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Comparative Sequence Analyses on the 16S Rrna INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1992, p. 263-269 Vol. 42, No. 2 0020-7713/92/020263-07$02.00/0 Copyright 0 1992, International Union of Microbiological Societies Comparative Sequence Analyses on the 16s rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoten-estris, and Bacillus cycloheptanicus and Proposal for Creation of a New Genus, Alicyclobacillus gen. nov. JEFFREY D. WISOTZJSEY,' PETER JURTSHUK, JR.,' GEORGE E. FOX,2* GABRIELE DEIN€VIRD,~AND KARL PORALLA3 Department of Biology' and Department of Biochemical and Biophysical Sciences, University of Houston, Houston, Taas 77204-5934, and Botanisches Institut, Mikrobiologie, Universitat Tubingen, D- 7400 Tiibingen, Germany3 Comparative 16s rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16s rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organism Bacillus stearother- mophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess w-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16s rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus. Phenotypically, the genus Bacillus is a large and hetero- will eventually be established. Among those bacilli that are geneous collection of aerobic, rod-shaped, gram-positive (to not members of the main groups, 16s rRNA catalog data (36) gram-variable), endospore-forming bacteria (19, 20, 34). The have indicated that one of the taxa that branch most deeply diversity that exists in this genus is demonstrated by the is Bacillus acidocaldarius . The proper taxonomic placement enormous range of genomic guanine-plus-cytosine contents of this obligately acidophilic thermophile, along with two (32 to 69 mol%), as well as the variety of interesting other phenotypically related species (Bacillus acidotewestris phenotypes, that are found in the various Bacillus species (7, and Bacillus cycloheptanicus), is the focus of this paper. 19,27,30). These phenotypes include (but are not limited to) More than 1,400 nucleotides in the 16s rRNA genes of each the ability to fix molecular nitrogen and growth under of these three species were determined. Our data indicate extreme conditions, including growth in thermophilic, psy- that these thermoacidophilic species should be grouped chrophilic, acidophilic, alcalophilic, and halophilic environ- together in a distinct cluster; these organisms appear to be ments, (Le., hot environments, cold environments, acidic related, and they are distinctly different from the other environments, alkaline environments, and environments species that make up the genus Bacillus. All three species in containing high salt concentrations, respectively) (5, 9, 10, this cluster, B. acidocaldarius, B. acidotewestris, and B. 43). Many Bacillus species also utilize a wide assortment of cycloheptanicus, contain a unique type of lipid (w-alicyclic carbon sources for heterotrophic growth, ranging from meth- fatty acids) as the major membrane fatty acid component in anol to complex natural polymers, such as like cellulose and their cells (11, 12, 23). This lipid has not been found in any chitin (34). Even facultatively autotrophic hydrogen-oxidiz- other Bacillus species examined to date. Therefore, we ing sporeformers have been isolated and studied (5). Exten- propose that this group of three related organisms should be sive multiphenotypic and molecular analyses have been used placed in a new genus, Alicyclobacillus gen. nov., in the to identify species in this group, as well as to establish the family Bacillaceae. taxonomic relationships among Bacillus species (27, 30-32). A preliminary report of our findings has been published However, even the resulting data have not simplified the elsewhere (39). task of organizing the members of this group into meaningful taxa, and the diversity found among the species is great enough that the members of the genus no doubt should be MATERIALS AND METHODS subdivided into several genera (2, 33, 36). 16s rRNA catalog Bacterial strains and culture conditions. The Bacillus and subsequent 16s rRNA and rDNA sequence analyses strains used in this study are shown in Table 1. Three strains have clearly revealed the presence of at least three main of B. acidocaldarius (strains DSM 446T [T = type strain], clusters (Fig. 1) in the genus Bacillus (2, 33). In addition, a ATCC 43034, and ATCC 43035) were grown and maintained significant number of Bacillus species are known to fall on American Type Culture Collection medium 573 at 65°C. outside these three clusters. Thus, it is apparent (2) that at B. acidotewestris DSM 2923 and B. cycloheptanicus DSM least two and probably more additional well-defined groups 4006T were grown as described previously (10, 11, 25) and were lyophilized before their nucleic acids were extracted (39)- * Corresponding author. Amplification and cloning. Chromosomal DNA was pre- 263 264 WISOTZKEY ET AL. INT. J. SYST.BACTERIOL. B. acidocaldarius to allow for the extension of any incomplete products. The reaction mixture was kept at 4°C until further analysis. B. acidoterrestris The amplification product was purified and digested with appropriate restriction enzymes (3). BamHI and SalI were B. cycloheptanicus typicalIy used since EcoRI (21) and SmaI (38a) also cut at 8. ALVEl cluster conserved internal sites of Bacillus 16s rRNA genes. The digested product was cloned into the sequencing vector 8. BREWS cluster M13mp19 and was sequenced (28) by using the M13 univer- sal primer and seven internal primers that were complemen- 8. coagulans tary to phylogenetically conserved regions of the 16s rRNA gene. The sequencing primers which we used were comple- 8. subiilis mentary to regions on the Bacillus subtilis 16s rDNA near positions 109, 357, 520, 690, 910, 1110, and 1406. B. stearothermophilus rRNA purification and sequencing. 16s rRNA was isolated, Streptococcus cecorum purified, and sequenced as previously described by using a reverse transcriptase-mediated, primer-directed, dideoxynu- L Lactobacillus lactis cleotide chain-terminating sequencing technique (26). Data analysis. Autoradiographs were read twice by one ILeuconostoc mesenteroides investigator and reread by another investigator. The se- quence data were analyzed by using a VAXstation 3100 LClostridium innocuum instrument (Digital Equipment Corp.) running the VMS FIG. 1. Representative dendrogram showing the relationship of operating system. A specialized sequence set editor, SE- the three thermoacidophilic organisms (i.e., the members of the new QEDT, which was developed and kindly provided by Gary genus AZicycZobacillus) to the traditional members of the genus Olsen (University of Illinois), was used to align the rRNA Bacillus, as well as other gram-positive bacteria. The dendrogram sequence data and generate similarity values. Sequences was generated from 16s rRNA sequence data by using the UPGMA method. A very detailed tree showing the highly divergent position were aligned with the previously published sequence of the of B. cycloheptanicus (and hence by inference the highly divergent 16s rRNA of B. subtilis (22), as well as with the sequences of positions of B. acidocaldanus and B. acidotewestris) has been a variety of partially sequenced Bacillus species (1,2,18,33, published elsewhere (2). 39, 40). For sequence alignments we relied on highly con- served regions of the primary sequence and areas of known secondary structure as reference points. Nucleotide sequence accession numbers. The 16s rRNA pared and polymerase chain reaction gene amplification (11) sequences of B. acidocaldarius ATCC 43034 and DSM 446T, was carried out as previously described (35). The two B. acidotewestris DSM 3923, and B. cycloheptanicus DSM primers used for amplification were complementary to phy- 4006T were deposited in the EMBL electronic data base logenetically conserved portions of the 5' and 3' ends of the under accession numbers X60741, X60742, X60743, and 16s rRNAs and contained 5 ' restriction site polylinker X51928, respectively. regions to facilitate cloning. Primer A (5'-CCG AAT TCG TCG ACA GAG TTT GAT CCT GGC TAG-3') was com- plementary to the 5' end of the 16s rRNA gene and con- RESULTS tained the restriction sites for EcoRI and SulI, while primer The sequences of 16s rRNA segments totalling more than B (5'-CCC GGG ATC CAA GCT TAG AAA GGA GGT 1,400 nucleotides each were determined for B. acidocaldar- GAT CCA-3') was complementary to the 5' end of the ius ATCC 43034 and DSM 446T, B. acidotewestris DSM opposite strand of the 16s rRNA gene and contained restric- 3923, and B. cycloheptanicus DSM 4006T. In addition, tion sites for SmaI, HindIII, and BamHI. A DNA thermal several hundred nucleotides of the B. acidocaldarius ATCC cycler (Perkin-Elmer-Cetus)
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