Hongia Gen. Nov., a New Genus of the Order Actinomycetales
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International Journal of Systematic and Evolutionary Microbiology (2000), 50, 191–199 Printed in Great Britain Hongia gen. nov., a new genus of the order Actinomycetales Soon Dong Lee, Sa-Ouk Kang and Yung Chil Hah Author for correspondence: Yung Chil Hah. Tel: 82 2 880 6700. Fax: 82 2 888 4911. e-mail: hahyungc!snu.ac.kr Department of An aerobic, nocardioform actinomycete, named LM 161T, was isolated from a Microbiology, College of soil sample obtained from a gold mine in Kongiu, Republic of Korea. This Natural Sciences and Research Center for organism formed well-differentiated aerial and substrate mycelia and Molecular Microbiology, produced branched hyphae that fragmented into short or elongated rods. The Seoul National University, cell wall contains major amounts of LL-diaminopimelic acid, alanine, glycine, Seoul 151-742, Republic of Korea glutamic acid, mannose, glucose, galactose, ribose and acetyl muramic acid. The major phospholipids of this isolate are phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, and the major isoprenologue is a tetrahydrogenated menaquinone with nine isoprene units. The whole-cell hydrolysate of strain LM 161T contains 12- methyltetradecanoic and 14-methylpentadecanoic acids as the predominant fatty acids, but does not contain mycolic acids. The GMC content of the DNA is 71<3 mol%. The phylogenetic position of the test strain was investigated using an almost complete 16S rDNA sequence. The isolate formed the deepest branch in the clade encompassing the members of the suborder Propionibacterineae Rainey et al. 1997. On the basis of chemical, phenotypic and genealogical data, it is proposed that this isolate be classified within a new genus as Hongia koreensis gen. nov., sp. nov. in the order Actinomycetales. The type strain is LM 161T (¯ IMSNU 50530T). Keywords: Hongia gen. nov., actinomycetes, Propionibacterineae, phylogeny INTRODUCTION Luteococcus (Tamura et al., 1994), Microlunatus (Nakamura et al., 1995), Propioniferax (Yokota et al., Phylogenetic analysis based on 16S rDNA (rDNA) 1994) and Propionibacterium (Cummins & Johnson, sequence data is currently one of the most effective 1986). The recently described genus Friedmanniella methods for the delineation of bacteria. The suborder (Schumann et al., 1997) was shown to be a new Propionibacterineae Rainey et al. (Stackebrandt et al., member of the family Propionibacteriaceae. All of the 1997) of the order Actinomycetales (Buchanan) members, except for the genus Propionibacterium Stackebrandt et al. (Stackebrandt et al., 1997) encom- (Cummins & Johnson, 1986), possess cell wall peptido- passes the type family Propionibacteriaceae (Delwiche) glycan based on -diaminopimelic acid (-DAP) as Rainey et al. (Stackebrandt et al., 1997) and the family the diagnostic diamino acid and exhibit a range of cell Nocardioidaceae (Nesterenko et al.) Rainey et al. morphology from coccus- or rod-shaped forms to (Stackebrandt et al., 1997), on the basis of 16S rDNA nocardioforms. Recently, the phylogenetic relation- studies. The family Nocardioidaceae contains the ship between members of the family Nocardioidaceae genera Aeromicrobium (Miller et al., 1991; Tamura & and related taxa was re-evaluated on the basis of 16S Yokota, 1994) and Nocardioides (Collins et al., 1989, rDNA sequence comparisons (Yoon et al., 1998). 1994; O’Donnell et al., 1982a; Prauser, 1976, 1984). The family Propionibacteriaceae contains the genera In addition, the following -DAP-containing actino- mycete genera, which belong to other phylogenetic ................................................................................................................................................. lineages (Stackebrandt et al., 1997), have been Abbreviations: LL-DAP, LL-diaminopimelic acid; ISP, International described: Streptomyces (Williams et al., 1989), Streptomyces Project. Sporichthya (Rainey et al., 1993), Kineosporia (Itoh et The EMBL accession number for the 16S rDNA sequence of strain LM 161T al., 1989), Intrasporangium (Kalakoutskii et al., 1967), is Y09159. Terrabacter (Collins et al., 1989) and Terracoccus 01146 # 2000 IUMS 191 S. D. Lee, S.-O. Kang and Y. C. Hah (Prauser et al., 1997). The members of the suborder Catalase activity was determined by the production of Propionibacterineae are readily differentiated from bubbles after the addition of a drop of 3% (v}v) hydrogen each other and from the other -DAP-containing peroxide solution on to a slide. Growth was tested over a genera mentioned above by a combination of mor- range of temperatures (10–45 mC) and pH values (5–10). phological, physiological and chemotaxonomic Nitrate reduction, urease activity, hydrogen sulfide pro- duction, decomposition of hippurate and hydrolysis of properties (Prauser et al., 1997; Schumann et al., arbutin, casein, aesculin, gelatin and starch were tested as 1997). described by Mac Faddin (1980). Hydrolysis of the DNA During the taxonomic study of soil actinomycetes was determined using Bacto DNase test agar (Difco). T from natural environments, strain LM 161 (T ¯ type Pectinolytic activity was examined according to the method strain) was isolated from a gold-mine cave in Kongju, of Hankin et al. (1971). Acid production from carbohydrates Republic of Korea. Strain LM 161T contained - was determined by means of a colour change from green to yellow in Bacto OF basal medium (Difco). The utilization of DAP as the diagnostic diamino acid in the peptido- organic acids as carbon sources and the decomposition of glycan, and the substrate- and aerial mycelia showed adenine, hypoxanthine, -tyrosine and xanthine were a tendency to fragment. In this work, we have de- examined using previously described methods (Gordon et termined the taxonomic and phylogenetic position of al., 1974). The utilization of carbohydrates as the sole the isolate by examining its morphological, physio- carbon source was studied according to the method of logical and chemotaxonomic properties and by ana- Pridham & Gottlieb (1948). NaCl-tolerance studies were lysing its 16S rDNA. Our results indicate that the performed on nutrient agar containing NaCl at final isolate should be placed in a new species of a novel concentrations of 3, 5, 7 and 10% (w}v). Susceptibility to genus, for which the name Hongia koreensis is antibiotics and chemical inhibitors was determined as proposed. Strain LM 161T has been deposited in the described previously (Williams et al., 1983). To determine sensitivity to lysozyme, a 0±1% solution of lysozyme was Culture Collection Centre of the Institute of Micro- sterilized by membrane filtration and added to nutrient agar biology, Seoul National University (IMSNU) as strain T at a final concentration of 0±01%. Antimicrobial activity was IMSNU 50530 . tested for nine target organisms by using the overlay technique of Williams et al. (1983). METHODS Cell wall analysis. Purified cell wall peptidoglycan was Micro-organisms and culture conditions. Strain LM 161T was prepared according to the method of Yamada & Komagata isolated, using the dilution plating method, from soil (1972) and its amino acid composition was analysed by two- collected at a gold-mine cave in Kongju, Republic of Korea. dimensional, ascending TLC on cellulose plates by using the The soil suspensions were pretreated at 30 mC for 20 min solvent systems described previously (Schleifer & Kandler, before plating (Lee, 1996). The medium used for selective 1972). The molar ratio of amino acids was determined by isolation was a tap-water agar (pH 7 0) supplemented with GC of N-heptafluorobutyryl amino acid isobutyl esters " ± " 50 g cycloheximide ml− and 50 g nystatin ml− . After the (O’Donnell et al., 1982b). The isomers of diaminopimelic purity had been checked, the organism was subcultured on acid were determined according to the method of Staneck & yeast extract}malt extract agar [medium 2 of the Inter- Roberts (1974). Cell wall sugars as alditol acetates were national Streptomyces Project (ISP)] (Shirling & Gottlieb, determined by GC (Saddler et al., 1991). GC analyses were 1966) at 28 mC. The following reference strains were used performed with a Hewlett Packard model HP5890A gas to compare chemotaxonomic properties: Aeromicrobium chromatograph equipped with a flame-ionization detector erythreum DSM 8599T, Aeromicrobium fastidiosum IFO and integrator. The acyl type of the cell wall was determined 14987T, Nocardioides albus IFO 13917T, Nocardioides jensenii using the colorimetric method of Uchida & Aida (1984). T T KCTC 9134 , Nocardioides luteus IFO 14491 , Nocardioides Lipid analysis. T T Cellular fatty acid methyl esters were prepared simplex IFO 12069 , Luteococcus japonicus IFO 12422 , from cells grown on yeast extract}glucose broth at 30 mC for Microlunatus phosphovorus JCM 9379T, Propioniferax T T 3 d by alkaline methanolysis (Minnikin, 1988) and were innocua DSM 8251 and Kineosporia aurantiaca IFO 13890 . analysed with a Hewlett Packard model 5890A gas Morphological and cultural characteristics. Growth and chromatograph equipped with a SPB-1 fused silica capillary morphology were examined by using the ISP media de- column (0±25 mm¬30 m; Supelco). The column tempera- scribed by Shirling & Gottlieb (1966) and the media ture was programmed according to the manufacturer’s described by Waksman (1961). The formation of melanin instructions. The fatty acid methyl esters were identified by was investigated on peptone}yeast extract}iron agar (ISP comparison with the Bacterial Acid Methyl Ester CP MIX medium 6) and