Ureibacillus Suwonensis Sp. Nov., Isolated From Cotton Waste Composts

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Ureibacillus suwonensis sp. nov., isolated fromcotton waste composts

Byung-Yong Kim,1 Seon-Young Lee,1,2 Hang-Yeon Weon,3

Soon-Wo Kwon,1 Seung-Joo Go,1 Yong-Keun Park,2 Peter Schumann4and Dagmar Fritze4

Correspondence

Soon-Wo Kwon

[email protected]

1Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Instituteof Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707,Korea

2School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea

3Applied Microbiology Division, National Institute of Agricultural Science and Technology,RDA, Suwon 441-707, Korea

4Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b,D-38124 Braunschweig, Germany

The taxonomic position of two spore-forming strains 6T19T and 6T29, isolated from cotton

composts for the cultivation of oyster mushroom (Pleurotus ostreatus), was investigated by a

polyphasic approach. Cells of strains 6T19T and 6T29 were rod-shaped, Gram-negative and

strictly aerobic. Sequencing and comparative analyses for the 16S rRNA genes of these strains

clearly showed their phylogenetic affiliation to the genus Ureibacillus. Their closest relatives

Ureibacillus thermosphaericusand Ureibacillus terrenushave sequence similarity of 96?9 and

97?5 %, respectively. The isoprenoid quinones of isolate 6T19T were MK-9, MK-8, MK-7, MK-10

and MK-6 (45 : 27: 18: 5 : 4 %), the peptidoglycan type wasL-LysrD-Asp and the main cellular

fatty acid was i-C16: 0. DNADNA hybridization experiments resulted in relatedness values of 37 %

between 6T19T andU. thermosphaericusDSM 10633T and 41% between 6T19T and U. terrenus

DSM 12654T. Based on the polyphasic data, strains 6T19T and 6T29 can be described as

members of a novel species of the genus Ureibacillus, for which the nameUreibacillussuwonenesissp. nov. is proposed. The type strain is 6T19T (=KACC 11287T=DSM 16752T).

Ureibacilluswas first described by Fortinaet al. (2001) andit comprises two ureolytic thermophilic bacilli species,Ureibacillus thermosphaericusand Ureibacillus terrenus.

In Korea, composted cotton wastes are frequently used asmedium for cultivation of oyster mushroom, Pleurotusostreatus. During the sterilization and composting of cottonwastes, the temperature of composts is raised graduallyfrom room temperature to 65 uC. Two bacterial strains wereobtained from the cotton wastes during the compostingprocess.

These strains were isolated from trypticase soy agar (TSA) atpH 7?0 incubated at 50 uC and maintained in TSA medium.Colonies were not detected as single entities because of thesmeared growth on both TSA and nutrient agar (Oxoid).Gram staining was done by using a Gram stain kit (Difco)according to the manufacturers recommended protocol.KOH test and L-alanine aminopeptidase assay were alsoused (Gregersen, 1978). The presence of flagella was sought

according to Heimbrook et al. (1989). Cell morphologywas observed in a phase-contrast microscope after incuba-tion for 2 days on CASO agar (DSMZ medium no. 220;http://www.dsmz.de/media/media.htm) supplemented with10 mg MnSO4 l

21. Cells grown on TSA for 24 h wereexamined by scanning electron microscopy. An agar blockwas cut from the plate, fixed in 1 % osmium tetroxide andobserved under a scanning electron microscope (HitachiS-2460N).

The following physiological tests were carried out accord-ing to Gordon et al. (1973) and Claus & Berkeley (1986):catalase test, anaerobic growth, VogesProskauer (VP) test,

Published online ahead of print on 25 November 2005 as DOI10.1099/ijs.0.63703-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNAgene sequences of strains 6T19T and 6T29 are AY850379 andAY850380.

Supplementary figure showing the morphological shapes of strain6T19T and a supplementary table showing the cellular fatty-acidcontent of Ureibacillus suwonensis and other Ureibacillus species areavailable in IJSEM Online.

63703 G 2006 IUMS Printed in Great Britain 663

International Journal of Systematic and Evolutionary Microbiology(2006), 56, 663666 DOI 10.1099/ijs.0.63703-0


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temperature range for growth (570 uC, increments of 5 uC),egg-yolk reaction, resistance to lysozyme, growth in thepresence of NaCl (0, 2, 5, 7 and 10 %), growth at pH 5?7,acids from carbohydrates (D-glucose, L-arabinose, D-xyloseand D-mannitol), formation of gas from glucose, hydrolysisof starch, utilization of citrate and propionate, nitrate reduc-tion, production of indole, deamination of phenylalanine,decomposition of casein and tyrosine and liquefaction ofgelatin. Oxidase test and hydrolysis of aesculin were con-ducted according to Smibert & Krieg (1994). Motility testwas performed on 1/10 CESP agar (1?5 g casitone, 0?5 gyeast extract, 0?3 g soytone, 0?2 g peptone, 0?015 g MgSO4,0?007 g FeCl2, 0?002 g MnCl2, made up to 1 l with distilledwater, pH 7?2) (Fortinaet al., 2001).

The 16S rRNA gene sequences were determined by PCRamplification (Kwon et al., 2003) and direct sequencing(Hiraishi, 1992). The 16S rRNA gene sequences were alignedby using the MEGALIGNprogram ofDNASTAR. An evolution-ary distance matrix was generated as described by Jukes &Cantor (1969). The evolutionary tree for the datasets wasinferred from the neighbour-joining method of Saitou &Nei (1987) by using MEGAversion 2.1 (Kumaret al., 2001).The stability of relationships was assessed by performingbootstrap analyses of the neighbour-joining data based on1000 resamplings.

DNADNA hybridization was carried out as a filter-hybridization method described by Seldin & Dubnau(1985). Probe labelling was conducted by using the non-radioactive DIG-High prime system (Roche) and hybridizedDNA was visualized by using the DIG luminescent detectionkit (Roche). DNADNA relatedness was quantified by using

a densitometer (Bio-Rad). The DNA G+C content (mol%)was determined by HPLC analysis of deoxyribonucleosidesas described by Mesbahet al. (1989) using a reverse-phasecolumn (Supelcosil LC-18-S; Supelco).

Peptidoglycan structure, menaquinones and polar lipidswere analysed according to Fortina et al. (2001). Aftergrowth of cells on TSA for 24 h at 50 uC, fatty acid methylesters were extracted and prepared by the standard protocolof the Microbial Identification system (MIDI; MicrobialID).

Supplementary Fig. S1 showing the morphological shapesof strain 6T19T is available in IJSEM Online. Comparisonsof phenotypic properties among Ureibacillus species arepresented in Table 1. In the report of the novel genusUrei-bacillus, Fortina et al. (2001) characterized the genus asureolytic, aerobic bacilli. For the urease test of the Urei-bacillusspecies, Fortina et al. (2001) used the method byAtlas (1993), which relies on the demonstration of alkal-inity. However, alkalinity can be produced from the use ofpeptone or other proteins in the medium, showing false-positive results (MacFaddin, 2000). In our tests, based ontwo different media, the medium used by Fortina et al.(2001) changed from yellow to pinkred colour, indicatinga positive reaction. However, both control media inoculatedwith and without urea also showed a colour change topink, indicating it to be an inappropriate test for urease ofUreibacillusstrains. On the other hand, the cultures whichwere inoculated in Rustigian and Stuarts urea broth showedno colour change, indicating a negative reaction in all thestrains tested. The phenotypic comparison amongUreibacil-lusspecies is shown in Table 1.

For the phylogenetic analysis of the 16S rRNA gene sequences,representative sequences retrieved from GenBank werealigned from position 37 (59) to 1431 (39) (Escherichia colinumbering; Brosiuset al., 1978). Strains 6T19T and 6T29

fell within the radiation of cluster comprising the twoUreibacillusspecies and Bacillus thermocloacaeDSM 5250T

(Fig. 1). The 16S rRNA gene sequence similarity betweenstrains 6T19T and 6T29 was 99?8 %. The closest relatives of

Table 1. Differentiating phenotypic characteristics of Ureibacillus species

Species: 1, U. suwonenesis sp. nov.; 2, U. thermosphaericus; 3, U. terrenus. Data from Fortina et al.

(2001) and this study. All strains were negative for urease according to our results. +, Positive; 2,

negative; V, variable among strains.

Characteristic 1 2 3

Spore shape* S/O S S

Growth at/in:

35 uC + + 2

65 uC 2 2 +

5% NaCl + + V

Aesculin hydrolysis 2 + +

Major menaquinones MK-9, MK-8, MK-7,

MK-10, MK-6

MK-7 MK-9, MK-8, MK-10,

MK-7, MK-11

DNA G+C content (mol%) 41?5 35?739?2 39?641?5

*S, Spherical; O, oval.

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strain 6T19T were U. terrenus (97?5%) and U. thermo-sphaericus (96?9 %). On the other hand, strain 6T19T wasvery distinct from species classified in other genera (


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Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules.In Mammalian Protein Metabolism, pp. 21132. Edited by H. N.Munro. New York: Academic Press.

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Ureibacillus Suwonensis Sp. Nov., Isolated From Cotton Waste Composts

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