Evaluation of PCR Primer Selectivity and Phylogenetic Specificity by Using Amplification of 16S rRNA Genes from Betaproteobacterial Ammonia-Oxidizing Bacteria in Environmental Samples

The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the βAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations βAMOf/βAMOr, βAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.     

基于线粒体12S和16S rRNA基因部分序列的角蟾亚科部分属种的系统发育关系

测定了角蟾亚科2属8种(亚种)和外群3种的线粒体12S和16S rRNA基因部分DNA序列,比对后序列长共949bp,其中变异位点数320,简约位点数206。邻接法和最大简约法分析的系统关系树一致表明内群为一单系群,其中腺角蟾首先与其他物种分开;沙坪角蟾与宽头短腿蟾聚为一支;余下的5种(亚种)角蟾组成一支,其中小角蟾短肢亚种的广西种群和香港种群聚为一亚支,另一亚支包括峨眉角蟾、小角蟾指名亚种、尾凸角蟾和重庆武隆的角蟾种,后两种角蟾进化关系最近。本结果支持短肢角蟾为有效种,同时提示腺角蟾、沙坪角蟾与宽头短腿蟾可能隶属3个不同的亚属或属。     

Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10⁷ per g of dry soil for the H₂-CO₂-utilizing methanogens and of up to 10⁶ for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H₂-CO₂, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

基于12S和16S rRNA序列的湍蛙属部分物种的系统发育关系

测定了湍蛙属 6个种共 10个种群 ,以及 4个外群种的线粒体 12S和 16SrRNA基因片段 ,比对后有94 0bp序列 ,发现 35 2个变异位点、 186个简约性位点。运用NJ法、MP法、ML法构建了系统关系树 ,各系统树一致表明内群为一单系群 ,分为两组 :第一组中 ,四川湍蛙两种群先聚合 ,再和棕点湍蛙聚为一支 ;第二组中 ,香港湍蛙和戴云湍蛙聚为一支 ,而香港大屿山离岛湍蛙种群首先与华南湍蛙相聚 ,再与武夷湍蛙构成姐妹支。研究结果表明 :香港地区增加 1种湍蛙分布 ;戴云湍蛙是一有效种 ;四川湍蛙的石棉和洪雅种群间遗传差异达到或超过其他种间的分歧水平。     

Microbial Populations Responsive to Denitrification-Inducing Conditions in Rice Paddy Soil,as Revealed by Comparative 16S rRNA Gene Analysis

Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes, including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.Rice is one of the most important agronomic plants in the world (20). More than 135 million ha are used for rice cultivation worldwide, 88% of which consists of paddy fields (i.e., flooded fields) (16). Since rice paddy soil has limited available oxygen, various anaerobic biochemical processes can occur, including methane production, Mn⁴⁺ and Fe³⁺ reduction, nitrate respiration, and denitrification.Denitrification is a microbial respiratory process during which soluble nitrogen oxides (NO₃⁻ and NO₂⁻) are reduced to gaseous products (NO, N₂O, and N₂) (14, 43). Reduction of nitrate (NO₃⁻) to nitrite (NO₂⁻) is part of the denitrification process; however, this reaction can also be performed by nondenitrifiers. Reduction of nitrate to nitrite as an end product is called nitrate respiration (43). The emission of N₂O from rice paddy soils is less than that from upland crop fields (2), which is probably due to complete nitrate-nitrite reduction to N₂, since rice paddy soil is known to have strong denitrifying activity (28). However, the microbes responsible for denitrification in rice paddy soil are not well known.Denitrifying ability is sporadically distributed among taxonomically diverse groups of bacteria, as well as some archaea and fungi (14, 33, 43). Therefore, it is difficult to identify denitrifying organisms based only on their 16S rRNA gene sequences (33). However, culture-independent 16S rRNA gene analysis can be used to identify microbial populations responsive to denitrification-inducing conditions if they are properly differentiated from background populations. The 16S rRNA gene can provide taxonomic information about organisms which cannot be obtained from analyses targeting nitrite reductase genes (nirS and nirK) alone (34).One approach to differentiate functionally active populations from background populations is to use stable-isotope probing (SIP) (35). SIP was previously used to identify succinate-assimilating bacterial populations under denitrifying conditions in rice paddy soil, using nitrate and succinate as the electron acceptor and donor, respectively (37). Although SIP analysis can provide solid evidence that links function with taxonomy, it requires assimilation of isotopically labeled substrates. This may limit the application of SIP in studies of dissimilatory processes, such as nitrate respiration and denitrification. For example, previous SIP studies targeted bacteria assimilating ¹³C-labeled acetate, methanol, or succinate under denitrifying conditions (13, 30, 37).Another approach is to detect specifically enriched microbial populations under certain conditions by comparative analysis of 16S rRNA gene sequences (9). This approach does not necessarily require addition of isotopically labeled substrates and therefore has the potential to identify microbes performing dissimilatory processes. Furthermore, the community structure of the total population can also be elucidated in this manner (10, 25, 36). However, the usefulness of comparative analysis of 16S rRNA gene sequences has not been thoroughly tested. In addition, this approach has not been used to study nitrate respirators and denitrifiers.Consequently, the objectives of this study were (i) to characterize the soil bacterial population in rice paddy soil by clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene and (ii) to identify active bacterial populations under denitrification-inducing conditions by comparing clone libraries.     

Phylogenetic Analysis of Sequences of the 12S and 16S rRNA Mitochondrial Genes in the Family Bovidae: New Evidence

The phylogeny of the family Bovidae has been inferred from our data on the 12S and 16S rRNA mtDNA gene sequences and from the results of other authors. A considerable (2460 bp) length of the analyzed fragments of these conserved genes and the use of different methods of cladogram construction allowed us to verify the systematic position of the genera Saiga, Pantholops, Procapra, and Oreamnos. Saigas were shown to be phylogenetically far closer to gazelles than black-tailed gazelles and pygmy antelopes. In general, the genetic analysis data are in agreement with the results of morphological studies.     

基于线粒体16S rRNA基因对中国狼蛛科(Lycosidae)系统发育研究

将自测的中国狼蛛科Lycosidae4亚科6属26种和从GenBank中检索到的北美2种豹蛛的mtD-NA-16S rRNA序列进行比较;以漏斗蛛科1种蜘蛛作为外群,对碱基序列的组成和遗传距离进行了分析,采用Bayesian方法和最大简约法(MP)构建分子系统树。研究结果表明:16SrRNA基因的部分序列为340bp到360bp,A T含量平均为75%,存在较强的A T含量偏向性;序列共有157个碱基存在变异,其中79个简约信息位点。狼蛛科各属间的遗传距离介于0.026￣0.200之间。2种建树方法均表明:科内的属及属内的种优先聚在一起;水狼蛛属相对马蛛属是狼蛛科中较为原始的类群,分化较早;獾蛛属作为1个单系群与熊蛛属合为1个并系,属于狼蛛亚科。狼蛛科6属间的分子系统关系为(Pirata(Hippasa(Trochsa Arctosa(Pardosa Wadicosa))))。     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

Phylogenetic Diversity and Population Densities of Culturable Cellulolytic Soil Bacteria across an Agricultural Encatchment

Abstract A typical, small encatchment (catena B?lkendorf) in the moraine, northeast German agricultural landscape Schorfheide-Chorin was studied with respect to summit, midslope, and foot-slope positions at northern and southern slope exposure, respectively, including a central noncultivated kettle hole position (pot hole). Across the sequence of seven distinct sampling positions, soil organic carbon and total nitrogen contents, soil gravimetric water content, and soil microbial biomass displayed maxima at the kettle hole position. Soil pH revealed a decreasing trend at the northern exposed slope and a minimum at the kettle hole position. Against this background, the population density of total culturable bacteria clearly displayed a minimum at the kettle hole position, whereas the population density of carboxymethylcellulose decomposing bacteria was not clearly differentiated in relation to sampling positions. To study the phylogenetic diversity of culturable cellulolytic bacteria, 311 isolates were obtained from the sampling positions across the entire encatchment and examined by restriction analysis of PCR-amplified 16S rDNA. Using the restriction enzyme ScrFI, isolates were classified into 31 pattern groups. Although the ratio of actinomycetes within total isolates ranged from 0.73 to 0.94, only 16 pattern groups originated from actinomycetes, but 15 from other bacteria. At all sampling positions, a dominant pattern group was identified, containing 38 to 65% of total isolates. Two site-specific pattern groups could be identified, representing significant parts of the total population, which were highly specific for the kettle hole (19% of total isolates) and for foot- and midslope positions (15–18% of total isolates), respectively. In general, the composition of cellulolytic isolates across the encatchment displayed differences with respect to slope positions, but was not significantly affected by soil properties. Based on 16S rDNA sequence analysis, isolates of the dominant as well as the specific pattern groups could be assigned to the genus Streptomyces. Furthermore, sequencing of 16S rDNA of isolates of another three pattern groups revealed a high phylogenetic diversity among these isolates, including cellulomonads and bacilli. Received: 12 August 1998; Accepted: 1 February 1999     

Prevalence of Lysogeny among Soil Bacteria and Presence of 16S rRNA and trzN Genes in Viral-Community DNA

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.     

Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA

In a previous study (S. G. Acinas, F. Rodríguez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria, Alteromonas macleodii, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated γ Proteobacteria predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the α Proteobacteria of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the A. macleodii cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.     

16S rRNA Sequences and Differences in Bacteria Isolated from the Muztag Ata Glacier at Increasing Depths

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs Plateau (China). Only 11% were psychrophiles (grew at 2°C or −2°C up to ~20°C), although the majority (82%) were psychrotolerant (grew at 2°C or −2°C up to 37°C). The majority of the isolates had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% were γ-Proteobacteria, 14.7% were α-Proteobacteria, 14.7% were Flavobacteria, and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with Proteobacteria, HGC/Cytophaga-Flavobacterium-Bacteroides (CFB), Proteobacteria, LGC/CFB/HGC, Cryobacterium psychrophilum, HGC/CFB, Proteobacteria/HGC/CFB, and HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and nutrient availability within the ice.     

基于12S rRNA和16S rRNA基因序列探讨中国蚤蝇科部分属间的系统发育关系

基于线粒体12S rRNA和16S rRNA基因序列联合分析,采用最大简约法、最大似然法和贝叶斯法分别构建了中国蚤蝇科14属的系统发育树.结果表明:联合分析序列总长度为819 bp,其中可变位点277个,简约信息位点200个;A+T平均含量为77.7%,具A、T偏倚性.系统发育分析显:中国蚤蝇科为单系发生,分为蚤蝇亚科和裂蚤蝇亚科两个单系群.蚤蝇亚科内脉蚤蝇属、锥蚤蝇属和刺蚤蝇属亲缘关系较近,栅蚤蝇属与栓蚤蝇属亲缘关系较近;裂蚤蝇亚科中虼蚤蝇属与裂蚤蝇属互为姐妹群,寡蚤蝇属与伐蚤蝇属互为姐妹群.     

Genus- and Species-Specific PCR-Based Detection of Dairy Propionibacteria in Environmental Samples by Using Primers Targeted to the Genes Encoding 16S rRNA

PCR assays with primers targeted to the genes encoding 16S rRNA were developed for detection of dairy propionibacteria. Propionibacterium thoenii specific oligonucleotide PT3 was selected after partial resequencing. Tests allowed the detection of less than 10 cells per reaction from milk and cheese and 10² cells per reaction from forage and soil.     

Intervening Sequences in 16S rRNA Genes of Campylobacter sp.: Diversity of Nucleotide Sequences and Uniformity of Location

We found and sequenced intervening sequences (IVSs) in the PCR-amplicons of 16S rRNA genes of 3 strains of Campylobacter rectus, 2 strains of C. curvus and 2 strains of C. sputorum. The lengths of the IVSs were 140 to 233 bp. The IVSs of C. rectus were identical and had a sequence homology of 55 to 79% against those of C. curvus and C. helveticus. The IVSs of C. sputorum were 97.9-100% homologous but poorly homologous to the other IVSs. In spite of the diversities of the lengths and the nucleotide sequences, all of the IVSs were located at the same position in the 16S rRNA genes.     

Analysis of Ammonia-Oxidizing Bacteria from Hypersaline Mono Lake, California, on the Basis of 16S rRNA Sequences

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the β subdivision of the division Proteobacteria (β-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different β-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the γ subdivision of the Proteobacteria, did not amplify target from any samples.     

Rhizobial 16S rRNA and dnaK Genes: Mosaicism and the Uncertain Phylogenetic Placement of Rhizobium galegae

The phylogenetic relatedness among 12 agriculturally important species in the order Rhizobiales was estimated by comparative 16S rRNA and dnaK sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of Mesorhizobium loti and Mesorhizobium ciceri, and the other group consisted of Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, and Rhizobium leguminosarum. Although bootstrap support for the placement of the remaining six species varied, A. tumefaciens, Agrobacterium rubi, and Agrobacterium vitis were consistently associated in the same subcluster. The three other species included Rhizobium galegae, Sinorhizobium meliloti, and Brucella ovis. Among these, the placement of R. galegae was the least consistent, in that it was placed flanking the A. rhizogenes-Rhizobium cluster in the dnaK nucleotide sequence trees, while it was placed with the other three Agrobacterium species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of R. galegae, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between R. galegae and certain other species, suggesting that the R. galegae genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of R. galegae, and they also illustrate a potential pitfall in the use of partial sequences for species identification.