Scientific Publications - Work Done by Microbiology Reader Bioscreen C

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1731-1739

Subtercola boreus gen. nov., sp. nov. and Subtercola frigoramans sp. nov., two new psychrophilic actinobacteria isolated from boreal groundwater

M. K. Mannisto, P. Schumann, F. A. Rainey, P. Kampfer, I. Tsitko, M. A. Tiirola and M. S. Salkinoja-Salonen

ABSTRACT

Psychrophilic actinobacterial isolates from permanently cold groundwater in Finland were characterized using a polyphasic approach. Growth on agar plates was observed at temperatures down to -2 °C, with an optimum at 15-17 °C, but no growth was observed at 30 °C. The peptidoglycan type was B2μ and the characteristic diamino acid was diaminobutyric acid. The cell wall sugars of strain K265^T were rhamnose, ribose, xylose and mannose and those of strain K300^T were glucose, rhamnose and xylose. The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and two glycolipids. The main whole-cell fatty acids were 12-methyltetradecanoic acid, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid. Large amounts of anteiso-1,1-dimethoxy-pentadecane and also iso-1,1-dimethoxy­hexadecane were present as diagnostic markers. The predominant menaquinones were MK-9 and MK-10. The GMC content of the DNA of strains K265^T and K300^T was 64.4 and 67.8 mol%, respectively. Comparison of 16S rRNA gene sequences revealed that strains K265^T and K300^T represent a new lineage among the type-B-peptidoglycan actinomycetes. The closest relatives were Clavibacter michiganensis, Frigoribacterium faeni and Rathayibacter rathayi. On the basis of 16S rDNA sequence, G+C content and chemotaxonomical and physiological characteristics, K265^T and K300^T clearly represent a new genus. The genus Subtercola gen. nov. is described, together with two species, namely Subtercola boreus sp. nov. (type strain K300^T =DSM 13056^T =CCUG 43135^T) and Subtercola frigoramans sp. nov (type strain K265^T =DSM 13057^T =CCUG 43136^T).

Keywords: Subtercola boreus gen. nov., sp. nov., Subtercola frigoramans sp. nov., psychrophilic actinobacteria, 1,1- dimethyl acetal

INTRODUCTION

The few psychrophilic or psychrotolerant actino­bacterial species so far described belong to the genera Cryobacterium (Suzuki et al., 1997) and the recently described Frigoribacterium (Kämpfer et al., 2000). Analysis of whole-cell fatty acid composition and partial 16S rRNA gene sequencing indicated that several taxa of actinomycetes were present among 190 isolates from Finnish groundwater in which the tem­perature is stable at 7 °C (Mä nnisto$ et al., 1999; unpublished results). This paper is a polyphasic des­cription of two novel actinomycetes, isolated from the groundwater, capable of growth on solid medium at temperatures down to ®2 °C. The phylogenetic, chemotaxonomic and physiological data show that the two isolates represent a new genus within B-type­peptidoglycan-containing members of the family Microbacteriaceae. We propose a new genus, Subter­cola gen. nov., and the species Subtercola boreus sp. nov. and Subtercola frigoramans sp. nov. for these strains.

METHODS

Isolation. Strains K265^T and K300^T were isolated from the groundwater of a shallow aquifer located under a glacial gravel ridge in Southern Finland. The water was pumped from a subsurface depth of 18 m. It was highly humic (with 4-13 mg dissolved organic carbon l-1), rich in iron (15 mg l-1) and had a stable temperature of 7±1 'C. The strains were isolated on PYGV agar plates (Staley, 1968) at 7± 1 'C. The cultures for characterization were grown on PYGV, TSA (BBL Microbiology Systems) or R2A (BBL Micro­biology Systems) agar.

Morphology. The cultures were studied by phase-contrast microscopy (8, 24, 48 and 72 h at 20 °C) using an Olympus BH-2 light microscope. Gram-staining was performed using the Hucker method (Gerhardt et al., 1994). For the prep­aration of thin sections, the cultures were grown for 7 d on TSA agar at 8 'C. The cells were prexed with 4% (v/v) glutaraldehyde in 0^.1 M sodium phosphate buffer (pH 7^.2) for 2 h at room temperature and washed three times in the same buffer. Thin sections were prepared and examined as described elsewhere (Väisänen et al., 1994).

Physiological characteristics. Growth on TSA plates at -2, 0, 2, 4, 8, 10, 15, 20, 25, 28 and 30 'C was observed and recorded after 14 d and the growth rates on PYGV and trypticase soy broth were determined at 13, 15, 17, 20 and 23 'C by automated kinetic turbidometry (Bioscreen; Lab-Systems). The inoculum (50l) was grown on PYGV plates for 34 d and suspended in PYGV broth to an optical density (600 nm) of 0^.3; 300l PYGV or TSA was pipetted into each well. Growth, with medium shaking, was measured as turbidity using a wide-bandlter (450580 nm) and BIOLnvK (Labsystems) software. Physiological tests were performed on microtitre plates as described elsewhere (Kaämpfer et al., 1991) and read after 3 d at 20 'C.

Chemotaxonomic analyses. For whole-cell fatty acid analy­sis, the strains were grown on TSA at 4, 10, 15, 20 and 25 'C for 312 d. Fatty acid methyl esters were prepared and analysed as described previously (Vaäisainen et al., 1994). Fatty acid methyl esters were identied using the MIDI aerobic library (TSBA, version 3.9; MIDI). 1,1-Dimethyl acetals were identied as described by Kaämpfer et al. (2000). Peaks in the whole-cell methanolysate not identifed with the MIDI aerobic library version 3.9, were rerun using the BHIBLA anaerobic library and also analysed by GC-MS using the Wiley 138K and NIST mass-spectral libraries. Methanolysates of Propionibacterium freudenreichii DSMZ 20271^T and Propionibacterium jensenii DSMZ 20535^T were used as references for 1,1-dimethyl acetals.

Puried cell wall preparations were obtained as described by Schleifer & Kandler (1972). Amino acids and peptides in cell wall hydrolysates were analysed by two-dimensional TLC on cellulose plates, using the solvent systems described by Schleifer & Kandler (1972). The molar ratios of cell wall amino acids were determined by GC and GC-MS of N-heptauorobutyryl amino acid isobutyl esters (MacKenzie, 1987). Cell wall sugars were determined by GC of alditol acetals as described by Groth et al. (1996).

Menaquinones were extracted as described by Collins et al. (1977) and analysed by HPLC (Groth et al., 1996). Polar lipids extracted by the method of Minnikin et al. (1979) were identied by two-dimensional TLC followed by spraying with specic reagents (Collins & Jones, 1980).

Base composition of DNA. DNA was extracted after lysis with proteinase K and puried with phenol}chloroform and chloroform-isoamyl alcohol extractions, 2-propanol pre­cipitation and caesium chloride gradient purication as described by Wilson (1994). Hydrolysis, dephosphorylation and HPLC measurement were performed as described by Johnson (1994). The HPLC column was a Purospher RP-18 endcapped reversed-phase column (250 mmx 4^.0 mm i.d., 5^.0m particle size; Merck). The mobile phase was 20 mM triethylamine phosphate (pH 5^.1) in 12% methanol. The flow rate was 1 ml min^w". Hydrolysed lambda phage DNA was used as a standard.

16S rRNA gene sequence determination and phylogenetic analyses. The extraction of genomic DNA, PCR ampli­cation of the 16S rRNA gene and sequencing of the puried PCR products were carried out as described previously (Rainey et al., 1996). Sequence reaction products were puried by ethanol precipitation and electrophoresed with a model 310 Genetic Analyzer (Applied Biosystems). The 16S rRNA gene sequences obtained in this study were aligned against the previously determined actinobacterial sequences available from the public databases, using the ae2 editor (Maidak et al., 1999). The programs of the PHYLIP package, including DNADIST and NEIGHBOR, were used for the phylo­genetic analyses (Felsenstein, 1993). The method of Jukes & Cantor (1969) was used to calculate evolutionary distances. The tree topology was re-analysed using 1000 bootstrapped data sets and the programs, DNADIST and coNsENsE of the PHYLIP package (Felsenstein, 1993).

Nucleotide sequence accession numbers. The accession numbers and strain designations of the reference 16S rRNA gene sequences used in the phylogenetic analyses are as follows: Agrococcus jenensis DSM 9580^T (X92492), Agro­myces ramosus DSM 43045^T (X77447), Agromyces medio­lanus DSM 20152^T (X77449), Arthrobacter globiformis DSM 20124^T (M23411),Brevibacterium helvolum ' DSM 20419 (X77440), Clavibacter michiganense subsp. michiganense DSM 46364^T (X77435),Corynebacterium aquaticum ' DSM 20146^T (X77450), Cryobacterium psychrophilum IAM12024^T (D45058), Curtobacterium citreum DSM 20528^T (X77436), Curtobacterium luteum DSM 20542^T (X77437), Frigori­bacterium faeni DSM 46346^T (Y18807), Leucobacter koma­gatae IFO 15245^T (D17751), Microbacterium aurum IFO 15204^T (D21340), Microbacterium barkeri DSM 20145^T (X77446), Microbacterium imperiale DSM 20530^T (X77442), Microbacterium lacticum DSM 20427^T (X77441), Micro­bacterium liquefaciens DSM 20638^T (X77437), Rathayibacter rathayi DSM 7458^T (X77439), Rathayibacter toxicus JCM 9669^T (D84127).

RESULTS

Morphological and physiological characteristics

Strains K265^T and K300^T formed yellow colonies within 34 d at 20 °C. K265^T formed large, mucoid, pale yellow colonies on PYGV or R2A agar; K300^T was less slimy. On TSA agar, the strains formed smooth, round, convex, non-slimy, yellow colonies. The microscopic investigations showed that the cells of strain K300^T were irregular, short, sometimes slightly curved rods 0^.2-0^.3μm in width and 0^.6-1^.0μm in length. The cells of strain K265^T were irregular, pleomorphic rods 0^.3-0^.4μm in width and 0^.9-1^.5μm in length. Young cells of K265^T were frequently swollen at the pole or in the middle. The non-motile cells stained Gram-positive at the early stages of growth (24 h at 20 °C). Both strains turned into short, coccoid rods in the late stages of growth (" 48 h at 20 °C). When grown in liquid medium, v- and y-forms were frequently seen. Micrographs of thin sections of strains K300^T and K265^T are shown in Fig. 1. Cells of both strains had the typical morphology of Gram­positive bacteria, i.e. a cell membrane surrounded by an amorphous layer. The cells exhibited a perpen­dicular type of division. Cells of strain K300^T had an irregular, electron-dense outer layer.

Growth was optimal at 15-17 °C. The growth rates of strain K265^T on PYGV medium were 0.017 and 0.018 1/h at 15 and 17 °C, respectively; the growth rate of strain K300^T was 0.023 1/h at both temperatures. Visible colonies formed within 14 d at +2 °C and, in the case of strain K265^T, even at -2 °C. The colony morphology was similar at -2 °C and 20 °C.

The metabolic properties of strains K265^T and K300^T are shown in Table 1. A wide variety of sugars were assimilated but none of the 11 amino acids tested was used. The strains differed from each other in their ability to assimilate substrates such as rhamnose, ribose, acetate, propionate, cis-aconitate and citrate.

Fig. 1. Electron micrographs of thin sections of strains K300^T (top) and K265^T (bottom) grown on tryptic soy agar plates for 7 d at 8 °C. Bar, 200 nm.

Table 1. Physiological characteristics of the strains K265^T and K300^T

Both strains assimilated-arabinose,-cellobiose,-fructose, n-galactose, gluconate,-glucose,-mannose,-maltose, sucrose,-trehalose,-xylose, adonitol,-mannitol, fumarate,-3-hydroxybutyrate and 4-hydroxybenzoate. Neither strain utilized N-acetyl--galactosamine, N-acetyl--glucosamine,--melibiose, maltitol,-sorbitol, putrescine, trans-aconitate, adipate, 4-aminobutyrate, azelate, glutarate, itaconate,-lactate,-malate, mesaconate, oxoglutarate, pyruvate, suberate,-alanine,-alanine,-aspartate, L-histidine,-leucine,-ornithine,-phenylalanine,-proline, L-serine,-tryptophan or phenylacetate. Both strains hydrolysed aesculin, pNP---glucopyranoside and pNP---glucopyranoside. Neither strain hydrolysed pNP-phenyl­phosphonate, pNP---galactopyranoside, pNP-phosphoryl­choline, 2-deoxythymidine-5^«-pNP-phosphate,-alanine-para­nitroanilide,-glutamate--3-carboxy-pNA or-proline-para­nitroanilide. Abbreviation: pNP, para-nitrophenyl.

Chemotaxonomic characteristics

The polar lipids in K265^T and K300^T consisted of phosphatidylglycerol, diphosphatidylglycerol, one un­identied phospholipid and two unidentied glyco­lipids.

The diamino acid in the peptidoglycan of strains K265^T and K300^T was diaminobutyric acid (DAB). The molar ratio of alanine:glycine:glutamic acid:DAB was, for strain K265^T, 0^.6:1^.2:0^.1:2^.0; for strain K300^T, the ratio was 0^.6:1^.1:0^.1:2^.0. The amino acid ratio (except that of glutamic acid) is consistent with peptidoglycan type B2 y (Schleifer & Kandler, 1972). This peptidoglycan type was conrmed by characteristic two-dimensional thin-layer chromato­graphic peptide patterns of partial hydrolysates of cell walls (data not shown). The low content of glutamic acid in the hydrolysates was due to nearly complete substitution of-glutamic acid at position 2 of the peptide subunit by threo-3-hydroxyglutamic acid, as revealed by two-dimensional TLC and GC-MS.

The cell wall sugars of strain K265^T included rham­nose, ribose and small amounts of xylose and mannose. Glucose, rhamnose and small amounts of xylose were present in the cell wall of strain K300^T.

The major isoprenoid quinones of K265^T were MK-9 (36%), MK-10 (32%), MK-12 (18%), MK-13 (4%), MK-11 (4%) and MK-8 (4%) and those of K300^T were MK-10 (58%), MK-9 (30%), MK-11 (8%) and MK-8 (2%). The G­C content of the genomic DNA of strain K265^T and strain K300^T was 64±4 and 67±8 mol%, respectively.

Table 2. Composition of whole-cell methanolysates of strains K265^T and K300^T grown at different temperatures

Fig. 2. (a) Total-ion gas chromatogram of a whole-cell methanolysate of strain K265^T grown on tryptic soy agar plates at 6 °C; i, iso-branched; a, anteiso-branched; DMA, 1,1-dimethyl acetal. (b) Mass fragmentogram of 1,1-dimethoxy-hexadecane (i-16:0 DMA).

Cellular fatty acid compositions

The results of the gas chromatographic data for whole­cell methanolysates of strains K265' and K300' are shown in Table 2. It shows that in both strains all identied signicant fatty acids were of the branched­chain type, mainly 12-methyl tetradecanoic acid (a-15:0) and 14-methyl pentadecanoic acid (i-16:0). In addition the strain K265^T contained 12-methyl tri­decanoic acid (i-14:0) and both strains contained un­saturated 12-methyl tetradecanoic acid (a-15:1) when grown at temperatures of ≤ 15 °C. In addition to fatty acids, several major peaks in the whole-cell methano­lysates (Fig. 2a) were identied as branched-chain C15, C16 and C17 1,1-dimethyl acetals. The BHIBLA anaerobic library and mass-spectral analysis, using P. freudenreichii DSMZ 20271' and P. jensenii DSMZ 20535' as standards for 1,1-dimethyl acetals, proved that the major peak of K265' and K300' (with an equivalent chain length of 15-197) was 1,1-dimethoxy­anteiso-pentadecane (Fig. 2a). Methanolysates of the strains K265^T and K300^T contained several peaks with the mass fragment at m/z 75 diagnostic for branched­chain 1,1-dimethyl acetals (Fig. 2b). Such peaks in the K265^T methanolysates were identied, on the basis of their mass fragments, as 1,1-dimethoxy-anteiso-penta­decane (a-15:0 DMA), 1,1-dimethoxy-iso-hexadecane (i-16:0 DMA) and 1,1-dimethoxy-anteiso-hepta­decane (a-17:0 DMA) (Table 2). In addition, small amounts of 1,1-dimethoxy-iso-pentadecane (i-15:0 DMA) and a straight-chain 1,1-dimethoxy-hexa­decane (16:0 DMA) were identied in strain K300^T. Three peaks in the methanolysates of strains K265^T and K300^T remained unidentied.

Fig. 3. Phylogenetic tree based on 16S rRNA gene sequence comparison, demonstrating the positions of strains K265^T and K300^T. Bar, 10 nucleotide substitutions per 100 nucleotides.

Effect of temperature on cellular fatty acid compositions

The effect of cultivation temperature on the whole-cell fatty acid composition of the new isolates was unusual. Strain K300^T reduced its anteiso-15:0 content and both strains reduced their anteiso-17:0 content as the growth temperature was reduced (Table 2). When grown at 4 °C, K265^T and K300^T contained 6-10% anteiso-15:1. Moreover, cultivation temperature in­uenced the 1,1-dimethyl acetal composition of strains K265^T and K300^T. As the growth temperature was lowered from 25 to 4 °C, the contribution of the anteiso-15:0 DMA, the main DMA in the whole-cell methanolysates, increased in both strains from 1011 to 14-20%, whereas the contributions of anteiso-17:0 DMA and iso-16:0 DMA fell accordingly. Thus, it appears that a low growth temperature favoured the synthesis of DMAs with shorter carbon chain lengths, a shift from iso-branched DMAs to anteiso-branched congeners, or both.

16S rRNA gene sequence comparison

Almost complete 16S rRNA gene sequences 1467 (strain K300^T) and 1472 (strain K265^T) nucleotides in length were determined. Phylogenetic analyses based on a data set comprising 1330 unambiguous nucleo­tides between positions 38 and 1478 but excluding positions 97-193 [due to the absence of this region in the reference sequence of L. komagatae (D17751)] (Escherichia coli positions; Brosius et al., 1978) showed that the new isolates cluster together as distinct lineages within the radiation of the actinomycete genera with group-B-peptidoglycan that comprises the family Microbacteriaceae (Stackebrandt et al., 1997) (Fig. 3). Strains K300^T and K265^T share 97^.0% 16S rRNA gene sequence similarity (data not shown). The new isolates show the highest levels of 16S rRNA gene sequence similarity with species of the genera Frigori­bacterium (95^.5-96^.7%), Clavibacter (95^.1-97^.1%) and Rathayibacter (94^.3-96^.7%) (data not shown). Species of the genera Clavibacter, Frigoribacterium and Rathayibacter share 96^.1-97^.0% 16S rRNA gene sequence similarity (data not shown). The bootstrap analyses indicate no signicance in the branching pattern of the lineage comprising the new isolates and any previously described generic lineage of this family (Fig. 3).

Table 3. Diagnostic and differentiating characteristics of the genera Clavibacter, Rathayibacter, Frigoribacterium, Cryobacterium and strains K265^T and K300^T

DISCUSSION

The characteristics that differentiate the strains K265^T and K300^T from the genera Clavibacter, Rathayibacter, Cryobacterium and Frigoribacterium are shown in Table 3. Clavibacter and Frigoribacterium species contain MK-9 as the predominant menaquinone, whereas Rathayibacter and Cryobacterium species contain MK-10 (Sasaki et al., 1998; Kämpfer et al., 2000; Suzuki et al., 1997). Strains K265^T and K300^T contained both MK-9 and MK-10 in almost equal proportions. In addition, K265^T contained MK-12 and MK-13, which are absent in strain K300^T and in species of Clavibacter, Frigoribacterium and Rathayi­bacter. The principal phospholipids of K265^T and K300^T were similar to those of Clavibacter (Collins & Jones, 1980), Rathayibacter (Zgurskaya et al., 1993), Frigoribacterium (Kämpfer et al., 2000) and Cryo­bacterium (Suzuki et al., 1997).

The whole-cell methanolysates of strains K265^T and K300^T contained predominantly iso- and anteiso­branched fatty acids and 1,1-dimethyl acetals. The whole-cell methanolysates of strains K265^T and K300^T were quite similar to that of F. faeni, only differing in their content of several different iso- and anteiso­branched 1,1-dimethyl acetals (Table 3). However, the peptidoglycan type of Frigoribacterium was B2, whereas that of strains K265^T and K300^T was B2.

The amino acid ratio in the peptidoglycan of strains K265^T and K300^T differed from that of other DAB-containing genera (Table 3) in the low levels of glutamic acid. In strains K265^T and K300^T, nearly all of the glutamic acid residues at position 2 of the peptide subunit were replaced by hydroxy-glutamic acid. More or less complete hydroxylation of­glutamic acid has been observed also in other repre­sentatives of the peptidoglycan-B-type cross-linkage, e.g. in Microbacterium species (Schleifer et al., 1967).

The optimal growth temperatures of strains K265^T and K300^T were around 10 degrees lower, down to -2 °C for K265^T, than those in the genera Clavibacter (Davis et al., 1984) and Rathayibacter (Zgurskaya et al., 1993). The new genus Frigoribacterium (Kämpfer et al., 2000), recently described and isolated from a Finnish farming environment, showed a similar growth-temperature range, whereas Cryobacterium (Suzuki et al., 1997) did not grow above 18 °C. Psychrophilic actinobacteria proposed as species of Arthrobacter have been reported recently (Vasilevaet al., 1998; Loveland-Curtze et al., 1999).Arthrobacter crygenae ' strain Z-0064 had an optimal growth tem­perature of 6 °C and did not grow above 15 °C (Vasileva et al., 1998), whereasArthrobacter psy­chrolactophilus ' strains had growth ranges of 0-30°C (Loveland-Curtze et al., 1999).

Decreases in temperature inuenced both the fatty acid and dimethyl acetal compositions of the strains K265^T and K300^T. Strains K265^T and K300^T increased their content of anteiso 15:1 fatty acid at cold temperatures, as reported for C. psychrophilum (Suzuki et al., 1997) and F. faeni (Kämpfer et al., 2000).

1,1-Dimethyl acetals are formed by the methanolysis of alk-1^«-enyl glyceryl ethers (plasmalogens) (Jantzen & Hofstad, 1981). Plasmalogens are known to occur as signicant components of animal cell membranes and in many obligately anaerobic bacteria such as Fuso­bacterium spp. (Jantzen & Hofstad, 1981), clostridia (Johnston & Goldne, 1994), Megasphaera elsdenii (Kaufman et al., 1990) and Eubacterium lentum (Verhulst et al., 1987). 1,1-Dimethyl acetals were detected in the anaerobic actinobacterial species Pro­pionibacterium freudenreichii and P. jensenii (Kämpfer et al., 2000).1,1-Dimethyl acetals were recently detected also in the aerobic psychrophilic F. faeni belonging to the same actinobacterial family (Micro­bacteriaceae) as strains K265^T and K300^T (Kämpfer et al., 2000).

The biological function of plasmalogens is not known but Kaufman et al. (1990) showed that membranes of M. elsdenii (with high plasmalogen content) were more ordered than membranes devoid of plasmalogens. This may indicate that the changes in the plasmalogen­derived 1,1-dimethyl acetal composition may reect a role in membraneuidity.

On the basis of the presence of several unusual 1,1-dimethyl acetals, other distinctive chemotaxonomic features and the 16S rRNA gene sequence, we propose that strains K265^T and K300^T should be placed in a new genus, namely Subtercola gen. nov. The 16S rRNA gene sequence comparisons clearly indicate that the strains K265^T and K300^T represent new taxa within the family Microbacteriaceae. It should be noted that the degree of 16S rRNA gene sequence divergence between these two strains (3%) is more than that found between strain K265^T and Clavibacter michi­ganense subsp. michiganense (which share 97±1% similarity) and is the same as that found between F. faeni and C. michiganense subsp. michiganense (3%). The inuence of this sequence divergence is also seen in the results of phylogenetic analyses in which the bootstrap values indicate that there is no signicance in the branching point joining the K265^T and K300^T lineages. Based on the results of 16S rRNA gene sequence analyses it could be proposed that the two strains represent different genera. However, there is little chemotaxonomic and phenotypic data to support this and we therefore propose, at this time, that these two strains should be included in a single genus as two novel species, for which we propose the names Sub­tercola boreus gen. nov., sp. nov. and Subtercola frigoramans gen. nov., sp. nov.

Description of Subtercola gen. nov.

Subtercola (sub.ter^«co.la. L. prep. subter below, under­neath; L. masc. suffix n. -cola inhabitant; M.L. masc.n. subtercola the one who lives underneath).

Aerobic, Gram-positive, irregular rods. Cells occur singly or in v-forms. Endospores are not produced. The colonies are pale to bright yellow depending on the culture medium. The colonies are circular, convex and smooth. Grows optimally at 15-17 °C. Growth is observed in the temperature range 2-28 °C. The cell wall diamino acid is DAB. The main cell wall amino acids are alanine, glycine, threo-3-hydroxy-glutamic acid and DAB. The peptidoglycan type is B2y. The polar lipids are phosphatidylglycerol, diphosphatidyl­glycerol, one unknown phospholipid and two gly­colipids. The main cellular fatty acids are 12-methyl­tetradecanoic acid (a-15:0), 14-methylpentadecanoic acid (i-16:0) and 14-methylhexadecanoic acid (a-17:0). The whole-cell methanolysates of Subtercola sp. contain 1,1-dimethoxy-anteiso-pentadecane (a-15:0 DMA) and 1,1-dimethoxy-iso-hexadecane (i-16:0 DMA) as major components in amounts comparable to those of fatty acids. The major isoprenoic quinones are MK-9 and MK-10. The G+ C content of the DNA is 64-68 mol%. No mycolic acids are present. The type species is Subtercola boreus.

Description of Subtercola boreus sp. nov.

Subtercola boreus (bo.re'us. L. adj. boreus Northern, referring to the boreal groundwater aquifer in Finland, from which the organism was isolated).

Cells are short, irregular rods 0^.2-0^.3μm in width and 0^.6-1^.0μm in length. Colonies are yellow, circular, convex and smooth. Growth occurs at 2-28 °C. The major cellular fatty acids are 12-methyltetradecanoic acid (a-15:0), 14-methylpentadecanoic acid (i-16:0), 14-methylhexadecanoic (a-17:0) acid and 13-methyl­tetradecanoic acid (i-15:0). 1,1-Dimethoxy-iso-penta­decane (i-15:0 DMA), 1,1-dimethoxy-anteiso-hepta­decane (a-17:0 DMA) and 1,1-dimethoxy-hexadecane (16:0 DMA) are found in whole-cell methanolysates. The cell wall contains glucose, rhamnose and xylose. Other chemotaxonomic characteristics are as described for the genus. Good carbon sources include L-arabinose,-cellobiose,-fructose,-galactose, gluconate,-glucose,-mannose,-maltose,-rhamnose,-ribose, sucrose,-trehalose,-xylose, adonitol, i-inositol,-mannitol, acetate, propionate, cis-aconitate, citrate, fumarate,-3-hydroxybutyrate, 3-hydroxybenzoate and 4-hydroxybenzoate. Amino acids are mostly not utilized. The G+ C content of the DNA is 68 mol%. Isolated from groundwater. The type strain is K300^T (= DSM 13056^T = CCUG 43135^T).

Description of Subtercola frigoramans sp. nov.

Subtercola frigoramans (fri.gor.a'mans. L. neut. n. frigus the cold; L. gen. neut. n. frigoris of the cold; L. part. pres. amans loving; M.L. part. pres. frigoramans loving the cold).

Cells are pleomorphic, irregular rods 0^.30^.4m in width and 0^.91^.5m in length. In liquid culture the cells may be arranged in v- or y-forms. Colonies are pale yellow to bright yellow depending on the culture medium. Large, mucoid colonies are formed on glucose-containing medium. Growth occurs at -2 °C to 28 °C. The major cellular fatty acids are 12-methyltetradecanoic acid (a-15:0), 14-methylpenta­decanoic acid (i-16:0), 14-methylhexadecanoic acid (i-17:0) and 12-methyltridecanoic acid (i-14:0). 1,1-Dimethoxy-anteiso-heptadecane (a-17:0 DMA) is found in whole-cell methanolysates. The cell wall contains rhamnose, ribose, xylose and mannose. The main isoprenoid quinones are MK-10 and MK-9. Other chemotaxonomical characteristics are the same as described for the genus. Good carbon sources include-arabinose, p-arbutin,-cellobiose,-fruc­tose,-galactose, gluconate,-glucose,-mannose, D-maltose, sucrose, salicin,-trehalose,-xylose, adonitol and-mannitol. Acetate, propionate, aconi­tate and most amino acids are not assimilated. Fumarate, but not citrate, is assimilated. The G+ C content of the DNA is 64 mol%. Isolated from groundwater. The type strain is K265^T (= DSM 13057^T = CCUG 43136^T).

ACKNOWLEDGEMENTS

This work was supported by the Academy of Finland (grant nos 34519 and 46569). We thank Maria Anderson for advice on whole-cell fatty acid analysis, Raimo Mikkola for expertise with the GC-MS analysis and Arja Strandell for thin-section preparation. We thank the Division of Electron Microscopy at the Institute of Biotechnology at Helsinki University for the lease of equipment. We are grateful to H. G. Tru$ per for the Latin construction of the genus and species names. We thank Jani Salminen for the water-quality monitoring data for the aquifer.

REFERENCES

Brosius, J., Palmer, M. L., Kennedy, J. P. & Noller, H. F. (1978). Complete nucleotide sequence of the ribosomal RNA gene from Escherichia coli, Proc Natl Acad Sci USA 75, 48014805.

Collins, M. D. & Jones, D. (1980). Lipids in the classication and identication of coryneform bacteria containing peptidoglycans based on 2,4-diaminobutyric acid, J Appl Bacteriol 48, 459470.

Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and coryne­bacteria, J Gen Microbiol 100, 221230.

Davis, M. J., Gillespie, A. G., Jr, Vidaver, A. K. & Harris, R. W. (1984). Clavibacter: a new genus containing some phyto­pathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. Cynodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease, Int J Syst Bacteriol 34, 107117.

Felsenstein, J. (1993). PHYLIP (Phylogenetic Inference Package) version 3.5.1 Department of Genetics, University of Washington, Seattle, WA, USA.

Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.

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, 234239.

Jantzen, E. & Hofstad, T. (1981). Fatty acids of Fusobacterium species: taxonomic implications, J Gen Microbiol 123, 163172.

Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular Bacteriology, pp. 655682. Edited by P. R. Gerhardt, G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.

Johnston, N. C. & Goldne, H. (1994). Isolation and character­ization of new phosphatidylglycerol acetals of plasmalogens. A family of ether lipids in clostridia, Eur J Biochem 223, 957963.

Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein mol­ecules. In Mammalian Protein Metabolism, vol. 3, pp. 21132. Edited by H. N. Munro. New York: Academic Press.

Kämpfer, P., Steiof, M. & Dott, W. (1991). Microbiological characterization of a fuel-oil contaminated site including numerical identication of heterotrophic water and soil bac­teria, Microb Ecol 21, 227251.

Kämpfer, P., Rainey, F. A., Andersson, M. A., Nurmiaho-Lassila, E.-L., Ulrych, U., Busse, H.-J., Weiss, N., Mikkola, R. & Salkinoja-Salonen, M. (2000). Frigoribacterium faeni gen. nov., sp. nov., a novel psychrophilic genus of the family Microbacteriaceae, Int J Syst Bacteriol 50, 355363.

Kaufman, A. E., Goldne, H., Narayan, O. & Gruner, S. M. (1990). Physical studies on the membranes and lipids of plasmalogen­decient Megasphaera elsdenii, Chem Phys Lipids 55, 4148.

Loveland-Curtze, J., Sheridan, P. P., Gutshall, K. R. & Brenchley, J. E. (1999). Biochemical and phylogenetic analyses of psychro­philic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov, Arch Microbiol 171, 355363.

MacKenzie, S. L. (1987). Gas chromatographic analysis of amino acids as the N-heptauorobutyryl isobutyl esters, J Assoc Off Anal Chem 70, 151160.

Maidak, B. L., Cole, J. R., Parker, C. T., Jr & 11 other authors (1999). A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27, 171173.

Männistö , M. K., Tiirola, M. A., Salkinoja-Salonen, M. S., Kulomaa, M. & Puhakka, J. A. (1999). Diversity of chlorophenol­degrading bacteria isolated from contaminated boreal ground­water, Arch Microbiol 171, 189197.

Minnikin, D. E., Collins, M. D. & Goodfellow, M. (1979). Fatty acid and polar lipid composition in the classication of Cellulomonas, Oerskovia and related taxa, J Appl Bacteriol 47, 8795.

Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stacke­brandt, E. (1996). The genus Nocardiopsis represents a phylo­genetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsiaceae fam. nov, Int J Syst Bacteriol 46, 2896.

Sasaki, J., Chijimatsu, M. & Suzuki, K. (1998). Taxonomic signicance of 2,4-diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassication of Clavibacter toxicus as Rathayibacter toxicus comb. nov, Int J Syst Bacteriol 48, 403410.

Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications, Bacteriol Rev 36, 407477.

Schleifer, K. H., Plapp, R. & Kandler, O. (1967). Identication of threo-3-hydroxyglutamic acid in the cell wall of Microbacterium lacticum, Biochem Biophys Res Commun 28, 566570.

Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997).

Proposal for a new hierarchic classication system, Actino­bacteria classis nov, Int J Syst Bacteriol 47, 479491.

Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new prosthecate freshwater bacteria, J Bacteriol 95, 19211942.

Suzuki, K.-I., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. (1997). Cryobacterium psychrophilum gen. nov., sp. nov., nov. rev., comb. nov., an obligately psychrophilic actinomycete to accommodateCurtobacterium psychrophilum ' Inoue and Komagata 1976, Int J Syst Bacteriol 47, 474478.

Vä isä nen, O. M., Nurmiaho-Lassila, E. L., Marmo, S. A. & Salkinoja-Salonen, M. S. (1994). Structure and composition of biological slimes of paper and board machines, Appl Environ Microbiol 60, 641653.

Vasileva, L. V., Saveleva, N. D., Omelchenko, M. V., Lysenko, A. M., Pusheva, M. A., Trotsenko, Y. A. & Zavarzin, G. A. (1998). Arthrobacter crygenae sp. nov. and Acidovorax psychrofacilis sp. nov., psychrophilic hydrogen bacteria from tundra soil, Microbiology 64, 195200.

Verhulst, A., Van Hespen, H., Symons, F. & Eyssen, H. (1987). Systematic analysis of the long-chain components of Eubacterium lentum, J Gen Microbiol 133, 275282.

Wilson, K. (1994). Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology, pp. 2.4.12.4.5. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Wiley.

Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N. & Kalakoutskii, L. V. (1993). Rathayibacter gen. nov., including the species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus comb. nov., and six strains from annual grasses, Int J Syst Bacteriol 43, 143149.

(order Full Text from publisher)

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005