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Int J Syst Evol Microbiol 55 (2005), 1933-1938; DOI  10.1099/ijs.0.63594-0
© 2005 International Union of Microbiological Societies

Proposal of Yaniaceae fam. nov. and Yania flava sp. nov. and emended description of the genus Yania

Wen-Jun Li1, Peter Schumann2, Yu-Qin Zhang1,{dagger}, Ping Xu1, Guo-Zhong Chen1, Li-Hua Xu1, Erko Stackebrandt2 and Cheng-Lin Jiang1

1 The Key Laboratory for Microbial Resources of Ministry of Education, Yunnan Institute of Microbiology and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, 650091, P. R. China
2 DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

Correspondence
Wen-Jun Li
wjli{at}ynu.edu.cn
Cheng-Lin Jiang
lihxu{at}ynu.edu.cn


   ABSTRACT
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A coccoid actinobacterium strain (designated YIM 70178T) was isolated from a soil sample collected in Qinghai Province, China. The isolate grew well with an optimum salt concentration of 10–15 % (KCl, w/v) but scarcely or not at all without salt. The cell-wall peptidoglycan type was A4{alpha}, L-lys–gly–L-Glu. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. The predominant menaquinones were MK-8 and MK-9. The major fatty acid was anteiso-C15 : 0. The DNA G+C content was 57·9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 70178T was most closely related to the type strain of Yania halotolerans. DNA–DNA hybridization and comparison of physiological and chemotaxonomic characteristics demonstrated that strain YIM 70178T was different from Yania halotolerans. The name Yania flava sp. nov. is proposed, with strain YIM 70178T (=DSM 16377T=KCTC 19047T) as the type strain. Based on the phenotypic characteristics and phylogenetic position, as determined by 16S rRNA gene analysis and 16S rRNA signature nucleotide data, the genus description of Yania is therefore emended and strains YIM 70085T and YIM 70178T represent a novel family of the suborder Micrococcineae, for which the name Yaniaceae fam. nov. is proposed.

Published online ahead of print on 13 May 2005 as DOI 10.1099/ijs.0.63594-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Yania flava YIM 70178T is AY684123.

A detailed phylogenetic tree based on 16S rRNA gene sequences displaying the position of Yania flava YIM 70178T, Yania halotolerans YIM 70085T and related taxa in the suborder Micrococcineae is available as supplementary material in IJSEM Online.

{dagger}Present address: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, P. R. China. Back


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The suborder Micrococcineae was proposed by Stackebrandt et al. (1997)Go based on their newly established hierarchic classification system for actinomycetes, the class Actinobacteria (high DNA G+C Gram-positive bacteria) and contained the families Micrococcaceae, Brevibacteriaceae, Cellulomonadaceae, Promicromonosporaceae, Dermatophilaceae, Dermabacteraceae, Intrasporangiaceae, Jonesiaceae and Microbacteriaceae. With the subsequent addition of novel taxa into the suborder Micrococcineae, the phylogenetic coherence of some families was disrupted, leading to the proposal of another five families, Bogoriellaceae, Dermacoccaceae, Rarobacteraceae, Sanguibacteraceae (Stackebrandt & Schumann, 2000Go) and ‘Beutenbergiaceae’ (Garrity & Holt, 2001Go).

The genus Yania has been described recently and placed within the suborder Micrococcineae (W.-J. Li et al., 2004aGo). Although the described isolate exhibited low (<94·3 %) similarity to its phylogenetic neighbours and its 16S rRNA gene signature nucleotides differed significantly from those of members of the suborder Micrococcineae, a new family was not proposed because we were aware that a description based on a single isolate would probably not reflect the phenotypic diversity of the taxon.

Recently, another Gram-positive actinobacterium (designated YIM 70178T) of the genus Yania was isolated from a hypersaline habitat in Qinghai Province, China. The 16S rRNA gene sequence of strain YIM 70178T was closest to Yania halotolerans YIM 70085T, the type species of the genus Yania. Subsequent DNA–DNA hybridization and comparison of physiological and chemotaxonomic data demonstrated that strain YIM 70178T was different from Yania halotolerans YIM 70085T. The name Yania flava sp. nov. is proposed for this novel strain. In addition, the family Yaniaceae fam. nov. is proposed based on its distinct phylogenetic lineage and 16S rRNA gene sequence signature nucleotides within the suborder Micrococcineae.

Strain YIM 70178T was isolated by the dilution plating method from a saline soil sample from Qinghai Province, in north-west China. The medium used for selective isolation was SGA agar (Al-Tai & Ruan, 1994Go) (pH 7·2) supplemented with 20 % (w/v) KCl. The culture was incubated at 28 °C for about 2 weeks. The strain was maintained on ISP5 agar slants containing 10 % (w/v) KCl at 4 °C and in glycerol suspensions (20 % v/v) at –20 °C. Biomass for chemical and molecular systematic studies was obtained by cultivation in shaken flasks (about 150 r.p.m.) of modified ISP5 medium (KCl 10 % w/v, pH 7·0) broth at 28 °C for 1 week.

Cell morphology and metabolic properties were studied as described previously (Chen et al., 2004Go; W.-J. Li et al., 2004aGo, bGo). The colour of colonies was determined with colour chips from the ISCC-NBS colour charts standard samples no. 2106 (Kelly, 1964Go). In addition, acid production from carbohydrates was examined by a slightly modified method after Gordon et al. (1974)Go.

Morphological observation of 24–48 h cultures of strain YIM 70178T grown on ISP5 medium supplemented with 10 % KCl (w/v) revealed that the cells were similar to those of strain YIM 70085T, i.e. non-motile cocci, 0·4–0·8 µm in diameter. The colony characteristics were also similar to those of strain YIM 70085T; light yellow, circular, lubricous and opaque. Some physiological and biochemical characteristics of strain YIM 70178T are presented in Table 1Go.


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  		Table 1. Differential characteristics between strain YIM 70178T and Yania halotolerans YIM 70085T

The following characteristics are the same for both strains: Gram-positive, non-motile cocci. The optimum pH for growth is 7·0–8·0. Catalase-positive and oxidase-negative. Methyl red and Voges-Proskauer tests, melanin production, H2S and indole production are negative; Tweens 20 and 80, casein and starch are not decomposed. Positive for lysine decarboxylase, arginine dihydrolase and lipase activity. Negative for ornithine decarboxylase, L-aspartic arylamidase, {alpha}-galactosidase activities and growth on cellulose. The following substrates are utilized as sole carbon sources for growth in both strains: maltose, glucose, mannose, fructose, salicin, acetamide and galactose, while mannitol, adonitol, arabinose, arabitol, inositol and sorbitol are not utilized. +, Positive; –, negative; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PL, unidentified phospholipid; GL, unidentified glycolipid.

 
Chemotaxonomic properties, including peptidoglycan type, polar lipids, menaquinones and whole-cell fatty acid pattern, were analysed as described previously (Chen et al., 2004Go; W. -J. Li et al., 2004aGo, bGo). The DNA G+C content was determined by reverse-phase HPLC according to Mesbah et al. (1989)Go. The cellular fatty acid profile contained anteiso-C15 : 0 (58·20 %), iso-C14 : 0 (12·52 %), iso-C15 : 1 (9·29 %), iso-C16 : 0 (8·03 %), anteiso-C17 : 0 (5·79 %), iso-C13 : 0 (2·08 %), iso-C17 : 0 (1·88 %), C16 : 0 (0·59 %), anteiso-C15 : 1 (0·51 %), anteiso-C13 : 0 (0·38 %), C16 : 1{omega}7c (0·35 %), C18 : 1{omega}9c (0·25 %) and C14 : 0 (0·13 %). Additional chemotaxonomic data are shown in Tables 1 and 2GoGo.


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  		Table 2. Differential chemotaxonomic characteristics of genera of the families Yaniaceae and Micrococcaceae

Data for reference taxa were taken from Stackebrandt & Schumann (2000)Go; Reddy et al. (2000)Go; Liu et al. (2000)Go; Reddy et al. (2002)Go; Fan et al. (2002)Go; Altenburger et al. (2002)Go; Wieser et al. (2002)Go; Y. Li et al. (2004)Go; Gupta et al. (2004)Go; W.-J. Li et al. (2004aGo, bGo, 2005)Go; Margesin et al. (2004)Go and this study. MCAvar, Variable monocarboxylic amino acid; DCAvar, variable dicarboxylic amino acid; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PE, phosphatidylethanolamine; DMDG, dimannosyldiacylglycerol; PL, unidentified phospholipid(s); GL, unidentified glycolipid(s). Abbreviations for menaquinones exemplified by MK-8(H2), partially saturated menaquinone with one of eight isoprene units hydrogenated and MK-9, unsaturated menaquinone with nine isoprene units. All taxa contain L-Lys as diamino acid of the cell-wall peptidoglycan.

 
Extraction of genomic DNA and amplification of the 16S rRNA gene were performed according to Xu et al. (2003)Go. Phylogenetic analysis was conducted using the MEGA version 2.1 software package (Kumar et al., 2001Go) after multiple alignment of data by CLUSTAL_X (Thompson et al., 1997Go). Distances (distance options according to the Kimura two-parameter model) (Kimura, 1980Go, 1983Go) and clustering were performed using the neighbour-joining (Saitou & Nei, 1987Go) and maximum-likelihood (Felsenstein, 1981Go) methods. Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by performing 1000 resamplings (Felsenstein, 1985Go).

The almost-complete 16S rRNA gene sequence of strain YIM 70178T (1493 bp) was determined. Phylogenetic analysis, based on a dataset consisting of 1376 unambiguous nucleotides between positions 42 and 1417, showed that the novel isolate was most closely related to Yania halotolerans YIM 70085T. A dendrogram (Fig. 1Go) confirmed that strains YIM 70178T and YIM 70085T are phylogenetic neighbours with 98·4 % 16S rRNA gene sequence similarity and a bootstrap value of 100 %. Similarity values with sequences of neighbouring taxa were significantly lower (89·2–94·0 %). A detailed phylogenetic tree displaying the phylogenetic position of strain YIM 70178T, Yania halotolerans YIM 70085T and related taxa in the suborder Micrococcineae is available as Supplementary Fig. S1 in IJSEM Online.



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  		Fig. 1. Phylogenetic dendrogram obtained by distance matrix analysis of 16S rRNA gene sequences, showing the position of strain YIM 70178T among phylogenetic neighbours. The sequence of Streptomyces megasporus DSM 41476T (Z68100) was used as the outgroup (not shown). Numbers on branch nodes are bootstrap percentages (1000 resamplings, only values over 50 % are given). Bar, 1 % sequence divergence.

 
The isolate YIM 70178T was different from Yania halotolerans YIM 70085T in some physiological, biochemical and chemotaxonomic characteristics (Table 1Go). DNA–DNA relatedness tests were performed between strain YIM 70178T and Yania halotolerans YIM 70085T using the optical renaturation method (De Ley et al., 1970Go; Huß et al., 1983Go; Jahnke, 1992Go) and DNA–DNA relatedness between the strains was 35·4 %. DNA–DNA relatedness provided decisive evidence that strain YIM 70178T and Yania halotolerans YIM 70085T are members of different genomic species (Wayne et al., 1987Go). Therefore, based on the above phenotypic and genotypic results, we consider strain YIM 70178T to represent a novel species of the genus Yania, for which we propose the name Yania flava sp. nov.

The results of 16S rRNA gene sequence comparisons clearly demonstrated that strains YIM 70178T and YIM 70085T are members of the suborder Micrococcineae. Both strains have some unique 16S rRNA gene signature nucleotides compared with other families of the suborder Micrococcineae, such as 140–223 (A–G), 142–221 (C–A), 615–625 (G–U), 839–847 (A–A) and 1134–1140 (A–U) (this study and W.-J. Li et al., 2004aGo). Strain YIM 70178T was isolated from Qinghai Province, north-west China and strain YIM 70085T originated from Xinjiang Province, in the north of China. Since the two strains were isolated from independent and geographically distant sources, they provide a first estimation of the phenotypic diversity of the genus Yania. As discussed previously (W.-J. Li et al., 2004aGo), the unique set of 16S rRNA gene signature nucleotides and chemotaxonomic markers distinguish the genus Yania from members of the closest family Micrococcaceae in the suborder Micrococcineae (Table 2Go). Thus, the family Yaniaceae fam. nov. is proposed.

Description of Yaniaceae fam. nov.
Yaniaceae (Ya'ni.a'ce.ae. N.L. fem. n. Yania type genus of the family; -aceae ending to denote a family; N.L. fem. pl. n. Yaniaceae the Yania family).

The pattern of 16S rRNA gene sequence signatures consists of nucleotides at positions 140–223 (A–G), 142–221 (C–A), 615–625 (G–U), 839–847 (A–A) and 1134–1140 (A–U) (W.-J. Li et al., 2004aGo). The type genus is Yania.

Emended description of the genus Yania Li et al. 2004Go
The description of the genus Yania (W.-J. Li et al., 2004Go) is emended as follows. Moderately halophilic or halotolerant. The polar lipids contain diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. The predominant menaquinone(s) are MK-8 and MK-9 or MK-8. The major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0 or anteiso-C15 : 0. The DNA G+C content is 53–58 mol%.

Description of Yania flava sp. nov.
Yania flava (fla'va. L. fem. adj. flava golden yellow, referring to the colour of the colonies).

Morphological, chemotaxonomic and general characteristics are as described for the genus. Colonies are light yellow, circular, lubricous and opaque. Acid is produced from glucose, maltose and fructose. It is negative for milk peptonization, milk coagulation, urease and nitrate reduction, gelatin liquefaction, growth in cellulose, H2S and melanin production. Some other physiological and biochemical characteristics are listed in Table 1Go. The cell-wall peptidoglycan type is A4{alpha}, L-lys–gly–L-Glu. The polar lipids contain diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. The predominant menaquinones are MK-8 and MK-9. The major cellular fatty acid is anteiso-C15 : 0. The DNA G+C content is 57·9 mol% (HPLC method).

The type strain, YIM 70178T (=DSM 16377T=KCTC 19047T), was isolated from a saline soil collected from Qinghai Province, north-west China.


   ACKNOWLEDGEMENTS
 
This research was supported by National Basic Research Program of China (project no. 2004CB719601), National Natural Science Foundation of China (project no. 30270004) and Yunnan Provincial Natural Science Foundation (project no. 2004C0002Q).


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