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Int J Syst Evol Microbiol 55 (2005), 871-875; DOI  10.1099/ijs.0.63414-0
© 2005 International Union of Microbiological Societies

Agromyces italicus sp. nov., Agromyces humatus sp. nov. and Agromyces lapidis sp. nov., isolated from Roman catacombs

Valme Jurado1, Ingrid Groth2, Juan M. Gonzalez1, Leonila Laiz1, Barbara Schuetze2 and Cesareo Saiz-Jimenez1

1 Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, E-41080 Sevilla, Spain
2 Hans-Knöll-Institute for Natural Products Research, Member of the Leibniz Association, D-07745 Jena, Germany

Correspondence
Cesareo Saiz-Jimenez
saiz{at}irnase.csic.es


   ABSTRACT
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A polyphasic study was carried out to clarify the taxonomic positions of three Gram-positive isolates from the Catacombs of Domitilla, Rome (Italy). 16S rRNA gene sequence comparisons placed these strains within the genus Agromyces. The morphological and chemotaxonomic characteristics of these isolates were consistent with the description of the genus Agromyces. The three isolates could be readily distinguished from one another and from representatives of all Agromyces species with validly published names by a broad range of phenotypic characteristics and DNA–DNA relatedness studies. Therefore, these isolates are proposed to represent three novel species of the genus Agromyces, Agromyces italicus sp. nov. (type strain CD1T=HKI 0325T=DSM 16388T=NCIMB 14011T), Agromyces humatus sp. nov. (type strain CD5T=HKI 0327T=DSM 16389T=NCIMB 14012T) and Agromyces lapidis sp. nov. (type strain CD55T=HKI 0324T=DSM 16390T=NCIMB 14013T).

Published online ahead of print on 5 November 2004 as DOI 10.1099/ijs.0.63414-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains CD1T, CD5T and CD55T are AY618215, AY618216 and AY618217, respectively.


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Strains of the genus Agromyces are common inhabitants of different soils ranging from fertile meadows to deserts [Agromyces ramosus Gledhill and Casida 1969Go, Agromyces cerinus subsp. cerinus Zgurskaya et al. 1992Go, Agromyces cerinus subsp. nitratus Zgurskaya et al. 1992Go, Agromyces fucosus Zgurskaya et al. 1992Go emend. Ortiz-Martinez et al. 2004Go, Agromyces hippuratus (Zgurskaya et al. 1992Go) Ortiz-Martinez et al., 2004Go, Agromyces mediolanus Suzuki et al. 1996Go and Agromyces aurantiacus Li et al. 2003Go]. The rhizosphere of plants harbours a wide diversity of these organisms, as has been shown by Takeuchi & Hatano (2001)Go with the description of three species, Agromyces bracchium, Agromyces luteolus and Agromyces rhizospherae, from mangrove rhizosphere soil. Dorofeeva et al. (2003)Go isolated Agromyces albus from the leaves and inflorescences of members of the Primulaceae, while Agromyces ulmi was isolated from the decayed stump of an elm tree, Ulmus nigra (Rivas et al., 2004Go). Recently, two novel species of this genus, Agromyces salentinus and Agromyces neolithicus, have been isolated from the Grotta dei Cervi, a cave in southern Italy with singular neolithic rock art paintings (Jurado et al., 2005Go).

Strains CD1T and CD5T were obtained from the wall of a tomb located in the Little Apostle cubicle, Domitilla Catacombs, Rome, Italy (Sanchez-Moral et al., 2004Go). Strain CD55T was isolated by touching the stone wall of a tomb (first arcosolium on the left side behind the entrance of Domitilla Catacombs) with a sterile cotton swab and suspending the attached bacteria in 1 : 10 diluted organic medium 79 (OM79) (Prauser & Falta, 1968Go; Jurado et al., 2005Go). The strains were isolated on nutrient agar (Difco) or, in the case of strain CD55T, on peptone/yeast extract/brain heart infusion agar (Yokota et al., 1993Go), using a standard dilution plate procedure.

All the methods used in this study have been recently described by Jurado et al. (2005)Go. Range of pH for growth was established using liquid OM79 medium adjusted to initial pH values between 4 and 11 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. albus VKM Ac-1800T, A. cerinus subsp. cerinus IMET 11525T, A. fucosus IMET 11529T, A. mediolanus VKM Ac-1388T, A. neolithicus DSM 16197T, A. ramosus IMET 11027T and A. salentinus DSM 16198T. Morphological and physiological traits are summarized in the species descriptions and Table 1Go.


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  		Table 1. Characteristics that differentiate strains CD1T, CD5T and CD55T and their closest relatives within the genus Agromyces

Strains: 1, A. italicus sp. nov. CD1T; 2, A. humatus sp. nov. CD5T; 3, A. lapidis sp. nov. CD55T; 4, A. albus VKM Ac-1800T; 5, A. cerinus subsp. cerinus IMET 11525T; 6, A. fucosus IMET 11529T; 7, A. mediolanus VKM Ac-1388T; 8, A. neolithicus DSM 16197T; 9, A. ramosus IMET 11027T; 10, A. salentinus DSM 16198T. Data for A. cerinus subsp. cerinus and A. ramosus were taken from Groth et al. (1996)Go. –, Negative; +, positive; (+) weakly positive; V, variable; ND, not determined. Strains CD1T, CD5T and CD55T share the following properties. They produce acid from starch, arbutin, D-fructose, glycogen, maltose, D-mannose, but not from adonitol, D-arabitol, L-arabitol, dulcitol, erythritol, D-fucose, gluconate, 2-ketogluconate, 5-ketogluconate, {beta}-gentiobiose, inositol, D-lyxose, melibiose, melezitose, sorbitol, L-sorbose; D-tagatose, L-xylose, methyl {beta}-xyloside or xylitol. They produce alkaline phosphatase, esterase (C1), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, {alpha}-glucosidase and {beta}-glucosidase, but not {alpha}-galactosidase, N-acetyl-{beta}-glucosaminidase, lipase (C14), trypsin or {alpha}-fucosidase. They are sensitive to chloramphenicol (30 µg), imipenem (10 µg), ofloxacin (10 µg), oxytetracycline hydrochloride (30 µg), rifampicin (5 µg) and vancomycin hydrochloride (30 µg), but not to kanamycin (30 µg), lincomycin (2 µg), norfloxacin (10 µg) or sulfonamide (200 µg). They hydrolyse aesculin and starch, but not adenine or Tween 80. Voges–Proskauer, methyl red and indole tests give negative results. They are positive for H2S production.

 
Differences referring to the compositions of whole-cell sugars, menaquinones and polar lipids are shown in Table 2Go. Cell-wall amino acids in strains CD1T, CD5T and CD55T were diaminobutyric acid, glycine, glutamic acid and alanine. Acyl type for these three strains was acetyl. The predominant fatty acids of strains CD1T, CD5T and CD55T were anteiso-C15 : 0 (50·9, 41·5 and 48·1 %, respectively), anteiso-C17 : 0 (15·3, 34·0 and 13·0 %), iso-C16 : 0 (14·7, 15·5 and 16·5 %), iso-C15 : 0 (14·2, 5·4 and 10·5 %) and C16 : 0 (1·7, 1·3 and 5·9 %).


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  		Table 2. Chemotaxonomic characteristics of strains CD1T, CD5T and CD55T

Components are listed in decreasing order of abundance.

 
Sequence comparisons of 16S rRNA genes from strains CD1T, CD5T and CD55T showed a close phylogenetic relationship to A. ramosus, A. cerinus subsp. cerinus, A. salentinus, A. neolithicus, A. albus, A. mediolanus and A. fucosus with percentages of similarity ranging from 94 to 98 %. Strains CD1T and CD55T showed high similarity (97 %). The recently described species A. salentinus (Jurado et al., 2005Go) was highly related to strains CD1T and CD55T with 97 % similarity. A. neolithicus was the closest relative to strain CD5T (98 %). A phylogenetic tree generated by the neighbour-joining method showing the relationships between members of the genus Agromyces and the three novel isolates, CD1T, CD5T and CD55T, is shown in Fig. 1Go.



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  		Fig. 1. Neighbour-joining phylogenetic tree showing the relationships between species of the genus Agromyces and isolates CD1T, CD5T and CD55T based on 16S rRNA gene sequences. Bar, 1 % sequence divergence. The outgroup (not shown) was Corynebacterium sp. QSSC3-5 (AF170740).

 
The DNA G+C contents of strains CD1T, CD5T and CD55T were 70·8, 70·6 and 70·4 mol%, respectively. DNA–DNA relatedness studies showed significant differences between the three isolates as well as with their closest phylogenetic neighbours. In all cases, differences in melting temperatures were >7·7 °C (roughly <51 % DNA–DNA relatedness; Rosselló-Mora & Amann, 2001Go), which is well above the 5 °C cut-off point recommended for the delineation of species (Stackebrandt & Goebel, 1994Go).

The genotypic and phenotypic characteristics of strains CD1T, CD5T and CD55T are consistent with their classification in the genus Agromyces (Gledhill & Casida, 1969Go; Zgurskaya et al., 1992Go). The isolates can be readily distinguished from representatives of Agromyces species with validly published names, and from one another, by a broad range of phenotypic properties (Table 1Go) and composition of whole-cell sugars (Table 2Go). DNA–DNA relatedness data support the above distinctions between strains CD1T, CD5T and CD55T and their closest relatives within the genus Agromyces. Based on the results of this polyphasic approach, we propose that the three studied isolates are classified within novel species of the genus Agromyces, Agromyces italicus sp. nov. (strain CD1T), Agromyces humatus sp. nov. (strain CD5T) and Agromyces lapidis sp. nov. (strain CD55T).

Description of Agromyces italicus sp. nov.
Agromyces italicus (i.ta'li.cus. L. masc. adj. italicus from Italy, the origin of the type strain).

Gram-positive, aerobic to microaerophilic actinomycete that forms branching hyphae (width 0·4–0·6 µm) which break up into irregular diphtheroid and rod-like, non-motile fragments. Colonies are circular, convex, smooth and cream. Colony diameter is about 1 mm. Growth occurs between 10 and 37 °C (optimal growth at 28 °C) and at pH 5–9·5. NaCl is tolerated up to 4 % (w/v), but not at 6 % (w/v). Phenotypic characteristics including antibiotic susceptibility and enzymic activities are reported in Table 1Go. Chemotaxonomic characteristics are reported in Table 2Go. Cell-wall amino acids are diaminobutyric acid, glycine, glutamic acid and alanine. Acyl type is acetyl. Predominant fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0; mycolic acids are absent. G+C content is 70·8 mol%.

The type strain is CD1T (=HKI 0325T=DSM 16388T=NCIMB 14011T), isolated from the wall of a tomb located in the Little Apostle cubicle, Domitilla Catacombs, Rome, Italy.

Description of Agromyces humatus sp. nov.
Agromyces humatus (hu.ma'tus. L. masc. part. adj. humatus buried).

Gram-positive, aerobic actinomycete that forms branching hyphae (width 0·3–0·5 µm) which break up into irregular diphtheroid and rod-like, non-motile fragments. Colonies are circular, convex, smooth and yellow. Colony diameter is about 1 mm. Growth occurs between 15 and 37 °C (optimal growth at 28 °C) and at pH 5–9·5. NaCl is tolerated up to 2 % (w/v), but not at 4 % (w/v). Phenotypic characteristics including antibiotic susceptibility and enzymic activities are reported in Table 1Go. Chemotaxonomic characteristics are reported in Table 2Go. Cell-wall amino acids are diaminobutyric acid, glycine, glutamic acid and alanine. Acyl type is acetyl. Predominant fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0; mycolic acids are absent. G+C content is 70·6 mol%.

The type strain is CD5T (=HKI 0327T=DSM 16389T=NCIMB 14012T), isolated from the wall of a tomb located in the Little Apostle cubicle, Domitilla Catacombs, Rome, Italy.

Description of Agromyces lapidis sp. nov.
Agromyces lapidis (la.pi'dis. L. gen. n. lapidis of a stone).

Gram-positive, aerobic to microaerophilic actinomycete that forms branching hyphae (width 0·4–0·6 µm) which break up into irregular diphtheroid and rod-like, non-motile fragments. Colonies are circular, convex, smooth and yellow. Colony diameter is about 1 mm. Growth occurs between 10 and 37 °C (optimal growth at 28 °C) and at pH 5–9·5. NaCl is tolerated up to 4 % (w/v), but not at 6 % (w/v). Phenotypic characteristics including antibiotic susceptibility and enzymic activities are reported in Table 1Go. Chemotaxonomic characteristics are reported in Table 2Go. Cell-wall amino acids are diaminobutyric acid, glycine, glutamic acid and alanine. Acyl type is acetyl. Predominant fatty acids are anteiso-C15 : 0 and iso-C16 : 0; mycolic acids are absent. G+C content is 70·4 mol%.

The type strain is CD55T (=HKI 0324T=DSM 16390T=NCIMB 14013T), isolated from a carved stone wall of the Domitilla Catacombs, Rome, Italy.


   ACKNOWLEDGEMENTS
 
V. J. and L. L. are grateful to fellowships from the Spanish Ministry of Education and Science (MEC) I3P programme and J. M. G. to an MEC contract from the ‘Ramón y Cajal’ programme. This study was supported by project CATS (EVK4-CT2000-00028) and MEC project BTE2002-04492-C02-01. We thank Christiane Weigel, Carmen Schult and Renate Schön for their technical assistance.


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Gledhill, W. E. & Casida, L. E. (1969). Predominant catalase-negative soil bacteria. III. Agromyces, gen. nov., microorganisms intermediary to Actinomyces and Nocardia. Appl Microbiol 18, 340–349.

Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–239.[Abstract/Free Full Text]

Jurado, V., Groth, I., Gonzalez, J. M., Laiz, L. & Saiz-Jimenez, C. (2005). Agromyces salentinus sp. nov. and Agromyces neolithicus sp. nov. Int J Syst Evol Microbiol 55, 153–157.[Abstract/Free Full Text]

Li, W. J., Zhang, L. P., Xu, P., Cui, X. L., Xu, L. H., Zhang, Z., Schumann, P., Stackebrandt, E. & Jiang, C. L. (2003). Agromyces aurantiacus sp. nov., isolated from a Chinese primeval forest. Int J Syst Evol Microbiol 53, 303–307.[Abstract/Free Full Text]

Ortiz-Martinez, A., Gonzalez, J. M., Evtushenko, L. I., Jurado, V., Laiz, L., Groth, I. & Saiz-Jimenez, C. (2004). Reclassification of Agromyces fucosus subsp. hippuratus as Agromyces hippuratus sp. nov., comb. nov. and emended description of Agromyces fucosus. Int J Syst Evol Microbiol 54, 1553–1556.[Abstract/Free Full Text]

Prauser, H. & Falta, R. (1968). Phagensensibilität, Zellwand-Zusammensetzung und Taxonomie von Actinomyceten. Z Allg Mikrobiol 8, 39–46 (in German).[Medline]

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Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. (1996). Agromyces mediolanus sp. nov., nom. rev., comb. nov, a species for "Corynebacterium mediolanum" Mamoli 1939 and for some aniline-assimilating bacteria which contain 2,4-diaminobutyric acid in the cell wall peptidoglycan. Int J Syst Bacteriol 46, 88–93.[Abstract/Free Full Text]

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