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 5Ϫ13ϫ106 cells mLϪ1 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 mLϪ1, 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 DSR1Fϩ their distribution in the lake. The only study was conducted (5Ј-ACSCACTGGAAGCACGGCGG-3Ј) (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 (5Ј-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 DSR1Fϩ; 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, 1ϫPCR culture-dependent method and show a diverse group of 2 buffer, 1ϫQ-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°35ЈN, 135°53ЈE) 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 1ϫTAE 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 LϪ1; 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- Ϫ 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 4Ј,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 mLϪ1, 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 Ϫ85°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).