Yoshikazu Koizumi

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

ABSTRACT A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Characterization of depth-related microbial community structure in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rDNA and reversely transcribed 16S rRNA fragments

Vertical changes of bacterial community structure in a mesophilic lake sediment were investigated by denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA and reversely transcribed 16S rRNA fragments. Comparison of community structure analyses based on 16S rDNA and rRNA revealed that the diversity indices from the 16S rDNA-based DGGE profiles were greater than those from the 16S rRNA-based DGGE profiles. In addition, the diversity indices based on 16S rDNA did not change drastically throughout the layers, but the diversity indices based on 16S rRNA decreased with sediment depth. This result indicates that the diversity of active bacteria decreases and inactive bacteria accumulate in association with sedimentation. Dendrograms deduced from DGGE profiles of either 16S rDNA or rRNA were different, and the rRNA-based dendrogram showed a significant difference between the upper layers (0-2, 2-5, and 5-8 cm) and lower layers (8-11, 11-14, 14-17, and 17-20 cm). The sequences of 13 DGGE bands were determined. Phylogenetic analysis of these rDNA fragments revealed that the most conspicuous band in both rDNA- and rRNA-based DGGE was closely related to the genus Nitrospira (95% sequence similarity), and the relative signal intensity was strong especially in the deep layers. Membrane hybridization with a probe targeting the genus Nitrospira also supported the observation that 16S rRNAs derived from Nitrospira-like microorganisms were abundant in this sediment (8.6-16.8% of bacterial 16S rRNA) and that the relative abundance increased with depth. Overall, our results demonstrated that parallel characterization of community structure based on both 16S rDNA and rRNA is important for assessing whole microbial populations and active microbial populations in sediments.

Vertical distributions of sulfate-reducing bacteria and methane-producing archaea quantified by oligonucleotide probe hybridization in the profundal sediment of a mesotrophic lake

Abstract Vertical distributions of sulfate-reducing bacteria and methane-producing archaea were investigated in the profundal sediment of a freshwater lake using membrane-immobilized small subunit rRNA hybridization with group- and genus-specific oligonucleotide probes. The annual average of the relative abundance of small subunit rRNA hybridized with all probes for sulfate-reducing bacteria to total small subunit rRNA was 2.3% at 0-2 cm and increased with depth up to 22.9% at 8-14 cm where sulfate concentration was less than 10 nmol ml(-1) in interstitial water, suggesting that these bacteria may survive on alternative metabolisms. The signal of probe Dsv687 (the family Desulfovibrionaceae and some Geobacteraceae) was the main factor in this increase. The relative abundance of methane-producing archaea to total small subunit rRNA was highest (7.8%) at 8-14 cm, dominated by the order Methanosarcinales. The metabolic rates measured in the sediments demonstrated that the peaks of sulfate reduction and methane production were separated vertically, and were not linked to their small subunit rRNA distributions. Our data indicate that sulfate-reducing bacteria can coexist with methane-producing archaea from 0 to 20 cm in the freshwater lake sediment.

Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization

ABSTRACT Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two δ-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the δ-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.

Depth-related change in archaeal community structure in a freshwater lake sediment as determined with denaturing gradient gel electrophoresis of amplified 16S rRNA genes and reversely transcribed rRNA fragments

Vertical changes in archaeal community structure in mesophilic freshwater lake sediment were investigated using denaturing gradient gel electrophoresis of amplified 16S rRNA genes and reversely transcribed 16S rRNA fragments. Electrophoretic band pattern of archaeal community did not drastically change with depth. The archaeal 16S rRNA-based denaturing gradient gel electrophoresis band pattern was very similar to the 16S rDNA-based pattern. These results indicate that archaeal community structure does not drastically change throughout the 0-20 cm in depth, and most archaeal populations in the sediment retain extractable rRNA. Phylogenetic analysis of seven sequences retrieved from both DNA- and RNA-based denaturing gradient gel electrophoresis revealed that these sequences were divided into four major lineages; the kingdom Crenarchaeota, the order Methanomicrobiales, the family Methanosaetaceae, and an uncultured cluster within Euryarchaeota. The order Methanomicrobiales and the family Methanosaetaceae were the dominant methane-producing archaea in this profundal freshwater lake sediment. Moreover, we found Euryarchaeota that are not related to methanogens, and non-thermophilic Crenarchaeota. This suggests that archaeal populations other than methanogens in low-temperature sediment have been previously underestimated.

Community structure of bacteria associated with sheaths of freshwater and brackish thioploca species.

Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.