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1 Vakgroep BFM WE10V, Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
3 BCCM/LMG Bacteria Collection, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
Jeroen Heyrman
Jeroen.Heyrman{at}UGent.be
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ABSTRACT |
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A figure showing phylogenetic positions of representative novel isolates and Arthrobacter species based on neighbour-joining of 16S rRNA gene sequences and details of DNA–DNA relatedness values and fatty acid profiles of the novel taxa are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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. Despite this knowledge, a reliable characterization of bacteria associated with such damage is often lacking (Heyrman & Swings, 2001). To obtain greater insight into the bacterial community involved in biodeterioration of mural paintings, three sites (from the necropolis at Carmona, Spain, and the Saint-Catherine chapel of the castle at Herberstein, Austria) were sampled. In previous studies from these sites, 385 isolates were analysed by fatty acid methyl ester GC (Heyrman et al., 1999) and representatives of different fatty acid clusters were further studied by 16S rRNA gene sequencing (Heyrman & Swings, 2001). Several of these representative strains showed the highest sequence similarity with members of the genus Arthrobacter. This paper presents the results of further investigation of 21 strains belonging to the fatty acid clusters that were attributed to Arthrobacter based on the previous studies. A polyphasic taxonomic examination on these isolates resulted in the description of six novel species. At the time of writing, there are 44 recognized species of the genus Arthrobacter (http://www.bacterio.cict.fr/bacterio/a/arthrobacter.html). Of these established species, three have an uncertain position according to Keddie et al. (1986): Arthrobacter duodecadis, Arthrobacter mysorens and Arthrobacter viscosus.
The 21 strains studied originate from three samples taken at two different mural painting sites (Fig. 1). Two samples were taken at the Servilia tomb of the Roman necropolis at Carmona (Spain), one from the wall (C1) and one from the ceiling (C2) at the right side of the entrance to the tomb. The third sample (H) was a small piece of a rosy biofilm that almost completely covered the north wall of the chancel of the Saint-Catherine chapel in the castle of Herberstein (Austria). The strains were isolated as described by Heyrman et al. (1999). All isolates were further subcultured on nutrient agar (NA) at 28 °C.
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, as described by Heyndrickx et al. (1996). rep-PCR genomic fingerprinting was performed with the REP- and (GTG)5 primers (Versalovic et al., 1994) using the PCR conditions described previously by Rademaker & de Bruijn (1997). Electrophoresis and pattern analysis were further performed as described by Heyrman et al. (2003). 16S rRNA gene sequencing and phylogenetic analysis were performed as described by Heyrman & Swings (2001). Phylogenetic trees were constructed using the BioNumerics software (Applied Maths) by applying the neighbour-joining and maximum-parsimony methods on a multiple alignment similarity matrix. The stability of relationships was assessed by a bootstrap analysis of 1000 datasets. For determination of the G+C content and levels of DNA–DNA hybridization, approximately 1 g biomass was harvested from agar plates. DNA was purified by using a combination of the protocols of Marmur (1961) and Pitcher et al. (1989), as described by Logan et al. (2000). The G+C content of the DNA was determined by HPLC (Mesbah et al., 1989) using the further specifications given by Logan et al. (2000). DNA–DNA hybridization experiments were performed using a modification of the microplate method of Ezaki et al. (1989), as described by Willems et al. (2001). A hybridization temperature of 40 °C was used.
REP- and (GTG)5-PCR were used to determine the genotypic diversity of the isolates and to select strains for further analyses. Based on the combined REP and (GTG)5 electrophoretic patterns, a dendrogram using Pearson's correlation was constructed (Fig. 1
). At more than 75 % similarity, all strains except one (LMG 22283T) could be organized into five groups. The clustering corresponds well with the origin of the strains, with groups 1–3 originating from Carmona sample 1, group 4 from sample 2, and group 5 and ungrouped strain LMG 22283T originating from Herberstein.
Based on the rep-clustering, one representative of each group and the ungrouped strain LMG 22283T were analysed for their nearly complete 16S rRNA gene sequence. For the larger groups (more than two strains), the partial (first ±400 bp) or nearly complete 16S rRNA gene sequence of one additional strain was analysed. Strain pairs LMG 19502T (1484 bp) and R-5514 (381 bp) of rep-group 1 and LMG 19501T (1475 bp) and R-5363 (428 bp) of rep-group 2 showed 100 % partial sequence similarity. In rep-group 4, LMG 22282T (1476 bp) and LMG 22285 (1480 bp) shared 99·9 % 16S rRNA gene sequence similarity. These results confirmed the accuracy of the rep-clustering and the delineation of the groups. In Fig. 2, the nearly complete 16S rRNA gene sequences of representatives of each rep-group and of strain LMG 22283T are clustered together with those of closely related Arthrobacter species. Supplementary Fig. S1 (available in IJSEM Online) shows the phylogenetic position of the novel species within the genus Arthrobacter. Because no 16S rRNA gene sequence was published for A. viscosus, the type strain of this species was also sequenced. In a FASTA search (Pearson & Lipman, 1988), the 16S rRNA gene sequence of LMG 19502T (rep-group 1) was most closely related to Arthrobacter luteolus (96·8 % similarity) and Arthrobacter citreus (96·3 %). Strains LMG 22281T (rep-group 2) and LMG 19501T (rep-group 3) both showed highest similarity to Arthrobacter ramosus and Arthrobacter pascens (both 97·1 % similarity), and LMG 22282T (rep-group 4) was most closely related to Arthrobacter globiformis (97·3 %) and A. pascens (97·1 %). Ungrouped strain LMG 22283T and one representative of rep-group 5, strain LMG 22284T, were most similar to Arthrobacter cumminsii (95·6 and 95·4 %, respectively) and Arthrobacter albus (95·4 and 95·2 %, respectively). LMG 22281T, LMG 19501T and LMG 22282T grouped closest to Arthrobacter agilis (pairwise similarity of 97·7–98·0 %), although this species was not mentioned in the list of the 30 most closely related sequences obtained by using a FASTA search. The pairwise similarity between the sequenced mural painting isolates is above 97 % for LMG 22281T, LMG 19501T and LMG 22282T (between 98·7 and 99·5 %) and for LMG 22283T and LMG 22284T (98·1 %).
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) have indeed been incorrectly assigned to the genus Arthrobacter. For Arthrobacter duodecadis, a 16S rRNA gene sequence is available (AB072496) that places it phylogenetically in the neighbourhood of the genus Tetrasphaera. From the sequence data it can be hypothesized that A. duodecadis is a possible novel species within Tetrasphaera (16S rRNA gene sequence similarity value below 97 % to all recognized species of Tetrasphaera). However, conflicting data have been reported for A. duodecadis (e.g. on the cell wall peptidoglycan; Keddie et al., 1986) in the literature and these data should be re-evaluated before the reclassification can be taken into consideration. A. viscosus, for which no 16S rRNA gene sequence was previously available, is most closely related to members of the genus Rhizobium, e.g. Rhizobium gallicum (98·1 % 16S rRNA gene sequence similarity), Rhizobium mongolense (98·0 %) and Rhizobium leguminosarum (97·6 %). This is not unexpected, as Keddie et al. (1986) stated that the chemotaxonomic data for this species were in accordance with the view that it is indeed a Gram-negative rod. A. mysorens groups closely with Arthrobacter nicotianae (approx. 99·1 % 16S rRNA gene sequence similarity). This supports the data of Stackebrandt et al. (1983), who considered A. mysorens as forming a distinct species within the A. nicotianae group. They reported a DNA–DNA relatedness level between A. mysorens ATCC 31021 and A. nicotianae DSM 20123T of 40 %. As no DNA–DNA relatedness study and chemotaxonomic analyses were performed with the type strain, it remains unclear whether A. mysorens deserves separate species status.
Generally recommended and accepted criteria for delineating bacterial species state that strains with DNA–DNA relatedness below 70 % as measured by hybridization or with 16S rRNA gene sequence dissimilarity above 3 % are considered as belonging to separate species (Wayne et al., 1987; Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002). For LMG 19502T, representative of rep-group 1, 16S rRNA gene sequence similarity with all recognized Arthrobacter species was below 97 %. Consequently, the members of rep-group 1 can be attributed to a novel genospecies and this group will be further denoted as Arthrobacter monumenti sp. nov. For the remaining strains, two DNA–DNA relatedness experiments were performed. The first experiment included LMG 22281T (rep-group 2), LMG 19501T (rep-group 3), LMG 22282T (rep-group 4) and the type strains of A. agilis, A. globiformis, A. pascens and A. ramosus (Supplementary Table S1 available in IJSEM Online). Results showed that the three rep-groups all represent novel genospecies. Rep-group 2 will be further denoted as Arthrobacter parietis sp. nov., group 3 as Arthrobacter tumbae sp. nov. and group 4 as Arthrobacter tecti sp. nov. The second DNA–DNA relatedness experiment included ungrouped strain LMG 22283T and one representative of rep-group 5, strain LMG 22284T. The level of DNA–DNA relatedness between these strains was 26 %, which suggests that they belong to different genospecies. LMG 22283T will be further denoted as Arthrobacter castelli sp. nov. and rep-group 5 as Arthrobacter pigmenti sp. nov.
The G+C content of the DNA of the type strains of the novel species (Table 1) falls within the range for the genus Arthrobacter (59–70 mol%) reported by Keddie et al. (1986).
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, starting from cells grown on trypticase soy agar (TSA) or marine agar (MA). Purified cell wall preparations were obtained by the method of Schleifer & Kandler (1972). The amino acids and peptides in peptidoglycan hydrolysates were analysed as described by Groth et al. (1999). Menaquinones were extracted as described by Collins et al. (1977) and were analysed by HPLC (Groth et al., 1997). Polar lipids extracted by the method of Minnikin et al. (1979) were identified by two-dimensional TLC and spraying with specific reagents (Collins & Jones, 1980). Sugar analysis of purified cell walls was performed with the chemical method as described by Staneck & Roberts (1974). Cell morphology and Gram staining were examined by phase-contrast and normal light microscopy, respectively. Catalase and oxidase tests were performed according to the methods described by Smibert & Krieg (1994). Optimal growth temperature was tested on NA (incubation at 4, 10, 15, 20, 28, 37, 45 and 50 °C); growth at various pH and salt range was tested in nutrient broth (NB) (with 0, 5, 10 and 15 % salt added and pH adjusted to 4·0–10·0 at 0·5 intervals). Anaerobic growth was recorded in an anaerobic chamber on NA. API ZYM and API CORYNE strips (bioMérieux) were used according to the manufacturer's instructions.
The novel species described here have a type A3
peptidoglycan, have dihydrogenated menaquinones with nine isoprene units [MK-9(H2)] as the major menaquinone, and contain the polar lipids diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. They share these features with most of the recognized Arthrobacter species (Keddie et al., 1986). The novel Arthrobacter species can be distinguished on the basis of peptidoglycan type, relative amounts of menaquinones and cell wall sugars (Table 1).
The fatty acid compositions of the novel species (Supplementary Table S2 available in IJSEM Online) are in good agreement with the genus description (Keddie et al., 1986): they contain major amounts of iso- and anteiso-methyl-branched acids (iso-C15 : 0 and anteiso-C15 : 0) together with small amounts of straight-chain saturated acids (C16 : 0); monounsaturated acids are absent or present only in trace amounts.
Results of the physiological characterization are given in the species descriptions below. Characters that distinguish the novel species are listed in Table 1. The phenotypic analyses seem to support the division suggested by the genomic data. Together, these data support the delineation of six novel Arthrobacter species.
Description of Arthrobacter castelli sp. nov.
Arthrobacter castelli (cas.tel'li. L. gen. n. castelli of the castle).
Cells are Gram-positive, short rods and cocci (diameter 0·8–1 µm) occurring in pairs and chains. They are non-motile and do not form endospores. Colonies on NA after 48 h are small (<1 mm), light yellow, round with entire margins, of low convexity, opaque and smooth. No growth in an anaerobic chamber on NA. Optimal temperature for growth is 22–37 °C. Weak growth at 15 °C and no growth at 10 or 45 °C. Poor growth on media without NaCl. Catalase-positive and oxidase-negative. By using the API CORYNE system, positive reactions are observed for pyrazinamidase, alkaline phosphatase, 
-glucuronidase, -galactosidase, -glucosidase and urease. Negative reactions are obtained for nitrate reduction, pyrrolidonyl arylamidase, N-acetyl--glucosaminidase, aesculin (-glucosidase), gelatinase and fermentation of glucose, ribose, xylose, mannitol, maltose, lactose, sucrose and glycogen. By using the API ZYM system, activity is detected for alkaline phosphatase, acid phosphatase (weak), esterase C4 (weak), esterase lipase C8, lipase C14 (weak), leucine arylamidase, trypsin (weak), phosphoamidase (weak) and -glucosidase. No activity is detected for valine arylamidase, cystine arylamidase, chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase or -fucosidase. Predominant fatty acids are anteiso-C15 : 0 and iso-C15 : 0 (approximately 51 and 23 %, respectively). Peptidoglycan type, menaquinone content and cell wall sugars are given in Table 1. Polar lipids are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and one unknown glycolipid.
The G+C content of the DNA, determined for the type strain LMG 22283T (=DSM 16402T), is 68·1 mol%. Isolated from a rosy biofilm overgrowing a mural painting in the Saint-Catherine chapel (castle of Herberstein, Austria).
Description of Arthrobacter monumenti sp. nov.
Arthrobacter monumenti (mo.nu.men'ti. L. gen. n. monumenti of the monument).
Cells are Gram-positive, short rods and cocci (diameter 0·8–1 µm) occurring in pairs or clusters. They are non-motile and do not form endospores. Colonies on NA after 48 h are small (<1 mm), light yellow, round with entire margins, of low convexity, opaque and smooth. No growth in an anaerobic chamber on NA. Optimal temperature for growth is 22–30 °C. Weak growth at 4 and 37 °C, and no growth at 45 °C. Growth on medium with 15 % NaCl. Optimum pH for growth is 7–8. Catalase-positive and oxidase-negative. Using the API CORYNE system, positive reactions are observed for nitrate reduction, pyrazinamidase, -glucuronidase, -galactosidase, -glucosidase, aesculin (-glucosidase) and gelatinase. Negative reactions are obtained for N-acetyl--glucosaminidase and fermentation of ribose, xylose, mannitol and glycogen. Variable reactions are obtained for pyrrolidonyl arylamidase, urease and fermentation of glucose, lactose, maltose and sucrose. Using the API ZYM system, activity is detected for alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, trypsin, acid phosphatase (weak) and phosphoamidase (weak). No activity is detected for lipase C14, valine arylamidase, cystine arylamidase, chymotrypsin, -galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase or -fucosidase. Variable reactions are obtained for -galactosidase, -glucosidase and -mannosidase. Predominant fatty acids are anteiso-C15 : 0 and iso-C15 : 0 (approximately 57 and 25 %, respectively). Peptidoglycan type, menaquinone content and cell wall sugars are given in Table 1. Polar lipids of the type strain are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and one unknown glycolipid.
The G+C content of the DNA, determined for the type strain LMG 19502T (=DSM 16405T), is 62·2 mol%. In the variable characters listed above for the API CORYNE tests, the type strain is positive for pyrrolidonyl arylamidase but negative for urease and fermentation of glucose, lactose, maltose and sucrose. In API ZYM the type strain is positive for -galactosidase and -glucosidase (weak) but negative for -mannosidase. Isolated from a biofilm overgrowing a mural painting in the Servilia tomb (Roman necropolis of Carmona, Spain).
Description of Arthrobacter parietis sp. nov.
Arthrobacter parietis (pa.ri.et'is. L. gen. n. parietis of a wall).
Cells are Gram-positive, short rods and cocci (diameter 0·8–1 µm) occurring in pairs or clusters. They are non-motile and do not form endospores. Colonies on NA after 48 h are 1–2 mm in diameter, yellow–orange, round with entire margins, of low convexity, opaque and smooth. No growth in an anaerobic chamber on NA. Optimal temperature for growth is 22–30 °C. No or only weak growth at 37 °C and no growth at 45 °C. Good growth after 1 week at 4 °C. Growth on medium with 15 % NaCl. Growth occurs at pH 6–9, with an optimum of 7–8. Catalase-positive and oxidase-negative. Using the API CORYNE system, positive reactions are observed for nitrate reduction, pyrazinamidase, -galactosidase, -glucosidase, aesculin (-glucosidase), gelatinase and fermentation of glucose. Negative reactions are obtained for alkaline phosphatase, -glucuronidase, N-acetyl--glucosaminidase and fermentation of ribose, xylose, mannitol and glycogen. Variable reactions are obtained for pyrrolidonyl arylamidase, urease, and fermentation of maltose, lactose and sucrose. Using the API ZYM system, activity is detected for acid phosphatase (weak), esterase C4, leucine arylamidase, trypsin, phosphoamidase (weak), -galactosidase, -galactosidase and -glucosidase. No activity is detected for alkaline phosphatase, esterase lipase C8, lipase C14, valine arylamidase, chymotrypsin, -glucuronidase, N-acetyl--glucosaminidase and -fucosidase. Variable results (if positive, weak) were obtained for cystine arylamidase, -glucosidase and -mannosidase. Predominant fatty acids are anteiso-C15 : 0 and iso-C15 : 0 (approximately 51 and 29 %, respectively). Peptidoglycan type, menaquinone content and cell wall sugars are given in Table 1. Polar lipids of the type strain are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and two unknown glycolipids.
The G+C content of the DNA, determined for the type strain LMG 22281T (=DSM 16404T), is 63·8 mol%. The type strain is negative for the variable characters listed above. Isolated from a biofilm overgrowing a mural painting in the Servilia tomb (Roman necropolis of Carmona, Spain).
Description of Arthrobacter pigmenti sp. nov.
Arthrobacter pigmenti (pig.men'ti. L. gen. n. pigmenti of pigment or paint).
Cells are Gram-positive, short rods and cocci (diameter 0·8–1 µm) occurring in pairs, chains or clusters. They are non-motile and do not form endospores. Colonies on NA after 48 h are small (<1 mm), light yellow, round with entire margins, of low convexity, opaque and smooth. No growth in an anaerobic chamber on NA. Optimum temperature for growth is 22–30 °C. No or only weak growth at 37 or 10 °C. No growth at 45 or 4 °C. Growth on medium with 15 % NaCl; optimal growth at 10 % NaCl. Growth at pH 7–10; optimal at pH 8–9. Catalase-positive and oxidase-negative. Using the API CORYNE system, positive reactions are observed for pyrazinamidase, -glucuronidase, -galactosidase, -glucosidase and gelatinase. Negative reactions are obtained for pyrrolidonyl arylamidase, N-acetyl--glucosaminidase, urease, and fermentation of ribose, xylose, mannitol, lactose and glycogen. Variable reactions are obtained for nitrate reduction, aesculin (-glucosidase) and fermentation of glucose, maltose and sucrose. Using the API ZYM system, activity is detected for alkaline phosphatase, esterase lipase C8 (weak), leucine arylamidase, trypsin, phosphoamidase (weak), -galactosidase (weak) and -glucuronidase. No activity is detected for lipase C14, valine arylamidase, cystine arylamidase, chymotrypsin, -galactosidase, -glucosidase, N-acetyl--glucosaminidase or -fucosidase. Variable results (if positive, weak) were obtained for esterase C4, acid phosphatase and -mannosidase. Predominant fatty acids are iso-C15 : 0 and anteiso-C15 : 0 (approximately 48 and 40 %, respectively). Peptidoglycan type, menaquinone content and cell wall sugars are given in Table 1. Polar lipids of the type strain are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and one unknown glycolipid.
The G+C content of the DNA, determined for the type strain LMG 22284T (=DSM 16403T), is 61·6 mol%. In the variable characters listed above, the type strain is positive. Isolated from a rosy biofilm overgrowing a mural painting in the Saint-Catherine chapel (castle of Herberstein, Austria).
Description of Arthrobacter tecti sp. nov.
Arthrobacter tecti (tec'ti. L. gen. n. tecti from the ceiling).
Cells are Gram-positive, short rods and cocci (diameter 0·8–1 µm) occurring in pairs or clusters. They are non-motile and do not form endospores. Colonies on NA after 48 h are small (<1 mm), yellow, round with entire margins, of low convexity, opaque and smooth. No growth in an anaerobic chamber on NA. Optimum temperature for growth is 22–30 °C. Growth on medium with 15 % NaCl added. Growth at pH 6–9; optimal growth at pH 8. Catalase-positive and oxidase-negative. Using the API CORYNE system, a positive reaction is observed for N-acetyl--glucosaminidase. Most strains, including the type strain, test positive for gelatinase but negative for pyrrolidonyl arylamidase and fermentation of ribose and mannitol. Negative reactions are obtained for nitrate reduction, -glucuronidase, urease, and fermentation of ribose, xylose, maltose, lactose and glycogen. Variable reactions are obtained for pyrazinamidase, alkaline phosphatase, -galactosidase, -glucosidase, aesculin (-glucosidase), and fermentation of glucose and sucrose. Using the API ZYM system, activity is detected for alkaline phosphatase (weak), acid phosphatase (weak), leucine arylamidase and phosphoamidase (weak). Most strains test positive for esterase C4, esterase lipase C8, trypsin (negative for the type strain), N-acetyl--glucosaminidase (negative for the type strain) and -mannosidase (negative for the type strain). No activity is detected for lipase C14, valine arylamidase, cystine arylamidase, -galactosidase, -glucuronidase or -fucosidase. Variable reactions are obtained for chymotrypsin, -galactosidase, -glucosidase and -glucosidase. Predominant fatty acids are anteiso-C15 : 0 and iso-C15 : 0 (approximately 44 and 37 %, respectively). Peptidoglycan type, menaquinone content and cell wall sugars are given in Table 1. Polar lipids of the type strain are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and one unknown phospholipid.
The G+C content of the DNA, determined for the type strain LMG 22282T (=DSM 16407T), is 63·7 mol%. The type strain is negative for all variable reactions in the API CORYNE system listed above. The type strain is positive for chymotrypsin and -glucosidase, and negative for -galactosidase and -glucosidase in the API ZYM system. Isolated from a biofilm overgrowing the ceiling of the main room of the Servilia tomb (Roman necropolis of Carmona, Spain).
Description of Arthrobacter tumbae sp. nov.
Arthrobacter tumbae (tum'bae. L. gen. n. tumbae of the tomb).
Cells are Gram-positive, short rods and cocci (diameter 0·8–1 µm) occurring singly, in pairs or in clusters. They are non-motile and do not form endospores. Colonies on NA after 48 h are small (<1 mm), yellow–orange, round with entire margins, of low convexity, opaque and smooth. No growth in an anaerobic chamber on NA. Optimum temperature for growth is 22–30 °C. Weak growth at 37 °C, and no growth at 52 °C. Growth at 4 °C after 1 week of incubation. Growth on medium with 10 % NaCl, but not with 15 % NaCl. Catalase-positive and oxidase-negative. Alkaliphilic; pH range for growth of 7–10 with an optimum of 8–9. Using the API CORYNE system, positive reactions are observed for pyrazinamidase, -glucuronidase, -galactosidase and -glucosidase. Most strains, including the type strain, test positive for gelatinase. Negative reactions are obtained for N-acetyl--glucosaminidase and fermentation of ribose, xylose, mannitol and glycogen. Variable reactions are obtained for nitrate reduction, pyrrolidonyl arylamidase, alkaline phosphatase, aesculin (-glucosidase), urease, and fermentation of glucose, maltose, lactose and sucrose. Using the API ZYM system, activity is detected for esterase C4, leucine arylamidase and phosphoamidase (weak). Most strains, including the type strain, test positive for esterase lipase C8 and negative for alkaline phosphatase, valine arylamidase, -galactosidase and -galactosidase. No activity is detected for lipase C14, cystine arylamidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase or -fucosidase. Variable reactions are obtained for trypsin, chymotrypsin, acid phosphatase, -glucosidase and -mannosidase. Predominant fatty acids are anteiso-C15 : 0 and iso-C15 : 0 (approximately 57 and 17 %, respectively). Peptidoglycan type, menaquinone content and cell wall sugars are given in Table 1. Polar lipids of the type strain are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and one unknown phospholipid.
The G+C content of the DNA, determined for the type strain LMG 19501T (=DSM 16406T), is 64·7 mol%. The type strain is negative for all variable characters listed above, except for trypsin in the API ZYM system. Isolated from a biofilm overgrowing a mural painting in the Servilia tomb (Roman necropolis of Carmona, Spain).
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ACKNOWLEDGEMENTS |
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80 % positive (6/7 or 4/5 strains); V, variable; (–), 
20 % positive; –, all strains negative; W, all positive reactions are weak; NG, not given. Data for A. agilis and A. flavus are from Koch et al. (1995)