Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0207-3
ORIGINAL PAPER
Gordonia jinhuaensis sp. nov., a novel actinobacterium, isolated from a VBNC (viable but non-culturable) state in pharmaceutical wastewater Shan-Hui Li • Yi Jin • Juan Cheng • Dong-Jin Park • Chang-Jin Kim • Wael N. Hozzein • Mohammed A. M. Wadaan Wen-Sheng Shu • Lin-Xian Ding • Wen-Jun Li
•
Received: 9 April 2014 / Accepted: 23 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract A Gram-stain positive, aerobic, nonmotile and rod-shaped actinobacterial strain, designated as ZYR 51T, was isolated from pharmaceutical wastewater in Jinhua city, Zhejiang province, Eastern China. Isolation was aided by using a resuscitationpromoting factor, suggesting the strain was recovered from a viable but non-culturable state. Strain ZYR 51T was characterized by a polyphasic taxonomic approach. Growth was found to occur at 10–45 °C, pH 6.0–10.0 and 0–9 % NaCl (w/v). Based on the 16S rRNA gene sequence, phylogenetic analysis clearly demonstrated that strain ZYR 51T belongs to the genus Gordonia and showed low level similarities (below
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0207-3) contains supplementary material, which is available to authorized users. S.-H. Li J. Cheng W.-J. Li (&) Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China e-mail:
[email protected] Y. Jin L.-X. Ding (&) College of Geography and Environmental Sciences, Zhejiang Normal University, Jinhua 321004, People’s Republic of China e-mail:
[email protected]
97 %) with all other members of this genus. The strain was found to possess meso-diaminopimelic acid (meso-DAP), along with MK-9(H2) as the predominant menaquonine. Mycolic acids were found to be present. C16:0 (34.9 %), 10-methyl C18:0 (30.3 %), isoC18:0(8.2 %), and summed feature 3 (C16:1 x6c and/or C16:1x7c as define by MIDI; 18.8 %) were identified as the major cellular fatty acids. The polar lipid profile of strain ZYR 51T was found to consist of diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and some unknown lipids. The genomic DNA G?C content of strain ZYR 51T was determined to be 67.7 mol%. The combined genotypic and phenotypic data showed that the strain represents a novel species of the genus Gordonia, for which the name Gordonia jinhuaensis sp. nov. is proposed, with
W. N. Hozzein M. A. M. Wadaan Bioproducts Research Chair (BRC), College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Kingdom of Saudi Arabia W.-S. Shu W.-J. Li State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China
D.-J. Park C.-J. Kim Microbial Resource Center KRIBB, Daejeon 305-806, Republic of Korea
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the type strain is ZYR 51T (=CGMCC 1.12827T = NBRL B-59111T = NBRC 110001T). Keywords Gordonia jinhuaensis sp. nov Rpf VBNC Pharmaceutical wastewater Polyphasic taxonomy
Introduction The genus Gordonia (formerly Gordona; Tsukamura 1971), a mycolic acid-containing group of actinomycetes, belongs to the suborder Corynebacterineae (Zhi et al. 2009). Gordonia are Gram-stain positive, nonmotile short rods or cocci, containing peptidoglycan of the A1c-type with meso-diaminopimelic acid as the diamino acid and with arabinose and galactose or glucose as cell-wall sugars. Some members of the genus Gordonia were originally reported to be opportunistic pathogens that were isolated from various clinical materials such as sputa of humans suffering from pulmonary diseases, e.g. Gordonia bronchialis, Gordonia aichiensis, and Gordonia sputi (Kim et al. 2003); ear discharge from a patient with external otitis and pleural fluid patient with bronchitis, e.g. Gordonia otitidis (Iida et al. 2005); and other pathogenic species (Jannat-Khah et al. 2009; Kageyama et al. 2006). In addition, some members of the genus have been isolated from various environmental sources in recent years, such as activated sludge foam, estuarine sand, sewage sludge, soil and a polluted stream (Soddell et al. 2006; le Roes et al. 2008; Drzyzga et al. 2009; Park et al. 2009; Kim et al. 2009). At the time of writing, the genus Gordonia encompasses 34 recognized species with validly published names (http:// www.bacterio.net/g/gordonia.html). Several members of this taxon play important roles in biodegradation and bioremediation (Arensko¨tter et al. 2004; Ka¨mpfer et al. 2011; Kang et al. 2009; Kim et al. 2009; le Roes et al. 2008; Soddell et al. 2006; Yassin et al. 2007; Yoon et al. 2000; Linos et al. 2002; Kim et al. 1999, 2000). During our research on the viable but non-culturable (VBNC) state of microorganisms, a novel actinobacterium strain, designated ZYR 51T, was isolated from pharmaceutical wastewater in Jinhua city, Zhejiang province, Eastern China, using a strategy employing a ‘‘bacterial cytokine’’ i.e. the
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resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus (Mukamolova et al. 1998; Ding et al. 2012). The method aims to recover more microorganisms from possible existence in the VBNC state (Ding 2004; Ding et al. 2012), which may be a relevant state in environments such as activated sludge and sewage treatment systems (Su et al. 2012). Besides, microbes resuscitated from VBNC states have played increasingly important roles in industrial applications, such as biological deodorization, denitrification and as biosensors (Ding et al. 2011). On the basis of the results from polyphasic taxonomic study reported in this paper, the isolate ZYR 51T is considered to represent a novel species of the genus Gordonia, for which the name Gordonia jinhuaensis sp. nov. is proposed.
Materials and methods Isolation and maintenance of microorganism Strain ZYR 51T was isolated from a pharmaceutical wastewater sample in Jinhua city, Zhejiang province, Eastern China (E119.64°, N29.12°). In the study, Rpf (1 %, v/v) was added as a component of culture system to investigate the recovery of organisms assumed to be in a VBNC state that may respond to Rpf. The Rpf was obtained from Micrococcus luteus as described by Ding et al. (2012). The basic isolation medium consisted of KNO3 2.0 g, MgSO47H2O 0.2 g, K2HPO4 0.5 g and KNaC4H4O64H2O 20.0 g dissolved in 1 L distilled water with the pH adjusted to 7.2. After suspension (10 %, v/v), the wastewater sample was serially diluted and strains were isolated by using the plate isolation method at 30 °C for 3 weeks. Through comparing the strains recovered in the treatment group (with Rpf) with a control group (without Rpf) (Ding 2004; Ding et al. 2012), we concluded that strain ZYR 51T had been recovered from a possible VBNC state. Subsequently, colonies were picked and re-streaked repeatedly onto YIM 38# medium (Li et al. 2007) to obtain pure cultures. The purified strain was maintained on YIM 38# medium and stored as glycerol suspensions (20 %, v/v) at -80 °C. Biomass for chemical and molecular studies was prepared by growing the strain on ISP 2 medium (Yeast extract-malt extract agar; Shirling and Gottlieb 1966) at 30 °C for 3 days.
Antonie van Leeuwenhoek
Morphological, cultural, physiological and biochemical characteristics Cultural characteristics of strain ZYR 51T were tested on ISP 2, oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5) potatodextrose agar (PDA), Czapek’s agar and nutrient agar, which were prepared according to Dong and Cai (2001). Colony colour was determined by comparison with colour chips from the ISCC-NBS colour chart standard samples (Kelly, 1964). Cell morphology was observed by using a light microscope (Olympus BX43) and scanning electron microscopy (ESEM-TMP). The procedure of preparing the sample for scanning electron microscopy was as described by Ming et al. (2012). To observe the morphological changes at different growth phases, the strains were examined after 15 and 72 h culture. Gram staining was carried out by using the standard Gram reaction and confirmed by using the KOH lysis test method (Cerny 1978). Cell motility was tested by the development of turbidity throughout a tube containing semisolid medium (Leifson 1960). Growth at different temperatures (4–60 °C) was observed on ISP 2 agar medium for 2 weeks. Salt tolerance was examined at different NaCl concentrations (0–15 %, w/v) on the basic medium of ISP 2 agar at 30 °C for 2 weeks. The pH range for growth was tested at pH 4.0–12.0 using the buffer system described by Xu et al.(2005) at 30 °C for 2 weeks by culturing the strain in trypticase soy broth (TSB; Becton–Dickinson). Catalase activity was detected by the production of bubbles after the addition of a drop of 3 % H2O2 and oxidase activity was determined by the oxidation of tetramethyl-pphenyl-enediamine (Kovacs 1956). Milk coagulation and peptonization were determined by using 20 % (w/ v) skimmed milk as the medium with incubation for 4 weeks at 30 °C. Hydrolysis of starch, gelatin, cellulose, Tweens 20, 40, 60, 80 and utilization of urea and nitrate reduction were tested. Media and procedures used for determination of these physiological features were described by Williams (1989) and Gordon et al. (1974). Nitrogen source utilization was assessed according to Williams et al. (1989). Biolog GIII microplateTM was used for carbon source utilization and enzyme activities; other physiological characteristics were determined by using the API ZYM, API 20 NE and API ID 32GN test strips (bioMe´rieux, France) following the manufacturer’s
instructions. Antibiotic sensitivity to dispersible cotrimoxazole tablets (1.25 lg), chloramphenicol (30 lg), amikacin (30 lg), ampicillin(10 lg), norfloxacin (10 lg), penicillin (10 IU), polymyxin (300 IU), gentamicin (10 lg), piperacillin (100 lg), cefuroxime sodium (30 lg), vancomycin (30 lg), ceftriaxone (30 lg), cefoperazone (75 lg), clindamycin (2 lg), ofloxacin(5 lg), cefazolin (30 lg), erythromycin (15 lg), ciprofloxacin (5 lg) of strain ZYR 51T was examined after 5 days at 30 °C on ISP 2 medium using antibiotic discs (Himedia) (Groth et al. 2004). Chemotaxonomy Cell wall hydrolysates were obtained and hydrolysed as described by Schleifer et al. (1972). Amino acids in the cell-wall hydrolysate were analysed by precolumn derivatization with o-phthalaldehyde followed by HPLC (Tang et al. 2009). The sugars in the hydrolysate were detected by precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone followed by HPLC (Agilent 1100) according to the method described by Tang et al. (2009). Cellular fatty acid analysis was performed by using the microbial identification system (Sherlock Version 6.1; MIDI database: TSBA6) (Sasser, 1990). Polar lipids were extracted and analyzed by two-dimensional TLC as described by Minnikin et al. (1979). The quinones were extracted by using the method of Collins et al. (1977) and separated by HPLC (Kroppenstedt 1982). Mycolic acids were extracted and analysed according to the protocol of Minnikin et al. (1980). Molecular analysis Genomic DNA extraction and PCR amplification of 16S rRNA genes were performed as described by Li et al. (2007). The amplicon was purified by using a PCR purification kit (Sangon Biotech, China). The full-length 16S rRNA gene sequence (1524 bp) of strain ZYR 51T was compared with the sequences of cultured species via BLAST search (Altschul et al. 1990) and with type strains via EzTaxon-e (Kim et al. 2012). Multiple alignments of the sequence of strain ZYR 51T with those of type strains of members of the genus Gordonia were performed using the CLUSTAL_X software package (Thompson et al. 1997). The Kimura two-parameter model was used to
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Antonie van Leeuwenhoek Table 1 Growth and cultural characteristics of strain ZYR 51T on standard agar media after incubation for 10 days at 30 °C Medium
Growth
Color of colonies
Soluble pigment
Nutrient agar
??
light yellow
none
Czapek’s agar
?
yellow-white
none
Potato-glucose agar Yeast extract-malt extract agar (ISP 2)
??? ?
orange-yellow orange-yellow
none none
Oatmeal agar (ISP 3)
?
orange-yellow
none
Inorganic salts-starch agar (ISP 4)
??
light yellow
none
Glycerol asparagine agar (ISP 5)
???
orange-yellow
none
???
Good,
??
Moderate,
?
Weak growth
calculate the evolutionary distances (Kimura 1980, 1985). Phylogenetic trees were constructed by neighbour-joining (Saitou and Nei, 1987), maximum-parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981) tree-making algorithms. Phylogenetic and molecular evolutionary analyses were performed using the software package MEGA version 5.0 (Tamura et al. 2011) and the PHYML package (Guindon and Gascuel 2003). Bootstrap analysis was used to evaluate the topology of each tree with 1,000 replications (Felsenstein 1985). The strain Turicella otitidis DSM 8821T (X73976) was used as outgroup. The G?C content of genomic DNA was determined using the HPLC method (Mesbah et al. 1989).
Results and discussion Strain ZYR 51T was isolated using a strategy in which an Rpf was used to promote recovery of cells potentially in a VBNC state. The strain was observed to be Gram-positive, non-motile, aerobic and to form hyphae. The strain was observed to grow well on potato agar, ISP 2 agar, ISP 4 agar, ISP 5 agar, nutrient agar and TSA, wbut eakly on ISP 3 agar and Czapek’s agar. Hyphae with similar colours (light yellow, yellow-white or orange-yellow) and no soluble pigments were produced following growth on the different media (Table 1). Microscopic observation revealed that strain ZYR 51T is rod-shaped in every growth phase, but the cell sizes vary (Fig. 1). The strain had morphological properties that were consistent with its assignment to the genus Gordonia. Growth was found to occur at temperatures ranging from 10 to 45 °C (optimum 28–30 °C), but no growth was observed at 4 and 50 °C. Strain ZYR 51T showed
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Fig. 1 Scanning electron micrographs of strain ZYR 51T grown on ISP 2 medium for 15 h (a) and 72 h (b) at 30 °C
a broad pH range for growth (pH 6.0–11.0; optimum pH, 7.0–8.0) but no growth was observed at pH 5.0 and 12.0; Strain ZYR 51T was found to have a wide tolerance to NaCl (0–9 % w/v). Activities of oxidase, catalase and urease, nitrate reduction were found to be
orange yellow
rods
? ?
?
Colour
Morphology
Catalase Oxidase
Nitrate reduction
?
?
-
Urea
Aesculin
Starch
-
-
?
?
-
-
?
-
-
-
-
?
-
-
?
-
?
-
-
D-Galactose
L-Rhamnose
Sucrose
D-Turanos
D-Arabitol
myo-Inositol
Glycerol
N-Acetyl-D-glucosamine Quinate
Citrate
Caprate
3-Hydroxybenzoate
4-Hydroxybenzoate
4-Aminobutyrate
2-Oxoglutarate
L-Alanine
L-Aspartate
L-Leucine
L-Proline
L-Serine
Utilization of
-
Tween 80
Degradation of
1
Characteristic
?
?
?
–
?
?
?
?
-
-
-
? ?
ND
ND
?
?
?
-
-
ND
ND
ND
ND
ND
ND ND
rod-coccus cycle
-
-
-
-
-
?
-
-
-
?
?
-
ND
ND
-
-
-
-
?
ND
ND
?
?
?
? -
rods or coccids
salmon
3
-
-
-
?
?
?
-
-
-
?
?
?
?
ND
-
-
-
-
?
-
-
?
?
?
? ND
rod-coccus cycle
pink/orange
4
-
-
-
-
-
?
-
?
?
?
?
? ?
-
?
?
-
?
?
?
?
–
–
-
-
? -
rods
pink/orange
5
4, G. aichiensis NBRC 108223T (data from Klatte et al. 1994); 5, G. rhizosphera NBRC 16068T (data from Takeuchi and Hatano 1998)
white/apricot/pale orange
2
Table 2 Comparison of physiological properties of strain ZYR 51T and its nearest phylogenetic neighbours. Taxa: 1, strain ZYR 51T; 2, G. otitidis NBRC 100426T (data from Iida et al. 2005); 3, G. sputi NBRC 100414T (data from Riegel et al. 1994);
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66.8
123
(?) Positive reaction, (-) negative reaction, ND No data
Pharmaceutical wastewater Isolation source
Clinical isolates
MK-9(H2)
Mangrove rhizosphere
65 65.2 67.7 DNA G?C content (mol%)
64.9–65.2
-
MK-9(H2) MK-8(H2) MK-9(H2) MK-8(H2) MK-9(H2)
? ? L-Valine
Menaquinone (s)
MK-9(H2) MK-8(H2)
2 1 Characteristic
Table 2 continued
3
4
5
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positive, but negative for milk coagulation, milk peptonization and H2S production. Strain ZYR 51T was found to be able to hydrolyse cellulose, gelatin, Tweens 20, 40 and 60, but not Tween 80, tryptophan, gelatin and starch. In Biolog GIII microplateTM assays, strain ZYR 51T could resist nalidixic acid, potassium tellurite, aztreonam, sodium butyrate and 1 % sodium lactate. The strain was found to be resistant to Dispersible co-trimoxazole tablets (1.25 lg), but sensitive to disks containing chloroamphenicol (30 lg), amikacin (30 lg), ampicillin(10 lg), norfloxacin (10 lg), penicillin (10 IU), polymyxin (300 IU), gentamicin (10 lg), piperacillin (100 lg), cefuroxime sodium (30 lg), vancomycin (30 lg), ceftriaxone (30 lg), cefoperazone (75 lg), clindamycin (2 lg), ofloxacin (5 lg), cefazolin (30 lg), erythromycin (15 lg), ciprofloxacin (5 lg). Phenotypic characteristics that differentiate strain ZYR 51T from closely related type strains are given in Table 2. The chemical analyses indicate that strain ZYR 51T contains meso-diaminopimelic acid as the diagnostic diamino acid and aspartic acid, glutamic acid, threonine, alanine were also detected in the cell wall hydrolysate. Mannose, ribose, rhamnose, glucosamine, galactose and arabinose were found in the whole-cell hydrolysates. MK-9(H2) was detected as the predominant menaquinone. The polar lipids were found to comprise of diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and five unknown lipids (Fig. S1). Mycolic acids were found to be present (Fig. S2). All the above data are very similar to the properties of other recognized Gordonia species. The major cellular fatty acids ([5.0 %) were identified as C16:0 (34.9 %), 10-methyl C18:0 (30.3 %), iso-C18:0 (8.2 %) and summed feature 3 (C16:1 x6c and/or C16:1 x7c;18.8 %, as defined by MIDI). The DNA G?C content of strain ZYR 51T was determined to be 67.7 mol%, which is very similar to those of other Gordonia species (Table 2). All these chemotaxonomic markers support the assignment of the new isolate ZYR 51T to the genus Gordonia. Phylogenetic analysis on the basis of the complete 16S rRNA gene sequence (1524 bp; GenBank Accession Number KJ700483) revealed that strain ZYR 51T should be recognised as a member of the family Nocardiaceae and forms a distinct lineage within the genus Gordonia (Fig. 2). The sequence similarities between the isolate and other members of the genus
Antonie van Leeuwenhoek 100* Gordonia sihwensis NBRC 108236T (BANU01000043) 99* Gordonia cholesterolivorans Chol-3T (EU244645) Gordonia neofelifaecis NRRL B-59395T (AEUD01000036) Gordonia hydrophobica DSM 44015T (X87340) Gordonia shandongensis DSM 45094T (AUHE01000029) Gordonia humi CC-12301T FN561544 Gordonia hirsuta DSM 44140T (BANT01000069) Gordonia caeni MJ32T (JF806526) 100* Gordonia phosphorivorans Ca8T (HE574551) 50* 99* Gordonia alkaliphila CJ10T (JN008111) Gordonia defluvii J4T (AY650265) Gordonia malaquae NBRC 108250T (BAOP01000027) Gordonia araii NBRC 100433T (BAEE01000084) Gordonia effusa IFM 10200T (AB162799) Gordonia amarae NBRC 15530T (BAED01000079) 100* Gordonia terrae NBRC 100016T (BAFD01000032) Gordonia lacunae BS2T (EF151959) Gordonia paraffinivorans NBRC 108238T (BAOQ01000072) Gordonia desulfuricans NCIMB 40816T (AF101416) Gordonia amicalis NBRC 100051T (BANS01000072) 69 Gordonia namibiensis NBRC 108229T (BAHE01000050) Gordonia rubripertincta NBRC 101908T (BAHB01000127) 76* Gordonia alkanivorans NBRC 16433T (BACI01000096) 99* Gordonia westfalica Kb2T (AJ312907) T 76* Gordonia aichiensis NBRC 108223 (BANR01000046) T 100* Gordonia sputi NBRC 100414 (BAFC01000092) 61* Gordonia otitidis NBRC 100426T (BAFB01000010) 64 Gordonia jinhuaensis ZYR 51T (KJ700483) Gordonia rhizosphera NBRC 16068T (BAHC01000019) Gordonia bronchialis DSM 43247T (CP001802) Gordonia polyisoprenivorans NBRC 16320T (BAEI01000017) Gordonia soli NBRC 108243T (BANX01000052) 90* Gordonia hankookensis ON-33T (FJ572038) Gordonia sinesedis DSM 44455T (AF380834) Gordonia kroppenstedtii NP8-5T (AM883151) Skermania piniformis IFO 15059T (Z35435) Millisia brevis J81T (AY534742) Williamsia muralis MA140/96T (Y17384) Rhodococcus rhodochrous DSM 43241T (X79288) Smaragdicoccus niigatensis DSM 44881T (AB243007) 59 Micropolyspora brevicatena ATCC 15333T (AF430040) 57 100* Nocardia asteroides ATCC 19247T (AF430019) Turicella otitidis DSM 8821T (X73976) 0.01
Fig. 2 Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequence (1524 bp) of strain ZYR 51T and the other members of the genus Gordonia. Bootstrap values (expressed as percentages of 1,000 replications) above 50 % are shown at the
branch points. Turicella otitidis DSM 8821T was used as an outgroup. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. Bar 0.01 substitutions per nucleotide position
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Gordonia ranged from 96.96 % (Gordonia rhizosphera NBRC 16068T and G. otitidis NBRC 100426T) to 93.57 % (Gordonia caeni MJ32T). Strain ZYR51T displayed a stable subclade (bootstrap value C50 %) with the type strains of G. otitidis NBRC 100426T (96.96 % similarity), G. sputi NBRC 100414T (96.68 % similarity) and G. aichiensis DSM 43978T (96.75 % similarity). A distinct subclade was also displayed in the other phylogenetic trees, including maximum-parsimony and maximum-likelihood phylogenetic trees (data not shown). Therefore, the phylogenetic analysis clearly indicates that the new isolate represents a novel genomic species of the genus Gordonia. In view of the combination of morphological, physiological and phylogenetic data described above, it is evident that strain ZYR51T forms a novel taxon within the genus Gordonia. Therefore, it is proposed that this organism be recognized as a novel species of the genus Gordonia, for which the name Gordonia jinhuaensis sp. nov. is proposed.
Description of Gordonia jinhuaensis sp. nov Gordonia jinhuaensis (jin.hua.en’sis. N.L. masc. adj. jinhuaensis pertaining to Jinhua, a city of Zhejiang in Eastern China). Aerobic, Gram-stain positive, non-motile actinobacterium that forms rod-shaped cells (0.3–0.5 lm in diameter and 0.7–1.5 lm in length). After 3 days incubation at 30 °C on ISP 2 medium, colonies are circular, convex, nontransparent, orange yellow, rough and 1.5–5 mm in diameter with irregular margins. No soluble pigment is produced. Growth occurs at 10–45 °C (optimum 28–32 °C). The pH range for growth is 6.0–11 (optimum, pH 7.0–8.0). Growth occurs with 0–9 % (w/v) NaCl. Positive for oxidase, catalase, urease, nitrate reduction, and hydrolysis of cellulose, Tweens 20, 40 and 60. Milk peptonization and coagulation, H2S production, Tween 80, tryptophan, gelatin, starch hydrolysis are negative. Alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-glucosidase, b-glucosidase activities are positive, but cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase,
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a-fucosidase activities are negative. Able to assimilate dextrin, D-maltose, D-trehalose, sucrose, D-turanose, Dsalicin, a-D-glucose, D-mannose, D-fructose, glycerol, pectin, glucuronamide, L-lactic acid, c-amino-butryric acid, a-hydroxy-butyric acid, b-hydroxy-D, L butyric acid, a-keto-butyric acid, acetoacetic acid, propionic acid, acetic acid, suberate, valerate, 4-hydroxybenzoate as the sole carbon sources and energy sources. Utilizes L-alanine, L-threonine, Lvaline, L-glycine, L-ornithine as sole nitrogen sources, but not L-arginine, L-aspartic acid, D-aspartic acid, Lglutamic acid, L-glutamine, L-tyrosine, L-serine, Dserine, L-Proline, L-histidine, L-lysine, L-phenylalanine, hypoxanthine. The whole-cell hydrolysates contain meso-diaminopimelic acid (meso-DAP), mannose, ribose, rhamnose, glucosamine, galactose and arabinose. Aspartic acid, glutamic acid, threonine, alanine are also present in the cell wall. The polar lipids comprise diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and five unknown lipids. The predominant menaquinone is MK-9(H2) and mycolic acids are present. The major cellular fatty acids ([5.0 %) are C16:0, 10-methyl C18:0, iso-C18:0 and summed feature 3(C16:1x6c and/ or C16:1x7c). The genomic DNA G?C content of the type strain is 67.7 mol%. The type strain ZYR 51T (=CGMCC 1.12827T = NBRL B-59111T = NBRC 110001T), isolated from pharmaceutical wastewater sample in Jinhua city, Zhejiang province, Eastern China (E119.6°, N29.12°). The GenBank accession number for the 16S rRNA gene sequence of strain ZYR 51T is KJ700483. Acknowledgments This research was supported by the National Natural Science Foundation of China (31340071), National Basic Research Program of China (No. 2010 CB833801), Key Project of Yunnan Provincial Natural Science Foundation (2013FA004) and a Grant NRF-2006-08790 funded by Ministry of Science, ICT and Future Planning of Korean government. WH and WJL extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project (No. RGPVPP-205).
References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Arensko¨tter M, Bro¨ker D, Steinbu¨chel A (2004) Biology of the metabolically diverse genus Gordonia. Appl Environ Microbiol 70:3195–3204
Antonie van Leeuwenhoek Cerny G (1978) Studies on aminopeptidase for the distinction of Gram-negative from Gram-positive bacteria. Appl Microbiol Biotechnol 5:113–122 Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100:221–230 Ding LX (2004) Studies on the isolation of viable but nonculturable bacteria and the phylogenetic analysis of the genus Aquaspirillum. PhD Thesis, The University of Tokyo Ding LX, Su XM, Yokota A (2011) Research progress of VBNC bacteria—a review. Acta Microbiol Sin 51:858–862 Ding LX, Zhang PH, Hong HC, Lin HJ, Yokota A (2012) Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp. DS471. Acta Microbiol Sin 52:77–89 Dong XZ, Cai MY (2001) Manual of systematics and identification of general bacteria. Science Press, Beijing Drzyzga O, Navarro Llorens JM, de Las Ferna´ndez, Heras L, Garciı´ Ferna´ndez E, Perera J (2009) Gordonia cholesterolivorans sp. nov., a cholesterol-degrading actinomycete isolated from sewage sludge. Int J Syst Evol Microbiol 59:1011–1015 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Fitch WM (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Biol 20:406–416 Gordon RE, Barnett DA, Handerhan JE, Pang CH-N (1974) Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24:54–63 Groth I, Rodrı´guez C, Schu¨tze B, Schmitz P, Leistner E, Goodfellow M (2004) Five novel Kitasatospora species from soil: Kitasatospora arboriphila sp. nov., K. gansuensis sp. nov., K.nipponensis sp. nov., K. paranensis sp. nov. and K. terrestris sp. nov. Int J Syst Evol Microbiol 54:2121–2129 Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704 Iida S, Taniguchi H, Kageyama A, Yazawa K, Chibana H, Murata S, Nomura F, Kroppenstedt RM, Mikami Y (2005) Gordonia otitidis sp. nov., isolated from a patient with external otitis. Int J Syst Evol Microbiol 55:1871–1876 Ka¨mpfer P, Young CC, Chu JN, Frischmann A, Busse HJ, Arun AB, Shen FT, Rekha PD (2011) Gordonia humi sp. nov., isolated from soil. Int J Syst Evol Microbiol 61:65–70 Kang Y, Takeda K, Yazawa K, Mikami Y (2009) Phylogenetic studies of Gordonia species based on gyrB and secA1 gene analyses. Mycopathologia 167:95–105 Kelly KL (1964) Inter-Society Color Council-National Bureau of Standards Color-Name Charts Illustrated with Centroid Colors. US Government Printing Office, Washington Kim KK, Lee CS, Kroppenstedt RM, Stackebrandt E, Lee ST (2003) Gordonia sihwensis sp. nov., a novel nitratereducing bacterium isolated from a wastewater-treatment bioreactor. Int J Syst Evol Microbiol 53:1427–1433 Kim KK, Lee KC, Klenk HP, Oh HM, Lee JS (2009) Gordonia kroppenstedtii sp. nov., a phenol-degrading actinomycete
isolated from a polluted stream. Int J Syst Evol Microbiol 59:1992–1996 Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, Park S-C, Jeon YS, Lee J-H, et al (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 of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120 Kimura M (1985) The neutral theory of molecular evolution. Cambridge University Press, Newyork Klatte S, Rainey FA, Kroppenstedt RM (1994) Transfer of Rhodococcus aichiensis Tsukamura 1982 and Nocardia amarae Lechevalier and Lechevalier 1974 to the genus Gordona as Gordona aichiensis comb. nov. and Gordona amarae comb. nov. Int J Syst Bacteriol 44:769–773 Kovacs N (1956) Identification of Pseudomonas pyocyanea by oxidase reaction. Nature 178:703–704 Kroppenstedt RM (1982) Separation of bacterial menaquinones by HPLC using reverse phase (RP18) and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr 5:2359–2387 le Roes M, Goodwin CM, Meyers PR (2008) Gordonia lacunae sp. nov., isolated from an estuary. Syst Appl Microbiol 31:17–23 Leifson E (1960) Atlas of bacterial flagellation. Academic press, Newyork Li WJ, Xu P, Schumann P, Zhang YQ, Pukall R, Xu LH, Stackebrandt E, Jiang CL (2007) Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China), and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57:1424–1428 Linos A, Berekaa MM, Steinbu¨chel A, Kim KK, Spro¨er C, Kroppenstedt RM (2002) Gordonia westfalica sp. nov., a novel rubber-degrading actinomycete. Int J Syst Evol Microbiol 52:1133–1139 Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167 Ming H, Nie GX, Jiang HC, Yu TT, Zhou EM, Feng HG, Tang SK, Li WJ (2012) Paenibacillus frigoriresistens sp. nov., a novel psychrotroph isolated from a peat bog in Heilongjiang Northern China. Antonie van Leeuwenhoek 102:297–305 Minnikin DE, Collins MD, Goodfellow M (1979) Fatty acid and polar lipid composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Bacteriol 47:87–95 Minnikin DE, Hutchinson IG, Caldicott AB, Goodfellow M (1980) Thin layer chromatography of methanolysates of mycolic acid-containing bacteria. J Chromatogr A 188:221–233 Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB (1998) A bacterial cytokine. Proc Natl Acad Sci USA 95:8916–8921 Park S, Kang SJ, Kim W, Yoon JH (2009) Gordonia hankookensis sp. nov., isolated from soil. Int J Syst Evol Microbiol 59:3172–3175 Riegel P, Kamne-Fotso MV, De Briel D, Prevost G, Jehl F, Piemont Y, Monteil H (1994) Rhodococcus chubuensis
123
Antonie van Leeuwenhoek Tsukamura 1982 is a later subjective synonym of Gordona sputi (Tsukamura 1978) Stackebrandt 1989 comb. nov. Int J Syst Evol Microbiol 44:764–768 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477 Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340 Soddell JA, Stainsby FM, Eales KL, Seviour RJ, Goodfellow M (2006) Gordonia defluvii sp. nov., an actinomycete isolated from activated sludge foam. Int J Syst Evol Microbiol 56:2265–2269 Su XM, Shen XY, Ding LX, Yokota A (2012) Study on the flocculability of the Arthrobacter sp., an actinomycete resuscitated from the VBNC state. World J Microbiol Biotechnol 28:91–97 Takeuchi M, Hatano K (1998) Gordonia rhizosphera sp. nov.isolated from the mangrove rhizosphere. Int J Syst Bacteriol 48:907–912 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739 Tang SK, Wang Y, Chen Y, Lou K, Cao LL, Xu LH, Li WJ (2009) Zhihengliuella alba sp. nov., and emended
123
description of the genus Zhihengliuella. Int J Syst Evol Microbiol 59:2025–2031 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 Tsukamura M (1971) Proposal of a new genus, Gordona, for slightly acid-fast organisms occurring in sputa of patients with pulmonary disease and in soil. J Gen Microbiol 68:15–26 Williams S (1989) Genus Streptomyces Waksman and Henrici 1943. Bergey’s Man Syst Bacteriol 4:2452–2492 Xu P, Li WJ, Tang SK, Zhang YQ, Chen GZ, Chen HH, Xu LH, Jiang CL (2005) Naxibacter alkalitolerans gen. nov., sp. nov., a novel member of the family ‘Oxalobacteraceae’ isolated from China. Int J Syst Evol Microbiol 55: 1149–1153 Yassin A, Shen FT, Hupfer H, Arun A, Lai WA, Rekha P, Young CC (2007) Gordonia malaquae sp. nov., isolated from sludge of a wastewater treatment plant. Int J Syst Evol Microbiol 57:1065–1068 Yoon JH, Lee JJ, Kang SS, Takeuchi M, Shin YK, Lee ST, Kang KH, Park YH (2000) Gordonia nitida sp. nov., a bacterium that degrades 3-ethylpyridine and 3-methylpyridine. Int J Syst Evol Microbiol 50:1203–1210 Zhi XY, Li WJ, Stackebrandt E (2009) An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol 59:589–608
