyly of the Death Valley group has 98% boot­ based on five experimental trials. The recipient was transformed b.y its own RifT strap support (15). Strains IA2 and 2A2 represent the 168 and \V23 subgroups mutant's DNA at an average IoglO-transformed frequency of -2.18 0.14 (= of B. slIb/ifis. respectively. Horizontal distances correspond to levels of sequence 6.64 Hr') in five experimental trials.
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