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Cellulomonas Carbonis Sp. Nov., Isolated from Coal Mine Soil

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Cellulomonas Carbonis Sp. Nov., Isolated from Coal Mine Soil %paper no. ije034934 charlesworth ref: ije034934& New Taxa - Actinobacteria International Journal of Systematic and Evolutionary Microbiology (2012), 62, 000–000 DOI 10.1099/ijs.0.034934-0 Cellulomonas carbonis sp. nov., isolated from coal mine soil Zunji Shi, Guosheng Luo and Gejiao Wang Correspondence State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan, Hubei 430070, PR China [email protected] or [email protected] A Gram-positive, aerobic, motile, rod-shaped bacterium, designated strain T26T, was isolated from subsurface soil of Tianjin coal mine, China. Colonies were yellow–white, convex, circular, smooth and non-transparent on R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain T26T was closely related to members of the genus Cellulomonas and a member of the genus Actinotalea with 96.8–94.7 % and 96.7 % gene sequence similarities, T respectively. The peptidoglycan type of strain T26 was A4b, containing L-ornithine–D-glutamic acid as the interpeptide bridge. The cell-wall sugars were rhamnose, galactose, xylose and inositol. The major fatty acids (.10 %) were anteiso-C15 : 0 (33.6 %), anteiso-C15 : 1 A (22.1 %), C16 : 0 (14.4 %) and C14 : 0 (12.1 %). The predominant respiratory quinone was MK-9(H4) and the genomic DNA G+C content was 74.4 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol-mannosides and phosphatidylinositol. Comparison of phenotypic and phylogenetic characteristics between strain T26T and related organisms revealed that the new isolate represented a novel species of the genus Cellulomonas, for which the name Cellulomonas carbonis sp. nov. is proposed. The type strain is T26T (5CGMCC 1.10786T5KCTC 19824T5CCTCC AB2010450T). The family Cellulomonadaceae was originally defined by 2008), blood (Brown et al., 2005), cerebrospinal fluid Stackebrandt & Prauser (1991) and included the genera (Funke et al., 1995), a decayed elm tree (Rivas et al., 2004), Cellulomonas (18 species), Oerskovia (4 species) and sediment (Jones et al., 2005), air (Lee et al., 2008) and reed Actinotalea (1 species). The genus Cellulomonas was first periphyton (Rusznya´k et al., 2011), respectively. The described by Bergey et al. (1923) and later emended by typical characteristics of members of the genus Clark (1952) and Stackebrandt et al. (2006). At the time of Cellulomonas are Gram-positive, slender irregular rods writing, the 18 recognized species of the genus with cellulolytic activity, containing menaquinone MK- Cellulomonas were Cellulomonas flavigena (type species), 9(H4) as the predominant respiratory quinone, having C. biazotea, C. cellasea, C. fimi, C. gelida, C. uda (Bergey et anteiso-C15 : 0 and C16 : 0 as the major fatty acids al., 1923), C. hominis (Funke et al., 1995), C. humilata (Stackebrandt et al., 2006) and with a high DNA G+C (Collins & Pascual, 2000), C. iranensis, C. persica (Elberson content (68.5–76.0 mol%). Other diagnostic characteristics et al., 2000), C. xylanilytica (Rivas et al., 2004), C. terrae for the genus Cellulomonas include rhamnose (Rha) as the (An et al., 2005), C. denverensis (Brown et al., 2005), C. major cell-wall sugar in most of the strains, except for C. bogoriensis (Jones et al., 2005), C. composti (Kang et al., gelida, C. uda, C. composti (Kang et al., 2007) and C. 2007), C. aerilata (Lee et al., 2008), C. chitinilytica (Yoon et aerilata (Lee et al., 2008) (data are not available for C. al., 2008) and C. phragmiteti (Rusznya´k et al., 2011). These hominis, C. bogoriensis and C. phragmiteti). The diagnostic species were mostly isolated from soil (Collins & Pascual, diamino acid in position 3 of the peptide subunit of the 2000; Elberson et al., 2000; An et al., 2005). Others were peptidoglycan is ornithine (Orn) with the interpeptide isolated from compost (Kang et al., 2007; Yoon et al., bridge containing either D-aspartic acid (D-Asp) (for C. flavigena, C. persica, C. bogoriensis, C. iranensis and C. phragmiteti)orD-glutamic acid (D-Glu) (for all the other Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, 13 species) (Stackebrandt et al., 2006). The polar lipids phosphatidylinositol; PIM, phosphatidylinositol mannosides; PL, have been reported for six species of the genus phospholipid; PGL, phosphoglycolipid. Cellulomonas and included diphosphatidylglycerol (DPG) The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene and phosphatidylglycerol (PG) (for C. aerilata and C. sequence of strain T26T is HQ702749. chitinilytica), PG (for C. bogoriensis), DPG, phosphatidy- Four supplementary figures are available with the online version of this linositol (PI) and phosphatidylethanolamine (PE) (for C. paper. composti), DPG and PI (for C. flavigena) and DPG, PG, PI, 034934 G 2012 IUMS Printed in Great Britain 1 %paper no. ije034934 charlesworth ref: ije034934& Cellulomonas carbonis sp. nov. Cells of strain T26T were Gram-staining-positive, aerobic, in this study and showed very similar polar lipids to strain motile and rod-shaped. Colonies were yellow–white, T26T (Fig. S4). The polar lipid data and 16S rRNA gene convex, circular, smooth and non-transparent after 3 days sequence phylogenies of both strain T26T and C. bogoriensis incubation on R2A agar at 28 uC. The strain grew on R2A DSM 16987T were consistent with their assignment to the agar, nutrient agar, full-strength TSA and LB agar, but did genus Cellulomonas. The DNA G+C content of strain not grow on MacConkey agar. Detailed results of the T26T was 74.4 mol%. The cell-wall peptidoglycan type was morphological, physiological and biochemical character- A4b, containing L-Orn–D-Glu. The cell-wall sugars were istics are given in the species description. A scanning rhamnose (80.9 %), galactose (3.3 %), xylose (5.3 %) and electron and transmission electron micrographs showing inositol (6.3 %). All of these results for strain T26T were the general morphology of cells of strain T26T are available very similar to those of the type species of the genus as Fig. S1a and b, respectively. The main phenotypic Cellulomonas, C. flavigena DSM 20109T, and to other differences between strain T26T and closely related type species of the genus. Strain T26T could be differentiated strains are shown in Table 1. from members of the genera Actinotalea and Oerskovia on T the basis of differences in the respiratory quinone [MK- The 1442 bp 16S rRNA gene sequence of strain T26 was ] analysed to determine the phylogenetic position of the 9(H4) vs MK-10(H4) and peptidoglycan type (L-Orn–D- novel strain. Strain T26T shared gene sequence similarities Glu vs L-Lys–L-ThrrD-Asp or L-Lys–L-ThrrD-Glu), of 96.8–94.7 % with other species of the genus respectively. Cellulomonas and a member of the genus Actinotalea. On the basis of the close relationship and distinctive T Strain T26 showed the highest 16S rRNA gene sequence phenotypic and phylogenetic differences between strain T similarities with C. cellasea DSM 20118 (96.8 %), A. T26T and other closely related species, it is proposed that T T fermentans DSM 3133 (96.7 %), C. hominis DMMZ CE40 strain T26T represents a novel species of Cellulomonas, with T (96.6 %), C. denverensis W6929 (96.5 %) and C. the name Cellulomonas carbonis sp. nov. bogoriensis 69B4T (96.4 %). A phylogenetic tree constructed using the neighbour-joining algorithm showed that strain Description of Cellulomonas carbonis sp. nov. T26T was always very closely associated with C. bogoriensis 69B4T; other species of the genus Cellulomonas fell into Cellulomonas carbonis (car.bo9nis. L. gen. n. carbonis of other nearby clusters, but all in a large cluster (Fig. 1). This coal, of charcoal). cluster also included four strains of the genus Oerskovia T T Cells are Gram-positive-staining, aerobic, motile and rod- and Paraoerskovia marina CTT-37 , however, strain T26 shaped (0.5–0.862.0–2.4 mm). Colonies are yellow–white, was distinct from members of the genus Oerskovia based convex, circular, smooth and non-transparent after on the peptidoglycan structure (see below). In addition, P. u T T incubation on R2A agar at 28 C for 3 days. Grows on marina CTT-37 could be distinguised from strain T26 on R2A agar, nutrient agar, full-strength TSA and LB agar, but the basis of the cellular fatty acid content and peptidogly- does not grow on MacConkey agar. Temperature range for can structure (Khan et al., 2009). The maximum- growth is 4–45 uC, and optimal temperature for growth is parsimony and the maximum-likelihood trees (Fig. S2a, 28 uC. Growth occurs with NaCl concentrations in the b) also supported the phylogenetic position obtained with range 0–7 % and with pH 6–10 (optimum, pH 7.0). the neighbour-joining algorithm. Catalase-positive and oxidase-negative. Positive in tests Strain T26T gave a negative result in tests for urease activity for b-galactosidase activity but negative result for arginine but a positive result for the hydrolysis of carboxymethyl dihydrolase and urease. Hydrolyses carboxymethyl cel- cellulose and aesculin. The major cellular fatty acids lulose, starch, gelatin, aesculin, but not DNA, casein, (.10 %) were anteiso-C15 : 0 (33.6 %), anteiso-C15 : 1 A tyrosine or Tween 80. Does not produce indole from (22.1 %), C16 : 0 (14.4 %) and C14 : 0 (12.1 %) on R2A tryptophan, but H2S is produced from cysteine and NH3 is medium (details are shown in Table 2). The fatty acid produced from peptone. Positive in tests for nitrate profile of the novel strain was very similar to that of the reduction but negative result in methyl-red and Voges– type species of the genus Cellulomonas, C. flavigena DSM Proskauer tests. Assimilates D-glucose, L-arabinose, man- T 20109 . The major respiratory quinone was MK-9(H4) nose, N-acetylglucosamine, maltose, gluconate, sucrose, (Fig. S3), which was the same as that of members of the glycogen, salicin, melibiose, D-sorbitol, xylose, lactose, D- genus Cellulomonas and was different from A.
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