Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM 70185T is DQ372937.
Strain YIM 70185T was isolated by using the dilution plating method on marine agar (Difco 2216; MA) supplemented with 15 % NaCl (w/v). The strain was maintained on MA slants at 4 °C and as 20 % (w/v) glycerol suspensions at 20 °C. Biomass for chemical and molecular studies was obtained by cultivation in shaken flasks (about 150 r.p.m.) using marine broth (Difco 2216; MB) at 28 °C for 1 week.
Gram staining and the KOH lysis test were carried out according to Gram (1884) and Cerny (1978), respectively. Morphology and motility were examined by light microscopy (model BH2; Olympus) and electron microscopy (JEM-1010 electron microscope; JEOL) using cells from exponentially growing cultures. For transmission electron microscopy observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid after air-drying. Colony morphology was observed on MA or ISP5 medium (Shirling & Gottlieb, 1966) containing 10 % NaCl and trypticase soy agar (TSA) containing 10 % NaCl after incubation at 28 °C for 2 days. The colony colour was determined with the ISCCNBS colour charts (Kelly, 1964). Growth was tested at 4, 10, 28, 30, 37, 40, 45 and 55 °C on TSA medium containing 10 % NaCl. The ability of the strain to grow at different pHs and NaCl concentrations was examined according to Tang et al. (2003) except that MB was used instead of ISP5 as the basic medium. Metabolic properties were determined using API ID 32E test kits (bioMérieux) according to the manufacturer's instructions.
Strain YIM 70185T was aerobic and stained Gram-positive. Short, rod-shaped cells (0.61.0x1.52.0 µm) were observed, but neither flagella nor spores were found. Pale-yellow colonies with a smooth surface and a maximum diameter of 1 mm were formed on MA or TSA containing 10 % NaCl after incubation for 48 h at 28 °C. Strain YIM 70185T grew well at 28 and 30 °C, but slowly at 4 and 45 °C. Growth was observed at initial pH values between 6 and 10 and on TSA containing 025 % NaCl. The strain grew optimally at pH 8.09.0 and in the presence of 10 % NaCl. The detailed physiological and biochemical characteristics of the strain are given in the species description.
A purified cell-wall preparation was obtained after disruption of cells by shaking with glass beads and subsequent trypsin digestion by the method of Schleifer (1985). Amino acids and peptides in cell-wall hydrolysates were analysed by two-dimensional ascending TLC on cellulose plates, using the solvent systems of Schleifer & Kandler (1972). The N-terminal amino acid of the interpeptide bridge was determined by dinitrophenylation, as described by Schleifer (1985). Molar ratios of amino acids were determined by GC and GC-MS of N-heptafluorobutyryl amino acid isobutyl esters (MacKenzie, 1987). Analysis of enantiomers of peptidoglycan amino acids was performed by GC of N-pentafluoropropionyl amino acid isopropyl esters (Frank et al., 1980) on an L-ChirasilVal column (Macherey-Nagel) as described by Groth et al. (1997). Sugar analysis of the purified cell wall followed procedures described by Staneck & Roberts (1974). Polar lipids were extracted, examined by two-dimensional TLC and identified by using published procedures (Minnikin et al., 1979; Collins & Jones, 1980). Menaquinones were isolated by using the method of Collins et al. (1977) and analysed by HPLC (Groth et al., 1997). Analysis of the whole-cell fatty acid pattern followed the instructions of the MIDI System (Microbial ID) (Kroppenstedt, 1985) by using exponential phase cultures.
The peptidoglycan type of strain YIM 70185T was A4α, L-lysL-alaL-Glu. Glucose and tyvelose were detected in the purified cell wall. The polar lipids contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and an unknown glycolipid. The menaquinones were MK-9, MK-10, MK-8 (5 : 2 : 1). The major cellular fatty acids were ai-C15 : 0 (63.9 %) and i-C15 : 0 (15.0 %); the complete profile of cellular fatty acids is given in detail in the species description.
Extraction of genomic DNA and PCR amplification of the 16S rRNA gene were done as described by Xu et al. (2003). Multiple alignments with sequences of the most closely related actinobacteria and calculations of levels of sequence similarity were carried out using CLUSTAL X (Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining method of Saitou & Nei (1987) from Knuc values (Kimura, 1980) using MEGA version 2.1 (Kumar et al., 2001). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
Phylogenetic analysis of the 16S rRNA gene sequence (1475 bp) of strain YIM 70185T revealed relatively remote relatedness to members of the family Micrococcaceae (similarities ranging from 93.5 to 96.4 %), forming a distinct subclade within the radiation of this family (Fig. 1). Patterns of selected 16S rRNA gene signature nucleotides defined for the family Micrococcaceae (Stackebrandt et al., 1997; Stackebrandt & Schumann, 2000) were consistent with nucleotides determined for the 16S rRNA gene sequence of strain YIM 70185T except that G, CG and UC were determined at positions 640, 839 : 847 and 1025 : 1036, respectively. Positions 502 : 543 and 1310 : 1327, given as RY by Stackebrandt & Schumann (2000), were determined as GC. The G+C content of the DNA was determined to be 66.5 mol% by reverse-phase HPLC of nucleosides according to Mesbah et al. (1989).
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Chemotaxonomic characteristics that differentiate strain YIM 70185T from representatives of its closest phylogenetic neighbours detected by 16S rRNA gene sequence analysis are given in Table 1.
Table 1. Differential chemotaxonomic characteristics of the genus Zhihengliuella and related genera of the family Micrococcaceae All taxa contain L-Lys as the diamino acid of the peptidoglycan. Data for reference genera were taken from Stackebrandt & Schumann (2000), Liu et al. (2000) and Wieser et al. (2002) (Micrococcus), Keddie et al. (1986), Koch et al. (1995), Stackebrandt et al. (1995) and recent publications (Arthrobacter globiformis group), Collins & Kroppenstedt (1983), Keddie et al. (1986), Koch et al. (1995), Stackebrandt et al. (1995), Gupta et al. (2004), Margesin et al. (2004) and Chen et al. (2005) (Arthrobacter nicotianae group), Stackebrandt & Schumann (2000) and W.-J. Li et al. (2006) (Kocuria), Stackebrandt et al. (1995), Collins et al. (2002), W.-J. Li et al. (2004, 2005b) (Nesterenkonia), Sanders & Fryer (1980) (Renibacterium), Bergan & Kocur (1982), Gerencser & Bowden (1986), Collins et al. (2000), Fan et al. (2002) and Y. Li et al. (2004) (Rothia), Altenburger et al. (2002) and W.-J. Li et al. (2005a) (Citricoccus) and Pukall et al. (2006) (Acaricomes). Menaquinones are exemplified by MK-8(H2), partially saturated menaquinone with one of eight isoprene units hydrogenated. Fatty acids are exemplified by ai-C15 : 0, 12-methyltetradecanoic acid; i-C15 : 0, 13-methyltetradecanoic acid. Sugars and polar lipids in parentheses vary among species or strains. ND, No data available.
Although some species of the Arthrobacter nicotianae group, e.g. Arthrobacter nicotianae, Arthrobacter protophormiae, Arthrobacter uratoxydans (Stackebrandt et al., 1983), Arthrobacter rhombi (Osorio et al., 1999), Arthrobacter bergerei and Arthrobacter arilaitensis (Irlinger et al., 2005), possess A4α peptidoglycan with the interpeptide bridge AlaGlu, they have different cell-wall sugar compositions, quinone systems or DNA G+C contents compared with those of strain YIM 70185T.
Tyvelose has been reported to be present in the cell walls of only a few members of the family Microbacteriaceae, suborder Micrococcineae, such as Agromyces cerinus subsp. cerinus (Zgurskaya et al., 1992) and Agrococcus citreus (Wieser et al., 1999). The presence of tyvelose differentiates the new isolate from other taxa in the family Micrococcaceae, although the cell-wall sugars have been studied in only a limited set of species so far.
For the quinone system, Renibacterium salmoninarum and some Arthrobacter species, such as Arthrobacter gangotriensis (Gupta et al., 2004), have predominant menaquinones similar to those of the new isolate, but they differ in other chemotaxonomic characteristics such as peptidoglycan structure, cell-wall sugars, major fatty acids and DNA G+C content (Table 1).
16S rRNA gene sequence analysis, chemotaxonomic properties and the profile of metabolic properties (see species description) revealed that strain YIM 70185T represents a new genus and a novel species within the family Micrococcaceae, for which the name Zhihengliuella halotolerans gen. nov., sp. nov. is proposed.
Description of Zhihengliuella gen. nov.
Zhihengliuella [Zhi.heng.li.u.el'la. N.L. fem. dim. n. Zhihengliuella named after Zhi-Heng Liu (1940), a Chinese microbiologist who devotes himself to the study of actinomycete taxonomy].
Gram-positive, mesophilic and aerobic. Cells are non-motile, non-spore-forming, short rods (0.61.0x1.52.0 µm). Catalase-positive and oxidase-negative. The peptidoglycan type is A4α, L-lysL-alaL-Glu. The predominant menaquinones are MK-9 and MK-10; MK-8 occurs in smaller amounts. The major fatty acids are ai-C15 : 0 and i-C15 : 0. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol. The G+C content of genomic DNA is about 66 mol%. 16S rRNA gene sequence similarity reveals membership of the family Micrococcaceae. The type species is Zhihengliuella halotolerans.
Description of Zhihengliuella halotolerans sp. nov.
Zhihengliuella halotolerans (ha.lo.to'le.rans. Gr. n. hals salt; L. part. adj. tolerans tolerating; N.L. part. adj. halotolerans salt-tolerating, referring to the organism's ability to tolerate high salt concentrations).
In addition to the characteristics that define the genus, the species has the following characteristics. The pale-yellow colonies are circular, opaque and approximately 1.0 mm in diameter after 24 h at 28 °C. Optimum growth occurs at pH 8.09.0 and at 2830 °C with 10 % NaCl. Negative for ornithine decarboxylase, urease, N-acetylglucosaminidase, β-galactosidase, α-glucosidase, gelatin liquefaction, methyl red and VogesProskauer tests and nitrate reduction, but positive for arginine dihydrolase, lysine decarboxylase, lipase, β-glucosidase, α-galactosidase, ammonia production, starch, hydrolysis of Tweens 20 and 80 and milk peptonization. Malonate is utilized and acid is produced from maltose, glucose, sucrose, L-arabinose and trehalose. The cell wall contains glucose and tyvelose. Polar lipids include an unknown phospholipid and an unknown glycolipid. The menaquinones are MK-9, MK-10, MK-8 (5 : 2 : 1). The cellular fatty acid profile contains ai-C15 : 0 (63.9 %), i-C15 : 0 (15.0 %), ai-C17 : 0 (8.5 %), i-C16 : 0 (7.1 %), C16 : 0 (2.5 %), i-C17 : 0 (1.0 %), i-C14 : 0 (0.8 %), C14 : 0 (0.8 %), C15 : 0 (0.2 %) and ai-C15 : 1 (0.2 %). The G+C content of the DNA of the type strain is 66.5 mol%.
The type strain is YIM 70185T (=DSM 17364T=KCTC 19085T), isolated from a saline soil sample collected from Qinghai province, north-west China.
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