Agromyces subbeticus sp. nov., isolated from a cave in southern Spain

An actinomycete, strain Z33^T, was isolated from a cyanobacterial biofilm in the Cave of Bats, near Zuheros (Cordoba, southern Spain). 16S rRNA gene sequence analysis showed that strain Z33^T formed a distinct phyletic line within the genus Agromyces. This isolate could be readily distinguished from representatives of all recognized Agromyces species on the basis of a broad range of phenotypic characteristics and DNADNA relatedness data. Genotypic and phenotypic properties indicate that strain Z33^T represents a novel species, for which the name Agromyces subbeticus sp. nov. is proposed. The type strain is Z33^T (=HKI 0340^T=DSM 16689^T=NCIMB 14025^T).

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Published online ahead of print on 29 April 2005 as DOI 10.1099/ijs.0.63637-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Z33^T is AY737778.

Since the description of the genus Agromyces by Gledhill & Casida (1969) with the type species Agromyces ramosus, the number of recognized species in the genus has increased to 16, and studies have indicated that these are widely distributed in nature and might play a significant role in a variety of ecosystems. Agromyces cerinus subsp. cerinus and Agromyces cerinus subsp. nitratus were described by Zgurskaya et al. (1992), Agromyces mediolanus by Suzuki et al. (1996), Agromyces luteolus, Agromyces rhizospherae and Agromyces bracchium by Takeuchi & Hatano (2001), Agromyces aurantiacus by Li et al. (2003), Agromyces albus by Dorofeeva et al. (2003) and Agromyces ulmi by Rivas et al. (2004). A reclassification of Agromyces fucosus subsp. fucosus and Agromyces fucosus subsp. hippuratus as Agromyces fucosus and Agromyces hippuratus, respectively, was proposed by Ortiz-Martinez et al. (2004). Studies on the diversity and role of Agromyces species in hypogean environments resulted in the description of two novel species, Agromyces salentinus and Agromyces neolithicus, which were isolated from an Italian cave (Jurado et al., 2005a), and three novel species, Agromyces italicus, Agromyces humatus and Agromyces lapidis (Jurado et al., 2005b), originating from Roman catacombs.

The aim of the present study was to determine the taxonomic position of a further cave isolate, strain Z33^T. On the basis of the results presented below, strain Z33^T is considered to represent a novel species of the genus Agromyces.

Strain Z33^T was isolated from a bluegrey cyanobacterial biofilm covering the walls of the Cave of Bats (Zuheros, Cordoba, southern Spain) on PY-BHI medium (Yokota et al., 1993) at 28 °C. Laboratory cultivation of strain Z33^T was performed on medium 79 (OM79) (Prauser & Falta, 1968; Jurado et al., 2005a).

All experimental methods used in this study are as described by Jurado et al. (2005a). The range of pH for growth was established using liquid OM79 medium adjusted to initial pH values of 411 with either 1 M HCl or 20 % (w/v) Na₂CO₃ solution and incubated at 28 °C for up to 10 days.

The following type strains were used as references for comparative studies: A. fucosus IMET 11529^T, A. ramosus IMET 11027^T, A. albus VKM 1800^T, A. cerinus subsp. nitratus IMET 11532^T and A. cerinus subsp. cerinus IMET 11525^T.

Morphological and physiological traits are summarized under the species description below and in Table 1. Chemotaxonomic characteristics are given in Table 2.

Table 1. Characteristics that differentiate strain Z33T from its closest relatives within the genus Agromyces Taxa: 1, Z33T; 2, A. fucosus; 3, A. ramosus; 4, A. albus; 5, A. cerinus subsp. nitratus; 6, A. cerinus subsp. cerinus. , Negative; +, positive; (+), weakly positive; +/, variable; *, delayed; ND, not determined; tr, trace. Data from this study unless indicated.

Table 2. Chemotaxonomic characteristics of strain Z33T Abbreviations: Gal, galactose; Glc, glucose; Man, mannose; Rha, rhamnose; DAB, diaminobutyric acid; DPG, diphosphatidylglycerol; GL, unknown glycolipid; PG, phosphatidylglycerol; PL, unknown phospholipid.

16S rRNA gene sequence analysis showed that strain Z33^T had closest phylogenetic relationships to A. fucosus (97·6 % sequence similarity), A. ramosus (95·3 %), A. albus (95·1 %), A. cerinus subsp. cerinus (95·0 %) and A. cerinus subsp. nitratus (92·1 %). Sequence alignment was performed using the software suite ARB (Ludwig et al., 2004). Alignment was manually edited considering the expected sequence secondary structure. An unrooted phylogenetic tree was constructed by the neighbour-joining method through the ARB suite. The tree topology obtained was reconstructed by quartet-puzzling using the program TREEPUZZLE (Strimmer & von Haeseler, 1996) available within the ARB package. The quartet-puzzling tree represented a consensus tree showing well-supported branching, and was based on 1000 puzzling trials. The reliability value of each internal branch indicates as a percentage how often the corresponding cluster was found. Fig. 1 shows the proposed phylogenetic relationships between the members of the genus Agromyces and strain Z33^T.

(42K): Fig. 1. Phylogenetic tree showing the proposed relationships between the type strains of Agromyces species and the studied strain Z33T. Reliability values are given at branching points as percentages from 1000 trials. Corynebacterium sp. QSSC3-5 (GenBank accession number AF170740) was used as the outgroup (not shown). Strain names and accession numbers (in parentheses) for the species represented in the tree are shown. Bar, 0·01 nucleotide substitutions per site.

The DNA G+C content of strain Z33^T was 71·2 mol%, which is in agreement with the values observed within the genus Agromyces (68·972·8 mol%; Dorofeeva et al., 2003). The degree of DNADNA relatedness between the Agromyces type strains investigated and strain Z33^T was determined by two independent methods: the DNADNA hybridization method described by Ziemke et al. (1998) and by measuring the divergence between the thermal denaturation midpoint of homoduplex DNA and heteroduplex DNA (ΔT_m) as described by Gonzalez & Saiz-Jimenez (2005). These studies revealed significant differences between strain Z33^T and its closest phylogenetic neighbours within the genus Agromyces. Hybridization experiments revealed DNADNA relatedness levels of 66 % and a ΔT_m of 5·8 °C (equivalent to a DNADNA relatedness level of approximately 51 %, as proposed by Rosselló-Mora & Amann, 2001). These values were below the 70 % DNADNA relatedness and above the 5 °C ΔT_m recommended as cut-off points for the delineation of species (Wayne et al., 1987). These results indicate that strain Z33^T shows enough genomic coherence and low enough levels of DNADNA relatedness to its closest relatives to be considered as a novel species (Rosselló-Mora & Amann, 2001; Stackebrandt et al., 2002).

The genotypic and phenotypic characteristics of strain Z33^T are consistent with its classification in the genus Agromyces (Gledhill & Casida, 1969; Zgurskaya et al., 1992).

Strain Z33^T can be readily distinguished from representatives of recognized Agromyces species on the basis of a number of phenotypic properties (Table 1). DNADNA relatedness data support the differentiation between strain Z33^T and its closest relatives within the genus Agromyces. Based on the results of this polyphasic approach, we suggest the studied isolate represents a novel species of the genus Agromyces, for which the name Agromyces subbeticus sp. nov. is proposed.

Description of Agromyces subbeticus sp. nov.
Agromyces subbeticus (sub.be'ti.cus. N.L. masc. adj. subbeticus referring to the Subbetic Mountain Range, southern Spain, where the Cave of Bats is located).

Gram-positive, aerobic and microaerophilic actinomycete. Cells form branching hyphae (0·30·5 µm in width by 2·54·0 µm in length) that break up into irregular rod-shaped and diphtheroid fragments. Colonies are circular, convex, smooth and intense yellow. Colony diameter is about 1 mm. Growth occurs between 6 and 37 °C (optimally at 28 °C) and at pH values between 5 and 9·5. Growth occurs at up to 4 % NaCl. It hydrolyses aesculin and starch, but not adenine or Tween 80. Utilizes acetate, but not benzoate or DL-tartrate. Produces H₂S. Indole, VogesProskauer and methyl red tests are negative. It produces acid from starch, arbutin, aesculin, D-fructose, glycogen and rhamnose, but not from DL-arabitol, dulcitol, 2-ketogluconate, 5-ketogluconate, erythritol, β-gentiobiose, gluconate, inositol, D-lyxose, melezitose, sorbitol, L-sorbose, D-tagatose, D-turanose, L-xylose, methyl β-xyloside or xylitol. It produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and β-glucosidase, but not lipase (C14), trypsin, α-chymotrypsin, β-glucuronidase or α-mannosidase. It is sensitive to chloramphenicol (30 µg), imipenem (10 µg), ofloxacin (10 µg), oxytetracycline hydrochloride (30 µg), rifampicin (5 µg), streptomycin (10 µg) and vancomycin hydrochloride (30 µg), but not to lincomycin (2 µg), methicillin (5 µg), norfloxacin (10 µg), nalidixic acid (30 µg) or sulfonamide (200 µg). Additional phenotypic characteristics are given in Table 1. Whole-cell sugars are rhamnose, glucose, galactose and mannose. Cell-wall amino acids are diaminobutyric acid, glutamic acid, glycine and alanine. Major menaquinones are MK-12 and MK-13. Polar lipids are diphosphatidylglycerol, an unknown phospholipid and two unknown glycolipids. Acyl type is acetyl. Major fatty acids are anteiso-C_{15 : 0} (45·6 %), anteiso-C_{17 : 0} (20·4 %), iso-C_{15 : 0} (16·3 %), iso-C_{16 : 0} (12·4 %), iso-C_{17 : 0} (3·57 %) and C_{16 : 0} (1·01). G+C content of the DNA is 71·2 mol%.

The type strain, Z33^T (=HKI 0340^T=DSM 16689^T=NCIMB 14025^T), was isolated from a bluegrey cyanobacterial biofilm covering the walls of the Cave of Bats, Zuheros, Cordoba, southern Spain.