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Cellulomonas Persica Sp. Nov. and Cellulomonas Iranensis Sp. Nov

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Cellulomonas Persica Sp. Nov. and Cellulomonas Iranensis Sp. Nov International Journal of Systematic and Evolutionary Microbiology (2000), 50, 993–996 Printed in Great Britain Cellulomonas persica sp. nov. and NOTE Cellulomonas iranensis sp. nov., mesophilic cellulose-degrading bacteria isolated from forest soils Margaret A. Elberson,1 Fereydoon Malekzadeh,2 Mojtaba T. Yazdi,3 Naimeh Kameranpour,3 Mohammad R. Noori-Daloii,4 Maria H. Matte,5 Manoucher Shahamat,1 Rita R. Colwell1 and Kevin R. Sowers1 Author for correspondence: Kevin R. Sowers. Tel: j1 410 234 8878. Fax: j1 410 468 3912. e-mail: Sowers!umbi.umd.edu 1 Center of Marine Two newly described species of mesophilic, cellulose-degrading, aerobic Biotechnology, bacteria were isolated from forest humus soils along the southern border of University of Maryland Biotechnology Institute, the Caspian Sea. Cellulomonas persica and Cellulomonas iranensis are Columbus Center, Suite proposed as new specific epithets based on comparative sequence analyses of 236, 701 E. Pratt Street, 16S rDNA, DNA–DNA hybridization and phenotypic characteristics. Formal Baltimore, MD 21202, USA species descriptions are provided. 2,3,4 Department of Microbiology, Faculty of Science2, Faculty of Keywords: Cellulomonas persica, Cellulomonas iranensis, cellulase, soil, 16S rRNA Pharmacy3 and Department of Biochemistry, Faculty of Medicine4, Tehran University of Medical Sciences, Tehran, I. R. Iran 5 School of Public Health, University of Sao Paulo, Sao Paulo, Brazil Three strains of cellulolytic bacteria were isolated from molecular-weight DNA was precipitated by adding 0n6 forest humus soil as described previously (Malekzadeh vol. 2-propanol, spooled onto a glass rod, washed et al., 1993). Two of the isolates, strains GG and IT, twice with cold 70% (v\v) ethanol and allowed to air were characterized as Cellulomonas spp. based on dry. The DNA recovered was dissolved in TE buffer selected phenotypic characteristics, including coryne- (10 mM Tris, pH 8n0, 1 mM EDTA, pH 8n0). The form morphology, the ability to hydrolyse cellulose purity of each DNA preparation was such that the and the presence of ornithine in the cell wall A#'!\A#)! ratio was 1n8–1n9. The gene encoding 16S (Malekzadeh et al., 1993). An additional isolate not rRNA was amplified between positions 29 and 1541 previously described, strain OT, was isolated con- (Escherichia coli positions) by PCR using the primer T currently with strains GG and I and has phenotypic combination 5h-GAGTTTGATCCTGGCTCAG-3h T characteristics similar to those of strain I (Table 1). (forward primer) and 5h-AAGGAGGTGATCCAG- CC-3h (reverse primer) (Achenbach & Woese, Phylogeny of the isolated Cellulomonas spp. was 1995; Rainey et al., 1995). Reaction mixtures con- established by comparative analysis of 16S rDNA and tained the following components in 20 µl (final vol- by DNA–DNA hybridization. DNA was extracted ume) in a 200 µl dome top reaction tube: 1i PCR using the CTAB (cetylethylammonium bromide) buffer (Perkin Elmer), 200 µmol of each deoxyribo- method (Ausubel et al., 1995). Residual RNA was nucleoside triphosphate, 15 mM MgCl#,0n025% (v\v) removed by RNase A treatment. The resultant high- formamide, 20 pmol of each primer, 100 ng chromo- somal DNA and 0n25 U Taq (AmpliTaq; Perkin ................................................................................................................................................. Elmer). PCR reactions were controlled with a Peltier The GenBank/EMBL accession numbers for the 16S rDNA sequences of thermocycler (PTC 200; MJ Research), programmed strains GG, IT and OT are AF140036, AF064701 and AF064702, respectively. as follows: initial hold at 95 mC for 15 s; 30 cycles at 01217 # 2000 IUMS 993 M. A. Elberson and others Table 1. Phenotypic characteristics that differentiate strains of Cellulomonas ..................................................................................................................................................................................................................................... Strains: 1, strain GG; 2, strain IT; 3, strain OT;4,C. biazotea (type strain); 5, C. cellasea (type strain); 6, C. cellulans (type strain); 7, C. flavigena (type strain); 8, C. fimi (type strain); 9, C. gelida (type strain); 10, C. uda (type strain). , Not determined; j, positive; jj, strongly positive; w+, weakly positive; k, negative. Phenotypic character* Strain 12345678910 Peptidoglycan amino acid† OG OG OG OG OG LSA OA OG OG OG Cell wall composition Glucose jjjkkkjkjj Glucosamine jj jj jj jj k k jj jj jj j Mannose jjjkjkjkkk Rhamnose jj jj jj jj jj jj k jj k k Growth substrates Acetate w+ jjjjjjkjj Dextrin jjjkk jjjj Gluconate kkkkkjjkkk Lactate kkkjjjkjkk Lactose jkkj jjjjj Raffinose kkkjkkkkkk Mannose kjjj jjjjj -Ribose kkkkkjjkkk Gelatin hydrolysis w+ w+ w+ jj jjjj Urea hydrolysis kjjk kkkk Motility jjjjk kjjk Colony pigment‡ Y Y-W Y-W Y-W Y Y-W Y Y-W Y-W W * Phenotypic characteristics of described species are from Malekzadeh et al. (1993), Stackebrandt & Kandler (1979), Stackebrandt & Keddie (1986) and Yamada & Komagata (1972). † OG, -Ornithine--glutamyl; OA, -ornithine--aspartyl; LSA, -lysine--serine--aspartyl. ‡ Y, yellow; Y-W, yellow-white; W, white. 94 mC for 15 s, 53 mC for 30 s, 72 mC for 60 s; and final 10 min in 6i SSC (1i SSC is 0n15 M NaCl and hold at 72 mC for 5 min. Plasmid libraries were 0n015 M sodium citrate) and transferred onto Nylon generated by directly ligating PCR fragments into membranes (Magnagraph) using a dot blot apparatus plasmid pCRII (Invitrogen) according to the manu- (Bio-Rad). Prehybridization was carried out for facturer’s recommendations. The plasmid libraries 30 min at 60 mC and hybridization was performed at were screened for clones containing 16S rDNA inserts the same temperature in a buffer solution containing by direct PCR of colonies (Elberson & Sowers, 1997). 5i SSC and formamide (35%, v\v). Chromosomal For sequencing, plasmid DNA from selected clones DNA of the test strains was denatured by boiling and was purified with a Qiagen Plasmid Mini kit and labelled using DIG High Prime DNA Labelling and amplified with an ABI Prism Dye Terminator Cycle Detection Starter kit II (Boehringer Mannheim). The " Sequencing reaction kit (Perkin Elmer) according to resultant DNA was added (10 ng ml− ) to the pre- the manufacturer’s recommendations. Both strands of hybridized membranes in hybridization buffer and the 16S rDNA (1511 bp) were sequenced using flanking preparations were incubated for 16 h. Hybridization and nested primers. Flanking primers included T7 and was performed at 60 mC overnight and the hybridized SP6. Nested primers were universal for E. coli 16S membranes were washed according to the manu- rRNA positions 519–536f, 549–531r, 907–926f, 926– facturer’s instructions. Chemoluminescence was 907r and 1406–1392r (Achenbach & Woese, 1995; detected using a detection kit (Boehringer Mannheim) Lane et al., 1985). The reaction mixture was loaded and exposed to Hyperfilm (Eastman Kodak). The onto an ABI 373 automated sequencer and the chemoluminescent intensity of each blot was quanti- sequence was analysed using ABI Prism version 2.1.1. fied using a densitometer. The signal produced by self- Levels of genomic relatedness were determined by hybridization of the probe with homologous target DNA–DNA dot blot hybridization (Marteisson et al., DNA was taken as 100% and the percentage 1995). Duplicate aliquots containing 100 and 200 ng of hybridization values were calculated for the duplicated each genomic DNA were denatured by boiling for slots. 994 International Journal of Systematic and Evolutionary Microbiology 50 Cellulomonas spp. from forest soils ..................................................................................................... Fig. 1. Comparative sequence analyses of 16S rDNA from Cellulomonas flavigena GG, Cellulomonas persica IT, Cellulomonas 56 Cellulomonas flavigena iranensis OT and representative strains from 96 Cellulomonas sp. GGT GenBank. The dendrogram was constructed from evolutionary distance matrices with T 97 Cellulomonas sp. I TREECON software using parameters indicated in the text (Van de Peer & De Cellulomonas sp. O Wachter, 1994). The significance of each 92 branch is indicated by a bootstrap value Cellulomonas uda 85 87 calculated for 100 subsets. Bar, 0n02 Cellulomonas gelida substitutions/site. The GenBank accession numbers for the sequences used to generate Cellulomonas fermentans the phylogenetic tree are as follows: 97 Cellulomonas biazotea DSM 20112T, X83802; 61 Cellulomonas cellasea T 74 Cellulomonas cellasea DSM 20118 , X83804; T Cellulomonas fimi Cellulomonas fermentans DSM 3133 , 100 X83805; Cellulomonas fimi DSM 20113T, 100 Cellulomonas biazotea X83803; Cellulomonas flavigena DSM 20109T, X83799; Cellulomonas gelida DSM Cellulomonas sp. CE40 20111T, X83800; Cellulomonas uda DSM 20107T, X83801; Cellulomonas turbata DSM Cellulomonas turbata 20577T, X83806; and Cellulomonas sp. CE40, Cellulomonas cellulans X82598. The 16S rDNA sequences were aligned with rep- Table 2. DNA hybridization among strains of resentative 16S rDNA sequences in the GenBank\ Cellulomonas EMBL database using (Genetics Computer Group, V.8). Based on comparison of a 1450 bp Strain DNA hybridization (%) with DIG- segment of rDNA (E. coli positions 41–1512), isolate labelled reference DNA from strain: GG showed a high level of similarity (99n7%) to Cellulomonas flavigena. Evolutionary distances C. flavigena C. uda C. gelida expressed as estimated changes per 100 nucleotides were calculated from the percentage similarities
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