INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Low environmental temperatures characterize the habitat of many prokaryotes living in marine sediments, since 90% of the sea floor has a temperature of less than 4°C (15). While prokaryotic activity is commonly found to be lower during cold seasons in temperate environments (for a review, see reference 23), the current data suggest that in permanently cold habitats bacterial activity is comparable to that in temperate environments at the respective ambient temperature (2, 9, 23, 27). Arnosti et al. (2) determined the temperature dependence of microbial degradation of organic matter and showed that carbon turnover in the cold Arctic is not intrinsically slower than in temperate environments. Also, Sagemann et al. (27) and Glud et al. (9) found rates of sulfate reduction and benthic carbon mineralization in Arctic sediments to be comparable to those in temperate or even tropical sediments. Optimal temperatures for polysaccharide hydrolysis, oxygen consumption (2), and sulfate reduction (27) in permanently cold sediments were significantly higher than the ambient temperature; however, the relative activity at a low in situ temperature compared to optimum activity was generally higher than in samples from temperate habitats. These observations indicate that the bacterial community in these Arctic sediments is adapted to cold temperature. However, little is known about the diversity and composition of prokaryotic communities in cold marine sediments; only a few cold-adapted psychrophilic isolates from these environments have been studied so far (for a review, see reference 26). In addition, few cultivation-independent studies have been conducted in these habitats (28).

The aims of our project, conducted in the context of the above-mentioned studies, were to characterize the sulfate-reducing bacterial community of permanently cold habitats and to quantify the abundance of psychrophilic sulfate reducers. We chose two sites, off the coast of Spitsbergen (Hornsund and Storfjord), which are never exposed to temperatures higher than 3°C. We concentrated on sulfate-reducing prokaryotes because sulfate reduction is a major process of carbon mineralization in marine sediments (11). A set of probes is available for the main phylogenetic groups of gram-negative mesophilic sulfate reducers (7), and the different phylogenetic groups can be defined by distinct physiological features (35, 36). In a related study, most probable number (MPN) counts were determined and psychrophilic sulfate reducers were isolated to enumerate and identify the sulfate-reducing bacteria (SRB) (12). A new oligonucleotide probe was designed to target the largest cluster of these isolates. This newly developed probe, along with an established set of probes, was applied to quantify sulfate reducer rRNA in the sediment. The presence of the isolates was further evaluated by denaturing gradient gel electrophoresis (DGGE) analysis. The results of this study will be discussed in relation to data from a 16S ribosomal DNA (rDNA) clone library presented in an accompanying paper (22).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Study site and sampling procedure. Our study was conducted as part of a research cruise in the Arctic Sea from Tromsø (northern Norway) to Spitsbergen (Arctic Ocean) in September and October of 1995. Sediments from two different stations (Hornsund [76°58.2'N, 15°34.5'E] and Storfjord [77°33.0'N, 19°05.5'E]) were investigated. In situ temperatures and depths were 2.6°C and 155 m for Hornsund and 1.7°C and 175 m for Storfjord. Sediment samples were collected with a multicorer. Samples for MPN dilutions (12) and molecular analysis were taken from the same core. The individual subcores (our replicates A and B) derived from two different multicorer cores. The sediments were anoxic below a depth of approximately 8 mm (9). Five distinct vertical horizons of 2 to 3 cm in thickness were sectioned from the upper 30 cm of each core. The sediment of each section was carefully homogenized, and subsamples of 1 or 2 cm³ were immediately frozen in liquid N₂.

DNA extraction and amplification of 16S rDNA. After three cycles of freezing and thawing, DNAs were extracted directly by the method of Zhou et al. (38), which is based on lysis with a high-salt extraction buffer and extended heating in the presence of sodium dodecyl sulfate and hexadecyltrimethylammonium bromide. Lysis efficiency was checked by DAPI (4',6-diamidino-2-phenylindole) staining. In general, at least 90 to 96% of the cells were lysed. The DNA could be used for PCR without further purification. Primers GM5clamp (Escherichia coli positions 341 to 357) and 907R (19) were used to amplify variable regions V3 to V5 of the 16S rDNA in a touchdown PCR, as described by Buchholz-Cleven et al. (5). To amplify the nearly complete 16S rDNA, primers 8F and 1492R (5) were used in a 35-cycle PCR with an annealing temperature of 40°C. Bovine serum albumin (final concentration, 3 mg ml¹) was added routinely to the PCR mixtures to prevent interference by humic acids (24).

16S rDNA sequencing. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). A Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.) was used to directly sequence the purified PCR products. Sequencing reactions were analyzed on an Applied Biosystems model 373S DNA sequencer. Both strands of the amplification products were sequenced with primers 8F, 787F, 787R, 1175R, 1099F, and 1492R (5). Primer nomenclature refers to 5' ends of the respective target sites on the 16S rDNA according to the E. coli numbering of 16S rRNA nucleotides (4).

Phylogenetic analysis. The ARB program package and the ARB database (34) were used for phylogenetic analysis. Sequences were aligned to the 16S rRNA primary structures present in the ARB database by using the automatic aligner tool, and the results were corrected manually where necessary. Pairwise distance matrix analysis was performed with the 16S rRNA sequences by considering only those positions that were present in both sequences. Phylogenetic trees were reconstructed for all available sequences from the  subclass of Proteobacteria, and a selection of representatives of major groups outside this subclass was used as an outgroup. Only sequences with at least 1,350 nucleotides were used. Tree topology was evaluated by using neighbor-joining, maximum-parsimony, and maximum-likelihood algorithms on the full set of data or on a subset. Furthermore, we applied filters that excluded positions with less than 50% conservation within the  subclass. Branching orders that were not supported by all methods are shown as multifurcation (Fig. 1).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Phylogenetic affiliations of psychrophilic sulfate-reducing isolates and specificities of probes used in this study. The tree shows members of the  subclass of Proteobacteria and was constructed by using the neighbor-joining algorithm and a 50% conservation filter. The arcs comprise the respective probe target groups. Sval428 is described in this study. Other probes are as described by Devereux et al. (7). Strains isolated from Svalbard sediments are shown in boldface type. Strain names indicate the carbon sources on which the strains were isolated (A, acetate; L, lactate; P, propionate; and B, betaine) and the sampling site (Sv2 indicates Hornsund and Sv5 indicates Storfjord).

Oligonucleotide probes. Oligonucleotides were purchased from Biometra (Göttingen, Germany). The probe target sequence of Sval428 is 5' GTAAAATCCTGTCAGATGG 3' (E. coli positions 428 to 446). Probes used and their specificities are shown in Fig. 1.

RNA extraction and slot blot hybridization. Nucleic acids were isolated directly by bead beating, phenol extraction, and isopropanol precipitation as described by Sahm and Berniger (28). Between 10 and 100 ng of RNA was blotted on nylon membranes (Magna Charge; Micron Seperations, Westborough, Mass.) in triplicate and probed with radioactively labeled oligonucleotides as described previously (33). Membranes were prehybridized at 40°C and washed at different temperatures depending on the dissociation temperature (T_d) of the probe as follows: 54°C (EUB338 [1]), 45°C (687 [7]), 59°C (660 [7]), 46°C (804 [7]), or 52°C (Sval428). The T_d for probe Sval428 was determined according to the method of Raskin et al. (21), with rRNA from strain LSv20 being used as one mismatch control. Intensity of hybridization signal was measured with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) and quantified as described by Sahm et al. (29) with the program ImageQuant (Molecular Dynamics). rRNA isolated from Desulfovibrio salexigens (DSM 2638), Desulfobulbus elongatus (DSM 2908), Desulfococcus multivorans (DSM 2059), and strain LSv23 served as standards for hybridization with the probes specific for sulfate reducers.

DGGE and Southern hybridization analysis. DGGE was performed on a D-Gene system (Bio-Rad, Munich, Germany) as described previously (17, 18). PCR products were analyzed directly on a 1-mm-thick 6% polyacrylamide gel containing a denaturing gradient from 20 to 80%. Electrophoresis with 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA [pH 8]) was performed at 100 V for 20 h. After electrophoresis, the gels were stained in ethidium bromide and photographed on a UV transilluminator. DGGE gels were blotted onto nylon membranes via electroblotting as described by Muyzer et al. (18). Hybridization analysis was performed with probe Sval428 by the protocol described by Santegoeds et al. (30). The probe was end labeled with [-³²P]ATP, and the membrane was hybridized at 40°C overnight. Stringent washes were performed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% (wt/vol) sodium dodecyl sulfate at the previously determined T_d of 56°C. The T_d of the probe was determined by the method described by Raskin et al. (21) for DNA and RNA targets. The hybridized membranes were sealed in plastic bags and exposed for 1 to 7 days on an X-ray film or a PhosphorImager screen.

Nucleotide sequence accession numbers. The 16S rDNA sequences have been deposited in the GenBank database. Accession numbers are AF099054 to AF099065 and AF136008.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Phylogenetic affiliation of isolates. Psychrophilic sulfate reducers isolated from MPN enrichments (12) were phylogenetically analyzed by 16S rDNA sequencing. All isolates belonged to the  subclass of the Proteobacteria, as is the case for the majority of mesophilic sulfate reducers (32). Adaptation to cold temperatures as represented by our sulfate-reducing isolates was widely spread within this phylogenetic group (Fig. 1). On the basis of their phylogenetic distance, strains ASv25, BSv41, and LSv20 belong to the existing genera Desulfobacter, Desulfobacterium, and Desulforhopalus (3.6, 1.9, and 0.7% phylogenetic distance, respectively) (Fig. 1). The remaining 10 strains were only distantly related (phylogenetic distance, 6.4 to 11.6%) to known sulfate reducers. Five of these have recently been described as members of three new genera (13). The results suggest that psychrophily is a polyphyletic property within gram-negative sulfate reducers. Furthermore, the wide distribution of our isolates within the -Proteobacteria indicates that the diversity among psychrophilic SRB might be as high as among mesophiles.

Probe design. Since cultivation is inherently selective and often leads to the isolation of quantitatively less-important microbial groups, we estimated the abundance of these isolates by a cultivation-independent method, i.e., rRNA slot blot hybridization. Besides LSv55, all strains that were related to the Desulfobacteriaceae were targeted by an already existing probe (probe 804) designed by Devereux et al. (7). Of the remaining strains, six incomplete oxidizers formed a cluster related to Desulfocapsa sp., Desulfofustis glycolicus, and the only other known moderately psychrophilic sulfate reducer, "Desulforhopalus vacuolatus" ltk10 (10) (Fig. 1). Two strains of this cluster have recently been described as species of the new genus Desulfotalea (13). The cluster will in what follows be referred to as the Desulfotalea cluster. This cluster was chosen for the design of a new oligonucleotide probe (Sval428). In addition to the two Desulfotalea strains and strains LSv23, LSv24, and LSv53, one of the mesophilic sulfate reducers, Desulfofustis glycolicus (8), is targeted by the probe (Fig. 1). It has one mismatch to LSv20 and "Desulforhopalus vacuolatus" ltk10 (10). LSv55 and LSv20 are the only strains isolated from Spitsbergen sediments not targeted by the set of probes used.

rRNA quantification of sulfate reducers. rRNA slot blot hybridization revealed that the concentration of SRB rRNA for all target groups generally decreased with depth (Table 1), following the trend of total prokaryotic rRNA (28) and MPN counts (12). The relative contribution of SRB rRNA to total eubacterial rRNA (pooled signals from all probes), however, increased from 3.8% in core A and 4.0% in core B at the surface to 10.5% in core A and 17% in core B at a depth of 15 to 18 cm in Hornsund and from 10.5% in core A and 7% in core B to 58.6% in core A and 36.4% in core B at a depth of 27 to 30 cm in Storfjord (Table 2).

Relative abundance of SRB and physiological considerations. SRB can, in general, be divided into two major groups: those that oxidize the carbon source completely to CO₂ and those that oxidize the carbon source incompletely to acetate. rRNA hybridization revealed a predominance of incompletely oxidizing SRB in these sediments. We detected incompletely oxidizing groups targeted by probes 687 (Desulfovibrionaceae but also some Geobacteraceae [16]), 660 (Desulfobulbus sp.), and Sval428 (Desulfotalea cluster), while the completely oxidizing genera Desulfococcus, Desulfosarcina, Desulfobacterium, and Desulfobacter targeted by probe 804 were below the detection limit (Table 2). This result is in agreement with cultivation-dependent data, since cell numbers of complete oxidizers on acetate were 10- to 100-fold lower than those of lactate oxidizers (12). If this finding reflects the actual relation between complete and incomplete oxidizers, the RNA concentration for acetate oxidizers would be at or below the detection limit of slot blot hybridization. Another possible explanation for not detecting RNA of complete oxidizers is that these organisms were not targeted by the probe used; all completely oxidizing strains isolated in this study, however, had the target sequence of probe 804.

Occurrence of the new psychrophilic isolates. The Desulfotalea cluster (probe 428), containing many psychrophilic isolates, was the second largest group among the detected sulfate reducers. In Storfjord, up to 15.0 and 20.9% (27 to 30 cm) of the eubacterial RNA was related to our isolates (Table 2). To estimate whether potentially dominant strains of the target group had been isolated, DGGE and Southern hybridization with probe Sval428 were performed on community DNAs. Positions of the hybridization signals within the community pattern were compared to the positions of amplified 16S rDNAs from isolates belonging to this group (Fig. 2). In both stations, we could detect several positive bands. One of them had the same position as isolates LSv23, LSv24, and LSv53 (Fig. 2). These isolates are closely related, showing three to four bases' difference within the amplified DNA fragment, and cannot be distinguished by DGGE. The presence of additional positive bands showed that there are at least three additional RNA species of this group present in Hornsund (Fig. 2) and one in Storfjord (data not shown) sediments. The high abundance of Desulfotalea-related rRNAs and the identification of bands corresponding to isolates in the community DNA profile demonstrate that a quantitatively important group of sulfate reducers was isolated from Svalbard sediment. In addition, this group was the second most abundant of the detected SRB (Table 2). Since it is doubtful that the most abundant SRB group, target group 687, is really a group of sulfate reducers, the Desulfotalea cluster, containing mainly psychrophilic strains, may even be the most abundant of SRB. This observation relates to the question of whether the bacterial community in Arctic sediments consists of cold-adapted prokaryotes. Our results show that a major group of SRB in this habitat is psychrophilic.

View larger version (52K): [in this window] [in a new window]   FIG. 2.   DGGE-hybridization analysis of community 16S rDNA patterns with probe Sval428 for Hornsund sediments. (A) DGGE results; (B) corresponding hybridization results. Numbers give the depth ranges from which target DNAs were extracted. For each depth, two cores have been investigated. "LSv" followed by a number indicates a DGGE fragment of a psychrophilic sulfate-reducing isolate targeted by probe Sval428. The arrow indicates corresponding bands between the community profile and isolates.