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1 Microbial Gene Research and Resources Facility, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, QLD 4111, Australia
2 Center for Forestry and Horticultural Research, Faculty of Science, Griffith University, Brisbane, QLD 4111, Australia
Correspondence
Bharat Patel
b.patel{at}griffith.edu.au
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peptidoglycan, the dominant cellular fatty acid was 18 : 1
7c and the major hydroxy fatty acid was 2-OH 14 : 0. The major menaquinones were MK-8 (76 %) and MK-7 (24 %) and the glycolipids present were disphosphatidylglycerol, phosphatidylglycerol and three unidentified phospholipids. The chemotaxonomic properties of strain E1HC-02T were distinctly different to all of the 17 genera of the family Microbacteriaceae and hence strain E1HC-02T is designated as representing a novel species of a new genus, Frondicola australicus gen. nov., sp. nov. The type strain of the type species is E1HC-02T (=JCM 13598T=DSM 17894T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Frondicola australicus sp. nov. strain E1HC-02T is DQ52589.
Details of the cellular fatty acid content of strain E1HC-02T are available as a supplementary table with the online version of this paper.
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). Extensive studies on the culture-dependent and culture-independent microbial diversity of soils and the plant rhizosphere and, to a lesser extent, the leaf litter biosphere, have suggested that members of the phyla Actinobacteria and Proteobacteria dominate these environments (Sharma et al., 2005; Xu et al., 1996). During our culture-dependent and culture-independent bacterial diversity studies of the leaf litter of a slash pine forest located in southeast Queensland, Australia, we frequently isolated members of the phylum Actinobacteria. Here, we report on the characteristics of an irregular rod-shaped bacterium, designated strain E1HC-02T, which represents a novel species of a new genus. For the isolation of strain E1HC-02T, 10 g of moist leaf litter was mixed with 50 ml of sterile distilled water and vortexed for 5 min. The suspension was then rocked horizontally for 5 min, serially diluted ten-fold and 100 µl of the mixture was spread onto trypticase soy broth (TSB) Gelrite plates, pH 7.2. The Gelrite plates contained (g l–1) 3 g TSB, 2.033 g MgCl2.6H2O, 0.882 g CaCl2.2H2O and 20 g Gelrite. The plates were incubated at room temperature (approx. 25 °C) for up to 6 days. Several morphologically distinct single colonies that developed were picked and individually restreaked onto fresh TSB Gelrite plates at least twice before being considered pure. Colonies from pure cultures were resuspended in sterile preservation medium (0.3 g TSB, 15 ml glycerol and 85 ml distilled water) and stored at –80 °C. One of the isolates that produced cream, round, smooth colonies within 3 days of incubation was designated strain E1HC-02T and selected for further characterization.
Cell morphology was determined by phase-contrast and electron microscopy (Kanso & Patel, 2003). Cells of strain E1HC-02T were non-flagellated and non-sporulating irregular short rods (0.5–1.0x0.2–0.4 µm). Electron micrographs of thin sections revealed a Gram-positive cell-wall ultrastructure with an S-layer (Messner & Sleytr, 1992; Sleytr et al., 1988) and electron-dense inclusion bodies (Fig. 1).
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The metabolic profile of strain E1HC-02T was determined using GN2 Biolog plates (Hayward). For this, the novel strain was grown on Biolog Universal Growth medium agar plates. Colonies were resuspended in an inoculation fluid and this was used to inoculate the wells of GN2 Biolog plates as recommended by the manufacturer. After 24 h incubation at room temperature (approx. 25 °C), the absorbances in the wells of the microtitre plates were read and the results transformed into positive, borderline and negative scores by using Biolog MicroStation System software. The results of the analysis are provided in the species description.
Catalase activity was determined by dropping H2O2 on to colonies that developed on 1 % TSB agar plates and activity was determined to be positive. The oxidase test, determined by using Oxidase detection strips (Medvet Science Pty. Ltd.), was negative.
The cell-wall peptidoglycan of strain E1HC-02T was isolated and analysed at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Germany, using previously described procedures (MacKenzie, 1987; Schleifer, 1985; Schleifer & Kandler, 1972; Rhuland et al., 1955). The total hydrolysate was found to contain the amino acids ornithine, alanine, glycine, homoserine and glutamic acid in the molar ratio of 1.0 : 0.9 : 1.7 : 0.5 : 1.0. The presence of these amino acids was confirmed by GC and MS (MacKenzie, 1984). Hydroxyglutamic acid was not detected. The partial hydrolysate of the cell-wall peptidoglycan showed that strain E1HC-02T contained the peptides Gly–L-glu and D-orn–D-Ala. On the basis of amino acid and peptide analyses of cell-wall hydrolysates, strain E1HC-02T was determined to contain a peptidoglycan of type B2 {Gly} [L-hsr] D-glu–D-Orn (Schleifer & Kandler, 1972). No glycolate was detected in the cell-wall acid hydrolysate, which suggests that muramic acid occurs in the N-acetyl form.
Fatty acid methyl ester (FAME) analysis of the whole-cell was determined at DSMZ using GC (MIDI, Microbial ID). Strain E1HC-02T possessed a very large amount of fatty acid 18 : 17c (77.9 %) and smaller amounts of other fatty acids (see Supplementary Table S1 in IJSEM Online).
Polar lipid analysis was carried out by the identification services of DSMZ and Dr B. J. Tindall, DSMZ, Germany. Strain E1HC-02T possessed disphosphatidylglycerol, phosphatidylglycerol, at least 6 glycolipids, of which 3 were probably diglycosyl diglycerides (the nature of sugars and their linkages have not been identified), and 4 unidentified phospholipids.
For genomic DNA purification, a 50 ml sample of actively growing culture was centrifuged at 5400 r.p.m. at 4 °C for 20 min (4K15; Sigma) and the cell pellet was resuspended in 4 ml buffer A (5 mM Tris/HCl pH 8.0, 100 mM NaCl and 5 mg lysozyme). After incubation on ice for 5 min, 0.85 ml of buffer [300 mM EDTA, pH 7.5 and 5 % (w/v) SDS] and 100 µl of 20 mg ml–1 proteinase K was added. Following overnight incubation at 55 °C, an equal volume of phenol : chloroform : isoamylalcohol (25 : 24 : 1) was added and the mixture was agitated gently for 30 min. Three volumes of cold 100 % ethanol were then carefully overlaid and the flocculent DNA that developed was spooled onto a sterile disposable plastic loop. The spooled DNA was resuspended in 5 ml TE buffer (10 mM Tris/HCl pH 7.4, 1 mM EDTA pH 8) and the quality assessed by agarose gel electrophoresis, ethidium bromide staining and visualization under a Gel Documentation Analysis System (GDAS 1200; UVP). The mol% G+C content of the genomic DNA was determined by the thermal denaturation method (Marmur & Doty, 1961) using a spectrophotometer (Cintra20; GBC Scientific Equipment) as described by Spanevello et al., 2002 and was calculated to be 71±1 mol%.
For studies on the 16S rRNA gene, 10 ml of an overnight culture of strain E1HC-02T was centrifuged at 5400 r.p.m. at 4 °C for 20 min (4K15; Sigma). The pellet was resuspended in 100 µl TE buffer and the cells were lysed by boiling for 15 min. The lysate was centrifuged at 14 000 r.p.m. for 15 min in a microcentrifuge (model 1-15; Sigma) and 2 µl aliquots of the supernatant were used as a template for the amplification of the 16S rRNA gene (Andrews & Patel, 1996). The sequencing of the gene was performed essentially as described previously (Andrews & Patel, 1996). The partial sequences that were generated were assembled using BioEdit v5.0.1 (Hall, 1999) and the consensus sequence of 1433 nucleotides was manually corrected for errors. The most closely related sequences in GenBank (version 152) and the Ribosomal Database Project II (release 9.37) identified using BLAST (Altschul et al., 1997) and the Sequence Match program (Cole et al., 2005) were extracted, aligned and manually adjusted according to the 16S rRNA secondary structure using BioEdit. Nucleotide ambiguities were omitted and evolutionary distances were calculated by using the Jukes and Cantor option (Jukes & Cantor, 1969) in TreeCon (Van de Peer et al., 1997). Phylogenetic trees were constructed from evolutionary distances using the neighbour-joining method (Saitou & Nei, 1987). Tree topology was re-examined by the bootstrap method (1000 replications) of resampling (Felsenstein, 1985).
16S rRNA gene sequence analysis indicates that strain E1HC-02T is a member of the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Actinomycetales, suborder Micrococcineae, family Microbacteriaceae (Fig. 2). The inclusion of strain E1HC-02T as a member of the phylum to the suborder level is supported by the presence of the defining nucleotide signatures and, with the exception of an A:U at the positions 771–808 (Escherichia coli numbering) instead of a G:C, to the family level (Stackebrandt et al., 1997). Phylogenetic analysis undertaken with representative members of the family Microbacteriaceae placed strain E1HC-02T almost equidistantly between the cluster represented by the strains of the psychrophile Frigoribacterium faeni (Miteva et al., 2004; Kämpfer et al., 2000) and an isolate from alpine subnival plants (H. M. Sheng, L. Z. Ang, S. J. Xu, T. Chen & X. L. Zheng, unpublished, GenBank accession no. DQ339615) and a 16S rRNA clone from DNA extracted from volcanic deposits (J. Gomez-Alvarez, J. Baniak, G. Harrison, J. G. Medrano & K. Nusslein, unpublished, GenBank accession no. DQ490454). This relationship was not robust when the sequences of the 16S rRNA clone and the alpine subnival plant isolate sequences were excluded from the analysis, suggesting that strain E1HC-02T is only distantly related to Frigoribacterium faeni. The phylogenetic analysis, taken together with the information that strain E1HC-02T possesses a protein subunit cell-wall ultrastructure, is unable to grow at psychrophilic temperatures, has a higher optimum pH for growth and has a number of significant chemotaxonomic differences in its cell wall and cell membranes and in its metabolizable substrates (Table 1), clearly differentiates the novel strain from members of the genus Frigoribacterium.
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. Strain E1HC-02T, however, possesses traits that are atypical and have so far not been reported for any of the genera of the family Microbacteriaceae, including the genus Frigoribacterium (Table 1). Strain E1HC-02T contains a high concentration of the monounsaturated fatty acid 18 : 17c (78 %), a fatty acid reported so far only in a few members of the class Alphaproteobacteria (Kim et al., 2006; Martínez-Cánovas et al., 2004), possesses a type B2 cell-wall peptidoglycan, has MK-8 and MK-7 as the major menaquinones and contains the glycolipids disphosphatidylglycerol, phosphatidylglycerol and three unidentified phospholipids. Based on the phylogenetic, chemotaxonomic and phenotypic evidence presented, strain E1HC-02T cannot be placed in any of the 17 genera of the family Microbacteriaceae and hence we propose to create a new genus, Frondicola gen. nov., to accommodate this novel species. Strain E1HC-02T represents the type strain of the type species, Frondicola australicus sp. nov.
Description of Frondicola gen. nov.
Frondicola [Fron.di'co.la. L. fem. n. frons (genitive, frondis) fallen leaves; L. masc. suffix n. -cola from incola inhabitant; N.L. masc. n. Frondicola inhabitant of leaves, leaf dweller].
Cells are aerobic, non-endospore-forming, irregular-shaped rods which stain Gram-positive. No mycelium is produced. A range of carbohydrates, organic compounds and amino acids are metabolized. 16S rRNA gene sequence analysis indicates that the genus is a member of the family Microbacteriaceae within the radiation of the genus Frigoribacterium. The cell-wall peptidoglycan type is B2, the major cellular fatty acid is 18 : 17c and menaquinones MK-7 and MK-8 are present. The type species is Frondicola australicus.
Description of Frondicola australicus sp. nov.
Frondicola australicus (aus.tra'li.cus. N.L. masc. adj. australicus pertaining to Australia, from where the type strain was isolated).
Has the following properties in addition to those given in the description of the genus. Cells are non-motile, non-spore-forming, aerobic, irregular, short rods (0.5–1.0x0.2–0.4 µm) that stain Gram-positive. Cell wall appears to be made of a protein subunit layer and inclusion bodies are present. Growth occurs at temperature between 15 and 37 °C (optimum temperature, 25 °C) and at pH 6.0–9.5 (optimum, pH 9.1). The type strain metabolizes dextrin, Tween 40, Tween 80, maltose, D-melibiose, 
-D-glucose, sucrose, D-trehalose, turanose, succinic acid monomethyl ester, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, L-glutamic acid, L-pyroglutamic acid and urocanic acid. Does not metabolize -cyclodextrin, glycogen, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, myo-inositol, -D-lactose, lactulose, D-mannitol, D-mannose, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, xylitol, pyruvic acid methyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinamic acid, glucuronamide, L-alaninamide, L-alanine, D-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-serine, D-serine, L-threonine, DL-carnitine, -aminobutyric acid, inosine, uridine, thymidine, phenylethylamine, 2-aminoethanol, 2,3-butanediol, glycerol, DL--glycerol phosphate, -D-glucose 1-phosphate or D-glucose 6-phosphate. Catalase-positive and oxidase-negative. The type B2 {Gly} [L-hsr] D-glu–D-Orn peptidoglycan contains the amino acids ornithine, alanine, glycine, homoserine and glutamic acid in a molar ratio of 1.0 : 0.9 : 1.7 : 0.5 : 1.0, respectively. The major cellular fatty acid is 18 : 17c (78 %) and the major menaquinones detected are MK-8 (76 %) and MK-7 (24 %). The glycolipids disphosphatidylglycerol, phosphatidylglycerol and three unidentified phospholipids are present. The DNA base content is 71±1 mol% G+C.
The type strain, strain E1HC-02T (=JCM 13598T=DSM 17894T), was isolated from the decaying leaf litter of a slash pine forest located in southeast Queensland, Australia.
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