Microcella putealis gen. nov., sp. nov., a Gram-positive alkaliphilic bacterium isolated from a nonsaline alkaline groundwater

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Systematic and Applied Microbiology 28 (2005) 479–487 www.elsevier.de/syapm

Microcella putealis gen. nov., sp. nov., a Gram-positive alkaliphilic bacterium isolated from a nonsaline alkaline groundwater Igor Tiagoa, Carlos Piresa, Vı´ tor Mendesa, Paula V. Moraisb, Milton da Costac, Anto´nio Verı´ ssimoa, a

Departamento de Zoologia and Centro de Neurocieˆncias de Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal b Departamento de Bioquı´mica and Instituto do Ambiente e Vida Universidade de Coimbra, 3004-517 Coimbra, Portugal c Departamento de Bioquı´mica and Centro de Neurocieˆncias de Biologia Celular Universidade de Coimbra, 3004-517 Coimbra, Portugal Received 3 March 2005

Abstract Three Gram-positive bacteria designated CV-2T, CV-40 and AC-30 were isolated from a highly alkaline groundwater environment (pH 11.4). These organisms formed very small rod-shaped cells, are aerobic, non-sporeforming, catalase-positive, oxidase-negative, with an optimum growth temperature of 35 1C and optimum pH value of growth between 8.5 and 9.0. The strains possessed a novel B-type cell-wall peptidoglycan structure with lysine as the diamino acid; the major respiratory quinones were menaquinone 12 (MK12) and MK13. The G+C content of DNA was between 67.1 and 70.7 mol%. The phylogenetic analyses of the sequences of the 16S rRNA genes reveled that they formed a deep branch within the family Microbacteriaceae, with the highest similarity of
95.6% with members of the genera Agreia, Agrococcus, Cryobacterium, Clavibacter, Frigoribacterium, Leifsonia, Mycetocola, Rhodoglobus, Salinibacterium and Subtercola. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strains CV-2T, CV-40 and AC-30, represent a new species of a novel genus within the family Microbacteriaceae for which we propose the name Microcella putealis gen. nov., sp. nov. r 2005 Elsevier GmbH. All rights reserved. Keywords: Microbacteriaceae; Microcella; Microcella putealis; Alkaline groundwater

Introduction The family Microbacteriacea accommodates species of the following genera of Actinobacteria (high G+C Gram positive bacteria): Agromyces, Clavibacter, Curtobacterium, and Microbacterium [21], Agrococcus and Corresponding author. Tel.: 351 239824024; fax: 351 239826798.

E-mail address: [email protected] (A. Verı´ ssimo). 0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.03.004

Rathayibacter [28], Leucobacter [30], Cryobacter [29], Frigoribacterium [14], Subtercola [18], Leifsonia [5], Agreia [3], Mycetocola [34], Okibacterium [4], Plantibacter [1] , Rhodoglobus [26], Salinibacterium [9], Gulosibacter and Pseudoclavibacter [17], and Zimmermanella [16]. These organisms have B-type peptidoglycan structure [10,24] with lysine, ornithine, diaminobutyric acid (DAB), and more rarely g-aminobutyric acid (GABA) as the diamino acid. The respiratory quinones

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are fully saturated menaquinones with 8–14 isoprene units and the fatty acids are saturated straight- or branched-chain. Microbacteria are widespread in natural and artificial environments, such as plants, soil, dairy products, sewage, mushrooms, insects, and fresh and marine water. To our knowledge microbacteria have not been isolated from alkaline environments. Natural alkaline environments are uncommon geological features. Soda lakes and soda deserts are the most studied and represent the most stable naturally occurring alkaline environments on earth, with pH values generally higher than 10 [12]. Alkalinity of these environments is generated by the accumulation of sodium carbonate. However, in most of the known sites high concentrations of NaCl and other salts are also present giving rise to saline, and sometimes, hypersaline environments [12]. Nonsaline alkaline environments are much rarer. The genesis of these peculiar environments depends on complex geological formations that probably lead, in all cases to a single geochemical process known as serpentinization [32]. The alkalinity of these springs is generated by high levels of Ca(OH)2 and maintained, around pH 11, due to an equilibrium between the solid phase Ca(OH)2 and the soluble Ca2+ and OH. An artesian groundwater at Cabec¸o de Vide, in Southern Portugal, is generated in a similar geological context that suggests serpentinization. This groundwater has a high alkalinity (pH 11.4) with an extremely low ionic concentration where Ca2+ and OH are major chemical constituents [32]. During a survey of the bacterial diversity of the nonsaline alkaline groundwater of Cabec¸o de Vide in Southern Portugal, we found that the major population recovered was phylogenetically related to the genera of the family Microbacteriaceae. In this study, we describe the morphological, physiological, chemotaxonomic and phylogenetic characteristics of these organisms. On the basis of our results we propose that the alkaliphilic strains CV-2T, CV-40 and AC-30 represent a new species of a novel genus of the family Microbacteriaceae for which we propose the name Microcella putealis gen. nov., sp. nov. (CV-2T ¼ LMG 22692T ¼ CIP 108471T; CV-40 ¼ LMG 22691 ¼ CIP 108474; AC-30 ¼ LMG 22689 ¼ CIP 108472).

Materials and methods Bacterial strains and culture conditions Strains CV-2T, CV-40, and AC-30 were isolated as described previously and were routinely cultured in Alkaline Buffered Medium 2 (ABM2), adjusted to pH 9.0, at 35 1C, and maintained at 70 1C in the same

medium supplemented with 15% glycerol [32]. ABM2 contained the following components per liter of media: yeast extract (Difco), 5.0 g; triptone (Difco), 5.0 g; aketoglutaric acid monopotassium salt (Sigma), 1.0 g; agar (Difco), 15.0 g; 100 ml of a macronutrients solution 10  concentrated; 10 ml of a micronutrients solution 100  concentrated; and 100 ml of a specific buffer solution (listed below) at a concentration of 1 M, autoclaved separately and mixed after cooling. The 10  concentrated macronutrients solution contained per liter: nitrilotriacetic acid, 1.0 g; CaSO4  2H2O, 0.6 g; MgSO4  7H2O, 1.0 g; NaCl, 0.8 g; KNO3, 1.03 g; NaNO3, 6.89 g; NaHPO4, 1.11 g. The 100  concentrated micronutrients solution contained per liter: MnSO4.H2O, 0.22 g; ZnSO4  7H2O, 0.05 g; H3BO3, 0.05 g; CuSO4  5H2O, 0.0025 g; Na2MoO4  2H2O, 0.0025 g; CoCl2  6H2O, 0.0046 g. The following buffer solutions, prepared according to Gomory [7], were used to adjust the media to different pH values: citrate buffer was used to adjust the pH to 6.5, phosphate buffer was used to adjust the pH to 7.0 and 7.5, tris-(hydroxymethyl) aminomethane–Tris buffer was used to adjust pH between 8.0 and 8.5, carbonate–bicarbonate buffer was used to adjust pH between 9.0 and 10.0 and carbonate–KOH was used to for pH values higher than 10.0. The pH value of each lot of solid medium was verified prior to using a surfacetesting Sentixs Sur pH electrode (WTW, Hoskin Scientific).

Morphological, physiological and biochemical tests Cell morphology and motility were examined by phase-contrast microscopy. Gram reaction, the presence of cytochrome oxidase and catalase were determined as described by Smibert and Krieg [27]. All tests were performed after 24 h of incubation at 35 1C on ABM2, pH 9.0. The pH range for growth of strains CV-2T, CV-40 and AC-30 was examined by determining the turbidity (610 nm) of cultures incubated in 300 ml Erlenmeyer flasks containing 100 ml ABM2, between pH 6.5 and 11.0, in a reciprocal water bath (170 rpm) at 35 1C. The growth temperature range of these strains was examined in liquid medium at pH 7.5, 9.0 and 10.0 between 10 and 45 1C using the buffers mentioned above. Growth in the presence of NaCl (w/v) ranging up to 9.0%, was examined in liquid medium at pH value 9.0 and 35 1C. Single carbon source assimilation was determined using API 50 CH test strips (Analytab Products Inc., Biomerieux, France), using 0.1 M carbonate–bicarbonate buffer pH 9.0 supplemented with 0.3% (w/v) agar (Difco), 0.05% NH4Cl2 (Merck), the macronutrients solution and the micronutrients solution (described

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above). Microorganisms were ressuspended in sterilized water with a turbidity corresponding to the McFarland No. 5 and 6 standard [27]. The cell suspensions (3 ml) were added to 60 ml of the medium and added to the API 50 CH test strip wells, as recommended by the manufacturer. Results were recorded after 24 h, 48 h and 5 days incubation at 35 1C. Nitrate reduction, indole production and the presence of b-galactosidase were determined using the API 20NE system with the minimal medium described above. Results were recorded after 24, 48 and 72 h of incubation at 35 1C. The hydrolysis of arginine, elastin, arbutin, gelatin, casein, esculin, starch and hippurate the presence of urease, xylanase, DNAse and b-galactosidase, and the reduction of nitrate was assessed on ABM2 agar as described by Hudson et al. [11] and Smibert and Krieg [27] up to 6 days.

Cell wall peptidoglycan type Purified cell wall preparations were obtained by the method of Schleifer and Kandler [25]. The amino acids and peptides in cell wall hydrolysates were analyzed by two-dimensional ascending thin-layer chromatography on cellulose plates using previously described systems [25]. The amino terminal amino acid of the interpeptide bridge was determined by dinitrophenylation as described by Schleifer [24].

Lipoquinones and fatty acid composition Lipoquinones were extracted from 300 mg of freeze dried cells, purified by thin layer chromatography and separated with a Gilson high-performance liquid chromatography apparatus, using a reverse phase column (RP18, Spherisorb, S5 ODS2) with methanol/heptane (10:2, vol/vol) as the mobile phase and were detected at 269 nm [33]. Cultures for fatty acid analysis were grown on ABM2 plates adjusted at pH 9.0, in sealed plastic bags submerged in a water bath at 28 1C for 24 h. Fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass by saponification, methylation and extraction as described previously, and separated, identified and quantified with the standard MIS Library Generation Software (Microbial ID Inc., Newark, Delaware, USA) as described by the manufacturer.

Antibiotic sensitivity Antibiotic susceptibility was examined with disks (BioMe´rieux) containing amoxicillin (25 mg), virginiamicin (15 mg), ceftriaxon (30 mg), cephalothin (30 mg), ceftazidin (30 mg), chloramphenicol (30 mg), colistin (50 mg), doxycycline (30 mg), gentamicin (10 mg), kanamycin (30 mg), lincomycin (2 mg), nalidixic acid (30 mg),

481

ofloxacin (5 mg), penicillin G (10 U/IE), piperacilin (100 mg), polymyxin B (300 U/IE), streptomycin (10 mg), tetracycline (30 mg) on ABM2 (pH 9.0) at 35 1C for 72 h.

Determination of G+C content of DNA and 16S rRNA gene sequence determination and phylogenetic analyses The DNA for the determination of the G+C content was isolated as described by Nielsen et al. [20]. The G+C content of DNA was determined by highperformance liquid chromatography as described by Mesbah et al. [19]. The extraction of genomic DNA for 16S rRNA gene sequence determination, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR products were carried out as described by Rainey et al. [22]. Purified reactions mixtures were electrophoresed using a model 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). The quality of 16S rRNA gene sequences was checked manually using Bioedit editor [8] and aligned against representative reference sequences of the most closely related members, obtained from the Ribossomal Database Project [2] and EMBL, using the multiple-alignment CLUSTAL X software package [31]. The method of Jukes and Cantor [13] was used to calculate evolutionary distances; phylogenetic dendrograms were constructed using the neighbor-joining method [23], and trees topologies were evaluated by performing bootstrap analysis [6] of 1000 data sets by using the MEGA2 package [15].

Results Isolation, morphological and biochemical characteristics of strains CV-2T, CV-40 and AC-30 Several bacteria were isolated by Tiago et al. [32] from the artesian borehole feeding the spa at Cabec¸o de Vide in Southern Portugal during a prokaryotic diversity study. The borehole water had a temperature of 20.5 1C and a pH of 11.4. Strains CV-2T, CV-40 and AC-30 were chosen from 54 isolates constituting the major population on the basis of preliminary grouping by fatty acid analysis, RAPD typing and partial 16S rRNA gene sequence analysis [32]. These organisms formed yellowpigmented colonies, were Gram positive with small rodshaped cells (0.4 mm in width and 0.8–1.6 mm in length) and aerobic. Strains CV-2T, CV-40 and AC-30 formed colonies that were 0.5 mm, smooth and convex. The Cabec¸o de Vide strains had an optimum growth temperature of about 35 1C, and did not grow at 10 1C or 45 1C (Fig. 1b). The optimum pH for growth of strain CV-2T, CV-40 and AC-30 was between 8.5 and 9.0, no

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Specific growth rate (h-1)

(a)

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amounts of menaquinone 11 (4%) and menaquinone 14 (9%) were also present. The cellular fatty acids detected in all the strains analyzed were predominantly saturated branched iso-14:0, iso-15:0, anteiso-15:0, and iso-16:0; straight chain 16:0 was found in relatively low proportions, and only traces of unsaturated fatty acids were detected (Table 2).

0.4

0.3

0.2

0.1

0 6

6.5

7

7.5

8

8.5

9

9.5

10

10.5

11

pH

Specific growth rate (h-1)

(b)

0.4

pH7.5 pH7. pH9 pH9 pH10 pH10

0.3

0.2

0.1

0 0

5

10

15

20

25

30

35

40

45

50

Temperature ºC

Fig. 1. (a) Effect of pH on growth rates of strain CV-2T and (b) effect of temperature on growth rates of strain CV-2T.

growth was observed at pH 7.0 or at pH 10.5 (Fig. 1a). The specific growth rate of the organisms was higher in ABM2 without NaCl, but they grew in ABM2 containing up to 7.0% NaCl (results not shown). All strains were catalase positive and cytochrome oxidase negative. The organisms were strictly aerobic and heterotrophic, did not reduce nitrate and utilized several sugars and proteinaceus substrates (Table 1). All strains were resistant for the antibiotics tested except for chloramphenicol and ceftriaxon.

Chemotaxonomic characteristics Peptidoglycan analysis of the strain CV-2T revealed the presence of the following amino acids: alanine (Ala), glycine (Gly), glutamic acid (Glu) and lysine (Lys) with the respective molar ratio (1.2:1.5:2.4:1.0). The 2D-TLC of the partial hydrolysate of the peptidoglycan revealed the presence of only two characteristic peptides: Gly–DGlu, and Lys–D-Ala. Lysine was detected as the only Nterminal amino acid by dinitrophenylation. From these data the presence of a B-type peptidoglycan based on lysine as diagnostic diamino acid was identified [24]. However, the high content of glutamic acid and the absence of homoserine and hydroxyglutamic acid indicated a novel of B-type peptydoglycan structure. The major respiratory quinones found in all strains were menaquinone 12 (45%); menaquinone 13 (42%); small

16S rRNA gene sequence comparison and G+C content of DNA Partial 16S rRNA gene sequences comprising 1494–1509 nucleotides were determined for strains CV2T, CV-40, and AC-30. Comparison of these sequences with representatives of the main lines of descent within the domain Bacteria indicated that these strains were members of the family Microbacteriaceae of phylum Actinobacteria (Fig. 2). The pairwise 16S rRNA gene sequences similarity determined between these strains was 100% (Fig. 2 and Table 3b). The 16S rRNA gene sequence of these organisms showed highest pairwise similarity (95.6%) with the type strain of Frigoribacterium faeni. Sequence similarities with previously described taxa of the genera Agreia, Agrococcus, Clavibacter, Cryobacterium, Leifsonia, Mycetocola, Rhodoglobus, Salinibacterium and Subtercola were o95% (Fig. 2 and Table 3b). The G+C content of the DNA varied between 68.8 mol% determined for strains CV-2T and CV-30 and 70.7 mol% for strain CV-40 which is in agreement with the placement of these strains in the high G+C Gram-positive bacteria (Actinobacteria).

Discussion The organisms characterized during this study were isolated from a rare alkaline aqueous environment. Phenotypic analysis of strains CV-2T, CV-40, AC-30 demonstrated a very homogeneous group of organisms. Indeed, we could only detect small differences in the single carbon source assimilation profile of the three strains. Phylogenetic analysis, showed that these organisms had identical 16S rRNA gene sequences comprising a novel branch within the family Microbacteriaceae whose closest described relative is Frigoribacterium faeni. Strain CV2T differed from all other species that comprise the genera of the family Microbacteriaceae because this organism possesses a new B-type peptidoglycan and iso C14:0 that reaches about 14% of the total fatty acids. These results also indicate that the new organisms belong to a novel genus. Moreover, the phylogenetic analysis also supports this view since all of the most closely related genera have 16S rRNA gene sequence similarities that vary between 92.9% for Clavibacter michiganensis and Agrococcus baldri and

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Table 1.

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Physiological characteristics of strains CV-2T, CV-40 and AC-30

Characteristics

Presence of: Xylanase Urease DNAse bGalactosidase Reduction of nitrate to: Nitrite Nitrogen Hydrolise: Casein Elastin Gelatin Arbutin Starch Esculin Hippurate Arginine Production of: Indol Utilization of: Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Ribitol b-Methyl-xyloside Galactose D-Glucose D-Frutose D-Mannose L-Sorbose

Strains

Characteristics (cont.):

CV-2T

CV-40

AC-30

 w+ + +

 w+ + +

 w+ + +

 

 

 

+   + +   +

+   + +   +

+   + +   +







+   + + +    + + + + 

+   + + +     + + + 

+   + +     + + + + 

Utilization of (cont.): L-Rhamnose Galactitol Inositol Mannitol Sorbitol a-Methyl-D-mannoside a-Methyl-D-glucoside N-Acetyl glucosamine Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Sucrose Trehalose Inuline Melezitose D-Raffinose Starch Glycogen Xylitol b-Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-ketogluconate 5-ketogluconate

Strains CV-2T

CV-40

AC-30

   +         + +   +  +   + +          +  +

   +         + +   + +    + +          +  +

   +         + +   +     + +   +       +  +

+, positive result or growth; ‘, negative result or no growth; ‘w+, weak positive.

96.5% between Frigoribacterium faeni and Mycetocola saprophilus. It is not surprising that a borehole venting water with a pH of 11.4 yielded alkaliphilic strains CV-2T, CV-40 and AC-30. It is noteworthy that these organisms do not grow, in laboratory medium, at pH higher than 11.0 [32]. To our knowledge, the species represented by these organisms appears to be the first alkaliphilic representative of the family Microbacteriaceae since there is no overt indication that any of the other species of this family described are alkaliphilic. It is noteworthy that alkaliphilic species are commonly encountered in genera which contain many non-alkaliphilic species, leading us to speculate that the new genus represented

by strains CV-2T, CV-40 and AC-30, may contain other neutrophilic species. Moreover, these organisms possess the highest temperature range for growth of the most closely related genera (Table 3a) all of which have lower growth temperature ranges and some are psychrophilic. Menaquinone with isoprenoid chains with lengths between 8 and 14 are found in all of the species of the family Microbacteriaceae. Strains CV-2T, CV-40 and AC-30 possess MK 11, MK12, MK13 and MK14; but MK12 and MK13 were clearly in major amounts. We could detect the respiratory quinones, which are obviously found in low concentrations, when we increased the quantity of cellular mass used.

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On the basis of these findings, we propose that strains CV-2T, CV-40 and AC-30 represent a new genus for which we propose the name Microcella and a new

species for which we propose the name Microcella putealis gen. nov., sp. nov.

Description of Microcella gen. nov. Table 2. Fatty acid composition of strains of the Microcella gen. nov. Total fatty acid (%)a

i 13:0 a 13:0 i 14:0 14:0 i 15:0 G a 15:0 A i 15:0 a 15:0 15:0 i 16:0 16:0 i 17:0 a 17:0 17:1 o 5c 17:0 i 18:0

CV2T

CV40

AC30

0.2 0.1 12.1 0.2 0.3 1.6 10.4 27.4 0.4 34.6 2.2 3.3 3.2 0.3 0.4 0.2

0.2 0.2 17.0 0.3 0.3 1.5 10.5 24.7 0.4 35.9 1.5 2.5 2.2 0.1 0.3 0.1

0.1 0.1 11.1 0.4 —b 1.7 10.1 24.3 0.6 38.6 2.8 4.5 3.8 0.3 0.5 0.2

a The fatty acids shown are those present in at least 0.05% of the total; the predominant fatty acids are indicated in bold. b Not detected.

Microcella (Mi. cro. cel’ la; Gr. adj., micros, small; L. fem. N. cella, chamber/cell; N. L. fem. n. Microcella, a small cell). Microcella forms small rod-shaped cells that stain Gram-positive. Endospores are not formed. Mesophilic. Strictly aerobic, oxidase negative and catalase positive. Some species are alkaliphilic. Peptidoglycan is of the B-type and lysine is the diamino acid. Mycolic acids are not present. Fatty acids are predominantly iso- and anteiso-branched. This genus belongs to the family Microbacteriaceae of the phylum Actinobacteria. The type species is Microcella putealis.

Description of Microcella putealis sp. nov. Microcella putealis (pu. te. a’ lis; L. fem. adj., putealis, belonging to a well). Microcella putealis forms very small rod cells 0.4 mm in width by 0.8–1.6 mm in length. Gram stain is positive. Non-spore-forming. Aerobic and heterotrophic. Colonies are small, smooth, convex and yellow. Oxidase negative, catalase positive. The optimum temperature for growth is about 35 1C, no growth occurs at 10 1C or 45 1C; the optimum pH is between 8.5 and 9.0, no growth occurs at pH 7.0 or 10.5; growth is faster without added NaCl, but grows up to7.0% NaCl.

Leifsonia aurea DSM 15303T (AJ438586) Salinibacterium amurskyense KMM3673T (AF539697) Rhodoglobus vestalii CIP 107482 T (AJ459101) 99 Leifsonia rubra DSM 15304T (AJ438585) Subtercola frigoramans DSM 13057 T(AF224723) Subtercola boreus DSM 13056 T (AF224722) Agreia pratensis DSM 14246 T (AJ310412) Agreia bicolorata DSM 14575 T (AF159363) 100 99 Mycetocola tolassinivorans DSM 15179 T (AB012646) 100 Mycetocola lacteus DSM 15177 T (AB012648) Mycetocola saprophilus DSM 15178 T (AB012647) i faeni DSM 10309 T (Y18807) Frigoribacterium CV2T (AJ717388) CV40 (AJ717387) 100 AC30 (AJ717386) Clavibacter michiganensis i DSM 46364T (X77435) Clavibacter michiganensis subsp. insidiosum LMG 3663T (U09761) 100 100 Agrococcus jenensis DSM 9580T (X92492) T (AJ309928) 4 Agrococcus baldri DSM 14215 Cryobacterium psychrophilum DSM 4854T (AJ544063) Leifsonia poae DSM 15202T (AF116342) Leifsonia aquatica DSM 20146T (X77450) Leifsonia xyli JCM 9733T (AB016985) 100

100

77 80

100

0.01

Fig. 2. Phylogenetic dendrogram based on a comparison of the 16S rDNA sequences of strains CV-2T, CV-40, AC-30 and the closest phylogenetic relatives. The trees were created using the neighbour-joining method. The numbers on the tree indicate the percentages of bootstrap sampling, derived from 1000 replications. Isolates characterized in this study are indicated in bold. Scale bar, 10 inferred nucleotide substitutions per 100 nucleotides.

Table 3. (a) Comparison of Microcella gen. nov. with the phylogenetically related genera of the family Microbacteriaceaea and (b) Phylogenetic distances between representative strains of Microcella gen. nov. and the most related genera of family Microbacteriaceae. Characteristic

Lys 12, 13 A, I 68,8 35

Lys 9 S, A, Id 71.7 4–10

Lys 10 A 64-65 25

DAB 9, 10 A, I 64–68 15–17

DAB, Orn 10 S, A, I 67 28

Lys, Orn 11 S, A, I 61 25-28

DAB 11 A, I 64 22

DAB 11 S, A, I 66–73 24–28

DAB 11 A, I 66 15

Orn 11, 12 A, I 62 18

DAB 9, 10 S, A, I 67–78 28

DAB 10 S, A, I (12 H) 65 9–12

DAB 11, 12 S, A, I 74-75 28

100e 95.6f 94.5–94.8 93.6–94.7 93.9–94.2 94.9 93.7 93.5–93.8 93.4 93.7 94.5–94.7 94.5 93.3–93.4

— 100 96.2–96.5 95.1–96.4 95.7–95.9 95.9 95.4 94.8–95.5 95.6 95.0 95.9–96 95.4 94.3–94.6

— — 99.4–100 94.3–95.6 94.1–94.8 94.1–94.4 93.6–93.8 94.0–94.8 94.3–94.7 93.7–94.1 94.9–95 94.3–94.4 93.7–94.2

— — — 97.1–100 96.9–97.6 95.4–95.8 94.6–95.4 94.0– 95.8 95.6–96.1 95.2–95.7 94.4–96.0 93.6–94.7 93.0–94.1

— — — — 99.7–100 96.1–96.3 95.5–95.7 95.0–96.6 96.1–96.3 95.5–95.8 95.0–95.4 93.8–94.1 94.0–94.8

— — — — — 100 98.3 95.2–95.6 98.7 98.2 93.9 93.3 94.1

— — — — — — 100 94.5–95.2 98.1 97.7 93.2–93.3 93.1 93.3–93.4

— — — — — — — 97.8–100 95.3–95.9 94.7–95.3 94.1–95.6 94.2–95.2 94.5–95.6

— — — — — — — — 100 99.4 94.3–94.4 93.7 94.2–94.3

— — — — — — — —

— — — — — — — — —

— — — — — — — — — — — 100 94.8–95.2

— – — — — — —

100 94.1–94.3 93.5 93.9

99.6–100 95.5–95.6 92.9–93.7

Data from Ka¨mpfer et al. [14], Ma¨nnisto¨ et al. [18], Han et al. [9], Sheridan et al. [26], Tsukamoto et al. [34], Suzuki et al. [29] and Evtushenko et al. [4]. Lys, Lysine; Orn, D-ornithine; DAB, Diaminobutyric acid. c A, Anteiso-methyl-branched; 12H, 12-methyl tetradecenoic acid; I, iso-methyl-branched; S, straight-chain saturated. d Frigoribacterium strains contain unsaturated anteiso fatty acids when grown at low temperatures (o10 1C). e On bold—phylogenetic similarity between species within same genera. f Not bold—phylogenetic similarity (minimal–maximal) between species of different genera. b

— —

98.7–100

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a

Salinibacterium Leifsonia Leifsonia Leifsonia Rhodoglobus Clavibacter Cryobacterium Agrococcus aurea (senso stricto) rubra

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(a) Diamino acidb Major quinones (MK) Major fatty acidsc G+C content (mol%) Optimal temperature (1C) (b) Microcella gen. nov. Frigoribacterium Mycetocola Subtercola Agreia Salinibacterium Leifsonia aurea Leifsonia (senso stricto) Leifsonia rubra Rhodoglobus Clavibacter Cryobacterium Agrococcus

Microcella Frigoribacterium Mycetocola Subtercola Agreia gen. nov.

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The diamino acid of peptidoglycan is lysine with high content of glutamic acid, homoserine and hydroxyglutamic acid are absent from the peptidoglycan. Major respiratory quinones are menaquinones 12 (MK12) and 13 (MK13). The predominant fatty acids are i16:0, a15:0, i14:0 and i15:0. The strains of this species do not hydrolyse elastin, esculin or hippurate. Hydrolysed starch, gelatine, arginine and arbutin. The presence of urease, b-galactosidase and DNAse was detected, xylanase was not detected. Sensitive to chloramphenicol and ceftriaxon. The type strain of this species assimilates glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, frutose, mannose, mannitol, cellobiose, maltose, sucrose, inulin, starch, glycogen, gluconate and 5ketogluconate. Strain CV-40 also assimilates trehalose but does not assimilate galactose or inulin. Strain AC-30 also assimilates turanose, but does not assimilate Dxylose or inulin. The mole G+C ratio of the DNA of the type strain is 68.8 mol%. This bacterium was isolated from water taken from the borehole at Cabec¸o de Vide in Southern Portugal. The type strain, CV-2T, has been deposited in the Collection of the Institute Pasteur, Paris, France, as strain CIP 108471T and in the BCCM/LMG Bacteria Collection, Ghent, Belgium as strain LMG 22692T. Note: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number(s): AJ717388 for strain CV2T, AJ717386 for strain AC30 and AJ717387 for strain CV40.

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Acknowledgements This research was funded in part by FCT/FEDER project POCTI/BSE/42732/2001. We are indebted to Prof. J. Euze´bi (E´cole National Ve´te´rinaire, Toulouse, France) for the etymology of the new organisms’ names. We thank Dr. Peter Schumann (DSMZ, Germany) for determining peptidoglycan structure, Dr. Fernanda Nobre (Universidade de Coimbra, Portugal) for helping in the FAME analysis, we also thank to the Junta de Freguesia de Cabec¸o de Vide and Mr. Manuel Fontainhas for permission to collect the water samples.

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