International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2700–2705
DOI 10.1099/ijs.0.055426-0
Planomicrobium soli sp. nov., isolated from soil Xiaonan Luo,1 Jianli Zhang,1 Dai Li,1 Yuhua Xin,2 Di Xin1 and Lei Fan1 Correspondence
1
Jianli Zhang
2
[email protected]
School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
A Gram-staining-positive bacterium, designated strain XN13T, was isolated from a soil sample collected from ALaShan National Geological Park in Inner Mongolia Autonomous Region, China and subjected to a taxonomic study using a polyphasic approach. Strain XN13T was found to have a range of chemical and morphological properties consistent with its classification in the genus Planomicrobium. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain XN13T was related to members of the genus Planomicrobium. The closest phylogenetic relatives were Planomicrobium okeanokoites NBRC 12536T, Planomicrobium koreense JG07T, Planomicrobium mcmeekinii S23F2T and Planomicrobium flavidum ISL-41T with 98.2 %, 97.8 %, 97.8 % and 97.7 % 16S rRNA gene sequence similarity, respectively. The major cellular fatty acids were anteiso-C15 : 0, C16 : 1v7c alcohol, iso-C14 : 0 and C16 : 1v11c. The predominant menaquinones were MK-8 and MK-7. The DNA G+C content was 40.3 mol%. The DNA–DNA relatedness values between strain XN13T and Planomicrobium okeanokoites KCTC 3672T, Planomicrobium koreense KCTC 3684T, P. mcmeekinii CGMCC 1.2724T, Planomicrobium flavidum KCTC 13261T, Planomicrobium chinense CGMCC 1.3454T and Planomicrobium glaciei CGMCC 1.6846T were 36 %, 30 %, 34 %, 29 %, 30 % and 31 %, respectively. The organism is different from recognized species of the genus Planomicrobium in several phenotypic characteristics. On the basis of phenotypic and genotypic properties, strain XN13T represents a novel species of the genus Planomicrobium, for which the name Planomicrobium soli sp. nov. is proposed. The type strain is XN13T (5CGMCC 1.12259T5KCTC 33047T).
The genus Planomicrobium was proposed by Yoon et al. (2001). At the time of writing, the genus Planomicrobium comprises nine species with validly published names: Planomicrobium alkanoclasticum (Engelhardt et al., 2001; Dai et al., 2005), Planomicrobium chinense (Dai et al., 2005), Planomicrobium flavidum (Jung et al., 2009), Planomicrobium glaciei (Zhang et al., 2009), Planomicrobium mcmeekinii (Junge et al., 1998; Yoon et al., 2001), Planomicrobium okeanokoites (Nakagawa et al., 1996; Yoon et al., 2001), Planomicrobium koreense (Yoon et al., 2001), Planomicrobium psychrophilum (Reddy et al., 2002; Dai et al., 2005) and Planomicrobium stackebrandtii (Mayilraj et al., 2005; Jung et al., 2009). Members of the genus Planomicrobium are Gram-staining-positive to Gramstaining-variable, aerobic, motile and non-endosporeforming. Colony colour is yellow to orange or pale orange. The peptidoglycan type is A4a type. The menaquinone profile is characterized by the predominance of MK-8 followed by MK-7, or by the predominance of MK-8 The GenBank/EMBL/DDBJ accession number for the16S rRNA gene sequence of strain XN13T is JQ772482. Two supplementary figures and a supplementary table are available with the online version of this paper.
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followed by MK-7 and MK-6. The cellular fatty acids are mainly saturated, unsaturated and branched fatty acids. The G+C content of the genomic DNA is 35–49 mol% (Yoon et al., 2001; Zhang et al., 2009). A bacterial strain, designated XN13T, was isolated from a soil sample collected from ALaShan National Geological Park (30u 279 240 N 106u 129 030 E, 1038 m above sea level), located in Inner Mongolia Autonomous Region, China. The pH of the soil sample was pH 6. For isolation, the soil sample (1 g) was suspended in distilled water (100 ml) and spread on plates of trypticase soy agar (TSA; Difco) after serial dilution. The plates were incubated at 30 uC for 3 days. Purification was achieved by streaking single colonies on TSA. Strain XN13T was maintained on TSA at 4 uC and as glycerol suspensions (20 %, v/v) at 220 uC. The aim of the present work was to elucidate the taxonomic position of strain XN13T by means of phenotypic, genotypic and chemotaxonomic analyses. The following type strains of species of the genus Planomicrobium were used as reference strains: Planomicrobium okeanokoites KCTC 3672T, Planomicrobium koreense KCTC 3684T and Planomicrobium flavidum KCTC 13261T, obtained from the Korean Collection for Type 055426 G 2014 IUMS Printed in Great Britain
Planomicrobium soli sp. nov.
Cultures, Taejon, Korea; and Planomicrobium mcmeekinii CGMCC 1.2724T, Planomicrobium chinense CGMCC 1.3454T and Planomicrobium glaciei CGMCC 1.6846T, obtained from China General Microbiological Culture Collection Center. General cell morphology was studied by light microscopy (BH-2; Olympus) and transmission electron microscopy (JEM-1400; JEOL) with a 2-day-old culture of strain XN13T grown on TSA. Colonial properties were examined after 3 days of growth at 28 uC on TSA. Gram-staining was performed as described by Smibert & Krieg (1994). Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck), and catalase activity was evaluated by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution (Smibert & Krieg, 1994). Hydrolysis of starch was tested on agar plates containing 1 % (w/v) peptone, 1 % yeast extract and 0.5 % soluble starch. Gelatinase production was determined on Luria–Bertani media containing 3 % (w/v) gelatin. Hydrolysis of casein was examined on nutrient agar supplemented with 1.5 % skimmed milk (Sigma). Hydrolysis of aesculin and nitrate reduction were evaluated as described by La´nyi (1987). H2S production was tested as described previously (Bruns et al., 2001). Acid production from carbohydrates was assessed using methods described by Leifson (1963), and utilization of sole carbon and energy sources was determined according to Gordon & Mihm (1957). Hydrolysis of urea was determined on medium described by Goodfellow & Orchard (1974), comprising 75 ml urea shake flask medium [10 g KH2PO4, 9.5 g Na2HPO4, 1 g yeast extract, with 0.04 % (w/v) phenol red, 1000 ml distilled water, pH 7.2] and 15 ml of 15 % urea. Citrate utilization was tested on Simmons citrate agar (Sigma). Indole production was tested as previously described (Smibert & Krieg, 1994). The temperature range for growth was tested by growing cells at 4–45 uC on TSA. Growth at different salinities up to 10 % (w/v, in increments of 0.5 %) was tested by growing cells on TSA prepared according to the formula of the Difco medium except that NaCl was omitted. The pH range for growth (pH 4.0–11.0, in increments of 1.0 pH unit) was determined in trypticase soy broth by using appropriate biological buffers. In addition, biochemical traits of enzyme activities were examined by using API ZYM kits (bioMe´rieux) according to the manufacturer’s instructions; the API ZYM tests were read after incubation for 4 h at 37 uC. Strain XN13T was a Gram-staining-positive, aerobic, cocci or short rod-shaped, non-endospore-forming bacterium (Fig. S1, available in the online Supplementary Material). Colonies were smooth, circular, convex with entire margins, and pale orange after 3 days cultivation at 28 uC on TSA. Growth occurred at 4–38 uC, but not at 39 uC. Growth was observed at pH 6.0–10.0 and the optimum pH for growth was 7.0–8.0. Growth occurred in the presence of 0.5–7.5 % NaCl with optimal growth at 3–4 % NaCl. Cells grew aerobically and were not capable of reducing nitrate to nitrite. Catalase activity was positive but oxidase activity http://ijs.sgmjournals.org
was negative. The morphological, cultural, physiological and biochemical characteristics of strain XN13T are given in the species description and in Table 1. Cell biomass for isoprenoid quinone analysis was obtained following cultivation in marine 2216 broth (Difco) for 3 days at 30 uC. Menaquinones were extracted and purified using the methods of Collins et al. (1987); purified menaquinones were determined by reverse-phase HPLC (Wu et al., 1989). Preparation of cell walls and determination of peptidoglycan structure were carried out using the methods of Schleifer & Kandler (1972). Polar lipids were extracted by the method of Minnikin et al. (1979) and polar lipid profiles were analysed according to Collins & Jones (1980). For quantitative analysis of cellular fatty acid compositions, cell mass of strain XN13T was collected from marine 2216 broth (Difco) after cultivation for 3 days at 28 uC. Fatty acids were extracted, purified, methylated and identified by using the standard Microbial Identification System (MIDI; Sasser, 1990; Ka¨mpfer & Kroppenstedt, 1996) version 4.5. The G+C content of the DNA was determined by means of the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli K-12 as the reference. Cells of strain XN13T contained MK-8 (73.9 %) and MK-7 (18.9 %) as the major respiratory quinones. The cell-wall composition of strain XN13T contained L-lysine, glutamic acid and alanine, and this is consistent with the A4a type (Schleifer & Kandler, 1972) cell wall of the genus Planomicrobium. The polar lipids of strain XN13T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol (Fig. S2). The major cellular fatty acids were anteiso-C15 : 0 (36.0 %), C16 : 1v7c alcohol (14.3 %), iso-C14 : 0 (10.9 %) and C16 : 1v11c (10.1 %). No pronounced differences in the fatty acid profiles of strain XN13T and the reference strains were found, although there were minor differences in the proportions of some components (Table S1). The genomic DNA G+C content of strain XN13T was 40.3 mol%. Extraction of genomic DNA and amplification of the 16S rRNA gene were performed as described by Rainey et al. (1996). 16S rRNA gene sequences were amplified by PCR from chromosomal DNA using two universal primers as described previously (Yoon et al., 1998). The PCR product was sequenced directly using the method of Lu et al. (2001). The resultant 16S rRNA gene sequence was compared with those available in the GenBank database, using the BLAST program. Multiple alignments with closely related species were performed using the CLUSTAL X 1.8 software (Thompson et al., 1997). Sequence similarities were calculated using the EzTaxon-e server (http://eztaxone.ezbiocloud.net/, Kim et al., 2012). The phylogenetic consensus tree was reconstructed using the neighbourjoining (Saitou & Nei, 1987) and maximum-parsimony (Kluge & Farris, 1969) methods in MEGA software version 5.0 (Tamura et al., 2011), with bootstrap values based on 1000 replications. Evolutionary distances were calculated 2701
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Table 1. Differential properties of strain XN13T and type strains of closely related species of the genus Planomicrobium Strains: 1, XN13T; 2, Planomicrobium okeanokoites KCTC 3672T; 3, Planomicrobium koreense KCTC 3684T; 4, Planomicrobium mcmeekinii CGMCC 1.2724T; 5, Planomicrobium flavidum KCTC 13261T; 6, Planomicrobium chinense CGMCC 1.3454T; 7, Planomicrobium glaciei CGMCC 1.6846T. All data are from this study, except for strain 5 (Jung et al., 2009) and strain 6 (Dai et al., 2005). +, Positive; –, negative; W, weakly positive; ND, no data available; C, Cocci; R, rods; SR, short rods. Characteristic Cell shape Gram-stain Colony colour Oxidase Nitrate reduction: Growth with/at: 7 % NaCl 10 % NaCl 4 uC 37 uC Degradation of: Casein Aesculin Gelatin Tween 80 Acid production from: Cellobiose D-Fructose D-Glucose Lactose Maltose Melibiose Predominant menaquinone(s) DNA G+C content (mol%)*
1 C or SR Positive
2
3
R
C or SR/R Positive to variable Yellow–orange
2 2
4
5
6
7
or SR Positive
C or SR Positive
C or SR Positive
Pale orange
C or SR Positive to variable Light yellow
Yellow–orange
Yellow–orange
2 +
+ 2
2 +
2 +
R/C
2 2
Positive to variable Bright yellow –bright orange 2 2
+ 2 + +
2 2 2 +
2 + +
+ + + +
+ + + +
+ + 2 +
+ + + 2
+ + + 2
+ 2 + 2
+ + + 2
+ 2 2 2
W
2 + +
2 2 + 2
+ + 2
2 2 2 2 2 2 MK-8,7
2 + 2 2 2 2 MK-8,7
+ 2 2 + + + MK-8,7,6
2 + + 2 2 2 MK-8,7
2 + 2 2 2 2 MK-8,7
2
2 MK-8
2 + 2 2 2 2 MK-8,7
40.3
46.3a
47.0b
35.0c
45.9
34.8
49.0d
Pale Orange
W
ND
+ 2 ND
*Data from: a, Nakagawa et al. (1996); b, Yoon et al. (2001); c, Junge et al. (1998); d, Zhang et al. (2009).
by using distance options according to Kimura’s twoparameter model (Kimura, 1980).
(97.9 %)]. However, from the neighbour-joining tree (Fig. 1) strain XN13T fell within the evolutionary radius of the genus Planomicrobium, and the isolate should therefore not be assigned to the genus Planococcus. The maximumparsimony tree also supported the clustering of strain XN13T and species of the genus Planomicrobium.
The 16S rRNA gene sequence of strain XN13T was a continuous stretch of 1468 bp. It was apparent from the neighbour-joining tree (Fig. 1) that strain XN13T was classically defined as belonging to the genus Planomicrobium. Strain XN13T showed 96.5–98.2 % 16S rRNA gene sequence similarities with respect to the type strains of species of the genus Planomicrobium, the highest similarity values being found with the sequences of Planomicrobium okeanokoites NBRC 12536T (98.2 %), Planomicrobium koreense JG07T (97.8 %), Planomicrobium mcmeekinii S23F2T (97.8 %), Planomicrobium flavidum ISL-41T (97.7 %), Planomicrobium chinense DX3-12T (97.7 %) and Planomicrobium glaciei 0423T (97.6 %). In addition, strain XN13T also shared high 16S rRNA gene sequence similarities with respect to the type strains of species of the genus Planococcus [Planococcus salinarum ISL-16T (98.0 %) and Planococcus plakortidis AS/ASP6 (II)T
DNA–DNA hybridization values were determined from the initial DNA–DNA liquid reassociation rate as described by De Ley et al. (1970) and modified by Huß et al. (1983). The tests were performed on a Lambda 35 UV/Vis spectrophotometer equipped with a temperature program controller (Perkin-Elmer). Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as DNA–DNA relatedness values. The mean DNA–DNA relatedness values between strain XN13T and Planomicrobium okeanokoites KCTC 3672T, Planomicrobium koreense KCTC 3684T, Planomicrobium mcmeekinii CGMCC 1.2724T, Planomicrobium
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Planomicrobium soli sp. nov.
77 Planococcus citreus NCIMB 1493T (X62172) 61 Planococcus rifietoensis M8T (AJ493659) Planococcus columbae PgEx11T (AJ966515) Planococcus maitriensis S1T (AJ544622) 74 Planococcus plakortidis AS/ASP6 (II)T (JF775504) 50 Planococcus maritimus TF-9T (AF500007) Planococcus donghaensis JH1T (EF079063) 50 Planococcus kocurii NCIMB 629T (X62173) 64 53 Planococcus antarcticus CMS 26orT (AJ314745) Planococcus salinarum ISL-16T (FJ765415) Planomicrobium flavidum ISL-41T (FJ265708) 59 Planomicrobium stackebrandtii K22-03T (AY437845) Planomicrobium psychrophilum CMS 53orT (AJ314746)
0.005
Planomicrobium okeanokoites NBRC 12536T (D55729) Planomicrobium mcmeekinii S23F2T (AF041791) Planomicrobium chinense DX3-12T (AJ697862) Planomicrobium soli XN13T (JQ772482) 100 58 Planomicrobium glaciei 0423T (EU036220) Planomicrobium koreense JG07T (AF144750) Planomicrobium alkanoclasticum MAE2T (AF029364) Sporosarcina aquimarina SW28T (AF202056) 100 Sporosarcina ureae DSM 2281T (AF202057) Bacillus subtilis NCIMB 3610T (X60646) Bacillus sphaericus IAM 13420T (D16280) Caryophanon latum NCIMB 9533T (X70314) Kurthia zopfii NCIMB 9878T (X70321) Exiguobacterium aurantiacum NCDO 2321T (X70316) 55
93 51
87
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, displaying the relationships between strain XN13T and closely related species. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Numbers at nodes are bootstrap values (%) based on 1000 replications; only values ¢50 % are shown. Bar, 0.005 substitutions per nucleotide position.
flavidum KCTC 13261T, Planomicrobium chinense CGMCC 1.3454T and Planomicrobium glaciei CGMCC 1.6846T were 36 %, 30 %, 34 %, 29 %, 30 % and 31 %, respectively. These DNA–DNA relatedness values were significantly lower than the threshold value of 70 % that is commonly accepted for the definition of bacterial species (Wayne et al., 1987). Strain XN13T showed a range of phenotypic characteristics that differentiated it from recognized species of the genus Planomicrobium (Table 1). In addition, phylogenetic divergence (Fig. 1) and DNA–DNA relatedness values differentiate strain XN13T from recognized species of the genus Planomicrobium. It is apparent that strain XN13T is not affiliated to any recognized species of the genus Planomicrobium. Therefore, on the basis of the data presented above, strain XN13T represents a novel species of the genus Planomicrobium, for which the name Planomicrobium soli sp. nov. is proposed. Description of Planomicrobium soli sp. nov. Planomicrobium soli (so9li. L. gen. n. soli of soil). Cells are Gram-staining-positive, aerobic, cocci or short rods (0.861.0 mm) and non-endospore-forming. Colonies on TSA (Difco) are smooth, circular, convex with entire http://ijs.sgmjournals.org
margins, and pale orange at 28 uC. Growth occurs between 4 uC and 38 uC (optimal growth at 28–30 uC) and at pH 6.0–10.0 (optimal growth at pH 7.0–8.0). Growth occurs with 0.5–7.5 % NaCl (optimal growth with 3–4 % NaCl). Does not reduce nitrate to nitrite or nitrogen gas. Oxidase-negative and catalase-positive. Degrades casein, gelatin, urea and aesculin, but not Tween 80 or starch. H2S is not produced. Citrate does not support growth. Negative for indole production. Acid is not produced from cellobiose, D-fructose, D-glucose, D-galactose, lactose, maltose, D-mannose, melibiose, raffinose, L-rhamnose, Dsorbitol, sucrose or trehalose. D-Glucose and sucrose are used as sole carbon and energy sources, while cellobiose, D-fructose, D-galactose, lactose, maltose, D-mannose, melibiose, raffinose, L-rhamnose, D-sorbitol and trehalose are not. Positive reactions (API ZYM) are obtained for esterase (C4), esterase lipase, leucine arylamidase, naphthol-AS-BIphosphohydrolase, a-galactosidase, b-galactosidase and aglucosidase activities, but negative reactions are observed for alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities. The predominant quinones are MK-8 and MK-7. The major 2703
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polar lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major cellular fatty acids are anteiso-C15 : 0, C16 : 1v7c alcohol, iso-C14 : 0 and C16 : 1v11c. The cell-wall peptidoglycan type is A4a type. The type strain, XN13T (5CGMCC 1.12259T5KCTC 33047T) was isolated from a soil sample collected from ALaShan National Geological Park, Inner Mongolia Autonomous Region, China. The DNA G+C content of the type strain is 40.3 mol%. The species description is based on this single strain and hence serves as a description of the species.
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fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
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Acknowledgements This research was supported by the National Natural Science Foundation of China (NSFC; grant numbers 31070002, 30970009) and the Special Fund for Public Welfare Industrial (Agriculture) Research of China (grant number 200903001). The authors are grateful to Dr Yafang Tan, Chinese Academy of Military Science, for carrying out the quantitative fatty acid analysis on isolate XN13T.
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bacterium isolated from the Korean traditional fermented seafood jeotgal, and transfer of Planococcus okeanokoites (Nakagawa et al. 1996) and Planococcus mcmeekinii (Junge et al. 1998) to the genus Planomicrobium. Int J Syst Evol Microbiol 51, 1511–1520. Zhang, D.-C., Liu, H.-C., Xin, Y.-H., Yu, Y., Zhou, P.-J. & Zhou, Y. G. (2009). Planomicrobium glaciei sp. nov., a psychrotolerant
bacterium isolated from a glacier. Int J Syst Evol Microbiol 59, 1387– 1390.
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DOI 10.1099/ijs.0.055426-0
Planomicrobium soli sp. nov., isolated from soil Xiaonan Luo,1 Jianli Zhang,1 Dai Li,1 Yuhua Xin,2 Di Xin1 and Lei Fan1 Correspondence
1
Jianli Zhang
2
[email protected]
School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
A Gram-staining-positive bacterium, designated strain XN13T, was isolated from a soil sample collected from ALaShan National Geological Park in Inner Mongolia Autonomous Region, China and subjected to a taxonomic study using a polyphasic approach. Strain XN13T was found to have a range of chemical and morphological properties consistent with its classification in the genus Planomicrobium. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain XN13T was related to members of the genus Planomicrobium. The closest phylogenetic relatives were Planomicrobium okeanokoites NBRC 12536T, Planomicrobium koreense JG07T, Planomicrobium mcmeekinii S23F2T and Planomicrobium flavidum ISL-41T with 98.2 %, 97.8 %, 97.8 % and 97.7 % 16S rRNA gene sequence similarity, respectively. The major cellular fatty acids were anteiso-C15 : 0, C16 : 1v7c alcohol, iso-C14 : 0 and C16 : 1v11c. The predominant menaquinones were MK-8 and MK-7. The DNA G+C content was 40.3 mol%. The DNA–DNA relatedness values between strain XN13T and Planomicrobium okeanokoites KCTC 3672T, Planomicrobium koreense KCTC 3684T, P. mcmeekinii CGMCC 1.2724T, Planomicrobium flavidum KCTC 13261T, Planomicrobium chinense CGMCC 1.3454T and Planomicrobium glaciei CGMCC 1.6846T were 36 %, 30 %, 34 %, 29 %, 30 % and 31 %, respectively. The organism is different from recognized species of the genus Planomicrobium in several phenotypic characteristics. On the basis of phenotypic and genotypic properties, strain XN13T represents a novel species of the genus Planomicrobium, for which the name Planomicrobium soli sp. nov. is proposed. The type strain is XN13T (5CGMCC 1.12259T5KCTC 33047T).
The genus Planomicrobium was proposed by Yoon et al. (2001). At the time of writing, the genus Planomicrobium comprises nine species with validly published names: Planomicrobium alkanoclasticum (Engelhardt et al., 2001; Dai et al., 2005), Planomicrobium chinense (Dai et al., 2005), Planomicrobium flavidum (Jung et al., 2009), Planomicrobium glaciei (Zhang et al., 2009), Planomicrobium mcmeekinii (Junge et al., 1998; Yoon et al., 2001), Planomicrobium okeanokoites (Nakagawa et al., 1996; Yoon et al., 2001), Planomicrobium koreense (Yoon et al., 2001), Planomicrobium psychrophilum (Reddy et al., 2002; Dai et al., 2005) and Planomicrobium stackebrandtii (Mayilraj et al., 2005; Jung et al., 2009). Members of the genus Planomicrobium are Gram-staining-positive to Gramstaining-variable, aerobic, motile and non-endosporeforming. Colony colour is yellow to orange or pale orange. The peptidoglycan type is A4a type. The menaquinone profile is characterized by the predominance of MK-8 followed by MK-7, or by the predominance of MK-8 The GenBank/EMBL/DDBJ accession number for the16S rRNA gene sequence of strain XN13T is JQ772482. Two supplementary figures and a supplementary table are available with the online version of this paper.
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followed by MK-7 and MK-6. The cellular fatty acids are mainly saturated, unsaturated and branched fatty acids. The G+C content of the genomic DNA is 35–49 mol% (Yoon et al., 2001; Zhang et al., 2009). A bacterial strain, designated XN13T, was isolated from a soil sample collected from ALaShan National Geological Park (30u 279 240 N 106u 129 030 E, 1038 m above sea level), located in Inner Mongolia Autonomous Region, China. The pH of the soil sample was pH 6. For isolation, the soil sample (1 g) was suspended in distilled water (100 ml) and spread on plates of trypticase soy agar (TSA; Difco) after serial dilution. The plates were incubated at 30 uC for 3 days. Purification was achieved by streaking single colonies on TSA. Strain XN13T was maintained on TSA at 4 uC and as glycerol suspensions (20 %, v/v) at 220 uC. The aim of the present work was to elucidate the taxonomic position of strain XN13T by means of phenotypic, genotypic and chemotaxonomic analyses. The following type strains of species of the genus Planomicrobium were used as reference strains: Planomicrobium okeanokoites KCTC 3672T, Planomicrobium koreense KCTC 3684T and Planomicrobium flavidum KCTC 13261T, obtained from the Korean Collection for Type 055426 G 2014 IUMS Printed in Great Britain
Planomicrobium soli sp. nov.
Cultures, Taejon, Korea; and Planomicrobium mcmeekinii CGMCC 1.2724T, Planomicrobium chinense CGMCC 1.3454T and Planomicrobium glaciei CGMCC 1.6846T, obtained from China General Microbiological Culture Collection Center. General cell morphology was studied by light microscopy (BH-2; Olympus) and transmission electron microscopy (JEM-1400; JEOL) with a 2-day-old culture of strain XN13T grown on TSA. Colonial properties were examined after 3 days of growth at 28 uC on TSA. Gram-staining was performed as described by Smibert & Krieg (1994). Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck), and catalase activity was evaluated by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution (Smibert & Krieg, 1994). Hydrolysis of starch was tested on agar plates containing 1 % (w/v) peptone, 1 % yeast extract and 0.5 % soluble starch. Gelatinase production was determined on Luria–Bertani media containing 3 % (w/v) gelatin. Hydrolysis of casein was examined on nutrient agar supplemented with 1.5 % skimmed milk (Sigma). Hydrolysis of aesculin and nitrate reduction were evaluated as described by La´nyi (1987). H2S production was tested as described previously (Bruns et al., 2001). Acid production from carbohydrates was assessed using methods described by Leifson (1963), and utilization of sole carbon and energy sources was determined according to Gordon & Mihm (1957). Hydrolysis of urea was determined on medium described by Goodfellow & Orchard (1974), comprising 75 ml urea shake flask medium [10 g KH2PO4, 9.5 g Na2HPO4, 1 g yeast extract, with 0.04 % (w/v) phenol red, 1000 ml distilled water, pH 7.2] and 15 ml of 15 % urea. Citrate utilization was tested on Simmons citrate agar (Sigma). Indole production was tested as previously described (Smibert & Krieg, 1994). The temperature range for growth was tested by growing cells at 4–45 uC on TSA. Growth at different salinities up to 10 % (w/v, in increments of 0.5 %) was tested by growing cells on TSA prepared according to the formula of the Difco medium except that NaCl was omitted. The pH range for growth (pH 4.0–11.0, in increments of 1.0 pH unit) was determined in trypticase soy broth by using appropriate biological buffers. In addition, biochemical traits of enzyme activities were examined by using API ZYM kits (bioMe´rieux) according to the manufacturer’s instructions; the API ZYM tests were read after incubation for 4 h at 37 uC. Strain XN13T was a Gram-staining-positive, aerobic, cocci or short rod-shaped, non-endospore-forming bacterium (Fig. S1, available in the online Supplementary Material). Colonies were smooth, circular, convex with entire margins, and pale orange after 3 days cultivation at 28 uC on TSA. Growth occurred at 4–38 uC, but not at 39 uC. Growth was observed at pH 6.0–10.0 and the optimum pH for growth was 7.0–8.0. Growth occurred in the presence of 0.5–7.5 % NaCl with optimal growth at 3–4 % NaCl. Cells grew aerobically and were not capable of reducing nitrate to nitrite. Catalase activity was positive but oxidase activity http://ijs.sgmjournals.org
was negative. The morphological, cultural, physiological and biochemical characteristics of strain XN13T are given in the species description and in Table 1. Cell biomass for isoprenoid quinone analysis was obtained following cultivation in marine 2216 broth (Difco) for 3 days at 30 uC. Menaquinones were extracted and purified using the methods of Collins et al. (1987); purified menaquinones were determined by reverse-phase HPLC (Wu et al., 1989). Preparation of cell walls and determination of peptidoglycan structure were carried out using the methods of Schleifer & Kandler (1972). Polar lipids were extracted by the method of Minnikin et al. (1979) and polar lipid profiles were analysed according to Collins & Jones (1980). For quantitative analysis of cellular fatty acid compositions, cell mass of strain XN13T was collected from marine 2216 broth (Difco) after cultivation for 3 days at 28 uC. Fatty acids were extracted, purified, methylated and identified by using the standard Microbial Identification System (MIDI; Sasser, 1990; Ka¨mpfer & Kroppenstedt, 1996) version 4.5. The G+C content of the DNA was determined by means of the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli K-12 as the reference. Cells of strain XN13T contained MK-8 (73.9 %) and MK-7 (18.9 %) as the major respiratory quinones. The cell-wall composition of strain XN13T contained L-lysine, glutamic acid and alanine, and this is consistent with the A4a type (Schleifer & Kandler, 1972) cell wall of the genus Planomicrobium. The polar lipids of strain XN13T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol (Fig. S2). The major cellular fatty acids were anteiso-C15 : 0 (36.0 %), C16 : 1v7c alcohol (14.3 %), iso-C14 : 0 (10.9 %) and C16 : 1v11c (10.1 %). No pronounced differences in the fatty acid profiles of strain XN13T and the reference strains were found, although there were minor differences in the proportions of some components (Table S1). The genomic DNA G+C content of strain XN13T was 40.3 mol%. Extraction of genomic DNA and amplification of the 16S rRNA gene were performed as described by Rainey et al. (1996). 16S rRNA gene sequences were amplified by PCR from chromosomal DNA using two universal primers as described previously (Yoon et al., 1998). The PCR product was sequenced directly using the method of Lu et al. (2001). The resultant 16S rRNA gene sequence was compared with those available in the GenBank database, using the BLAST program. Multiple alignments with closely related species were performed using the CLUSTAL X 1.8 software (Thompson et al., 1997). Sequence similarities were calculated using the EzTaxon-e server (http://eztaxone.ezbiocloud.net/, Kim et al., 2012). The phylogenetic consensus tree was reconstructed using the neighbourjoining (Saitou & Nei, 1987) and maximum-parsimony (Kluge & Farris, 1969) methods in MEGA software version 5.0 (Tamura et al., 2011), with bootstrap values based on 1000 replications. Evolutionary distances were calculated 2701
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Table 1. Differential properties of strain XN13T and type strains of closely related species of the genus Planomicrobium Strains: 1, XN13T; 2, Planomicrobium okeanokoites KCTC 3672T; 3, Planomicrobium koreense KCTC 3684T; 4, Planomicrobium mcmeekinii CGMCC 1.2724T; 5, Planomicrobium flavidum KCTC 13261T; 6, Planomicrobium chinense CGMCC 1.3454T; 7, Planomicrobium glaciei CGMCC 1.6846T. All data are from this study, except for strain 5 (Jung et al., 2009) and strain 6 (Dai et al., 2005). +, Positive; –, negative; W, weakly positive; ND, no data available; C, Cocci; R, rods; SR, short rods. Characteristic Cell shape Gram-stain Colony colour Oxidase Nitrate reduction: Growth with/at: 7 % NaCl 10 % NaCl 4 uC 37 uC Degradation of: Casein Aesculin Gelatin Tween 80 Acid production from: Cellobiose D-Fructose D-Glucose Lactose Maltose Melibiose Predominant menaquinone(s) DNA G+C content (mol%)*
1 C or SR Positive
2
3
R
C or SR/R Positive to variable Yellow–orange
2 2
4
5
6
7
or SR Positive
C or SR Positive
C or SR Positive
Pale orange
C or SR Positive to variable Light yellow
Yellow–orange
Yellow–orange
2 +
+ 2
2 +
2 +
R/C
2 2
Positive to variable Bright yellow –bright orange 2 2
+ 2 + +
2 2 2 +
2 + +
+ + + +
+ + + +
+ + 2 +
+ + + 2
+ + + 2
+ 2 + 2
+ + + 2
+ 2 2 2
W
2 + +
2 2 + 2
+ + 2
2 2 2 2 2 2 MK-8,7
2 + 2 2 2 2 MK-8,7
+ 2 2 + + + MK-8,7,6
2 + + 2 2 2 MK-8,7
2 + 2 2 2 2 MK-8,7
2
2 MK-8
2 + 2 2 2 2 MK-8,7
40.3
46.3a
47.0b
35.0c
45.9
34.8
49.0d
Pale Orange
W
ND
+ 2 ND
*Data from: a, Nakagawa et al. (1996); b, Yoon et al. (2001); c, Junge et al. (1998); d, Zhang et al. (2009).
by using distance options according to Kimura’s twoparameter model (Kimura, 1980).
(97.9 %)]. However, from the neighbour-joining tree (Fig. 1) strain XN13T fell within the evolutionary radius of the genus Planomicrobium, and the isolate should therefore not be assigned to the genus Planococcus. The maximumparsimony tree also supported the clustering of strain XN13T and species of the genus Planomicrobium.
The 16S rRNA gene sequence of strain XN13T was a continuous stretch of 1468 bp. It was apparent from the neighbour-joining tree (Fig. 1) that strain XN13T was classically defined as belonging to the genus Planomicrobium. Strain XN13T showed 96.5–98.2 % 16S rRNA gene sequence similarities with respect to the type strains of species of the genus Planomicrobium, the highest similarity values being found with the sequences of Planomicrobium okeanokoites NBRC 12536T (98.2 %), Planomicrobium koreense JG07T (97.8 %), Planomicrobium mcmeekinii S23F2T (97.8 %), Planomicrobium flavidum ISL-41T (97.7 %), Planomicrobium chinense DX3-12T (97.7 %) and Planomicrobium glaciei 0423T (97.6 %). In addition, strain XN13T also shared high 16S rRNA gene sequence similarities with respect to the type strains of species of the genus Planococcus [Planococcus salinarum ISL-16T (98.0 %) and Planococcus plakortidis AS/ASP6 (II)T
DNA–DNA hybridization values were determined from the initial DNA–DNA liquid reassociation rate as described by De Ley et al. (1970) and modified by Huß et al. (1983). The tests were performed on a Lambda 35 UV/Vis spectrophotometer equipped with a temperature program controller (Perkin-Elmer). Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as DNA–DNA relatedness values. The mean DNA–DNA relatedness values between strain XN13T and Planomicrobium okeanokoites KCTC 3672T, Planomicrobium koreense KCTC 3684T, Planomicrobium mcmeekinii CGMCC 1.2724T, Planomicrobium
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Planomicrobium soli sp. nov.
77 Planococcus citreus NCIMB 1493T (X62172) 61 Planococcus rifietoensis M8T (AJ493659) Planococcus columbae PgEx11T (AJ966515) Planococcus maitriensis S1T (AJ544622) 74 Planococcus plakortidis AS/ASP6 (II)T (JF775504) 50 Planococcus maritimus TF-9T (AF500007) Planococcus donghaensis JH1T (EF079063) 50 Planococcus kocurii NCIMB 629T (X62173) 64 53 Planococcus antarcticus CMS 26orT (AJ314745) Planococcus salinarum ISL-16T (FJ765415) Planomicrobium flavidum ISL-41T (FJ265708) 59 Planomicrobium stackebrandtii K22-03T (AY437845) Planomicrobium psychrophilum CMS 53orT (AJ314746)
0.005
Planomicrobium okeanokoites NBRC 12536T (D55729) Planomicrobium mcmeekinii S23F2T (AF041791) Planomicrobium chinense DX3-12T (AJ697862) Planomicrobium soli XN13T (JQ772482) 100 58 Planomicrobium glaciei 0423T (EU036220) Planomicrobium koreense JG07T (AF144750) Planomicrobium alkanoclasticum MAE2T (AF029364) Sporosarcina aquimarina SW28T (AF202056) 100 Sporosarcina ureae DSM 2281T (AF202057) Bacillus subtilis NCIMB 3610T (X60646) Bacillus sphaericus IAM 13420T (D16280) Caryophanon latum NCIMB 9533T (X70314) Kurthia zopfii NCIMB 9878T (X70321) Exiguobacterium aurantiacum NCDO 2321T (X70316) 55
93 51
87
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, displaying the relationships between strain XN13T and closely related species. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Numbers at nodes are bootstrap values (%) based on 1000 replications; only values ¢50 % are shown. Bar, 0.005 substitutions per nucleotide position.
flavidum KCTC 13261T, Planomicrobium chinense CGMCC 1.3454T and Planomicrobium glaciei CGMCC 1.6846T were 36 %, 30 %, 34 %, 29 %, 30 % and 31 %, respectively. These DNA–DNA relatedness values were significantly lower than the threshold value of 70 % that is commonly accepted for the definition of bacterial species (Wayne et al., 1987). Strain XN13T showed a range of phenotypic characteristics that differentiated it from recognized species of the genus Planomicrobium (Table 1). In addition, phylogenetic divergence (Fig. 1) and DNA–DNA relatedness values differentiate strain XN13T from recognized species of the genus Planomicrobium. It is apparent that strain XN13T is not affiliated to any recognized species of the genus Planomicrobium. Therefore, on the basis of the data presented above, strain XN13T represents a novel species of the genus Planomicrobium, for which the name Planomicrobium soli sp. nov. is proposed. Description of Planomicrobium soli sp. nov. Planomicrobium soli (so9li. L. gen. n. soli of soil). Cells are Gram-staining-positive, aerobic, cocci or short rods (0.861.0 mm) and non-endospore-forming. Colonies on TSA (Difco) are smooth, circular, convex with entire http://ijs.sgmjournals.org
margins, and pale orange at 28 uC. Growth occurs between 4 uC and 38 uC (optimal growth at 28–30 uC) and at pH 6.0–10.0 (optimal growth at pH 7.0–8.0). Growth occurs with 0.5–7.5 % NaCl (optimal growth with 3–4 % NaCl). Does not reduce nitrate to nitrite or nitrogen gas. Oxidase-negative and catalase-positive. Degrades casein, gelatin, urea and aesculin, but not Tween 80 or starch. H2S is not produced. Citrate does not support growth. Negative for indole production. Acid is not produced from cellobiose, D-fructose, D-glucose, D-galactose, lactose, maltose, D-mannose, melibiose, raffinose, L-rhamnose, Dsorbitol, sucrose or trehalose. D-Glucose and sucrose are used as sole carbon and energy sources, while cellobiose, D-fructose, D-galactose, lactose, maltose, D-mannose, melibiose, raffinose, L-rhamnose, D-sorbitol and trehalose are not. Positive reactions (API ZYM) are obtained for esterase (C4), esterase lipase, leucine arylamidase, naphthol-AS-BIphosphohydrolase, a-galactosidase, b-galactosidase and aglucosidase activities, but negative reactions are observed for alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities. The predominant quinones are MK-8 and MK-7. The major 2703
X. Luo and others
polar lipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major cellular fatty acids are anteiso-C15 : 0, C16 : 1v7c alcohol, iso-C14 : 0 and C16 : 1v11c. The cell-wall peptidoglycan type is A4a type. The type strain, XN13T (5CGMCC 1.12259T5KCTC 33047T) was isolated from a soil sample collected from ALaShan National Geological Park, Inner Mongolia Autonomous Region, China. The DNA G+C content of the type strain is 40.3 mol%. The species description is based on this single strain and hence serves as a description of the species.
2005 to the genus Planomicrobium as Planomicrobium stackebrandtii comb. nov. Int J Syst Evol Microbiol 59, 2929–2933. Junge, K., Gosink, J. J., Hoppe, H.-G. & Staley, J. T. (1998).
Arthrobacter, Brachybacterium and Planococcus isolates identified from antarctic sea ice brine. Description of Planococcus mcmeekinii, sp. nov. Syst Appl Microbiol 21, 306–314. Ka¨mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of
fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. Kimura, M. (1980). A simple method for estimating evolutionary rates
Acknowledgements This research was supported by the National Natural Science Foundation of China (NSFC; grant numbers 31070002, 30970009) and the Special Fund for Public Welfare Industrial (Agriculture) Research of China (grant number 200903001). The authors are grateful to Dr Yafang Tan, Chinese Academy of Military Science, for carrying out the quantitative fatty acid analysis on isolate XN13T.
of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120. Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Zool 18, 1–32. La´nyi, B. (1987). Classical and rapid identification methods for
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