Analysis of the large bowel microbiota of colitic mice using PCR/DGGE

AIM: To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis. METHODS AND RESULTS: The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations ('Bacteroides sp.', Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent (Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed. CONCLUSIONS: PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis.     

Supplementation of the diet with high-viscosity beta-glucan results in enrichment for lactobacilli in the rat cecum

BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley beta-glucans of high [HV] or low viscosity [LV] in order to measure the prebiotic effects of these different sources of dietary fiber. The dietary impact on the composition of the cecal microbiota was determined by the generation of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene sequences. The DGGE profiles produced from the cecal microbiota of rats within each dietary group were similar, but consensus profiles generated from pooled bacterial DNAs showed differences between rat groups. Animals fed HV glucans (HV-fed rats) had DGGE consensus profiles that were 30% dissimilar from those of the other rat groups. A 16S rRNA gene fragment that was more conspicuous in the profiles of HV-fed animals than in those of cellulose-fed rats had sequence identity with Lactobacillus acidophilus. Measurements of L. acidophilus rRNA abundance (DNA-RNA hybridization), the preparation of cloned 16S rRNA gene libraries, and the enumeration of Lactobacillus cells (fluorescent in situ hybridization) showed that lactobacilli formed a greater proportion of the cecal microbiota in HV-fed rats. In vitro experiments confirmed that some lactobacilli utilize oligosaccharides (degree of polymerization, 3 or 4) present in beta-glucan hydrolysates. The results of this study have relevance to the use of purified beta-glucan products as dietary supplements for human consumption.     

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Supplementation of the Diet with High-Viscosity Beta-Glucan Results in Enrichment for Lactobacilli in the Rat Cecum

BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley β-glucans of high [HV] or low viscosity [LV] in order to measure the prebiotic effects of these different sources of dietary fiber. The dietary impact on the composition of the cecal microbiota was determined by the generation of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene sequences. The DGGE profiles produced from the cecal microbiota of rats within each dietary group were similar, but consensus profiles generated from pooled bacterial DNAs showed differences between rat groups. Animals fed HV glucans (HV-fed rats) had DGGE consensus profiles that were 30% dissimilar from those of the other rat groups. A 16S rRNA gene fragment that was more conspicuous in the profiles of HV-fed animals than in those of cellulose-fed rats had sequence identity with Lactobacillus acidophilus. Measurements of L. acidophilus rRNA abundance (DNA-RNA hybridization), the preparation of cloned 16S rRNA gene libraries, and the enumeration of Lactobacillus cells (fluorescent in situ hybridization) showed that lactobacilli formed a greater proportion of the cecal microbiota in HV-fed rats. In vitro experiments confirmed that some lactobacilli utilize oligosaccharides (degree of polymerization, 3 or 4) present in β-glucan hydrolysates. The results of this study have relevance to the use of purified β-glucan products as dietary supplements for human consumption.     

Specific response of a novel and abundant Lactobacillus amylovorus-like phylotype to dietary prebiotics in the guts of weaning piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 +/- 5.5) than in the ileum (9.7 +/- 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Selection of bacteria originating from a human intestinal microbiota in the gut of previously germ-free rats

Denaturing gradient gel electrophoresis (DGGE) was applied to separate PCR-amplified 16S rRNA genes originating from human microbiota associated (HMA) rat faeces as well as from the human faecal sample used for inoculation of the animals. Subsequently, a total of 15 dominant bands were excised from the DGGE gels, cloned and sequenced. Comparison of the obtained sequences with the Ribosomal Database revealed that species of Bacteroides/Prevotella and Faecalibacterium gave rise to the majority of the dominant bands in the human sample and in the HMA rats. In the HMA rats, two dominant bands, which were not present in the human DGGE profile, originated from species of Ruminococcus. With the exception of the Ruminococcus sequences, sequences originating from both rats and human samples were represented in all major branches of a maximum parsimony tree, indicating that the rat feed and gut environment allows colonization of the dominant taxonomic units from the human microbiota, but additionally selects for Ruminococci. Bands representing Prevotella and Faecalibacterium, which were found in identical positions of the DGGE gels originating from human and HMA rat faecal samples, originated from completely identical sequences, indicating that the same strains of these species were dominating in the human and rat samples.     

<Emphasis Type="Italic">Methanobrevibacter</Emphasis> Phylotypes are the Dominant Methanogens in Sheep from Venezuela

Rumen methanogens in sheep from Venezuela were examined using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE) profiles prepared from pooled and individual PCR products from the rumen contents from 10 animals. A total of 104 clones were examined, revealing 14 different 16S rRNA gene sequences or phylotypes. Of the 14 phylotypes, 13 (99 of 104 clones) belonged to the genus Methanobrevibacter, indicating that the genus Methanobrevibacter is the most dominant component of methanogen populations in sheep in Venezuela. The largest group of clones (41 clones) was 97.9-98.5% similar to Methanobrevibacter gottschalkii. Two sequences were identified as possible new species, one belonging to the genus Methanobrevibacter and the other belonging to the genus Methanobacterium. DGGE analysis of the rumen contents from individual animals also revealed 14 different bands with a range of 4-9 bands per animal.     

Specific Response of a Novel and Abundant Lactobacillus amylovorus-Like Phylotype to Dietary Prebiotics in the Guts of Weaning Piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Development of group-specific PCR-DGGE fingerprinting for monitoring structural changes of Thauera spp. in an industrial wastewater treatment plant responding to operational perturbations

A Thauera-specific nested-PCR denaturing gradient gel electrophoresis (DGGE) method was developed, and its usefulness was demonstrated by monitoring the structural shifts of Thauera spp. in an anaerobic-anoxic-oxic fixed-biofilm coking wastewater treatment plant (WWTP) responding to operational perturbations. The specificity of the PCR method was confirmed by the fact that all 16 S rRNA gene sequences, cloned from the amplicons of a biofilm sample, belonged to Thauera genus. 16 S rRNA gene V3 region was then amplified from the first round Thauera-specific PCR product and applied for DGGE analysis. All Thauera clones, with 13 different V3 regions, migrated into 10 positions on DGGE gel, which demonstrated the high resolution of this DGGE method. When the WWTP experienced a gradual deterioration in chemical oxygen demand (COD) removal function due to a mechanical failure of the recirculation pump, biofilm samples were collected from the reactor and analyzed by this method. Principal component analysis (PCA) of the DGGE fingerprinting data showed that the composition of Thauera group exhibited a time related trajectory when the plant's COD removal rate decreased from 84.1+/-2.7% in the first 4 weeks to less than 75% at week 5 and 6, suggesting a concomitant shift of Thauera composition and the system's COD removal function. This group-specific PCR DGGE fingerprinting technology has the potential to be a profiling tool for monitoring structural shifts of Thauera spp. in industrial WWTPs.     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1

16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no significant difference was observed between the two groups.     

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.     

昆明盐矿古老岩盐沉积中的原核生物多样性

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物多样性,含有大量未知的、未培养或不可培养的原核生物,但在原核生物物种组成和丰度上,免培养与此前的纯培养研究结果存在一定差异。因此,结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。     

Increase in terminal restriction fragments of Bacteroidetes-derived 16S rRNA genes after administration of short-chain fructooligosaccharides

It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.     

Diversity of gut microbiota increases with aging and starvation in the desert locust

Here we report the effects of starvation and insect age on the diversity of gut microbiota of adult desert locusts, Schistocerca gregaria, using denaturing gradient gel electrophoretic (DGGE) analysis of bacterial 16S rRNA genes. Sequencing of excised DGGE bands revealed the presence of only one potentially novel uncultured member of the Gammaproteobacteria in the guts of fed, starved, young or old locusts. Most of the 16S rRNA gene sequences were closely related to known cultured bacterial species. DGGE profiles suggested that bacterial diversity increased with insect age and did not provide evidence for a characteristic locust gut bacterial community. Starved insects are often more prone to disease, probably because they compromise on immune defence. However, the increased diversity of Gammaproteobacteria in starved locusts shown here may improve defence against enteric threats because of the role of gut bacteria in colonization resistance.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.     

Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus.

The phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy which does not rely on cultivation of the resident microorganisms. Small-subunit rRNA genes (16S rDNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial 16S rDNA sequences showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rDNA sequence data are available. Of 55 clones sequenced, 45 were phylogenetically affiliated with four of the major groups of the domain Bacteria: the Proteobacteria, the spirochete group, the Bacteroides group, and the low-G+C-content gram-positive bacteria. Within the Proteobacteria, the 16S rDNA clones showed a close relationship to those of cultivated species of enteric bacteria and sulfate-reducing bacteria, while the 16S rDNA clones in the remaining three groups showed only distant relationships to those of known organisms in these groups. Of the remaining 10 clones, among which 8 clones formed a cluster, there was only very low sequence similarity to known 16S rRNA sequences. None of these clones were affiliated with any of the major groups within the domain Bacteria. The 16S rDNA gene sequence data show that the majority of the intestinal microflora of R. speratus consists of new, uncultured species previously unknown to microbiologists.     

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.     

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.