Transcription of [FeFe]-Hydrogenase Genes during H2 Production in Clostridium and Desulfovibrio spp. Isolated from a Paddy Field Soil

Changes in the relative abundances of the transcripts of hydA gene paralogs for [FeFe]-hydrogenase in Clostridium sp. strain H2 and Desulfovibrio sp. strain A1 isolated from paddy field soil were analyzed during H2 production. Strains H2 and A1 had at least five and two phylogenetically different hydA genes, respectively. The relative abundances of their hydA transcripts differed among the paralogs and H2 production activity changed in a manner that depended on the growth phase and conditions. Increases or decreases in the relative abundances of the transcripts of two out of five hydA genes in strain H2 correlated with changes in H2 production rates, whereas those of the others remained unchanged or decreased. In strain A1, the relative abundances of the transcripts of two hydA genes differed between monoculture, sulfate-reducing, and syntrophic, methanogenic conditions. The relative abundance of the transcripts of one hydA gene, predicted to encode a cytosolic [FeFe]-hydrogenase, was higher under syntrophic, methanogenic conditions than sulfate-reducing conditions, while that of the transcripts of the other hydA gene decreased with time under both conditions. This study showed that the transcription of the hydA gene during growth with active H2 production was differently regulated among the paralogs in H2 producers isolated from paddy field soil.

of 1.5 % agar-containing 50 mg L -1 L-cysteine hydrochloride hydrate were overlaid to the solidified mixture, bottom face of the sterilized inner glass petri dish was pressed onto the agar to shut off air supply (double-petri dish method: (16)). The petri dish was covered with a plastic wrap and incubated at 30°C for 1 month. Bacterial colonies formed under the bottom face on the medium were picked up by sterilized Pasteur pipettes under filtered N 2 gas atmosphere and inoculated into 5 mL of VL liquid medium in test tubes, which were prepared anoxically by the Hungate technique (6,12,14). The test tube was closed with a butyl rubber cap and a screw cap and incubated at 30°C under N 2 gas phase.
The culture after growth was purified by the roll-tube technique, and then well-isolated colonies formed on the agar in the roll-tube were picked up and transferred into a new VL liquid medium. To select H 2 producers, H 2 production activity was monitored by gas chromatography as described in Baba et al. (5). This purification cycle was repeated at least three times, and then a pure culture was obtained finally. Purity was confirmed by preparing roll tubes inoculated with serial dilutions of the culture and examining homogeneity of colony morphology, in addition to the homogeneity of cell morphology by microscopic observations. DNA extraction, PCR amplification, cloning and sequencing steps for direct determination of 16S rRNA gene sequence were described previously (4,13).
The primer set of 27f and 1492r (19) was used for the analysis (Table S1). Gram staining was checked by the Hucker method. Cell morphology was observed by a phase contrast microscope (BX50, Olympus, Tokyo, Japan). The pure culture was designated strain H2. Nucleotide sequence of 16S rRNA gene has been deposited to the DDBJ database under the accession number LC194786.

Isolation of Desulfovibrio sp. A1 and Methanobacterium sp. AH1
A sulfate-reducing bacterium and a methanogenic archaeon were isolated from a methanogenic consortium enriched on acetate from a paddy field soil.
Enrichment was performed in a 1 L serum bottle containing 100 mL medium sealed with a butyl rubber stopper and an aluminum seal. A moist soil sample (about 20 g), which was collected from a paddy field plot (Anthraquic Yellow Soil; Oxyaquic Dystrudepts) in the Aichi-ken Anjo Research and Extension Center, Anjo, Aichi, Japan on 28 July 2003 (18), was inoculated into the medium and the bottle was incubated statically at 30˚C. About 90% of the culture was replaced by fresh medium about once per one to two months with monitoring methane production. Methane production was determined by gas chromatography as described previously (2) with a GC-9A gas chromatograph equipped with a frame ionization detector (Shimadzu, Kyoto, Japan). The enrichment culture was scaled down to a 20 mL medium in a 120 mL serum bottle since the fourth transfer. After the following two transfers, roll tubes were prepared. For roll tubes, 1.7% (w/v) agar was added to the medium. The well-isolated colonies were inoculated into 5 mL of liquid medium in test tubes and methane production was followed with time after incubation. Two mL of a culture that formed methane was inoculated into the fresh medium in a 120 mL serum bottle. After two successive transfers as described above, the culture was subjected to microscopic observation and denaturing gradient gel electrophoresis (DGGE) to examine the archaeal/bacterial communities in the culture. Epifluorescent microscopy was conducted with a BX50 microscope (Olympus, Tokyo, Japan). DGGE analysis of methanogenic archaeal 16S rRNA gene was carried out for the extracted DNA from the culture by bead-beating method with a primer set of 1106F-GC/1378R (17,18). DGGE analysis of bacterial 16S rRNA gene in the culture was conducted for the extracted DNA as described by Asari et al. (3).
One mL of the culture was transferred into 10 mL of the basal medium without acetate and incubated under H 2 /CO 2 (mixing ratio was 4:1, 203 kPa) atmosphere to isolate a hydrogenotrophic methanogenic archaeon. Roll tubes were prepared for the culture on H 2 /CO 2 and well-isolated colonies were picked up and inoculated into 5 mL liquid medium in test tubes. A culture that grew with methane production was subcultured in a 120 mL serum bottle in 10 mL medium.
To test for purity, the culture was inoculated into three kinds of media as follows: 1) the basal medium containing 1% (w/v) glucose, 0.1% Bacto Yeast Extract (Difco) and 0.1% Polypepton (Nihon Pharmaceutical, Osaka, Japan), 2) the basal medium containing 30 mM Na 2 SO 4 and 20 mM sodium lactate and 3) VL medium under N 2 /CO 2 (4:1, 203 kPa) atmosphere for anaerobic heterotrophs as described previously (1). The preparations were incubated statically for 30 days at 30˚C. No growth was observed and no contaminants were detected microscopically. The pure culture was designated strain AH1.
The basal medium without acetate but containing 8.8 g L -1 sodium pyruvate was used to isolate a bacterium from the enrichment culture. Two mL of the enrichment culture were inoculated into 20 mL of the liquid medium and incubated at 30˚C. The medium became turbid within 3 days and proliferation of curved rods was observed in the culture. Roll tubes were then prepared for the culture and well-isolated colonies were picked up to 5 mL liquid medium in the test tubes. A grown culture was subcultured in 20 mL of the basal medium.
Isolation of colonies from roll tubes was repeated for purification and well-isolated colonies were inoculated into the liquid medium. Purity was confirmed by preparing roll tubes inoculated with serial dilutions of the culture and examining homogeneity of colony morphology and proportional changes in number of colonies according to dilution. The pure culture was designated strain A1.
DNA extraction, PCR amplification, cloning and sequencing steps for determination of 16S rRNA gene sequences of strains AH1 and A1 were described previously (13,17). The primers were same as those described by Watanabe et al. (17) and 1106F and 1378R (18) were also used for strain AH1.
Morphology of cells of the isolates was observed by phase contrast and epifluorescent microscopy as described above and also by transmission electron microscopy. Cells for electron microscopy were collected from a late logarithmic culture at 10,000 g for 2 min. Resuspended cells were stained negatively with 20 g L -1 uranyl acetate after treating with 0.5 % (vol/vol) glutaraldehyde solution and observed with a Hitachi H-7500 AMT Advantage HR electron microscope (Hitachi High-Technology, Tokyo, Japan). Gram staining was carried out by the Hucker method. Substrate utilization of strain A1 was tested in the basal media with and without sodium sulfate (29 mM).
To examine syntrophic methanogenesis of strains AH1 and A1, the strains were cultivated in 5 mL basal media in test tubes or 20 mL basal media in serum bottles containing acetate, pyruvate, lactate, ethanol, malate or fumarate as described above. Growth was measured turbidimetrically at 660 nm and methane production was determined as described above. medium, strain A1 grew on pyruvate (80 mM), lactate (30 mM) and ethanol (0.2% [v/v]), but not acetate (50 mM).
Cell morphology of strain AH1 was long rod with the size 0.3 -0.6 µm  2 -8 µm (Fig. S5). The 16S rRNA gene sequence showed more than 99% similarity with Methanobacterium palustre DSM 3108 T (Fig. S6). Strain AH1 produced methane from H 2 /CO 2 and formate. The co-culture of strains AH1 and A1 utilized pyruvate, lactate and ethanol associated with methane production under the condition without sulfate, but not acetate, malate and fumarate.