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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 (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 of Bacillus hydrolysing or -synthesizing . 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) 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 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 macerans) ; X77846 (Paenibacillus durum) ; D78473 (Paenibacillus lautus NRRL NRS-666T); D85609 (Paenibacillus lautus NRRL B- 377); D85394 (P. lautus NRRL B-379); D78470 (Paeni- Fig. 7. Transmission electron micrograph of strain 324T from a bacillus glucanolyticus DSM 5 162T) ; D885 14 (P. glucanoly- 24 h culture. Bar, 1 pm.

International Journal of Systematic Bacterioloav 48 Paenibacillus campinasensis sp. nov.

Table 7. Substrates oxidized as sole carbon source by strain 324T

...... I...... , . . , ...... , . . . . , ...... , . , ...... , ...... , ...... , . . , ...... , . . . , . , . , ...... , ...... , .. . . , .. . + , Oxidized; - , not oxidized; w, weak reaction.

~~ ~~ ~~ ~~ ~~ ~ ~~ ~ Substrate Reaction Substrate Reaction Substrate Reaction

Carbohydrates : Psicose Propionic acid N-Acetyl-D-ghcosamine D- Pyruvic acid N-Acetyl-D- L-Rhamnose Succinamic acid mannosamine D-Ribose Succinic acid Amy gdalin Salicin Alcohols : L-Arabinose Sedoheptulosan 2,3-Butanediol Arabitol D- Glycerol Arbutin Stachyose Amino acids: Cellobiose D- Alanine a-C yclodextrin Tagatose L-Alanine P-C yclodextrin D- L- Alan y l-gl ycine Dextrin Turanose L- Asparagine D- Xylitol L-Glutamic acid D-Fucose D-Xylose Glycyl-L-glutamic acid L-Fucose Esters : L-Pyroglutamic acid D-Galactose Methylpyruvate L-Serine Gentiobiose Mono-methylsuccinate Nucleosides : D-Glucose D-Lactic acid methyl Adenosine Glycogen ester 2-Deoxy adenosine rn-Inositol Detergents : Inosine Tween 40 Thymidine Lactose Tween 80 Uridine Lactulose Carboxylic acids : Nucleotides : Adenosine 5'- Malt otriose N-Acetyl-L-glutamic acid monophosphate Mannan D-Galacturonic acid Thymidine 5'- D-Gluconic acid monophosphate D- a-Hydroxybutyric acid Uridine 5'- D-Melezitose P-Hydroxybutyric acid monophosphate Melibiose y-Hydroxybutyric acid Phosphorylated compounds : Methyl a-D-galactoside p-Hydroxyphenyl acetic Fructose 6-phosphate Methyl P-D-galactoside acid Glucose 1-phosphate 3-Methylglucose a-Ketoglutaric acid Glucose 6-phosphate Methyl a-D-glucoside a-Ketovaleric acid DL-a-Glycerol phosphate Methyl P-D-glucoside L-Lactic acid Putre scine Methyl a-D-mannoside D-Mak acid Alaninamide Palatinose L-Malic acid Lactamide

This strain grew in the presence of 7% (w/v) NaCl, important phenotypic difference that distinguished whereas most Paenibacillus species failed to grow in strain 324T from the known Paenibacillus species. The the presence of 5% (w/v) NaCl (Shida et al., 1997a). results of utilization of substrates as sole carbon Gelatin, casein, aesculin and starch were hydrolysed. sources for respiration (formation of NADH) on Strain 324T grew at 10 and 45 "C, but not at 5 and Biolog microplates are shown in Table 1. 50 "C. The optimum growth temperature was 40 "C. In contrast, the optimum growth temperature for all Chemotaxonomic characteristics and DNA base Paenibacillus species, except P. macquariensis, was composition 28-30 "C (Shida et al., 1997a). P. macquariensis had an optimum growth temperature of 20-23 "C (Shida et meso-Diaminopimelic acid was the diamino acid found al., 1997a). Strain 324Twas alkaliphilic. This strain did in the cell-wall peptidoglycan of strain 324T. This not grow at pH 7, but grew at pH 7.5-10.5. The diamino acid also appeared in the members of the optimum pH for growth was 10; members of the genus genus Paenibacillus (Shida et al., 1997a). Other aerobic Paenibacillus grew optimally at pH 7-0 (Shida et al., endospore formers such as Bacillus, 1997a). The alkaliphilic property of strain 324T was an and also contained meso-diaminopi-

International Journal of Systematic Bacteriology 48 835 J.-H. Yoon and others

almost complete 165 rDNA sequence (1 508 bp), which corresponds to a region between positions 28 and 1524 by comparison with the E. coli 16s rRNA gene, was directly sequenced. The phylogenetic tree (Fig. 2) Halobacillus halophilus constructed from the sequence data shows that strain 324T appeared within the evolutionary radiation en- Aneurinibacillus aneurinolyricus Brevibacillus brevis compassing the genus Paenibacillus and occupied a

Paenibacillus chibensis distinct phylogenetic position within the genus. Levels Paenibacillus macquariensis of 16s rDNA similarity between strain 324T and the Paenibacillus species were 90-6-95-9 YO.The highest 16s rDNA sequence similarities of 95.2-95.9 YOwere observed between strain 324T and P. lautus, and between strain 324T and P. glucanolyticus. The phylo- Paenibacillus glucanolyficus DSM 5 I 62T genetic definition states that ' strains with approxi- Paenibacillus glucanolyricus DSM 5 188 mately 70% or greater DNA-DNA relatedness' are members of the same species (Wayne et al., 1987).

Paenibacillus peoriac According to the available compilation of data by Paenibacillus polymyrn Stackebrandt & Goebel (1994), organisms that have Paenibacillus durum less than 97.0 YO16s rDNA or 16s rRNA similarities Paenibacillus macerans will not reassociate to more than 60%, no matter Poenibacillus fhiam'nolyricus which hybridization method is used. The phylogenetic

Paenibacillus apiarius study clearly established that strain 324T was a Paenibacillus species.

I 7851- Pamibacillus validus The phenotypic, chemotaxonomic and phylogenetic larvae subsp. pulvifaciens data showed that strain 324T belonged to the genus Paenibacillus larvae subsp. larvae Paenibacillus. Moreover, phenotypic uniqueness im- parted by the alkaliphilic and moderately thermophilic nature of the strain and genetic distinctiveness inferred Alicyclobacillus acidocaldarius from the phylogenetic study warranted the proposal of - Escherichia coli strain 324T as a new species, Paenibacillus campin- 0.02 K,,, asensis sp. nov.

Fig. 2. Phylogenetic tree showing the position of strain 324T in relation to Paenibacillus species and some rod-shaped, Description of Paenibaci//uscampinasensis sp. nov. endospore-forming based on 165 rRNA gene sequences. Bootstrap values are indicated. Bar, 2 nucleotide Paenibacillus campinasensis (cam. pi .na.sen'sis. M .L. substitutions per 100 nucleotides. adj. campinasensis referring to Campinas, the city where the College of Food Engineering, State Uni- versity of Campinas, Brazil, is located). melic acid in their cell-walls (Shida et al., 1997a). The major isoprenoid quinone was MK-7, which is the Cells are rods measuring 0-6-0.9 by 3-0-6.0 pm and -- - -. _. motile by means of peritrichous flagella. Ellipsoidal msinr mensniiinnne cwnerallv ----a-thiind in 2ernhir.v endn--*-uv ---- J------=------,------, spore-forming rods. Anteiso-C,,: (53 %), the pre- spores are formed in swollen sporangia. Colonies are dominant fatty acid found in the genus Paenibacillus, flat, smooth and opaque. Forms motile microcolonies on wet agar plates. Facultatively anaerobic and Gram- was also tne major component 01 strain 5.~4' Iatty acids. Although other aerobic, endospore-forming variable. Catalase-positive and oxidase- and urease- negative. Growth occurs in the presence of 7 % NaCl. genera also contained large amounts of anteiso-C,, :,, fatty acid, they simultaneously contained significant Gelatin, casein, aesculin and starch are hydrolysed. amounts of other fatty acids (Shida et al., 1997a). Utilizes L-arabinose, /3-cyclodextrin, D-fructose, D- glucose, melibiose, 3-methylglucose, psicose, D-ribose, The G+C content of strain 324T was 50.9 mo1Y0, a D-sorbitol, tagatose, D-xylose, Tween 40, acetic acid, level included within the range found in the members propionic acid, pyruvic acid, fructose 6-phosphate and of the genus Paenibacillus (Shida et al., 1997a). glucose 6-phosphate as sole carbon sources for res- piration. Substrates which are not utilized are shown Phylogenetic analysis in Table 1. Grows at 10 and 45 OC, but not at 5 and 50 "C; optimum temperature is 40 "C. Alkaliphilic. Although the preceding chemotaxonomic, G + C con- Does not grow at pH 7. Grows at pH 7.5-10.5; tent and biochemical analyses did not establish a optimum pH is 10. Cell-wall peptidoglycan contains definitive taxonomic position for strain 324T, they meso-diaminopimelicacid. The major isoprenoid quin- suggested Paenibacillus as a possible classification. 16s one is a menaquinone, MK-7. The major fatty acid is rDNA was sequenced to verify the suggestion. An anteiso-C,,: o, The G + C content is 50.9 mol YO (de-

836 International Journal of Systematic Bacteriology 48 Paenibacillus campinasensis sp. nov. termined by HPLC). The type strain is 324T,which was Shida, Om,Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, isolated in Brazil. The type strain has been deposited in K. (1 997b). Emended description of Paenibacillus amylolyticus the Korean Collection for Type Cultures as KCTC and description of Paenibacillus illinoisensis sp. nov. and 03 64BPT. Paenibacillus chibensis sp. nov. Int J Syst Bacteriol47,299-306. Skerman, V. B. D. (1967). A Guide to the Identification of the Genera of Bacteria, 2nd edn. Baltimore : Williams & Wilkins. ACKNOWLEDGEMENTS Slepecky, R. A. & Hemphill, H. E. (1991). The genus Bacillus - nonmedical. In The Prokaryotes, pp. 1663-1696. Edited by A. This work was supported by grant HS 1331 from the Ministry Balows, H. G. Triiper, M. Dworkin, W. Harder & K.-H. of Science and Technology of the Republic of Korea. We are Schleifer. New York: Springer. grateful to Dr Yong Kook Shin, Keunchul Lee and Dr Akio Spring, S., Ludwig, W., Marquez, M. C., Ventosa, A. & Schleifer, Seino for helpful discussions. K.-H. (1996). Halobacillus gen. nov., with description of Halo- bacillus litoralis sp. nov. and Halobacillus truperi sp. nov., and transfer of Sporosarcina halophilia to Halobacillus halophilus REFERENCES comb. nov. Int J Syst Bacteriol46, 492-496. Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a Ash, C., Priest, F. G. & Collins, M. D. (1993). Molecular identi- place for DNA-DNA reassociation and 16s rRNA sequence fication of rRNA group 3 (Ash, Farrow, Wallbanks, and analysis in the present species definition in bacteriology. Int J Collins) using a PCR probe test. Proposal for the creation of a Syst Bacteriol44, 846-849. new genus Paenibacillus. Antonie Leeuwenhoek 64, 253-260. Stackebrandt, E. & Liesack, W. (1993). Nucleic acids and Claus, D. & Berkeley, R. C. W. (1986). 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International Journal of Systematic Bacteriology 48 837