International Journal of Systematic Bacteriology (1 998),48,833-837 Printed in Great Britain Paenibacillus campinasensis sp. nov., a cyclodextrin-produci ng bacteriurn isolated in Brazi I Jung-Hoon Yoon,’I2 Dong Koo Yim,3 Jung-Sook Lee,’ Kee-Sun Shin,’ Helia Harumi sat^,^ Sung Taik Lee,’Yong Kun Park3 and Yong-Ha Park’ Author for correspondence: Yong-Ha Park. Tel: + 82 42 860 4620. Fax: + 82 42 860 4625. e-mail : yhpark@ kribb4680.kribb.re.kr Korean Collection for Type An alkaliphilic, endospore-forming bacterium isolated from Brazilian soil was Cultures (KCTC), Korea taxonomically studied and is proposed as a new Paenibacillusspecies. This Research Institute of Bioscience and organism (strain 3243 was particularly distinguishable from other Biotechnology (KRIBB), PO Paenibacillus species by its ability to grow optimally at pH 10 and 40 OC. The Box 115, Yusong, Taejon, DNA G+C content was 50.9 mol%. The diamino acid of the cell-wall Korea peptidoglycan was meso-diaminopimelicacid. MK-7 was the predominant * Department of Biological menaquinone and anteiso-C,,:, was the major fatty acid. Levels of 16s rDNA Sciences, Korea Advanced Institute of Science and similarity between strain 324Tand other Paenibacillus species were 906-959 %. Technology, Taejon, Korea Phylogenetically, strain 324Tformed an evolutionary lineage distinct from 3 State University of other species within the evolutionary radiation encompassing the genus Campinas, College of Food Paenibacillus. Based on phenotypic and chemotaxonomic properties, and Engineering (UNICAMP), phylogenetic inference, it is proposed that strain 324Tshould be placed in the Campinas, SP, CEP 13081- 970, Brazil genus Paenibacillus as a new species, Paenibacillus campinasensis. The type strain of the new species is strain 324T(= KCTC 0364BP”). Keywords : Paenibacillus campinasensis, alkaliphilic species, 16s rDNA analysis INTRODUCTION bacillus (Shida et al., 1996), Brevibacillus (Shida et al., 1996), Halobacillus (Spring et al., 1996) and Paeni- Traditionally, because of the lack of incisive differ- bacillus (Ash et al., 1993). entiating criteria, all aerobic, endospore-forming A bacterium was isolated in Brazil that was capable of organisms have been classified as Bacillus. This lack of cyclodextrin glycosyltransferase-mediated production differentiating criteria has also hampered recognition of cyclodextrin from starch (Yim et al., 1997). Based of new species and promoted grouping of organisms on phenotypic characterization, this organism (strain that failed to show clear-cut differences. Developments 324T) was tentatively identified as Bacillus Jirmus. in molecular biological methods have suggested that Shida et al. (1997a) reclassified many Bacillus species the genus is a phylogenetically heterogeneous taxon. as members of the genus Paenibacillus, many species of For example, analyses showed a wide range of G+C which excrete diverse assortments of polysaccharide- contents (32-69 mol %) among the DNAs of Bacillus hydrolysing or -synthesizing enzymes. This work species (Claus & Berkeley, 1986 ; Slepecky & Hemphill, suggested that strain 324T could be a Paenibacillus 1991; Stackebrandt & Liesack, 1993). DNA re- species. To rectify the tenuous classification and to association studies have shown that many Bacillus establish its correct taxonomic position, strain 324T species were composites of several genetically un- was characterized further using 16s rDNA sequencing, related species (Priest, 198 1; Slepecky & Hemphill, chemotaxonomic and additional phenotypic methods. 1991; Nakamura, 1996; Shida et al., 1997b). Finally, Based on data obtained, a new species, Paenibacillus 16s sequencing and resulting phylogenetic studies campinasensis, is proposed. revealed that the genus Bacillus could be separated into several phylogenetically distinct genera such as Alicyclobacillus (Wisotzkey et al., 1992), Aneurini- METHODS Bacterial strain and culture conditions. The isolation of The GenBank accession number for the sequence reported in this paper is strain 324T was described in a previous study (Yim et al., AFO2 1924. 1997). Strain 324Twas cultivated on a medium (pH 10) that 00742 0 1998 IUMS 833 J.-H. Yoon and others contained (1-l): 10 g starch; 5 g peptone; 5 g yeast extract; ticus DSM 5188); D85395 (Paenibacillus chibensis); X60625 1 g K2HP04;0-2 g MgS04.7H20;10 g Na,CO, (separately (Paenibacillus macquariensis) ; X60630 (Paenibacillus autoclaved); and 15 g agar (if needed). Strain 324T was pabuli) ;D85396 (Paenibacillusamylolyticus) ;D85397 (Paeni- cultivated at 37 "C for 24 h on trypticase soy agar for fatty bacillus illinoisensis) ; D783 17 (Paenibacillus alvei) ; U49247 acid methyl ester analysis and on BUGM (Biolog) supple- (Paenibacillus apiarius) ; D78475 (Paenibacillus thiaminoly- mented with 1 % (w/v) glucose for the Biolog substrate ticus) ; D78466 (Paenibacillus curdlanolyticus) ; D7847 1 utilization test. (Paenibacillus kobensis) ; D78320 (Paenibacillus validus) ; D82064 (Paenibacillus chondroitinus) ;D18465 (Paenibacillus Morphological and physiological tests. The morphology of cells was examined by phase-contrast microscopy. Flagellum aliginolyticus) ; X60636 (Paenibacillus larvae subsp. pulvi- faciens) ; X606 19 (P. larvae subsp. larvae) ; X60646 (Bacillus type was examined with transmission electron microscopy using cells from 24 h culture. The cells were negatively subtilis); D16266 (Bacillus cereus); D78312 (Bacillus circulans) ; X60629 (Bacillus megaterium) ; D783 13 (Bacillus stained with 1 % (w/v) phosphotungstic acid and, after air coagulans) ; X62 174 (Halobacillus halophilus) ; D82065 (Am- drying, the grids were examined using a model CM-20 transmission electron microscope (Philips). Motility was phibacillus xylanus) ; D78455 (Aneurinibacillus aneurinoly- observed by the hanging-drop method (Skerman, 1967). ticus) ; D18457 (Brevibacillus brevis) ; X60742 (Alicyclo- bacillus acidocaldarius) ; and V00348 (Escherichia coli). The Catalase activity was determined by bubble production in a 3 (v/v) solution. Oxidase activity was determined by 16s rDNA similarity values were calculated from the YO H202 alignments and the evolutionary distances were calculated oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine. using the Kimura two-parameter correction with the Hydrolyses of gelatin, casein and starch, and production of CLUSTAL w package (Thompson et al., 1994). A phylogenetic urease were determined as described previously (Cowan & Steel, 1965). Hydrolysis of aesculin was determined ac- tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) and the distance matrix data. A cording to the method of Kurup & Fink (1975). Tests for utilization of substrates as sole carbon source were per- bootstrap analysis with 1000 replications for evaluating the formed with GP Biolog microplates containing 95 different topology of the phylogenetic tree was performed with the carbon compounds. The results were checked over 48 h. CLUSTAL w package (Thompson et al., 1994). Isolation of DNA. Chromosomal DNA was isolated by a The GenBank accession number for the 16s rDNA previously described method (Yoon et al., 1996). sequence of strain 324T is AF021924. Chemotaxonomic characterizations. The diamino acid of the peptidoglycan was determined by a previously described RESULT AND DISCUSSION method (Komagata & Suzuki, 1987). Menaquinones were Morphological and physiological characteristics analysed as described previously (Komagata & Suzuki, 1987) by reversed-phase HPLC. Fatty acids were extracted Cells were rod-shaped measuring 0-6-0-9 by 3-0-6.0 pm and analysed according to the instructions of the Microbial in 48 h culture grown at 37 "C. They produced el- Identification System (MIDI ; Microbial ID). lipsoidal spores in swollen sporangia. Colonies were Determination of G+C content. The G+C content was flat, smooth and opaque. Strain 324T formed motile determined by a previously described method (Tamaoka & microcolonies during growth on wet agar plates. Komagata, 1984). DNA was enzymically hydrolysed and However, this characteristic was weak at 45 "C and on dephosphorylated ; the resultant nucleosides were analysed completely dried agar plates. Strain 324T was facul- by HPLC. tatively anaerobic, Gram-variable and motile by 16s rDNA sequencing. 16s rDNA sequencing of strain 324T means of peritrichous flagella (Fig. 1). Strain 324T had was performed as described previously (Yoon et al., 1997). catalase activity, but no oxidase and urease activities. The PCR products were recovered by precipitating with 2- propanol and the strands containing phosphorylated primer from PCR products were selectively digested using A exonuclease according to the instructions included with Strandase ssDNA Preparation kit (Novagen). The ssDNA templates produced were directly used for the sequencing reaction. The sequencing was performed as described pre- viously (Kim et al., 1995) using a-35S-labelleddATP and a DNA sequencing kit (US Biochemical). The sequencing primers were derived from conserved regions of previously described 16s rRNA gene sequences of eubacteria. Phylogenetic analysis. The 16s rDNA sequence of strain 324T was aligned with the 16s rRNA and 16s rDNA sequences of other Paenibacillus species and some other taxa using CLUSTAL w software (Thompson et a/., 1994). Other reference sequences, obtained from the GenBank database, had the following accession numbers : X60632 (Paenibacillus polymyxa) ; D783 18 (Paenibacillus azotojixans); D78476 (Paenibacillus peoriae) ; D783 19 (Paenibacillus