Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ~99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.     

T-RFLP技术在人肠道柔嫩梭菌类群的组成分析中的应用

目的建立能快速筛查和比较大规模人群肠道中柔嫩梭菌类群组成结构的T-RFLP（末端限制性片段长度多态性）。方法采用T-RFLP技术特异性地分析柔嫩梭菌类群的组成,考察该方法的重复性和灵敏度。用该方法对15个志愿者肠道中的柔嫩梭菌类群进行分析,并与DGGE方法分析的结果进行对比。结果T-RFLP方法重复性高,最低能检测到群落中1%的细菌,其结果与DGGE的分析结果具有较好的一致性。结论本研究建立的柔嫩梭菌类群特异性的T-RFLP方法能够对大量肠道样品中柔嫩梭菌类群的结构进行快速、有效的筛查和比较。     

Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis.

Besides lichens and bacteria, fungi play a crucial role in the biodeterioration of historical glass. In the present paper, the fungal diversity on the surface of two historical church window glasses was investigated by 18S rDNA-based denaturing gradient gel electrophoresis (DGGE) analysis. 566-bp 18S rDNA-specific clone libraries were constructed with primer set NS1/NS2+10. Positive clones were reamplified with primer sets EF4/518rGC (426-bp fragments) and NS26/518rGC (316-bp fragments), amplicons were screened by DGGE and clustered according to their position in DGGE. Results indicated that fungal 18S rDNA clone libraries should be screened with at least two different primer sets to obtain the maximum number of different clones. For phylogenetic sequence analyses, clone inserts were sequenced and compared with 18S rDNA sequences listed in the EMBL database. Similarity values ranged from 93.7% to 99.81% to known fungi. Analyses revealed complex fungal communities consisting of members and relatives of the genera Aspergillus, Aureobasidium, Coniosporum, Capnobotryella, Engyodontium, Geomyces, Kirschsteiniothelia, Leptosphaeria, Rhodotorula, Stanjemonium, Ustilago, and Verticillium. The genera Geomyces and Aureobasidium were present on both glass surfaces. Some genera had not been detected on historical glass so far.     

Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla, Firmicutes and Proteobacteria. Within Firmicutes, the majority of the clones belonged to the genus Bacillus. Within Proteobacteria, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were Actinobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadetes, Chlorobi, Bacteroidetes, Chlamydiae, candidate division TM7, Ferribacter, Cyanobacteria, and Deinococcus. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.     

Predominant <Emphasis Type="Italic">Bacillus</Emphasis> spp. in Agricultural Soil under Different Management Regimes Detected via PCR-DGGE

A PCR system for studying the diversity of species of Bacillus and related taxa directly from soil was developed. For this purpose, a specific 24-bp forward primer located around position 110 of the 16S ribosomal RNA gene was designed and combined with a reverse bacterial primer located at the end of the gene. The specificity of this PCR system for bacilli and related taxons was confirmed on the basis of tests with diverse strains as well as with soil DNA. Analysis of a soil DNA derived clone library showed that the amplified fragments affiliated exclusively with sequences of gram-positive bacteria, with up to 95% of the sequences originating from putative Bacillus species. In particular, sequences affiliated to those of B. mycoides, B. pumilus, B. megaterium, B. thuringiensis, and B. firmus, as well as to related taxa such as Paenibacillus, were obtained. A minority, i.e., less than 6%, of the clones affiliated with other gram-positive bacteria, such as Arthrobacter spp., Frankia spp., and uncultured gram-positives. The amplified fragments were used as templates for a second PCR using bacterial 16S rDNA primers, yielding PCR products of about 410 bp, which were separated by denaturing gradient gel electrophoresis (DGGE). Amplicons indicating Bacillus spp. were found in the gel between 45% and roughly 60% denaturant, whereas those representing other, high-G+C% bacteria, were localized in gel regions with denaturant concentrations exceeding about 60%, thus allowing the distinction between these two groups of sequences. We applied this system to compare the group-specific diversity in bacterial communities in an agricultural soil under different regimes, i.e., permanent grassland, grassland recently turned to arable land, and arable land under agricultural rotation. Differences in the Bacillus-related community structures between the treatments were clearly detected. Higher diversities, as judged by Shannon–Weaver indices calculated on the basis of the molecular profiles, were consistently observed in the permanent grassland and the grassland turned into arable land, as compared to the arable land.     

Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria

Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned, and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several sequences that were not related to the known Nitrosospira-Nitrosomonas group and, thus, not likely to be ammonia oxidizers. However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few Nitrosospira-like sequences were detected.     

Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA.

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and WEISSELLA: Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant's life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.     

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, >?99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n?=?382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n?=?96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.     

High bacterial diversity in permanently cold marine sediments.

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones.     

Phylogenetic diversity and dietary association of rumen Treponema revealed using group-specific 16S rRNA gene-based analysis

Treponema spp. are a commonly detected bacterial group in the rumen that are involved in the degradation of soluble fibers. In this study, a ruminal Treponema group-specific PCR primer targeting the 16S rRNA gene was designed and used to assess the phylogenetic diversity and diet association of this group in sheep rumen. Total DNA was extracted from rumen digesta of three sheep fed a diet based on alfalfa/orchardgrass hay or concentrate. The real-time PCR quantification indicated that the relative abundance of the Treponema group in the total rumen bacteria was as high as 1.05%, while the known species Treponema bryantii accounted for only 0.02%. Fingerprints of the Treponema community determined by 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE) analysis tended to differ among the diets. Principal component analysis of the DGGE profiles distinguished those Treponema associated with either the hay or the concentrate diets. Analysis of a Treponema 16S rRNA gene clone library showed phylogenetically distinct operational taxonomic units for a specific dietary condition, and significant (P=0.001) differences in community composition were observed among clone libraries constructed from each dietary regimen. The majority of clones (75.4%) had <97% sequence similarity with known Treponema. These results suggest the predominance of uncultured Treponema that appear to have distinct members related to the digestion of either hay or concentrate diet.     

Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% +/- 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% +/- 7% and 14% +/- 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Diversity of geobacteraceae species inhabiting metal-polluted freshwater lake sediments ascertained by 16S rDNA analyses

The abundance, distribution, and phylogenetic diversity of members of the Fe(III)-reducing family Geobacteraceae were studied along a gradient of metal contaminants in Lake Coeur d’Alene, Idaho. Partial 16S rRNA gene fragments were amplified by PCR using primers directed toward conserved regions of the gene within the family Geobacteraceae. Analysis of amplicons separated by denaturing gradient gel electrophoresis (DGGE) suggested within-site variation was as great as between-site variation. Amplicons were cloned and grouped by RFLP type and DGGE migration distance and representatives were sequenced. Grouping clones with 3% or less sequence dissimilarity, 15 distinct phylotypes were identified compared to 16 distinct DGGE bands. Only 1 phylotype was recovered from all sites. This clone, B14, is most closely related to Geobacter metallireducens and constituted a greater portion of the pristine community than of the contaminated communities. A second phylotype, Q2, predominated in the contaminated communities and was notably absent from the pristine libraries. Clone Q2 presents a high degree of sequence similarity to two Geobacter spp. previously isolated from this region of Lake Coeur d’Alene. Six phylotypes were unique to the contaminated sediments, whereas two were found only in the pristine sediments. Indices of diversity (Shannon and Simpson) were consistently higher when calculated with DGGE data than when clone library data were used. Most-probable-number PCR and real-time PCR suggested that the Geobacteraceae phylotypes were spread relatively evenly across all three sites along the gradient. Our data indicate that the Geobacteraceae are diverse and abundant in Lake Coeur d’Alene sediments, regardless of metals content. These results provide insight into the ability of dissimilatory Fe(III)-reducing bacteria to colonize habitats with elevated metal concentrations, and they have important implications for the management and remediation of metal-contaminated sites.     

Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis

Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease.     

Phylogenetic composition of bacterioplankton assemblages from the Arctic Ocean

We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domain Bacteria: alpha(36%)-, gamma(32%)-, delta(14%)-, and epsilon(1%)-Proteobacteria; Cytophaga-Flexibacter-Bacteroides spp. (9%); Verrucomicrobium spp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 and Roseobacter groups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related to Cytophaga and Polaribacter species were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) with Alteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) with Oceanospirillum sp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to alpha-Proteobacteria (dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to gamma-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.     

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.     

Assessment of the diversity of Paenibacillus species in environmental samples by a novel rpoB-based PCR-DGGE method

A specific PCR system based on the gene encoding the RNA polymerase beta subunit, rpoB, was developed for amplification and denaturing gradient gel electrophoresis (DGGE) fingerprinting of Paenibacillus communities in environmental samples. This gene has been previously proven to be a powerful identification tool for the discrimination of species within the genus Paenibacillus and could avoid the limitations of 16S rRNA-based phylogenetic analysis. Initially, the PCR system based on universal rpoB primers were used to amplify DNAs of different Paenibacillus species. A new reverse primer (rpoBPAEN) was further designed based on an insertion of six nucleotides in the Paenibacillus sequences analyzed. This semi-nested PCR system was evaluated for specificity using DNAs isolated from 27 Paenibacillus species belonging to different 16S rRNA-based phylogenetic groups and seven non-Paenibacillus species. The non-Paenibacillus species were not amplified using this PCR approach and one group of Paenibacillus species consisting of strains without the six-base insert also were not amplified; these latter strains were found to be distinct based on 16S rRNA gene phylogeny. In addition, a clone library was generated from the rpoB fragments amplified from two Brazilian soil types (Cerrado and Forest) and all 62 clones sequenced were closely related to one of the 22 sequences from Paenibacillus previously obtained in this study. To assess the diversity of Paenibacillus species in Cerrado and Forest soils and in the rhizosphere of different cultivars of maize, a PCR-DGGE system was used. The Paenibacillus DGGE fingerprints showed a clear distinction between communities of Paenibacillus in Forest and Cerrado soils and rhizosphere samples clustered along Cerrado soil. Profiles of cultivars CMS22 and CMS36 clustered together, with only 53% of similarity to CMS11 and CMS04. The results presented here demonstrate the potential use of the rpoB-based Paenibacillus-specific PCR-DGGE method for studying the diversity of Paenibacillus populations in natural environments.     

Application of a novel Paenibacillus-specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus spp. in the maize rhizosphere

In this study, a Paenibacillus-specific PCR system, based on the specific primer PAEN515F in combination with bacterial primer R1401, was tested and used to amplify specific fragments of the 16S rRNA gene from rhizosphere DNA. The amplicons were used in a second (semi-nested) PCR for DGGE, in which bacterial primers F968GC and R1401 were used. The resulting products were separated into community fingerprints by DGGE. To assess the reliability of the method, the diversity of Paenibacillus species was evaluated on the basis of DNA extracted directly from the rhizospheres of four different cultivars of maize (Zea mays), i.e. CMS04, CMS11, CMS22 and CMS36, sown in two Brazilian field soils (Cerrado and Várzea). In addition, a clone library was generated from the PCR-generated 16S rDNA fragments, and selected clones were sequenced.The results of the bacterial community analyses showed, at the level of clone libraries, that considerable diversity among Paenibacillus spp. was present. The most dominantly found sequences clustered into 12 groups, each one potentially representing a species complex. Sequences closely affiliated with the P. macerans and P. azotofixans complexes were found in all samples, whereas other sequences were scarcer. Clones affiliated with the latter species complex were most abundant, representing 19% of all clones analysed.The Paenibacillus fingerprints generated via semi-nested PCR followed by DGGE showed a clear distinction between the maize plants grown in Cerrado versus Várzea soils. Thus, soil type, instead of maize cultivar type, was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the rhizospheres investigated. At a lower level (subcluster), there was a trend for maize cultivars CMS11 and CMS22 on the one hand, and CMS36 and CMS04 on the other hand, to cluster together, indicating that these respective pair of cultivars were similar in their Paenibacillus species composition. This trend was tentatively linked to the growth characteristics of these maize cultivars. These results clearly demonstrated the efficacy of the Paenibacillus-specific PCR-DGGE method in describing Paenibacillus species diversity in rhizosphere soils.     

Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.

The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.     

Molecular cloning of a human immunoglobulin lambda chain variable sequence

We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences.     

16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化

用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。