Genus-specific primers targeting the 16S rRNA gene for PCR detection of members of the genus <Emphasis Type="Italic">Verrucosispora</Emphasis>

Little is known about the genus Verrucosispora though it does contain organisms which produce novel antibiotics. A set of genus-specific oligonucleotide primers was generated to gain an insight into the presence, distribution and taxonomic diversity of members of this genus in diverse samples taken from marine habitats. In silico and pure culture studies showed that the primers matched perfectly with target sequences of the 16S rRNA genes of representatives of the genus Verrucosispora. The primers, designated S-G-Verr-0195-a-S-20 and S-G-Verr-1152-a-A-18, amplified an ≈960 bp stretch of the 16S rRNA genes of Verrucosispora strains but not those of representatives of other genera classified in the family Micromonosporaceae. Genus-specific amplicons were detected from 17 out of 20 community DNA samples prepared from diverse marine sediments and coastal soils. Phylogenetic analysis of over 40% of clones derived from five of the samples indicated they belonged to novel Verrucosispora species. The primers were also used to confirm the identity of Verrucosispora-like strains isolated from two of the environmental samples. The primers can be used to facilitate the isolation of novel Verrucosispora strains by allowing prescreening of environmental samples and the subsequent identification of verrucosisporae on selective isolation plates. For this purpose, a novel medium facilitating the recovery of Verrucosispora strains was formulated and used to recover novel isolates validated using the novel PCR primers. This medium may be useful as the basis for development of a selective medium.     

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.     

Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea

In this study, we report on first 16S rRNA gene sequences from highly saline brine sediments taken at a depth of 1,515 m in the Kebrit Deep, northern Red Sea. Microbial DNA extracted directly from the sediments was subjected to PCR amplification with primers specific for bacterial and archaeal 16S rRNA gene sequences. The PCR products were cloned, and a total of 11 (6 bacterial and 5 archaeal) clone types were determined by restriction endonuclease digestion. Phylogenetic analysis revealed that most of the cloned sequences were unique, showing no close association with sequences of cultivated organisms or sequences derived from environmental samples. The bacterial clone sequences form a novel phylogenetic lineage (KB1 group) that branches between the Aquificales and the Thermotogales. The archaeal clone sequences group within the Euryarchaeota. Some of the sequences cluster with the group II and group III uncultivated archaea sequence clones, while two clone groups form separate branches. Our results suggest that hitherto unknown archaea and bacteria may thrive in highly saline brines of the Red Sea under extreme environmental conditions. Received: 5 February 1999 / Accepted: 14 July 1999     

Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the γ- Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of δ- Proteobacteria , most closely related to the Bdellovibrio . The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria , Myxobacteria and Dinoflagellata .     

Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization(1)

We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel 'non-extreme' environmental Archaea in the pelagic water of a boreal forest lake.     

硝尔库勒湖沉积物中非培养放线菌多样性

[目的]认识和了解盐湖沉积环境中放线菌的多样性,为今后的开发和利用奠定基础.[方法]应用免培养技术和基于16S rRNA基因序列的系统发育分析对新疆硝尔库勒盐湖沉积物中放线菌的多样性进行了研究.实验采用SDS-CTAB法提取土样中总DNA,利用放线菌特异性引物对土样总DNA进行16S rRNA基因扩增,并构建16S IRNA基因克隆文库;对随机挑选的160个克隆通过酶切筛选出51个不同图谱的重组克隆,并对其测序.[结果]所获得的51个克隆序列属于39个OTUs,其中52.9%的克隆序列分布于放线菌门(phylum Actinobacteria)放线菌亚(Actinobacteridae)的5个亚目和酸微菌亚纲(Acidimicrobidae)中,并且在这两个亚纲中有大量克隆序列属于放线菌的新类群,另外47.1%的克隆序列以极高的自展值在放线菌门内支持形成一个独立的大分支,极有可能代表一个新亚目或更高级分类单元的类群.[结论]这些研究结果说明硝尔库勒盐湖中存在有较为丰富的放线菌系统发育多样性,并且潜藏着新类型的放线菌资源.     

云南江城和黑井盐矿沉积物未培养放线菌多样性比较

类群特异性引物的应用使得研究者可以对感兴趣的微生物类群进行针对性研究.围绕云南江城和黑井两个地区的3个盐矿样点沉积物中放线菌的多样性和群落组成,我们通过放线菌特异性引物对总DNA进行16S rRNA基因扩增,经过克隆文库构建,利用酶切并选择其中不同带型的133个克隆的16S rRNA基因插入片段进行测序.系统发育分析和统计学结果表明,两地放线菌16S rRNA基因克隆广泛分布于整个放线菌门,同时发现部分序列可能属于放线菌的新类群.分析结果还预示,江城和黑井两地盐矿虽处云南不同地域含盐区,但两地未培养放线菌物种多样性和系统发育关系均较为相似.     

Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes

The phylogenetic diversity of green nonsulfur bacteria in nine stratified freshwater lakes was investigated. A set of oligonucleotide primers was developed that permitted the selective amplification of 16S rRNA gene sequences of this group. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced, which yielded a total of 19 novel sequence types. Ten of the sequences were related to those of different cultivated members of the C hloroflexus assemblage, whereas nine fell into the T78 group of environmental clones. For the latter subgroup of the green nonsulfur bacteria, no molecular isolate from freshwater plankton has been reported so far. Several of the sequence types occurred in more than one lake, indicating that not only relatives of the C hloroflexus assemblage, but also bacteria of the clone T78 group represent indigenous bacteria of nonthermal stratified freshwater ecosystems. Our results indicate that the natural diversity in the phylum of the green nonsulfur bacteria has been significantly underestimated in the past.     

High-Density Universal 16S rRNA Microarray Analysis Reveals Broader Diversity than Typical Clone Library When Sampling the Environment

Molecular approaches aimed at detection of a broad-range of prokaryotes in the environment routinely rely on classifying heterogeneous 16S rRNA genes amplified by polymerase chain reaction (PCR) using primers with broad specificity. The general method of sampling and categorizing DNA has been to clone then sequence the PCR products. However, the number of clones required to adequately catalog the majority of taxa in a sample is unwieldy. Alternatively, hybridizing target sequences to a universal 16S rRNA gene microarray may provide a more rapid and comprehensive view of prokaryotic community composition. This study investigated the breadth and accuracy of a microarray in detecting diverse 16S rRNA gene sequence types compared to clone-and-sequencing using three environmental samples: urban aerosol, subsurface soil, and subsurface water. PCR products generated from universal 16S rRNA gene-targeted primers were classified by using either the clone-and-sequence method or by hybridization to a novel high-density microarray of 297,851 probes complementary to 842 prokaryotic subfamilies. The three clone libraries comprised 1391 high-quality sequences. Approximately 8% of the clones could not be placed into a known subfamily and were considered novel. The microarray results confirmed the majority of clone-detected subfamilies and additionally demonstrated greater amplicon diversity extending into phyla not observed by the cloning method. Sequences matching operational taxonomic units within the phyla Nitrospira, Planctomycetes, and TM7, which were uniquely detected by the array, were verified with specific primers and subsequent amplicon sequencing. Subfamily richness detected by the array corresponded well with nonparametric richness predictions extrapolated from clone libraries except in the water community where clone-based richness predictions were greatly exceeded. It was concluded that although the microarray is unreliable in identifying novel prokaryotic taxa, it reveals greater diversity in environmental samples than sequencing a typically sized clone library. Furthermore, the microarray allowed samples to be rapidly evaluated with replication, a significant advantage in studies of microbial ecology.     

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

16S rRNA phylogenetic analysis and quantification of <Emphasis Type="Italic">Korarchaeota</Emphasis> indigenous to the hot springs of Kamchatka,Russia

The candidate archaeal division Korarchaeota is known primarily from deeply branching sequences of 16S rRNA genes PCR-amplified from hydrothermal springs. Parallels between the phylogeny of these genes and the geographic locations where they were identified suggested that Korarchaeota exhibit a high level of endemism. In this study, the influence of geographic isolation and select environmental factors on the diversification of the Korarchaeota was investigated. Fourteen hot springs from three different regions of Kamchatka, Russia were screened by PCR using Korarchaeota-specific and general Archaea 16S rRNA gene-targeting primers, cloning, and sequencing. Phylogenetic analyses of these sequences with Korarchaeota 16S rRNA sequences previously identified from around the world suggested that all Kamchatka sequences cluster together in a unique clade that subdivides by region within the peninsula. Consistent with endemism, 16S rRNA gene group-specific quantitative PCR of all Kamchatka samples detected only the single clade of Korarchaeota that was found by the non-quantitative PCR screening. In addition, their genes were measured in only low numbers; small Korarchaeota populations would present fewer chances for dispersal to and colonization of other sites. Across the entire division of Korarchaeota, common geographic locations, temperatures, or salinities of identification sites united sequence clusters at different phylogenetic levels, suggesting varied roles of these factors in the diversification of Korarchaeota.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Review and re-analysis of domain-specific 16S primers

The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the "universal" primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective "universal" Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.     

Use of Subtractive Hybridization To Design Habitat-Based Oligonucleotide Probes for Investigation of Natural Bacterial Communities

We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

Miniprimer PCR, a New Lens for Viewing the Microbial World

Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide “miniprimers” expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.     

Complementary retrieval of 16S rRNA gene sequences using broad-range primers with inosine at the 3'-terminus: implications for the study of microbial diversity

We evaluated the impact of the base analogue inosine substituted at the 3'-terminus of broad-range 16S rRNA gene primers on the recovery of microbial diversity using terminal restriction fragment length polymorphism and clonal analysis. Oral plaque biofilms from 10 individuals were tested with modified and unmodified primer pairs. Besides a core overlap of shared terminal restriction fragments (T-RFs), each primer system provided unique information on the occurrence of T-RFs, with a higher number generally displayed with inosine primers. All clones sequenced were at least 99% identical to publicly available full-length sequences. Analysis of the corresponding primer-binding sites showed that most sequence types were 100% complementary to the unmodified primers so that the characteristic of inosine to bind with all four nucleotides was not crucial for the observed increase in microbial richness. Instead, differences in community compositions were correlated with the identity of the nearest-neighbor 3' of the primer-targeting region. By influencing the thermal stability of primer hybridization, this position may play a previously unrecognized role in biased amplification of 16S rRNA gene sequences. In conclusion, the combined use of inosine and unmodified primers enables the complementary retrieval of 16S rRNA gene types, thereby expanding the observed diversity of complex microbial communities.     

Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.     

Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses

Two specific primers were developed for the amplification of 16S rRNA genes of Desulfotomaculum lineage 1 to detect members of the genus Desulfotomaculum in rice field soil. The combination of both primers in PCR allowed the specific amplification and cloning of ten 16S rDNA sequences of this group from rice paddy soil DNA extracts. The phylogenetic analysis showed that these sequences formed a deeply branching cluster within Desulfotomaculum lineage 1, together with two sequences from the database and two sequences from a hydrocarbon-contaminated aquifer. Dissimilarity values to validly described species, including recently isolated strains of Desulfotomaculum from rice paddy microcosms, were higher than 12%. Within the new cluster the cloned sequences formed three separate groups which were each represented by at least two sequences with identities of >/=99% while one sequence represented an additional group. The sequences should represent sulfate-reducing organisms because they clearly fell into the physiologically coherent group of Gram-positive sulfate reducers. The relative abundance of bacteria of the Desulfotomaculum lineage 1 in rice paddy soil and root samples was estimated with rRNA dot blot hybridizations of extracted RNA. The relative RNA content of Desulfotomaculum lineage 1 was 0.55% in the bulk soil and 1% in the rice root samples, respectively, of the total 16S rRNA content (probe Eub338). Hybridization of rRNA with a probe targeting the new cluster represented by the cloned sequences confirmed the high abundance of 16S rRNA sequences from this cluster in the rice paddy field samples. Another hybridization probe detecting Desulfotomaculum acetoxidans and two closely related Desulfotomaculum isolates from rice paddy soil indicated that these bacteria were less abundant.