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1 Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK
2 Vakgroep BFM WE 10V Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Correspondence
N. A. Logan
N.A.Logan{at}gcal.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Graphs showing the effects of ionophores on glutamate uptake (Fig. A) and tables giving the fatty acid profiles (Table A) and the effects of various competitors (Table B) and energy inhibitors (Table C) on glutamate uptake in strains LMG 22481T and LMG 22483T are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Two such sites are Mt Melbourne and Mt Rittmann in northern Victoria Land, Antarctica (Fig. 1a). Mt Melbourne (2733 m; 74° 21' S, 164° 42' E) is a stratovolcano situated in the centre of a relatively young (probably 2–3x106 years B.P.) volcanic field that has been formed by a large number of small, individual eruptive centres (Broady et al., 1987) and some of the higher parts of the mountain bear large areas of hot ground and fumaroles. Located on the southern rim of the main summit crater of Mt Melbourne is a Specially Protected Area called Cryptogam Ridge (Fig. 1b). This is a deglaciated site with soil temperatures typically reaching 40–50 °C at depths of a few centimetres; it supports a unique community including algal and bryophyte species unknown elsewhere in Antarctica (Nicolaus et al., 1991). The flora of the geothermally heated area of the north-west slope of the mountain is less well developed than that of Cryptogam Ridge. Mt Rittmann (2600 m; 73° 27' S, 165° 30' E), which was discovered during the 4th Italian Expedition (1988–1989; Armienti & Tripodo, 1991), is located in the Mountaineer Range, about 110 km north of Mt Melbourne. During the 6th Italian Antarctic Expedition (1990–1991), fumaroles and heated ground were discovered in a minor caldera rim of this mountain (Bonaccorso et al., 1991). The soil surface temperature is 34·4–41·5 °C and there is patchy development of vegetation. Although the soils of the two mountains are comparable, Bargagli et al. (1996) showed that there were differences in the mineral contents of soil samples collected from Mt Melbourne (higher Cu and Zn) and Mt Rittmann (higher Cd and Pb). During the 11th and 14th Italian Antarctic Expeditions (1995–1996 and 1998–1999, respectively) samples of heated soils were collected from Cryptogam Ridge, the north-west slope of Mt Melbourne and Mt Rittmann, in order to study the aerobic endospore-forming bacterial floras of these sites (Logan et al., 2000). This report describes the characterization of six strains of a novel Aneurinibacillus species isolated from soils of Cryptogam Ridge and Mt Rittmann and seven strains of a novel Brevibacillus species from the north-west slope of Mt Melbourne. The names Aneurinibacillus terranovensis sp. nov. and Brevibacillus levickii sp. nov. are proposed for these novel taxa.
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). Currently, there are 12 Brevibacillus species and four Aneurinibacillus species. Given their poor reactivity in conventional identification tests, it has been difficult to distinguish between species of these genera (Logan et al., 2002); amplified rDNA restriction analysis is also unable to discriminate between all of the species (Logan et al., 2002). However, some techniques have proved successful, e.g. SDS-PAGE (Logan et al., 2002) and, more recently, sequencing of the hypervariable regions that are conserved within species and diverge between species (Goto et al., 2004). The unique environmental conditions from which these species have been isolated, the restriction of the Brevibacillus strains to the north-west slope of Mt Melbourne and the absence of Aneurinibacillus strains from that site stimulated us to compare their metabolism. The investigation of the metabolic pathways of these organisms could be of value in the characterization of these two species, given their poor reactivities in conventional identification tests. Glutamate uptake and metabolism were studied, given the probable availability of this substrate in these habitats from primary producers such as cyanobacteria and microalgae. This is not only the first metabolic study of this kind for members of these genera, but also for aerobic endospore-forming bacteria from Antarctic geothermal environments. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Strains LMG 22482, R-22010, R-12318, R-22009, R-12317, LMG 22481T and R-22013 (strains Logan B-1598, B-1606, B-1607, B-1608, B-1611, B-1657T and B-1659, respectively) were isolated from the north-west slope of Mt Melbourne. Strains LMG 22483T and R-12871 (strains Logan B-1599T and B-1624, respectively) were isolated from Cryptogam Ridge at Mt Melbourne. Strains R-12872 and R-12873 (strains Logan B-1633 and B-1634, respectively) were isolated from vents of Mt Rittmann and strains R-12874 and LMG 22484 (strains Logan B-1636 and B-1641, respectively) were isolated from the summit of Mt Rittmann.
Cultivation and maintenance of strains.
Strains were initially grown on a variety of media in order to identify which medium supported optimal growth. Media tested included full-strength, half-strength (1/2 BFA) and a quarter-strength version (1/4 BFA) of Bacillus fumarioli agar (BFA; Logan et al., 2000). BFA contained (l–1) 4 g yeast extract, 2·5 g (NH4)2SO4, 3 g KH2PO4, 5 mg MnSO4, 0·2 g MgSO4.7H2O, 0·25 g CaCl2.2H2O and 18 g agar, adjusted to pH 5·5. A defined medium was created, based on BFA and 1/2 BFA, in which the yeast extract and (NH4)2SO4 were omitted and replaced with 1 ml vitamin solution described by Dijkhuizen et al. (1988) and 0·06 g each of 13 L- and DL-amino acids (L-alanine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-isoleucine, L-leucine, L-lysine, L-methionine, DL-norleucine, DL-norvaline, L-threonine and L-valine). The amino acids to be included in this defined medium were determined by creating a broth consisting of all 23 amino acids and then creating subsequent broths in which families of these amino acids or single amino acids were omitted. Those amino acids that were omitted from broths that yielded no growth were deemed as being essential for growth and included in the defined medium. This defined medium was also prepared without the vitamin solution. A variation of Davis and Mingioli minimal medium (Cruickshank et al., 1975) was also used; it contained (l–1) 20 ml sterile 10 % glucose solution, 3·5 g K2HPO4, 1·5 g KH2PO4, 0·5 g sodium citrate (Na3C6H5O7.2H2O), 0·1 g MgSO4.7H2O, 1 g (NH4)2SO4, 20 g agar and 2 g yeast extract. A further variation of this medium lacked both glucose and (NH4)2SO4. The most rapid and profuse growth was achieved through the use of 1/2 BFA at pH 5·5 and 40 °C; this medium was used throughout the remainder of the study along with the broth version, 1/2 BFB (Bacillus fumarioli broth), which lacked agar and MnSO4. A broth version of the defined medium, lacking agar and MnSO4, was used for the metabolic studies.
DNA preparation, DNA base composition and 16S rRNA gene sequencing.
These were carried out as described by Logan et al. (2000).
SDS-PAGE of whole-cell proteins.
Cells were obtained after 48 h growth on 1/2 BFA at 28 °C. The SDS protein extracts were prepared and electrophoresed according to Pot et al. (1994) and data were collected and interpreted as described by Vauterin & Vauterin (1992).
DNA–DNA relatedness.
DNA–DNA hybridization was performed using a modification of the microplate method of Ezaki et al. (1989), as described by Willems et al. (2001).
GC analysis of methylated fatty acids.
Cells were grown for 48 h on 1/2 BFA at 28 °C and subsequently analysed as described by Logan et al. (2000).
Phenotypic analysis.
Isolates were grown on 1/2 BFA at pH 5·5 and 40 °C for 24–72 h and vegetative cells and sporangial morphologies were observed as described by Logan et al. (2000). As colonies of some strains on agar media were tenacious and cohesive, capsule production was investigated using an adaptation of the India ink wet-film method described by Duguid (1951); initially the cells were stained with 1 % rose Bengal and India ink was substituted with 1 % nigrosin. Temperature ranges for growth were determined by incubating the organisms in 10 ml 1/2 BFB in water baths set at 15, 20, 25, 30, 37, 40, 45, 50 and 55 °C. The pH ranges for growth were determined by growing the organisms at 40 °C in 10 ml 1/2 BFB adjusted to pH 3·5, 4·0, 4·5, 5·0, 5·5, 6·0, 6·5 and 7·0 using HCl and NaOH. Turbidities of both series were determined using a spectrophotometer (Cecil CE1010) set at 680 nm, at 24 h intervals. The isolates were tested for anaerobic and microaerobic growth in GasPak jars (BBL) using aerobically grown isolates as controls; a Campylobacter sachet (BBL) was used to create microaerobic conditions. Haemolysis was tested using 5 % horse blood with Columbia Blood Agar base (Oxoid) and 1/2 BFA as a base. Egg-yolk reaction, starch hydrolysis and casein hydrolysis were tested using the methods of Gordon et al. (1973) with 1/2 BFA as the base. Gelatin hydrolysis was determined by aseptically removing the charcoal gelatin from API 20E strips (bioMérieux) and adding them to 1 ml 1/2 BFB before inoculation. Strains were also characterized using the API 20E kit as described by Logan & Berkeley (1984). All isolates were tested for carbon source utilization and for acid or alkali production from the same substrates using the Biotype 100 gallery (bioMérieux), which comprises 99 carbon sources, such as carbohydrates, amino acids and organic acids, and one control tube. 1/2 BFB (200 ml) was inoculated and incubated for 24 h at 40 °C. The cells were then centrifuged at 2000 g for 15 min, the supernatant was discarded, the pellet was resuspended in 10 ml of 10 mM phosphate buffer and recentrifuged (2000 g for 15 min). The resulting supernatant was discarded and the pellet was resuspended in 2 ml phosphate buffer. Drops of this bacterial suspension were added to sterile 0·45 % sodium chloride solution until a turbidity equivalent to 3 on the McFarland scale was achieved. For carbon source utilization testing, this suspension (2 ml) was homogenized in 60 ml Biotype medium 2 (bioMérieux) and for acid or alkaline reactions, 2 ml suspension was homogenized in 60 ml 1/2 BFB prepared without yeast extract or (NH4)2SO4, but containing phenol red at a final concentration of 0·006 %. In order to allow reading of acid and alkaline reactions, and direct comparison with other Aneurinibacillus and Brevibacillus species, the medium was adjusted to pH 7·0, although this lay outside the optimal pH range for the Antarctic isolates. These suspensions were used to inoculate both the tubes and cupules of the Biotype 100 strips. For utilization tests, results were read at 24 h intervals according to the manufacturer's instructions; for most substrates, utilization was indicated by turbidity. For acid or alkali production, results were read at 24 h intervals for 96 h with a yellow colour reaction scored as positive for acid reactions and a violet colour scored as positive for alkaline reactions. It was noted, however, that several tubes showed reactions immediately upon inoculation – two tubes produced alkaline reactions and several other tubes, mostly containing organic acids, gave acid reactions; these were not regarded as evidence of substrate utilization.
Metabolic studies.
A standard glutamate transport assay was performed on strains LMG 22483T and LMG 22481T to determine the uptake of glutamate under normal conditions. 1/2 BFB (200 ml) was inoculated and incubated for 24 h at 40 °C in an orbital incubator. Cells were then centrifuged at 2000 g for 15 min, the supernatant was discarded, and the pellet was resuspended in 10 ml of 10 mM phosphate buffer (KH2PO4/K2HPO4, pH 7) and recentrifuged (2000 g for 15 min). The resulting supernatant was discarded and the pellet was resuspended in 2 ml phosphate buffer. Defined medium (200 ml) was inoculated with this suspension (0·3 ml) and incubated at 40 °C for 17 h. Cells were harvested as before and resuspended in 2 ml phosphate buffer. Aliquots (800 µl) of the cell suspension were incubated with 195 µl phosphate buffer for 5 min at 40 °C and 5 µl of 0·05 µCi (1·85 kBq) [14C]L-glutamic acid (10 mM) was introduced to initiate transport measurement. Samples (150 µl) were taken from the 1 ml assay mixture at 30 s intervals for 150 s and cells were immediately filtered on glass microfibre GF/B discs (Whatman) and washed in 2x10 ml cold phosphate buffer. The filters were then dried and the 14C in the samples was assayed by liquid scintillation counting using 4 ml scintillation fluid (Optiscint HiSafe; Perkin Elmer).
Competition experiments were carried out using unlabelled amino acids (L-glutamic acid, D-glutamic acid, L-proline, L-arginine and DL-ornithine) to determine whether the uptake system used by the strains was specific for L-glutamate or was also involved in the uptake of other, metabolically related amino acids. To create a 50-fold molar excess of each amino acid, in comparison to [14C]L-glutamic acid, 100 µl of each amino acid (25 mM) with 95 µl phosphate buffer was introduced to the cell suspension and incubated for 5 min before adding [14C]L-glutamic acid. Secondary competition experiments involved testing the uptake of [14C]arginine and [14C]proline (0·05 µCi) in the presence of a 50-fold molar excess of unlabelled L-glutamic acid. The same volumes of amino acids, radiolabelled substrate and phosphate buffer were used as before and the cells were pre-grown in the presence of L-glutamic acid (to induce uptake).
To determine whether Na+ or K+ channels were involved in glutamate uptake, 1 ml assay mixtures were prepared with valinomycin (K+ ionophore), monensin (Na+ ionophore) or nigericin (K+/Na+ ionophore) (final concentrations of 10, 0·1 and 10 µM, respectively). The ionophores, 10 µl of 1 mM valinomycin (dissolved in DMSO), 10 µl of 10 µM monensin (dissolved in DMSO) or 10 µl of 1 mM nigericin (dissolved in ethanol), respectively, were introduced and incubated for 5 min before the addition of [14C]L-glutamic acid. Controls containing only 10 µl DMSO or 10 µl ethanol were used to ensure that the ionophores were the only differing factors in the experiments.
To create artificial Na+, K+ and H+ ion gradients, NaCl, KCl or HCl, respectively, were introduced to create final concentrations of 50 or 100 mM in the 1 ml assay mixtures, 5 min prior to the addition of [14C]L-glutamic acid. To determine whether glutamate uptake is linked to an osmotic effect, sucrose was introduced to create final concentrations of 50 and 100 mM in the 1 ml working suspensions; the resulting mixtures were incubated for 5 min before adding [14C]L-glutamic acid.
Energy inhibitors were employed to identify the system used by the micro-organisms for glutamate uptake. Sodium fluoride (glycolysis inhibitor), dinitrophenol (respiration uncoupler) or sodium arsenate (ATP synthesis inhibitor) were introduced to the 1 ml working stock to give 10 mM final concentrations and incubated for 5 min before adding [14C]L-glutamic acid. Iodoacetic acid (glycolysis inhibitor) or N-ethylmaleimide p-chloromercuribenzoate (S-H reagent, in ethanol) were introduced to the cell suspension to give 5 mM final concentrations and incubated for 5 min before adding [14C]L-glutamic acid. Carbonyl cyanide m-chlorophenyl hydrazone [respiration uncoupler (collapses ion gradients), in ethanol] or N-N-dicyclohexylcarbodiimide (ATPase inhibitor, in ethanol) were introduced to the cell suspension to give 0·1 mM final concentrations and incubated for 5 min before adding [14C]L-glutamic acid. An ethanol control was used to ensure that the energy inhibitors were the only differing factors in the experiment.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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16S rRNA gene sequencing
The nearly complete 16S rRNA gene sequences of all 13 strains were analysed. Clustering of the obtained sequences revealed two groups (Fig. 2): one containing strains isolated from the north-west slope of Mt Melbourne and one containing strains isolated from Cryptogam Ridge at Mt Melbourne, as well as the Mt Rittmann isolates. Both groups show high internal similarity (pairwise similarities of 99·7–99·9 and 99·9–100·0 %, respectively). According to a FASTA search (Pearson & Lipman, 1988), strains belonging to the first group show highest sequence similarity to the Brevibacillus borstelensis type strain (97·0–97·2 % similarity), but are distinct from it in SDS-PAGE analysis (see below). The sequence similarity to all other Brevibacillus species is well below 97·0 %. These low sequence similarities, on the borderline of species delineation (Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002), together with the specific growth conditions of the strains suggest that the strains possibly represent a novel Brevibacillus species. DNA–DNA relatedness measurements confirmed this suggestion, with Antarctic strain LMG 22841T and Brevibacillus borstelensis LMG 16009T showing reciprocal DNA–DNA association values of 18·3 and 18·9 %. These strains are subsequently referred to as Brevibacillus levickii sp. nov. (Fig. 2). Based on 16S rRNA gene sequences, the second group of strains is most closely related to members of the genus Aneurinibacillus (sequence similarities of 95–96 % according to a FASTA search) and forms a tight cluster with them (Fig. 2). According to these sequence similarities (<97 %), the strains can be attributed to a novel Aneurinibacillus genospecies (Stackebrandt et al., 2002); these strains are subsequently referred to as Aneurinibacillus terranovensis sp. nov. (Fig. 2).
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) also enabled discrimination of the 13 isolates in the two groups revealed by 16S rRNA gene sequence analysis. The Brevibacillus levickii strains grouped at 93–98 % similarity and the patterns represent members of a distinct and homogeneous taxon. Furthermore, the SDS-PAGE profile of Brevibacillus levickii was clearly different from that of the type strain of Brevibacillus borstelensis (below 65 % similarity; data not shown), the species with which the strains share 97·0–97·2 % 16S rRNA gene sequence similarity. The A. terranovensis strains grouped at 93–99 % similarity and the patterns represent members of a distinct and homogeneous taxon. The two clusters merged at a low and insignificant similarity, indicating that the taxa are not closely related.
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) and thus the medium could be responsible for the observed differences. The resulting profiles of the Brevibacillus levickii strains showed high amounts of anteiso-C15 : 0 (mean of 74·5 %), whereas A. terranovensis has high amounts of iso-C15 : 0 and anteiso-C15 : 0 (Supplementary Table A).
Phenotypic characterization
The strains were Gram-positive motile rods that lost the positive reaction of the Gram stain in 24–48 h and formed ellipsoidal spores in swollen sporangia (Fig. 4a, b). On 1/2 BFA plates, the Brevibacillus strains formed tough, adherent colonies, whereas colonies of the Aneurinibacillus strains were butyrous. The A. terranovensis and Brevibacillus levickii strains showed many phenotypic similarities and few biochemical characters useful for differentiation. Their reactions in the various tests are summarized in the species descriptions below and in Table 1 and Table 2. Sporangial morphologies were notably different between the species: sporangia of A. terranovensis were greatly swollen by central to subterminal spores, whereas those of Brevibacillus levickii were less swollen with subterminal and terminal spores (Fig. 4a, b). Strains of both species appeared pleomorphic and filamentous when grown in 1/2 BFB, whereas cultivation in defined medium generated cells of regular appearance. All strains were weakly positive for catalase and grew well in aerobic conditions but, as explained above, outgrowth was poor so restreaking on the same plate was necessary in order to obtain satisfactory growth; such outgrowth problems were not seen with plates incubated in microaerobic conditions. This is consistent with the observation that growth in broth media was always more abundant than growth on their solid counterparts. Although agar colonies of Brevibacillus were adherent and difficult to emulsify in liquid media, capsule production could not be demonstrated. Strains of both species showed negative or weak reactions in many of the phenotypic tests; for example, no hydrolysis was observed around colonies on starch agar and weak reactions only became apparent after the colonies had been scraped from the surface of the medium. In the API 20E tests, the two species could be distinguished from each other by the gelatin reaction; a reaction was evident after 48 h for Brevibacillus levickii, whereas no reaction was observed in A. terranovensis. However, when the charcoal gelatin was aseptically removed from the API 20E tube and introduced into 1/2 BFB, gelatin was hydrolysed by Brevibacillus levickii within 24 h and by A. terranovensis after 24 h, but within 48 h. Both species may also be separated on the basis of reaction intensities in the API 20E strip, with stronger reactions for arginine dihydrolase, Voges–Proskauer and nitrate reduction being observed for A. terranovensis and for citrate utilization in Brevibacillus levickii. Positive reactions for arginine dihydrolase in the API 20E strip distinguish these species from other members of both genera that have been tested. In the Biotype 100 utilization and acid/alkali production tests, both species demonstrated rapid performance, with substrate utilization (turbidity or acid/alkaline reaction) becoming evident within 24–48 h incubation. The results produced by the utilization tests were inconsistent within species, especially for the Aneurinibacillus strains; this problem has been seen with other groups of aerobic endospore-formers (Heyrman et al., 2003). The results obtained do, however, indicate that Brevibacillus strains are capable of utilizing a wide range of carbon sources and that the Aneurinibacillus strains utilize a more limited and less consistent range of substrates. The results of the acid/alkali production tests indicated that both species utilize similar kinds of carbon sources, mainly organic acids and amino acids; however, as noted above, these tests were done outside the optimal pH ranges of these organisms.
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). Glutamate is non-essential, however, as the organisms grew on a formulation of the defined medium from which it had been omitted. Both species appear to use similar glutamate uptake systems, as experimental outcomes were similar. When grown in the presence of a mixture of amino acids, Brevibacillus levickii strain LMG 22481T demonstrated uptake of [14C]L-glutamic acid at a mean rate of 0·105 nM s–1 mg–1, whereas A. terranovensis strain LMG 22483T demonstrated uptake at a mean rate of 0·0044 nM s–1 mg–1, over 150 s (Fig. 5). Perhaps Brevibacillus levickii has a higher affinity uptake system for glutamate on account of nutritional limitations in its habitat. The rate of uptake in the Brevibacillus levickii strain is greater than that of Bacillus sp. strain TA2.A1, which was isolated from a thermal spring and had an L-glutamate uptake of 0·0117 nM s–1 mg–1 in an external glutamate concentration of 50 µM (Peddie et al., 1999). In comparison, the A. terranovensis strain had a slower rate of uptake than that of Bacillus sp. strain TA2.A1. The uptake system of the Brevibacillus levickii strain appears to be specific for the L-glutamate isomer; this is evident from the fact that D-glutamate caused no inhibition of [14c]L-glutamate uptake and that the other amino acids related to L-glutamate (L-proline, L-arginine and DL-ornithine) were fourfold less efficient in inhibiting uptake of [14c]L-glutamate than unlabelled L-glutamate, despite being present in excess. This conclusion is supported by the observation that [14C]L-proline and L-arginine uptakes were not inhibited by an excess of L-glutamate, which implies that there are separate uptake systems for glutamate, proline and arginine (results not shown). The uptake system of the A. terranovensis strain appeared not to be specific for the L-glutamate isomer, but could transport most of the other related amino acids; the exceptions were L-proline, which caused partial inhibition, and D-glutamate, which caused no inhibition when present in excess (Supplementary Table B in IJSEM Online). No inhibition of [14C]L-proline or [14c]L-arginine uptake was observed in A. terranovensis pre-grown with L-glutamic acid when in the presence of an excess of unlabelled L-glutamic acid, thus implying that separate uptake systems for proline and arginine exist (results not shown). In Brevibacillus levickii, the ionophores nigericin and valinomycin decreased [14C]L-glutamic acid uptake two- and tenfold, respectively. The addition of monensin, however, did not cause any decrease in the rate of uptake (Supplementary Fig. Aa in IJSEM Online). In A. terranovensis, the addition of nigericin resulted in a 40-fold decrease in the rate of [14C]L-glutamic acid uptake and valinomycin caused complete inhibition. Very little inhibition (less than twofold) was observed in the presence of monensin (Supplementary Fig. Ab). Glutamate uptake inhibition by the ionophores valinomycin and nigericin implies that a K+ gradient is involved. However, slight inhibition by monensin in the A. terranovensis strain suggests that a charge gradient may also be involved in the uptake of glutamate in this species. In both strains, the rate of [14C]L-glutamic acid uptake in pre-grown cells decreased twofold in the presence of NaCl at 50 mM. In the presence of KCl at 50 mM, the rate of uptake in Brevibacillus levickii decreased fourfold and in A. terranovensis decreased eightfold. In the presence of HCl at 50 mM, cell death occurred in both strains (results not shown). The creation of an artificial K+ gradient resulted in a decrease in the rate of glutamate uptake in both species, thus suggesting that the uptake mechanism is a K+ antiport system, with the increased K+ concentration outside the cell inhibiting K+ export from inside the cell, hence resulting in decreased glutamate uptake. This is of particular interest given that previous studies on glutamate uptake metabolism in other thermophilic bacteria have shown it to be driven by Na+ gradients; Geobacillus stearothermophilus, for example, utilizes a sodium/proton symport mechanism (de Vrij et al., 1990). In the presence of 50 mM sucrose, pre-grown cells of both strains showed no decrease in the rate of [14C]L-glutamic acid uptake (data not shown). This lack of inhibition by sucrose demonstrates that the uptake of glutamate in both species is not osmotically sensitive. For both strains, high levels of inhibition of [14C]L-glutamic acid uptake occurred through the addition of the energy inhibitors dinitrophenol, iodoacetic acid, N-ethylmaleimide p-chloromercuribenzoate, carbonyl cyanide m-chlorophenyl hydrazone and N-N-dicyclohexylcarbodiimide (Supplementary Table C in IJSEM Online). Inhibition of [14C]L-glutamate uptake by energy inhibitors implies the presence of an active transport system for glutamate. Almost complete inhibition of glutamate uptake in both species by N-N-dicyclohexylcarbodiimide, an inhibitor of ATP synthesis, and iodoacetic acid, a glycolysis inhibitor, suggests a role for ATP in uptake. However, NaAsO4 and NaF, both of which directly inhibit ATP generation, cause only 10 % inhibition of uptake in Brevibacillus levickii strain LMG 22481T, thus suggesting that the system does not require ATP directly. This would imply that the mechanism of uptake is not a primary uptake system (directly coupled to ATP hydrolysis), but more likely a secondary transport system that utilizes a gradient. A similar system is probably utilized by A. terranovensis. However, a significant level of inhibition is apparent in the presence of sodium arsenate (Supplementary Table C). Many bacteria can utilize ATP to generate a transmembrane proton-motive force, directly by respiration, which can be coupled to solute uptake. A role for such a proton-motive force in the Brevibacillus and Aneurinibacillus strains is also implied by the inhibition of glutamate uptake by the ionophores. Collectively, the metabolic data indicate that the mechanism of glutamate uptake in Brevibacillus levickii is an energy-dependent, glutamate-specific K+ antiport system, whereas in A. terranovensis it is an energy-dependent K+ antiport system capable of transporting glutamate and other related amino acids.
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to isolate Bacillus fumarioli from the north-west slope of Mt Melbourne, although it could be cultivated from Cryptogam Ridge and Mt Rittmann. Although the soil and environmental conditions at each isolation site are superficially similar, our findings clearly imply that there are some important differences between the soil of the north-west slope of Mt Melbourne and those of other sites. Bargagli et al. (1996) found that although mean element concentrations from the fumarolic areas of the two mountains were broadly similar, there were some differences in trace element contents. Soils from Mt Melbourne had higher Cu and Zn contents, whereas soils from Mt Rittmann had higher Cd and Pb; no data for the north-west slope of Mt Melbourne have yet been reported. A notable feature of the north-west slope of Mt Melbourne is the near-absence of moss (Broady et al., 1987); this may indicate that this soil contains substances inhibitory to the mosses that otherwise grow so abundantly on the nearby Cryptogam Ridge, or else the soil lacks one or more essential nutrients. Such conditions may also affect the aerobic endospore-forming floras in these soils, and the paucity of moss growth on the north-west slope may limit the organic substrates available to heterotrophic bacteria. Our failure to identify our Antarctic strains by the genotypic and phenotypic methods applied and the similarities of the members of each group of strains to each other in these analyses support the proposal of two novel species; their descriptions are given below.
Description of Brevibacillus levickii sp. nov.
Brevibacillus levickii (le.vic.ki'i. N.L. adj. levickii of Levick, named after G. Murray Levick, surgeon and biological scientist of Captain R. F. Scott's Northern Party, the first scientific expedition to visit the vicinity of Mt Melbourne in 1912).
Cells are Gram-positive, becoming Gram-negative after 48 h, motile, round-ended rods (0·7–0·8x2·0–5·0 µm) occurring singly, in pairs and in chains. Endospores are ellipsoidal, occurring subterminally or terminally in swollen or slightly swollen sporangia (Fig. 4a
). After 48 h incubation at 40 °C on 1/2 BFA (pH 5·5), colonies are circular, flat, up to 3·0 mm in diameter and cream-coloured with a matt appearance. Colony consistency becomes tough and difficult to break with a loop. Minimum growth temperature lies between 15 and 20 °C, with optimum growth temperature of 40–45 °C and maximum growth temperature of 50–55 °C. Growth occurs between pH 4·5 and 6·5 and the optimum pH for growth is pH 5·0–5·5. Growth is inhibited by 2–3 % NaCl. The organisms are microaerophilic and weakly catalase-positive. Horse blood agar is partially haemolysed. Gelatin is hydrolysed in 24 h and starch hydrolysis is weakly positive. Casein hydrolysis is weakly positive or negative. In the API 20E strip reactions, arginine dihydrolase, citrate utilization and the Voges–Proskauer reaction are positive. Gelatin is hydrolysed and nitrate reduction is variable. Reactions for ONPG hydrolysis, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide production, urease, tryptophan deaminase and indole production are negative. The following carbon sources are utilized in the API Biotype 100 system: 
-D-glucose, adonitol, aesculin, -lactose, D-alanine, L-alanine, L-arabitol, L-aspartate, 
-D-fructose, cis-aconitate, citrate, fumarate, D-galacturonate, D-gluconate, D-glucuronate, L-glutamate, DL-glycerate, glycerol, 2-keto-D-gluconate, 5-keto-D-gluconate, DL-lactate, D-malate, L-malate, maltose, maltotriose, D-mannitol, D-mannose, meso-tartrate, 1-O-methyl--galactopyranoside, 1-O-methyl--D-glucopyranoside, N-acetyl-D-glucosamine, L-proline, putrescine, quinate, L-serine, D-sorbitol, succinate, sucrose and trans-aconitate. Utilization of the following substrates is variable: DL--amino-n-butyrate, 2-oxoglutarate, D-galactose, -gentobiose, mucate, D-ribose, D-saccharate, D-trehalose and L-tryptophan. The following carbon sources are not utilized: i-erythritol, DL--amino-n-valerate, -D-melibiose, L-arabinose, D-arabitol, benzoate, betaine, DL--hydroxybutyrate, caprate, caprylate, D-cellobiose, dulcitol, ethanolamine, -L-fucose, gentisate, D-glucosamine, glutarate, histamine, L-histidine, hydroxyquinoline--glucuronide, itaconate, lactulose, D-lyxose, malonate, maltitol, m-coumarate, D-melezitose, 1-O-methyl--D-glucopyranoside, 3-O-methyl-D-glucopyranose, 1-O-methyl--galactopyranoside, m-hydroxybenzoate, myo-inositol, palatinose, phenylacetate, 3-phenylpropionate, p-hydroxybenzoate, propionate, protocatechuate, D-raffinose, -L-rhamnose, L-sorbose, D-tagatose, D-tartrate, L-tartrate, tricarballyate, trigonelline, tryptamine, D-turanose, L-tyrosine, xylitol and D-xylose. The following substrates in API Biotype 100 produce alkaline reactions when inoculated with a suspension in 1/2 BFB prepared without yeast extract or (NH4)2SO4, but containing 0·006 % phenol red: 2-oxoglutarate, L-aspartate, fumarate, D-galacturonate, D-gluconate, L-glutamate, D-malate, L-malate, malonate, mucate, quinate, D-saccharate, succinate, D-tartrate, L-tartrate and tricarballyate. Alkaline reactions for the following substrates are variable: 2-keto-D-gluconate, L-alanine, caprylate, cis-aconitate, citrate, D-glucuronate, glutarate, DL-glycerate, L-histidine, itaconate, DL-lactate, meso-tartrate, propionate, L-serine, trans-aconitate and L-tryptophan.
The major cellular fatty acid is anteiso-C15 : 0, accounting for approximately 74 % total fatty acid content. The following fatty acids are present in smaller amounts (at least 1 %): iso-C14 : 0, iso-C15 : 0, C16 : 0, iso-C16 : 0, summed feature 4 (iso-C17 : 1 and/or anteiso-C17 : 1) and anteiso-C17 : 0 (detailed fatty acid data are given in Supplementary Table A).
The DNA G+C content is 48·3–50·3 mol% and the G+C content of the type strain (Logan B-1657T=LMG 22481T=CIP 108307T) is 50·3 mol%. In the variable reactions listed above, the type strain is weakly positive for casein hydrolysis and it utilizes DL--amino-n-butyrate, 2-oxoglutarate, D-galactose, D-ribose, D-trehalose and L-tryptophan; alkaline reactions are observed for D-glucuronate, itaconate, 2-keto-D-gluconate, meso-tartrate and L-tryptophan.
Description of Aneurinibacillus terranovensis sp. nov.
Aneurinibacillus terranovensis [terr.a.no.ven'sis. N.L. adj. terranovensis referring to Terra Nova Bay Station (Italy), northern Victoria Land, Antarctica, where the strains were first isolated].
Cells are Gram-positive, becoming Gram-negative after 48 h, motile, round-ended rods (0·8–1·0x2·0–8·0 µm), occurring singly, in pairs and in chains. Endospores are ellipsoidal, occurring paracentrally and subterminally in very swollen sporangia (Fig. 4b). After 48 h incubation on 1/2 BFA (pH 5·5) at 40 °C, colonies are circular, flat, up to 1·5 mm in diameter and cream-coloured with a slightly glossy appearance and butyrous consistency. Minimum growth temperature is 20–25 °C, with optimum growth at 37–45 °C and maximum growth temperature of 50–55 °C. Growth occurs between pH 3·5 and 6·0 and the optimum pH for growth is pH 5·0–5·5. Growth is inhibited by 2–3 % NaCl. Microaerophilic and weakly catalase-positive. Gelatin is hydrolysed after 48 h. Starch is weakly hydrolysed. No growth occurs on casein agar. In the API 20E strip reactions, arginine dihydrolase, the Voges–Proskauer reaction and nitrate reduction are positive. Citrate utilization is variable. Reactions for ONPG hydrolysis, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide production, urease, tryptophan deaminase, indole production and gelatin hydrolysis are negative. The following carbon sources are utilized in the Biotype 100 system: aesculin, -D-fructose, D-gluconate, -D-glucose, L-glutamate, glycerol, DL-lactate, D-mannose, 1-O-methyl--D-glucopyranoside and myo-inositol. Utilization of the following substrates is variable: adonitol, 2-oxoglutarate, -lactose, L-alanine, D-arabitol, L-arabitol, L-aspartate, DL--hydroxybutyrate, cis-aconitate, citrate, fumarate, D-galactose, D-galacturonate, D-glucosamine, DL-glycerate, 2-keto-D-gluconate, 5-keto-D-gluconate, D-malate, L-malate, D-mannitol, meso-tartrate, 1-O-methyl--galactopyranoside, N-acetyl-D-glucosamine, L-proline, putrescine, quinate, D-ribose, D-sorbitol, succinate, sucrose, trans-aconitate and D-trehalose. The following carbon sources are not utilized: 3-phenylpropionate, DL--amino-n-butyrate, DL--amino-n-valerate, -D-melibiose, D-alanine, -L-fucose, -L-rhamnose, L-arabinose, benzoate, betaine, -gentobiose, caprate, caprylate, D-cellobiose, dulcitol, ethanolamine, gentisate, D-glucuronate, glutarate, histamine, L-histidine, hydroxyquinoline--glucuronide, i-erythritol, itaconate, lactulose, D-lyxose, malonate, maltitol, maltose, maltotriose, m-coumarate, D-melezitose, 1-O-methyl--D-glucopyranoside, 1-O-methyl--galactopyranoside, 3-O-methyl-D-glucopyranose, m-hydroxybenzoate, mucate, phenylacetate, p-hydroxybenzoate, palatinose, propionate, protocatechuate, D-raffinose, D-saccharate, L-serine, L-sorbose, D-tagatose, D-tartrate, L-tartrate, tricarballyate, trigonelline, tryptamine, L-tryptophan, D-turanose, L-tyrosine, xylitol and D-xylose. The following substrates in API Biotype 100 produce alkaline reactions when inoculated with a suspension in 1/2 BFB prepared without yeast extract or (NH4)2SO4, but containing 0·006 % phenol red: 2-oxoglutarate, L-aspartate, cis-aconitate, citrate, fumarate, D-galacturonate, D-gluconate, L-glutamate, glutarate, DL-glycerate, malonate, mucate, quinate, D-saccharate, succinate, L-tartrate and D-tartrate. Alkaline reactions for the following substrates are variable: D-alanine, L-alanine, DL--hydroxybutyrate, caprylate, D-glucuronate, L-histidine, DL-lactate, D-malate, L-malate, meso-tartrate, L-proline, propionate, L-serine, trans-aconitate, tricarballyate and L-tryptophan.
The major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0, accounting for approximately 41 and 46 % total fatty acid content, respectively. The following fatty acids are present in smaller amounts (at least 1·0 %): C14 : 0, iso-C14 : 0, C16 : 0, iso-C16 : 0 and C16 : 1
7c alcohol (detailed fatty acid are given in Supplementary Table A).
The G+C content is 43·2–44·6 mol% and the G+C content of the type strain (Logan B-1599T=LMG 22483T=CIP 108308T) is 43·2 mol%. In the variable reactions listed above, the type strain utilizes citrate, fumarate, D-galactose, D-glucosamine, -lactose, L-malate, quinate and trans-aconitate; alkaline reactions are observed in DL-lactate, D-malate, L-malate and trans-aconitate.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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