Plankton Benthos Res 1(4): 165–177, 2006 Plankton & Benthos Research © The Plankton Society of Japan
Abundance and diversity of sulphate-reducing bacterioplankton in Lake Suigetsu, a meromictic lake in Fukui, Japan
RYUJI KONDO*, KYOKO OSAWA, LISA MOCHIZUKI, YUKIYASU FUJIOKA & JUNKI BUTANI
Department of Marine Bioscience, Fukui Prefectural University, Obama, Fukui 917–0003, Japan Received 10 July 2006; Accepted 14 September 2006
Abstract: The depth distribution of sulphate-reducing bacteria (SRB) in the water column of a meromictic lake, Lake Suigetsu, Fukui, Japan was investigated using quantitative competitive PCR targeting the gene coding for portions of the a-subunit of dissimilatory sulphite reductase (dsrA). The total bacterial cell density (DAPI count) was 5 13 106 cells mL 1 in the water column of the lake with maximum abundance occurring at the oxic-anoxic interface layer. SRB were not detected in oxic surface water using competitive PCR. SRB were found in the anoxic waters below the oxy- cline ranging from 104 to 105 cells mL 1, accounting for 0.3–8.9% of the total bacteria. The SRB cell densities were higher than previously estimated using the most-probable-number (MPN) method. Sequencing of the cloned PCR prod- uct of dsrA showed the existence of different SRB groups in the anoxic water. The majority of the dsrA sequences were associated with the Desulfosarcina-Desulfococcus-Desulfonema group and members of the Desulfobulbaceae family. Other dsrA clones belonged to the Desulfomicrobium and Desulfovibrio species as well as to a deeply branched group in the dsrA tree with no representatives from previously isolated SRB groups. These SRB species appear to be impor- tant for the sulphur and carbon cycle in the anoxic waters of Lake Suigetsu. Key words: competitive PCR, dissimilatory sulphite reductase gene, meromictic lake, sulphate-reducing prokaryotes
This is the final step of sulphate respiration, a reaction Introduction found only in dissimilatory sulphate-reducing prokaryotes. Microbial sulphate reduction is of great ecological and The ubiquity of DSR and its highly conserved sequence has biogeochemical importance in anaerobic environments as it made this enzyme ideal for assessing the diversity of sul- is the major terminal oxidation step for the flow of carbon phate-reducing prokaryotes in nature (Wagner et al. 1998, and electrons. Sulphate-reducing prokaryotes are widely Zverlov et al. 2005). Using new assays for the PCR amplifi- distributed in most aquatic and terrestrial environments that cation of fragments from genes coding for a- (dsrA) and b- are depleted of oxygen e.g. marine and freshwater sedi- subunits (dsrB) of DSR, studies of the diversity and distrib- ments, anoxic waters, sewage sludge digesters, water- ution of SRB in aquatic environments are occurring (Chang logged soils and the gastrointestinal tracts of humans and et al. 2001, Joulian et al. 2001, Pérez-Jiménez et al. 2001, animals (Postgate 1984, Widdel 1988). Sulphate-reducing Thomsen et al. 2001). Shorter fragments of dsrA have been bacteria (SRB) are obligate anaerobic bacteria that play a used to profile communities of SRB (Karr et al. 2005). We significant role in the mineralisation of organic matter in developed new PCR primers selective for dsrA genes of anaerobic environments as well as in the biogeochemical most mesophilic SRB belonging to d-Proteobacteria and cycling of sulphur. In environments rich in sulphate, sul- used quantitative competitive PCR to rapidly and repro- phate reduction dominates mineralisation accounting for up ducibly detect and count SRB in situ as an alternative to to 50% of the organic matter decomposition in estuarine culture-dependent methods (Kondo et al. 2004). and coastal marine sediments (Jørgensen 1982). Lake Suigetsu is a meromictic lake in Fukui, Japan, char- Dissimilatory sulphite reductase (DSR) is a key enzyme acterised by a permanent oxycline at a depth between 5 and in the dissimilatory sulphate reduction by SRB. DSR catal- 8m separating the aerobic freshwater epilimnion from the yses the six-electron reduction of (bi)sulphite to sulphide. anaerobic, saline, sulphidogenic hypolimnion (Kondo et al. 2000, Matsuyama 1973, Matsuyama & Saijo 1971, Taka- * Corresponding author: Ryuji Kondo; E-mail, [email protected] hashi & Ichimura 1968). Seawater from the Sea of Japan 166 R. KONDO et al. comes through Lakes Hiruga and Kugushi next to Lake After a final ethanol precipitation, the nucleic acid was re- Suigetsu. Thus the anoxic water chemistry of Lake Suigetsu suspended in 50 mL TE buffer (10 mM Tris-HCl, 1 mM is dominated by inorganic sulphur compounds with a high EDTA; pH 8.0). Nucleic acid purity and yield were deter- concentration of sulphate and steep gradients of sulphide at mined using scanning spectrophotometry (Sambrook et al. the chemocline (Kondo et al. 2000, Matsuyama 1973, Mat- 1989). suyama & Saijo 1971). Because Lake Suigetsu is highly sulphidic, we assume microbiological sulphate reduction is Competitive PCR to enumerate SRB responsible for the production of sulphide. Despite the im- Competitive PCR was carried out as described elsewhere portance of SRB in Lake Suigetsu, little is known about (Kondo et al. 2004). Briefly, the primers used were DSR1F their distribution in the lake. The only study was conducted (5 -ACSCACTGGAAGCACGGCGG-3 ) (an improved by Takeuchi & Takii (1987) who reported their vertical dis- primer than the DSR1F used by Wagner et al. 1998) and tribution in the water column of Lake Suigetsu by enumera- DSR-R (5 -GTGGMRCCGTGCAKRTTGG). Competitor tion using the most-probable-number (MPN) method. MPN DNA which was about 20% shorter than the targeted region cell counts may underestimate because MPNs are selective of dsrA was constructed using PCR with DNA from Desul- and represent only a minor fraction of the actual microbial fovibrio desulfuricans DSM642T as the template and the communities (Gibson et al. 1987, Jørgensen 1978). primer set of DSR1F ; and Comp-DSR which consisted of Here we examine the distribution and diversity of SRB in D. vulgaris DSM644T dsrAB sequence positions 559–578 the water column of Lake Suigetsu using a quantitative and the DSR-R primer sequence (position 622–644). PCR competitive PCR targeting dsrA genes. We conclude SRB reactions were performed in 50 mL containing 0.2 mM were more abundant than previously determined using a dNTPs, 3.5 mM MgCl , 0.4 mM each primer, 1 PCR culture-dependent method and show a diverse group of 2 buffer, 1 Q-solution, 2.5 U Taq DNA polymerase (QIA- SRB inhabit the anoxic waters of Lake Suigetsu. GEN) and the DNA from the water samples as the tem- plate. Also added were at least five dilutions of the serially Materials and Methods diluted competitor DNA for each sample. Amplification was performed using a thermal cycler (GeneAmp PCR Sys- Sample collection tem 2400, Applied Biosystems): initial denaturation at Water samples were collected from the central basin of 94°C for 1 min followed by 30 cycles: 94°C for 30 s, 60°C Lake Suigetsu (35°35 N, 135°53 E) on 30th July 2003 and for 30 s and 72°C for 60 s with a final elongation step at 26th January 2004 using a Kitahara’s water sampler 72°C for 7 min. Aliquots of PCR products were analysed by (Rigosha). The samples were immediately added to an au- electrophoresis on 3% (w/v) agarose gel in 1 TAE buffer toclaved BOD bottle to prevent contact with air. All sam- (40 mM Tris-acetate, 1 mM EDTA; pH 8.0); and stained ples were kept in an ice-cooled box and transported to the with ethidium bromide. The gels were photographed and laboratory within a few hours of sampling. Temperature, band intensity was measured by densitometry (CS-9300PC, salinity and dissolved oxygen (DO) concentration were Shimadzu). To correct for differences in the intensity of the measured using an oxygen metre (Model 85, YSI). Vertical PCR fragments (Piatak et al. 1993), the intensity of the profiles of turbidity (as kaolin mg L 1; ppm) were obtained competitor DNA was multiplied by the ratio 221/177. Copy using a turbidity metre (Model PT-1, Alec Electronics). numbers of dsrA in the samples were calculated using re- gression analysis between the band intensity ratio of the Bacterial counts PCR product from water DNA to those from competitor DNA using the known amounts of competitor DNA. The The water sample for bacterioplankton counts was pre- dsrA copy number was expressed as cells mL 1 of water served using buffered formaldehyde at a 2% (v/v) final con- (equivalent to cell counts assuming one dsrA copy per cell) centration. The bacterial cells in lake water were filtered was calculated using dilution factors and the volume of nu- onto black 0.2-mm polycarbonate membrane filters (Advan- cleic acid extract. tec), stained with 4 ,6-diamidino-2-phenylindole (DAPI) D. desulfuricans DSM642T was used to generate a cali- and counted using epifluorescence microscopy (Porter & bration curve by analysis of filtered samples. D. desulfuri- Feig, 1980). cans DSM642T was grown in Postgate’s C medium (Post- gate 1984) and a sample was counted using the DAPI stain DNA extraction method (described above). The cells were centrifuged at A 50-mL water aliquot was filtered through a sterile 14,400 g for 20 min at 4°C and resuspended to about 1011 polycarbonate membrane filter (0.2-mm, Advantec) to col- cells mL 1, serially diluted, and collected on sterile mem- lect microbial biomass for subsequent nucleic acid extrac- brane filters. DNA extracts were performed for each serially tion. The filters were stored at 85°C until processed. Nu- diluted sample (as described above). cleic acids were extracted from the filtered samples using the hydroxyapatite spin-column method (Purdy et al. 1996). Sulphate-reducing bacteria in Lake Suigetsu 167
esulfovibrio islandicus (99% similarity). Thus the taxa Sequencing and phylogenetic analysis (phylotypes) defined for this analysis may be distinct at After PCR amplification without competitor DNA, unpu- least to the species level (or higher). rified dsrA PCR products were cloned using a TA Cloning Coverage (C) was calculated using the following for- Kit (Invitrogen) with the pCR II vector and Escherichia coli mula: C 1 (n1/N), where n1 is the number of phylotypes INVaF’ competent cells according to the manufacturer’s that occurred only once in the clone library and N is the instructions. From each of these eight libraries, about 60 total number of clones examined (Mullins et al. 1995). Rar- white colonies were randomly selected and the cloned in- efaction curves (Heck et al. 1975) were produced using serts were reamplified using the vector primers M13 for- software available online at http://www.uga.edu/ strata/ ward and reverse (25 cycles of 94°C for 30 sec, 50°C for 30 software.html. The phylogenetic compositions of libraries sec and 72°C for 30 sec); and the PCR products were puri- were compared using the Sorensen similarity index, fied using Wizard SV Gel and PCR Clean-Up System Cs 2j/(a b), where j is the number of phylotypes common (Promega) according to the manufacturer’s instructions. Se- to both samples and a and b are the numbers of phylotypes lected clones (52 or 53 clones from each sample) were se- in libraries A and B, respectively (Magurran 1988). Statisti- quenced at Macrogen, Inc. (Seoul, Korea) using the M13 cal significance of differences in composition of pairs of li- forward primer. braries was tested using the LIBSHUFF programme (Sin- Partial dsrA sequences corresponding to D. vulgaris gleton et al. 2001) which is available online at http://www. DSM644T dsrAB sequence position 421–641 were deter- arches.uga.edu/~whitman/libshuff.html. mined and aligned using CLUSTALX (Thompson et al. 1997). Maximum parsimony (MP) and neighbor joining Nucleotide sequence accession numbers (NJ) analyses were performed using PAUP* 4.0b10 (Swof- Partial cloned dsrA sequences recovered from the water ford 2002). MP analysis was performed using the heuristic column of Lake Suigetsu were deposited in DDBJ under search algorithm with unordered unweighted characters; accession numbers AB240585 to AB240638. Only one rep- and gaps were treated as missing data. The likelihood ratio resentative sequence with 98% similarity was deposited. test was applied to select an appropriate substitution model in the maximum likelihood (ML) analysis using Modeltest 3.7 (Posada & Crandall 1998). The optimal model selected Results for the dsrA data set was GTR G I (general time re- Water column profiles versible model estimating gamma distribution and the pro- portion of invariable sites; Rodriguez et al. 1990) with the Figure 1 shows the depth distribution of the physico- following parameters: nucleotide frequencies A 0.1745; chemical properties of the central basin of Lake Suigetsu C 0.3053, G 0.2512, T 0.2691; gamma distribution when our samples were collected. A steep thermocline was with shape parameter 0.7690; substitution rate A→C evident between 5 and 10 m (regardless of sampling date). 2.2117, A→G 4.4578, A→T 2.6221, C→G 1.6916, C→ Surface water was saturated with DO and the DO concen- T 4.5735, G→T 1.00; proportion of invariable sites tration decreased rapidly below 3 m to the limit of detection 0.1341. This model was also used for the NJ analysis. The at 6 m in July 2003 and 7 m in January 2004. The salinity of ML analysis was carried out using TREEPUZZLE 5.2 the epilimnic water was 2–4 practical salinity units (psu) (Schmidt et al. 2002) with 5,000 puzzle steps. For bootstrap and 12–14 psu for hypolimnic water. This demonstrates analysis (Felsenstein 1985), 1000 bootstrap data sets were stagnation of the anoxic saline water in deeper layers from generated from resampled data for MP and NJ analyses, approximately 6 m to the bottom at 34 m. Turbidity was with all other settings set by default. about 2–5.6 ppm in the surface layer and increased to a maximum of 12.2 ppm at 6 m in July 2003 and 20.6 ppm at Statistical analysis and sequence population diversity 7m in January 2004. To assign sequences to distinct phylotypes, sequences Depth distribution of bacterioplankton with similarities greater than 98% were considered to repre- sent the same phylotypes. In previous similar analyses for The vertical distribution of total bacteria in the water col- 16S rDNA, the discriminator values are 97% or greater umn of Lake Suigetsu is shown in Fig. 2. Bacterioplankton (Sakano & Kerfhof 1998, Humayoun et al. 2003). Sequence densities in the epilimnion were 8.1 8.8 106 cells mL 1 similarities of the region amplified by PCR in this study did in July 2003 and 7.4 9.8 106 cells mL 1 in January 2004 not exceed 98% among pure SRB cultures available from with the peak at the oxycline. Bacterioplankton densities the databases, except for some species; e.g. Desulfovibrio were less in the hypolimnion (5.0 5.8 106 cells mL 1 in termitidis compared to Desulfovibrio vulgaris subsp. oxam- July 2003 and 5.5 7.8 106 cells mL 1 in January 2004) icus (99% similarity), Desulfomicrobium apsheronum com- than in the epilimnion. pared to Desulfomicrobium macestii (98% similarity) and Different known numbers of D. desulfuricans DSM642T Thermodesulfovibrio yellowstonii compared to Thermod- cells were collected on filters to generate a calibration curve 168 R. KONDO et al.
Fig. 1. Depth distribution of water temperature ( ), salinity ( ), dissolved oxygen concentration ( ) and turbidity ( ) in the Lake Suigetsu water column on 30th July 2003 (A) and 26th January 2004 (B).
Fig. 2. Depth profile of total bacterial counts (shaded bar) and SRB numbers (black bar) determined by competitive PCR in the Lake Suigetsu water column on 30th July 2003 (A) and 26th January 2004 (B). Error bars represent the standard error of the mean (n 3). by analysis of filtered samples. The advantage of measuring the calibration curve with whole cells onto the filter is the reduced potential biases such as incomplete cell lysis, DNA degradation and DNA adsorption onto the filter are in- cluded in the calibration curve. DNA from each of the sam- ples was coamplified under optimal competitive PCR con- ditions and the results show a linear relationship (r2 0.994) between the cell numbers collected on filters and those de- tected by PCR (Fig. 3). The assay slightly overestimated the number of D. desulfuricans cells filtered compared to the ideal (y x). This result indicates that PCR of DNA from 105 to 1010 cells could be used to quantify filtered samples. The depth distribution of SRB determined using compet- itive PCR is shown in Fig. 2. SRB were not detected in oxic surface water of Lake Suigetsu by PCR using the DSR1F and DSR-R primers. High densities of SRB from 1.9 104 cells mL 1 to 6.7 105 cells mL 1 were detected in the anoxic water layer using the competitive PCR. The highest Fig. 3. Calibration curve of SRB count by quantitative competi- densities were 2.2 105 cells mL 1 at 6 m depth in July tive PCR. Dotted line denotes y x. Error bars indicate the stan- 2003 and 6.7 105 cells mL 1 at 7 m depth in January 2004 dard error of the mean (n 3). Sulphate-reducing bacteria in Lake Suigetsu 169 where the maximum turbidity was found. The cell densities SRB groups on the dsrA tree. Clones in cluster C were estimated by competitive PCR were from 0.3 to 8.9% of the slightly more similar in sequence to dsrA from Desulfos- total bacterial cell densities. The ratios of SRB cells to total arcina variabilis (77–80%) than from Desulfomusa bacterial cells may have been higher in July 2003 (mean: hansenii (75–77%) (Table 1). Clusters C, D and E were af- 5.7%, range: 0.3–8.9%) than in January 2004 (mean: 1.3%, filiated with the Desulfococcus-Desulfonema-Desulfos- range: 0.7–2.1%). arcina group in the family Desulfobacteraceae. The second most abundant group, cluster G, comprises Diversity of SRB based on dsrA 91 sequences from the water column. This cluster was affil- iated with members of the Desulfobulbaceae family. Clone As expected, the 221-bp gene product was obtained from sequences in cluster G showed 81 to 82% similarities to a anoxic water samples using the primer set DSR1F /DSR- groundwater clone retrieved from a uranium mill tailing R (Kondo et al. 2004). Clone libraries were made from the site (Table 1). These sequences were recovered in higher dsrA PCR products from water samples at 6 m, 10 m, 20 m abundances from the water column in July 2003 as com- and 30 m depth in July 2003, and 7 m, 10 m, 20 m and 30 m pared to January 2004 (Table 1). depth in January 2004 to identify the various SRB in Lake Cluster H was 17% of all clones. These sequences were Suigetsu. A total of 419 clones (52 or 53 clones for each phylogenetically distant from any of the isolated SRB lin- sample) were sequenced. As several clones had identical se- eages but were related to dsrA clones recovered from uncul- quences, the clones were assembled into 134 different se- tured SRB in environmental samples including one from quences. Fifty-four distinct phylotypes were recognised uranium mill tailing groundwater (Chang et al. 2001), an (Table 1 and Fig. 4) using our definition ( 98% identity). acidic fen (Loy et al. 2004), Lake Fryxell (Karr et al. 2005), To obtain a reliable description of the phylogenetic rela- Guaymas Basin (Dhillon et al. 2003) and the Seine estuary tionship of the SRB population in the water column of Lake (Leloup et al. 2004, 2006). Suigetsu, we included in our analysis the most charac- Cluster F comprises 25 sequences from all samples ex- terised dsrA sequences of cultured SRB and uncultured en- cept for the 20 m sample in July 2003. Two sequences (phy- vironmental clones available in the databases. Several phy- lotype-46) were closely related to Desulfomonile tiedjei logenetic approaches were taken to analyse the partial dsrA (82% similar). However, phylotypes-5 and -51 were more sequences. Although differences in tree topologies were ob- similar to Desulfonema ishimotoi (73%) and the sulphate- tained among these approaches, a similar ordering of taxa reducing bacterium Hxd3 (80%), respectively, than to was found among the different phylogenetic analyses. Simi- Desulfomonile tiedjei (72–75%). lar orders of taxa were also found between our partial dsrA Cluster J contained 17 dsrA sequences unique to Lake sequences and almost complete dsrAB (Zverlov et al. Suigetsu. These sequences were phylogenetically distant 2005). This ensured that biases imposed from phylogenetic from any isolated SRB group. Clones in phylotype-14 analyses of shorter dsrA sequences are less evident in the showed low sequence similarity (70%) to dsrA from Desul- resulting tree. fotomaculum nigrificans being the closest cultured rela- Phylogenetic analyses revealed the presence of ten lin- tive; whereas clones of phylotype-49 showed 79% sequence eages of cloned dsrA sequences designated as clusters A to similarity to an estuarine sediment clone, CF5 (Table 1). J (Fig. 4). These groupings were stable and were consis- The remaining 11 sequences form the small clusters A, B tently recovered using the MP and ML methods. Although and I. Cluster A contains most of the dsrA sequences from some taxa were related to SRB reference cultures, others cultured Desulfovibrio within the d-Proteobacteria. A sin- represent previously undescribed SRB. gle clone from 10 m depth sample collected in January The largest cluster of clone sequences grouped in cluster 2004 was detected in this cluster. Cluster B comprises E (38% of all clones) includes Desulfococcus multivorans, seven sequences. This cluster is affiliated with the genus Desulfonema limicola and Desulfonema ishimotoi within Desulfomicrobium of the d-Proteobacteria. Six of these the d-Proteobacteria. This cluster also contains environ- clones were recovered from water samples collected in Jan- mental dsrA sequences retrieved from estuarine sediments uary 2004; whereas only one clone was detected from the (INOC-DSR3, INOC-DSR26, VN4, VN11), a mesophilic 30 m sample in July 2003. Cluster I contains no cultured sulphide-rich spring (ZDSR2), a salt marsh (PIM02A05) representatives. Cloned sequences in this cluster showed se- and a deep-sea hydrothermal vent chimney (INDO-40). quence similarities (72 to 80%) to an environmental clone These environmental clones were reported to be closely re- sequence retrieved from an acidic fen (Loy et al. 2004). lated to the genera Desulfococcus, Desulfonema and Desul- fosarcina (Bahr et al. 2005, Elshahed et al. 2003, Joulian et Depth distribution of SRB groups al. 2001, Leloup et al. 2006, Nakagawa et al. 2004). Cluster D was related to the genus Desulfosarcina. An environmen- The relative abundance of the different phylotypes in the tal clone from the shallow-water sediment in Kysing Fjord, libraries was calculated for all eight samples (Fig. 5). Of the INOC-DSR20 (Joulian et al. 2001), was included in this sequenced clones, 48% grouped with the Desulfosarcina- cluster. Cluster C was not clustered with any of the isolated Desulfococcus-Desulfonema group (clusters C, D and E) 170 R. KONDO et al.
Table 1. Assignment of dsrA clones from the water samples of Lake Suigetsu to distinct phylotypes.
No. of clones from samples collected at the following depth: Most similar dsrA sequence in DDBJ as Phylotype July 2003 January 2004 determined by BLAST searcha Clusterb (accession no., % identity) 6m 10m 20m 30m 7m 10m 20m 30m
1201516134723Uranium mill tailing clone G UMTRAdsr853-36 (AY015529, 81) 21 1Uranium mill tailing clone H UMTRAdsr617-8 (AY015542, 77) 36 79 65694Seine estuary clone VN11 E (AY953403, 92) 41 1 Deep-sea hydrothermal vent chimney E clone INDO-40 (AB124917, 81) 52 1 1 3 3 6 6Desulfonema ishimotoii F (AY626030, 73) 66 89 818714 8 Seine estuary clone VN4 E (AY953396, 82) 71Seine estuary clone VN4 E (AY953396, 80) 81 Guaymas basin clone B04P026 H (AY197455, 76) 91 11 42 12Kysing Fjord clone INOC-DSR20 D (AF360755, 91) 10 1 Petroleum-contaminated sediment E clone Nap51 (AF327309, 82) 11 3 8 7 7 5 4 7 9 Desulfotomaculum ruminis H (U58118, 74) 12 1 Desulfosarcina variabilis D (AF360643, 79) 13 1 3 1 1 2 Uranium mill tailing clone G UMTRAdsr853-36 (AY015529, 81) 14 2 1 3 3 1 3 1 Desulfotomaculum. nigrificans J (AF482466, 70) 15 1 Acidic fen clone dsrSbII-25 I (AY167481, 80) 16 1 2 2 1 1 Sulfide-rich spring clone ZDSR2 E (AY327244, 81) 17 2 1 1 2 Desulfosarcina variabilis C (AF360643, 80) 18 1 1 1 Uranium mill tailing clone H UMTRAdsr617-8 (AY015542, 74) 19 1 1 1 Seine estuary clone VN4 E (AY953396, 78) 20 1 2 1 1 2 Kysing Fjord clone INOC-DSR20 D (AF360755, 89) 21 1 Uranium mill tailing clone H UMTRAdsr617-8 (AY015542, 72) 22 1 Desulfotomaculum thermosapovorans H (AF271769, 71) 23 1 Desulfosarcina variabilis C (AF360643, 77) 24 1 1 Deep-sea hydrothermal vent chimney D clone INDO-40 (AB124917, 80) 25 1 1 Acidic fen clone dsrSbI-64 H (AY167474, 78) 26 1 1 1 1 Uranium mill tailing clone H UMTRAdsr624-20 (AY015544, 89) Sulphate-reducing bacteria in Lake Suigetsu 171
Table 1. continued.
No. of clones from samples collected at the following depth: Most similar dsrA sequence in DDBJ as Phylotype July 2003 January 2004 determined by BLAST searcha Clusterb (accession no., % identity) 6m 10m 20m 30m 7m 10m 20m 30m
27 1 Uranium mill tailing clone H UMTRAdsr624-20(AY015544, 89) 28 1 1 Seine estuary clone VN9 H (AY953401, 75) 29 1 1 1 Petroleum-contaminated sediment clone E Nap51 (AF327309, 71) 30 1 Guaymas basin clone B04P026 H (AY197455, 74) 31 1 Sulfide-rich spring clone ZDSR2 E (AY327244, 75) 32 1 SRB AK01 (AF327301, 87) D 33 1 1 1 Uranium mill tailing clone G UMTRAdsr853-36 (AY015529, 82) 34 1 SRB Hxd3 (AF327308, 85) E 35 1 1 3 2 Desulfovibrio longus (AB061540, 82) B 36 2 Kysing Fjord clone INOC-DSR20 D (AF360755, 91) 37 2 Deep-sea hydrothermal vent chimney E clone INDO-40 (AB124917, 81) 38 1 Guaymas basin clone B04P037 H (AY197458, 77) 39 2 3 3 Deep-sea hydrothermal vent chimney D clone INDO-40 (AB124917, 78) 40 1 Lake Fryxell clone LFdsrC24 H (AY273288, 84) 41 1 Desulfotomaculum thermosapovorans H (AF271769, 73) 42 1 Acidic fen clone dsrSbII-25 I (AY167481, 76) 43 1 Acidic fen clone dsrSbII-25 I (AY167481, 72) 44 1 Desulfovibrio aminophilus A (AY626029, 87) 45 2 1 Petroleum-contaminated sediment clone E Nap30 (AF327311, 97) 46 2 Desulfomonile tiedjei (AF334595, 82) F 47 2 1 New England salt marsh clone E PIMO2A05 (AY741562, 90) 48 1 2 Kysing Fjord clone INOC-DSR26 E (AF360761, 81) 49 1 2 Estuarine sediment clone CF5 J (AF442721, 79) 50 1 SRB AK01 (AF327301, 84) E 51 1 SRB Hxd3 (AF327308, 80) F 52 1 Uranium mill tailing clone H UMTRAdsr626-8 (AY015543, 87) 53 1 Desulfosarcina variabilis D (AF360643, 80) 54 1 Guaymas basin clone B04P026 H (AY197455, 76) a Altchul et al. (1997) b Cluster of dsrA clones as inferred from Fig. 4. 172 R. KONDO et al.
Fig. 4. Phylogenetic tree showing the relationships of the analysed dsrA clones retrieved from the waters of Lake Suigetsu with the dsrA from characterised sulphate-reducing prokaryotes. Environmental sequences determined in this study are shown in bold- face. Bootstrap values based on 1000 replicates for NJ and MP, and quartet-puzzling support values for ML are shown for branches with more than 50% support. The distance scale indicates the expected number of changes per sequence position. Num- bers in parentheses are the number of clones within each phylotype followed by DDBJ accession numbers. Sulphate-reducing bacteria in Lake Suigetsu 173
Fig. 5. Depth and seasonal distribution of dsrA clones in the water column of Lake Suigetsu in July 2003 (a) and January 2004 (b).
Discussion Lake Suigetsu has attracted considerable interest, espe- cially in bacterial sulphate reduction with reference to the carbon cycle, because of the large accumulation of sulphide in the deeper layers of the lake (Kondo et al. 2000); how- ever, little is known about the distribution and structure of the SRB population in the lake. Since no suitable media are available to enumerate all SRB simultaneously, we devel- oped a method to quantify the microorganisms responsible for sulphate reduction directly from natural samples (Kondo et al. 2004). We determined here the distribution and diversity of SRB in the water column of Lake Suigetsu Fig. 6. Rarefaction curves generated for dsrA in clone libraries using a competitive PCR. The technique was used to esti- from samples collected at 6 ( ), 10 ( ), 20 ( ) and 30 m ( ) in mate the dsrA copy number in the waters from Lake July 2003 and at 7 ( ), 10 ( ), 20 ( ) and 30 m ( ) in January Suigetsu. Assuming the cells have a single copy of the DSR 2004. gene, the copy number should indicate the cell number of SRB in the water samples. However, DSR gene copy num- within the d-Proteobacteria. Some sequences recovered ber may vary with species. Desulfobacter vibrioformis, from all samples, except for the sample collected at 6 m in Desulfobulbus rhabdoformis (Larsen et al. 2000), Desul- July 2003, fell within these groups. The second most abun- fovibrio vulgaris (Karkhoff-Schweizer et al. 1995), Desul- dant group, cluster G, comprises 91 sequences from the fobacula toluolica (Zverlov et al. 2005) and Archaeoglobus water column of the lake. Six metre samples were primarily fulgidus (Dahl et al. 1993) have only a single copy of the sequences related to cluster G of the Desulfobulbaceae DSR gene but SRB existing in nature may have multiple family. Cluster G was frequently detected from samples copies of the DSR gene. Furthermore, some bacterial collected in July 2003. The dsrA sequences from cluster H species incapable of sulphate reduction such as Desulfito- were also detected irrespective of water depth or season. bacterium halogenans (Klein et al. 2001), Desulfitobac- Using the 98% similarity cut-off value, the coverage val- terium hafniense (Nonaka et al. 2006), Bilophila wadswor- ues for each library were from 75 to 91% indicating that the thia (Lane et al. 2001) and Sporotomaculum hydroxyben- libraries were reasonably well sampled for diversity zoicum (Zverlov et al. 2005) have the DSR gene. Therefore, (Mullins et al. 1995). Analysis of the dsrA clonal libraries our competitive PCR analysis probably overestimates the in which rarefaction curves were used did not reveal a great SBR population in situ. change in the diversity of sulphate-reducing populations Collection of Desulfovibrio desulfuricans DSM642T cells among the samples (Fig. 6). The similarity of the phylotype on the filter, followed by competitive PCR quantification, populations in these libraries ranged from 39 to 65%. LIB- demonstrates the usefulness of the technique in situ. There SHUFF analysis indicates the libraries from all samples was a good correlation between the cell numbers filtered were not significantly different, indicating that there is no and those detected by the assay. The slope of the calibration difference in the diversity of SRB populations in Lake curve was found to be 1.05; suggesting D. desulfuricans Suigetsu in the water below the oxycline or during seasons. DSM642T has a single copy of the DSR gene. The detection 174 R. KONDO et al. limit of dsrA is approximately 103 copies in a PCR reaction a significant effect on the tree topology by reconstructing (Kondo et al. 2004). With the dilution factor of the water phylogenetic trees for alignments of different length dsrAB samples taken into account this would translate to a detec- sequences and found that the general topology of all trees tion limit of approximately 103 copies (cells) mL 1 using was consistent with the previous dsrAB tree based on full- our standard PCR conditions. By competitive PCR, SRB length dsrAB fragments (Minz et al. 1999, Wagner et al. were not detected in oxic surface water (Fig. 2); however, 1998). Karr et al. (2005) also found that branching patterns SRB were detected even in the oxic layer of the lake using of phylogenetic trees generated based on shorter dsrA se- the MPN method (Takeuchi & Takii 1987). Koizumi et al. quences were consistent with trees generated based on the (2004) detected SRB by quantitative oligonucleotide probe entire dsrAB operon. Although slight differences in tree membrane hybridisation in oxic and microaerophilic sur- topologies were obtained between our partial sequences and face waters of Lake Kaiike, a small saline meromictic lake complete dsrAB (Zverlov et al. 2005), similar orderings of in Japan similar to Lake Suigetsu. No amplification of dsrA taxa were found between these two analyses. Furthermore, was observed with DNA extracted from water samples of we include in our analysis well-defined environmental the oxic surface waters possibly because there was either a dsrAB sequences (e.g. Chang et al. 2001, Thomsen et al. low density of SRB in the oxic waters or there were mis- 2001, Leloup et al. 2006) which were chosen from the data- matches in the primer regions of the SRB existing in Lake bases based on BLAST (Altschul et al. 1997) similarities. Suigetsu. These indicate that the dsrA fragment we have targeted can High SRB densities (0.2 6.7 105 cells mL 1) were de- be used to analyse SRB communities in situ. Our molecular tected in the waters below the oxycline in Lake Suigetsu characterisation of SRB showed the presence of novel dsrA using the competitive PCR. Takeuchi & Takii (1987) report sequences related to the d-Proteobacteria and to a deeply densities of SRB in the water column of the lake were branched group in the dsrA tree with no representatives 100 102 cells mL 1 using the MPN method. Cell densities from previously isolated SRB. Most members of the Lake by competitive PCR were considerably higher than those Suigetsu SRB community are related to complete oxidizing estimated by the MPN method. Several studies demonstrate genera, Desulfococcus, Desulfonema and Desulfosarcina in the number of viable SRB in aquatic sediments are underes- the family Desulfobacteraceae (Fig. 4; clusters C, D and timated by a factor of more than 1,000 when standard MPN E). Some sequences recovered from all samples, except for methods are used with selective enriched media (Gibson et the 6 m samples in July 2003, fell primarily within this al. 1987, Jørgensen 1978). Thus, the cell density estimates group (Fig. 5). Sequences from this group were abundantly using the molecular techniques are several orders of magni- recovered from the surface sediment of Aarhus Bay, Den- tude higher than the MPN estimates. SRB cell densities es- mark (Thomsen et al. 2001), a New England salt marsh timated by competitive PCR ranged from 0.3 to 8.9% of the (Bahr et al. 2005) and the Colne estuary, UK (Kondo et al. total bacterial densities. This apparent high ratio may be 2004). Desulfonema and Desulfosarcina species are marine due to overestimation using the competitive PCR. However, organisms that require NaCl to grow, while Desulfococcus sulphate reducers within the d-Proteobacteria were de- species are freshwater organisms but also grow well in tected as intensely stained bands by rRNA-based denaturing brackish and marine media (Widdel & Bak 1992). Water gradient gel electrophoresis (DGGE) analysis (Kondo, un- salinity below the cline was approximately half that of sea- published data). Muyzer et al. (1993) found DGGE gel water (Fig. 1). If the physiological features of the dsrA phy- bands correspond to different 16S rRNA gene sequences lotypes in this study are similar to those of cultured species, and thus reflect distinct microbial populations in the com- we infer that SRB related to the NaCl-requiring complete munity. Moreover, only numerically dominant populations oxidizers, Desulfonema and Desulfosarcina, and primarily will be detected by DGGE. As bacterial sulphate reduction freshwater-inhabiting Desulfococcus could be present to- activity is correlated with the rRNA content in cells gether and may play an important role in the terminal oxi-
(Neletin et al. 2003), the intensely of the DGGE bands of dation of organic matter to CO2 in Lake Suigetsu. SRB from Lake Suigetsu rRNA samples indicate that the The second most abundant group, cluster G, comprises SRB must constitute a significant fraction of the bacterial 91 sequences (22% of all clones). Sequences recovered community and are active. Thus SRB in the water column from the 6 m sample in July 2003 were predominantly from of Lake Suigetsu appear to play an important role in the cluster G. This cluster is affiliated with incomplete oxidiz- anaerobic degradation of organic matter as well as the cy- ers of the Desulfobulbaceae family and has been referred to cling of sulphur. as the UMTRA DSR cluster D (Chang et al. 2001). Clone Clone libraries were made from the dsrA PCR products sequences in cluster G were closely related to an environ- at several water depths to identify the various SRB in the mental sequence retrieved from a uranium mill tailing site water column. We used a short fragment (221 bp) of dsrA to (Chang et al. 2001). Sequences from this cluster were also reconstruct phylogenetic trees. Sequence length has a pro- recovered from freshwater or brackish environments such found effect on reliable reconstruction of phylogenetic trees as estuarine sediments (Bahr et al. 2005, Kondo et al. 2004, (Kumar & Gadagkar 2000). Pérez-Jiménez et al. (2001) Leloup et al. 2006) and a wetland (Castro et al. 2002), but tested whether the length of dsrAB used for the analysis had not marine environments (Dhillon et al. 2003, Thomsen et Sulphate-reducing bacteria in Lake Suigetsu 175 al. 2001). Members of the Desulfobulbaceae family can use dependent methods; and show the highest cell densities of alternative electron acceptors leading to sulphate and can SRB were observed in water just below the oxycline in the disproportionate sulphur oxianions while Desulfobulbus is lake. Sequences of cloned PCR products show the different known to be able to grow by fermentation of lactate or SRB groups in the water. The complete oxidizers, Desulfo- ethanol and CO2 without sulphate. Sulphate concentrations coccus, Desulfonema and Desulfosarcina, species may be below the oxycline are 2–8 mM (Kondo et al. 2000) which important in the sulphur and carbon cycles in Lake is sufficiently high as not to limit sulphate reduction. How- Suigetsu as may be members within the family Desulfobul- ever, we previously reported that thiosulphate was detected baceae and a deeply branched group in the dsrA tree with in anoxic waters below the oxycline (6–7 m) of Lake no representatives from previously isolated SRB. These Suigetsu, ranging from 1 mM or less to 60 mM (Kondo et al. groups were the principal components of SRB existing in 2000). This suggests thiosulphate may be used as an elec- the anoxic waters of Lake Suigetsu; however, further study tron acceptor or may be disproportionated by microorgan- is needed to determine if these sulphate-reducing species isms belonging to cluster G, including the thiosulphate-dis- are active in situ. The quantification of dsrA mRNA expres- proportionating bacterium Desulfocapsa. sion by competitive RT-PCR analysis could be used to clar- Cluster H contains dsrA sequences that were phylogenet- ify this. ically distant from any isolated SRB group. Cluster H com- pressed 11.5–26.9% of the cloned sequences in each sam- Acknowledgements ple. This cluster has been referred to as the UMTRA DSR cluster F (Chang et al. 2001). The dominance of sequences We are grateful to S. Miura, Y. Momoki and M. Murako related to this cluster was reported in ground water from a from our laboratory for assistance in field sampling and to uranium mill tailing site (Chang et al. 2001), a freshwater M. Kamiya of Fukui Prefectural University for his help wetland (Castro et al. 2002), acidic fens (Loy et al. 2004) with phylogenetic analyses. This study was supported in and a hydrothermal vent site (Dhillon et al. 2003). This part by a Grant-in-Aid for Scientific Research (No. cluster is not related to any cultured SRB and its physiology 15580170) from the Japan Society for the Promotion of is unknown. As their importance is not sufficiently under- Science and Fukui Prefectural Fund for the Promotion of stood, further study is required to isolate SRB belonging to Science to RK. this cluster and to investigate whether this SRB cluster is active in the lake. The coverage values for each library were from 75 to References 91% indicating that the libraries were reasonably well sam- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller pled for diversity (Mullins et al. 1995). Although there were W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new no significant differences among the water depths and sam- generation of protein database search programs. Nucleic Acids pling dates, a minor change in the composition of dsrA phy- Res 25: 3389–3402. lotypes between seasons was recognised – that is, dsrA se- Bahr M, Crump BC, Klepac-Ceraj V, Teske A, Sogin L, Hobbie quences related to cluster G were recovered with relatively JE (2005) Molecular characterization of sulfate-reducing bacte- higher frequency from summer samples (29–40%) than ria in a New England salt marsh. Environ Microbiol 7: 1175– from winter samples (3.8–21.7%). The vertical profiles of 1185. water temperature, salinity and DO concentration clearly Castro H, Reddy KR, Ogram A (2002) Composition and function demonstrated the stagnation of anoxic saline water in the of sulfate-reducing prokaryotes in eutrophic and pristine areas deeper layers from approximately 6 m to the bottom of the of the Florida Everglades. Appl Environ Microbiol 68: 6129– lake at 34 m (Fig. 1). Salinity and temperature below the 6137. oxycline were stable regardless of the season (Fig. 2, Kondo Chang Y-J, Peacock AD, Long PE, Stephen JR, Mackinley JP, et al. 2000). This implies that environmental factors other Macnaughton SJ, Hussain AKMA, Saxton AM, White DC (2001) Diversity and characterization of sulfate-reducing bacte- than temperature and salinity influence the composition of ria in groundwater at a uranium mill tailings site. Appl Environ the SRB population. In the surface water of Lake Suigetsu, Microbiol 67: 3149–3160. cyanobacterial blooms of Microcystis and/or Anabaena Dahl C, Kredich NM, Deutzmann R, Trüper HG (1993) Dissimi- occur during the summer season while the dinoflagellate, latory sulphite reductase from Archaeoglobus fulgidus: physico- Heterocapsa dominates during the winter season. Thus, the chemical properties of the enzyme and cloning, sequencing and compositions of organic matter in the deeper layers sup- analysis of the reductase genes. J Gen Microbiol 139: plied from the euphotic layer of the lake may vary with sea- 1817–1828. son. Composition of organic matter available for the SRB Dhillon A, Teske A, Dillon J, Stahl DA, Sogin ML (2003) Molec- as an electron donor may cause minor differences in the ular characterization of sulfate-reducing bacteria in the Guay- composition of the SRB. mas Basin. Appl Environ Microbiol 69: 2765–2772. In conclusion, using dsrA-selective competitive PCR Elshahed MS, Senko JM, Najar FZ, Kenton SM, Roe BA, Dewers to enumerate SRB, we show SRB in Lake Suigetsu were TA, Spear JR, Krumholz LR (2003) Bacterial diversity and sul- more abundant than previously determined using culture- fur cycling in a mesophilic sulfide-rich spring. Appl Environ 176 R. KONDO et al.
Microbiol 69: 5609–5621. Leloup J, Quillet L, Oger C, Boust D, Petit F (2004) Molecular Felsenstein J (1985) Confidence limits on phylogenies: an ap- quantification of sulfate-reducing microorganisms (carrying proach using the bootstrap. Evolution 39: 783–791. dsrAB genes) by competitive PCR in estuarine sediments. Gibson GR, Parkers RJ, Herbert RA (1987) Evaluation of viable FEMS Microbiol Ecol 47: 207–214. counting procedures for the enumeration of sulfate-reducing Leloup J, Quillet L, Berthe T, Petit F (2006) Diversity of the bacteria in estuarine sediments. J Microbiol Methods 7: 201– dsrAB (dissimilatory sulfite reductase) gene sequences retrieved 210. from two contrasting mudflats of the Seine estuary, France. Heck KL Jr, Van Belle G, Simberloff D (1975) Explicit calcula- FEMS Microbiol Ecol 55: 230–238. tion of the rarefaction diversity measurement and the determi- Loy A, Küsel K, Lehner A, Drake HL, Wagner M (2004) Microar- nation of sufficient sample size. Ecology 56: 1459–1461. ray and functional gene analyses of sulfate-reducing prokary- Humayoun SB, Bano N, Hollibaugh JT (2003) Depth distribution otes in low-sulfate, acidic fens reveal cooccurrence of recog- of microbial diversity in Mono Lake, a meromictic soda lake in nized gnera and novel lineages. Appl Environ Microbiol 70: California. Appl Environ Microbiol 69: 1030–1042. 6998–7009. Joulian C, Ramsing NB, Ingvorsen K (2001) Congruent phyloge- Magurran AE (1988) Ecological diversity and its measurement. nies of most common small-subunit rRNA and dissimilatory Princeton University Press, Princeton, New Jersey, 192 pp. sulfite reductase gene sequences retrieved from estuarine sedi- Matsuyama M (1973) Changes in the limnological features of a ments. Appl Environ Microbiol 67: 3314–3318. meromictic Lake Suigetsu during the years, 1926–1967. J Jørgensen BB (1978) A comparison of methods for the quantifica- Oceanogr Soc Japan 29: 131–139. tion of bacterial sulfate reduction in coastal marine sediments. Matsuyama M, Saijo Y (1971) Studies on biological metabolism III. Estimation from chemical and bacteriological field data. in a meromictic Lake Suigetsu. J Oceanogr Soc Japan 27: 197– Geomicrobiol J 1: 49–63. 206. Jørgensen BB (1982) Mineralization of organic matter in the sea- Minz D, Flax JL, Green SJ, Muyzer G, Cohen Y, Wagner M, bed—The role of sulfate reduction. Nature 269: 643–645. Rittmann BE, Stahl DA (1999) Diversity of sulfate-reducing Karkhoff-Schweizer RR, Huber DP, Voordouw G (1995) Conser- bacteria in oxic and anoxic regions of a microbial mat charac- vation of the genes for dissimilatory sulfite reductase from terized by comparative analysis of dissimilatory sulfite reduc- Desulfovibrio vulgaris and Archaeoglobus fulgidus allows their tase genes. Appl Environ Microbiol 65: 4666–4671. detection by PCR. Appl Environ Microbiol 61: 290–296. Mullins TD, Britschgi TB, Krest RL, Giovannoni ST (1995) Ge- Karr EA, Sattley WM, Rice MR, Jung DO, Madigan MT, Achen- netic comparisons reveal the same unknown bacterial lineages bach LA (2005) Diversity and distribution of sulfate-reducing in Atlantic and Pacific bacterioplankton communities. Limnol bacteria in permanently frozen Lake Fryxell, McMurdo Dry Oceanogr 40: 148–158. Valleys, Antarctica. Appl Environ Microbiol 71: 6353–6359. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of com- Klein M, Friedrich M, Roger AJ, Hugenholtz P, Fishbain S, Abicht plex microbial populations by denaturing gradient gel elec- H, Blackall LL, Stahl DA, Wagner M (2001) Multiple lateral trophoresis analysis of polymerase chain reaction-amplified transfer of dissimilatory sulfite reductase genes between major genes coding for 16S rRNA. Appl Environ Microbiol 59: 695– lineages of sulfate-reducing prokaryotes. J Bacteriol 183: 6028– 700. 6035. Nakagawa T, Nakagawa S, Inagaki F, Takai K, Horikoshi K (2004) Koizumi Y, Kojima H, Fukui M (2004) Dominant microbial com- Phylogenetic diversity of sulfate-reducing prokaryotes in active position and its vertical distribution in saline meromictic Lake deep-sea hydrothermal vent chimney structures. FEMS Micro- Kaiike (Japan) as revealed by quantitative oligonucleotide biol. Lett 232: 145–152. probe membrane hybridization. Appl Environ Microbiol 70: Neletin LN, Schippers A, Pernthaler A, Hamann K, Amann R, 4930–4940. Jørgensen BB (2003) Quantification of dissimilatory (bi)sul- Kondo R, Kasashima N, Matsuda H, Hata Y (2000) Determina- phite reductase gene expression in Desulfobacterium autotroph- tion of thiosulfate in a meromictic lake. Fisheries Sci 66: icum using real-time RT-PCR. Environ Microbiol 5: 660–671. 1076–1081. Nonaka H, Keresztes G, Shinoda Y, Ikenaga Y, Abe M, Naito K, Kondo R, Nedwell DB, Purdy KJ, Silva SQ (2004) Detection and Inatomi K, Furukawa K, Inui M, Yukawa H (2006) Complete enumeration of sulphate-reducing bacteria in estuarine sedi- Genome Sequence of the Dehalorespiring Bacterium Desulfito- ments by competitive PCR. Geomicrobiol J 21: 145–157. bacterium hafniense Y51 and Comparison with Dehalococ- Kumar S, Gadagkar SR (2000) Efficiency of the neighbor-joining coides ethenogenes 195. J Bacteriol 188: 2262–2274. method in reconstructing deep and shallow evolutionary rela- Pérez-Jiménez JR, Young LY, Kerkhof LJ (2001) Molecular char- tionships in large phylogenies. J Mol Evol 51: 544–553. acterization of sulfate-reducing bacteria in anaerobic hydrocar- Lane H, Friedrich M, Lehner A, Drake HL, Cook AM (2001) Dis- bon-degrading and pure cultures using the dissimilatory sulfite similatory sulfite reductase (desulfoviridin) of the taurine-de- reductase (dsrAB) genes. FEMS Microbiol Ecol 35: 145–150. grading non-sulfate-reducing bacterium Bilophila wadsworthia Piatak M, Luk KC, Williams B, Lifson JD (1993) Quantitative RZATAU contains a fused DsrB-DsrD subunit. J Bacteriol 183: competitive polymerase chain reaction for accurate quantifica- 1727–1733. tion of HIV DNA and RNA species. BioTechniques 14: 70–80. Larsen Ø, Lien T, Birkeland N-K (2000) Characterization of the Porter KG, Feig YS (1980) The use of DAPI for identifying and desulforubidin operons from Desulfobacter vibrioformis and counting aquatic microflora. Limnol Oceanogr 25: 943–948. Desulfobulbus rhabdoformis. FEMS Microbiol Lett 186: 41–46. Posada D, Crandall KA (1998) Modeltest: testing the model of Sulphate-reducing bacteria in Lake Suigetsu 177
DNA substitution. Bioinformatics 14: 817–818. matter production of photosynthetic sulfur bacteria in Japanese Postgate JR (1984) The sulphate-reducing bacteria, 2nd ed. Cam- lakes. Limnol Oceanogr 13: 644–655. bridge University Press, Cambridge, 208 pp. Takeuchi J, Takii S (1987) Distribution of sulfate-reducing bacte- Purdy KJ, Embley TM, Takii S, Nedwell DB (1996) Rapid extrac- ria in the water of Lakes Hinuma and Suigetsu. Yosui to Haisui tion of DNA and rRNA from sediments by a novel hydroxyap- 29: 3–7 (in Japanese). atite spin-column method. Appl Environ Microbiol 62: 3905– Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG 3907. (1997) The Clustal X windows interface: flexible strategies for Rodriguez F, Oliver JF, Marin A, Medina JR (1990) The general multiple sequence alignment aided by quality analysis tools. stochastic model of nucleotide substitutions. J. Theor. Biol. 142: Nucleic Acids Res 25: 4876–4882. 485–501. Thomsen TR, Finster K, Ramsing NB (2001) Biogeochemical and Sakano Y, Kerfhof L (1998) Assessment of changes in microbial molecular signatures of anaerobic methane oxidation in a ma- community structure during operation of an ammonia biofilter rine sediment. Appl Environ Microbiol 67: 1646–1656. with molecular tools. Appl Environ Microbiol 64: 4877–4882. Wagner M, Roger AJ, Flax JL, Brusseau GA, Stahl DA (1998) Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a Phylogeny of dissimilatory sulfite reductases supports an early laboratory manual, 2nd ed. Cold Spring Harbor Laboratory origin of sulfate respiration. J Bacteriol 180: 2975–2982. Press, Cold Spring Harbor, New York. Widdel F (1988) Microbiology and ecology of sulfate- and sulfur- Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) reducing bacteria. In: Biology of anaerobic microorganisms (ed TREE-PUZZLE: maximum likelihood phylogenetic analysis Zehnder AJB). John Wiley and Sons, Inc, New York, pp. 469– using quartets and parallel computing. Bioinformatics 18: 502– 585. 504. Widdel F, Bak F (1992) Gram-negative mesophilic sulfate-reduc- Singleton D, Furlong MA, Rathbun SL, Whitman WB (2001) ing bacteria. In: The Prokaryotes, 2nd ed. (ed Balows A, Trüper Quantitative comparisons of 16S rRNA gene sequence libraries HG, Dworkin M, Harder W, Schleifer K-H). Springer-Verlag, from environmental samples. Appl Environ Microbiol 67: New York, pp. 3352–3378. 4374–4376. Zverlov V, Klein M, Lücker S, Friedrich MW, Kellermann J, Stahl Swofford DL (2002) PAUP*: Phylogenetic Analysis Using Parsi- DA, Loy A, Wagner M (2005) Lateral gene transfer of dissimi- mony (and other methods). Sinauer Associates, Sunderland, latory (bi)sulfite reductase revisited. J Bacteriol 187: 2203– Massachusetts. 2208. Takahashi M, Ichimura S (1968) Vertical distribution and organic