Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.     

Genotypic characterization of soil bacteria in the Umm Al-Namil Island,Kuwait

Microflora is an integral part of soil ecosystem, in which bacteria are the largest group of soil microbes. This is a pioneer study for establishing baseline data on the diversity of soil bacteria among different regions in Kuwait. The aim is to understand biodiversity in different settings, how bacteria adapt to different niches in the environment as well as in different hosts. The identification of bacterial 16S rRNA molecules from environmental soil samples was investigated. Genomic Deoxyribonucleic acid DNA was extracted from 25 soil samples derived from five different test regions in the Umm Al-Namil Island, Kuwait. After amplification of bacterial 16S rRNA molecules by the Polymerase chain reaction PCR, the products were characterized and complex band patterns were obtained, indicating high bacterial diversity. A sample of the 16 s rRNA amplicons were sequenced in order to identify the species. The spatial distribution of bacterial taxa in the different soil samples was homogeneous, suggesting a stable and widespread community. Forty-nine isolates from Umm Al-Namil island were identified by comparative analysis of partial 16S rRNA gene sequences. Phylogenetic analysis was carried out in order to study the connection between the isolates to identify species. A large proportion of these isolates represent correspond to known or novel species within the Pseudomonus and Bacillus genera, which are common soil bacteria. Our results provided a reference for future studies to facilitate bacterial identification and ecological research in Kuwait.     

Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 10⁶ Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.     

Shift in microbial population in response to crystalline cellulose degradation during enrichment with a semi-desert soil

Addition of crystalline cellulose to semi-desert soil shifts the microbial population; this was assessed by following the 16S rRNA gene, glycosyl hydrolase, and measuring its functional diversity in the bacterial population. Quantification of the glycosyl hydrolase gene showed an increase from 1 × 10⁴ g⁻¹ of unamended soil to 3 × 10⁴ g⁻¹ of crystalline-cellulose-amended soil by the 15th day of crystalline cellulose utilization. The indigenous glycosyl hydrolase community in unamended soil was dominated by the clone families that were closely related to the glycosyl hydrolases from Betaproteobacteria and Firmicutes. The addition of crystalline cellulose induced a shift in the glycosyl hydrolase population toward an increase in the relative abundance of the glycosyl hydrolase that was consistent with those of Bacteroidetes and Flavobacteria. The population shift of glycosyl hydrolase was also supported by the comparison of the 16S rRNA gene families in unamended and crystalline-cellulose-amended soil libraries. The most abundant 16S rRNA gene sequences retrieved in the unamended soil were identical to Pseudomonas, Massilia, Paenibacillus, and Bacillus spp., while Cytophaga and Flavobacterium spp. dominated in crystalline-cellulose-amended soil.     

Antibacterial activity of soil bacteria isolated from Kochi,India and their molecular identification

The present study, deal about the antibiosis activity of soil bacteria, isolated from 10 different locations of rhizosphere and diverse cultivation at Kochi, Kerala, India. The bacteria were isolated by standard serial dilution plate techniques. Morphological characterization of the isolate was done by Gram’s staining and found that all of them gram positive. Isolated bacteria were tested against 6 human pathogens viz., Escherichia coli, Enterococcus sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Acinetobacter sp. Primary screening was carried out by perpendicular streaking and seed overlay method. Based on the result of primary screening most potential isolates of S1A1 and S7A3 were selected for secondary screening. Both the isolates showed positive results against Enterococcus sp. and S.aureus. The maximum antagonistic activity of 20.98 and 27.08?mm zone of inhibition was recorded at S1A1 against Enterococcus sp. and S. aureus respectively, at 180?µl concentration. Molecular identification was carried out by 16S rRNA sequence. The 16S rRNA was amplified from the DNA samples by using PCR. The amplified 16S rRNA PCR products were purified and sequenced. The sequences were subjected to NCBI BLAST. The isolates S1A1 and S7A3 BLAST results showed 99% and 95% respectively, similarity with the available database sequence of Bacillus amyloliquefaciens. The sequences were deposited in GenBank and the accession numbers KY864390 (S1A1) and KY880975 (S7A3) were obtained.     

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.     

Characterization of Soil Bacterial Communities in Rhizospheric and Nonrhizospheric Soil of Panax ginseng

A culture-independent approach was used to evaluate the bacterial community in rhizospheric and nonrhizospheric soil in which Panax ginseng had grown for 3?years. For each sample, soil was randomly collected from multiple sampling points and mixed thoroughly before genomic DNA extraction. Universal primers 27f and 1492r were used to amplify 16S rRNA genes. Clone libraries were constructed using the amplified 16S rRNA genes, and 192 white clones were chosen for further sequencing. After digestion with restriction endonuclease, 44 operational taxonomic units (OTUs) were generated for rhizospheric and 21 OTUs for nonrhizospheric soils, and the clones of each OTU were sequenced. Blast analysis showed that bacillus, acidobacteria, and proteobacteria were the dominant populations in rhizospheric soil, and proteobacteria were dominant in nonrhizospheric soil. Phylogenetic results showed that bacillus and acidobacteria were clustered into the group of uncultured bacteria in rhizospheric soil; however, proteobacteria were the unique dominant in nonrhizospheric soil.     

Monitoring Precursor 16S rRNAs of Acinetobacter spp. in Activated Sludge Wastewater Treatment Systems

Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457–4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3′ region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

A ribosomal RNA operon from Pseudomonas stutzeri Zobell

A ribosomal RNA operon from the marine bacterium, Pseudomonas stutzeri Zobell, was cloned and characterized by Southern hybridization and sequence analysis. The 16S rRNA, 23S rRNA, 5S rRNA and 2 tRNA genes (alanine and isoleucine) were identified by homology with sequences in GenBank. The rRNA gene exhibited typical eubacterial organization (16S-tRNAs-23S-5S). A putative ribosomal promoter and anti-terminator regions were also identified and described. Significant differences in spacing of the anti-terminator regulatory elements were observed between P. stutzeri Zobell and Escherichia coli.     

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples.     

Bacilli community of saline–alkaline soils from the Ararat Plain (Armenia) assessed by molecular and culture-based methods

The bacterial community composition in the A horizon of a natural saline–alkaline soil located in Ararat Plain (Armenia) was studied using molecular and culture-based methods The sequence analysis of a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) profiles indicated dominance of Firmicutes populations. The majority of the sequences of the bacterial 16S rRNA gene library were close relatives of representatives belonging to the genera Halobacillus (41.2%), Piscibacillus (23.5%), Bacillus (23.5%) and Virgibacillus (11.8%). Eight novel moderately halophilic bacilli isolates were successfully obtained from the enriched cultures of the saline–alkaline soil samples. 16S rRNA gene sequence analyses of isolates revealed their affiliation (97.7–99.7% similarity) to representatives of the genera Bacillus, Piscibacillus and Halobacillus. All isolates were able to tolerate high concentrations of NaCl and highly alkaline conditions. This is the first study combining cultivation-independent and -dependent approaches to reveal the bacterial diversity of the saline–alkaline soils of Ararat Plain and it suggested an important role of bacilli as key microbes in biogeochemical cycles of these environments.     

Microbial Community Structure in Midgut and Hindgut of the Humus-Feeding Larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter. In a companion paper (T. Lemke, U. Stingl, M. Egert, M. W. Friedrich, and A. Brune, Appl. Environ. Microbiol. 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community. Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P. ephippiata larvae, determined with cultivation-independent techniques. Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P. ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil. The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides [CFB] phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper. Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum. The archaeal community, however, is less diverse. 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut. These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae.     

The Diversity of Phaseolus-Nodulating Rhizobial Populations Is Altered by Liming of Acid Soils Planted with Phaseolus vulgaris L. in Brazil

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on “species” types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS (“strain”) groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.     

Molecular Evolution of Mycoplasma capricolum subsp. capripneumoniae Strains, Based on Polymorphisms in the 16S rRNA Genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

Phylogenetic analysis based evolutionary study of 16S rRNA in known Pseudomonas sp

Molecular evolution analysis of 16S rRNA sequences of native Pseudomonas strains and different fluorescent pseudomonads were conducted on the basis of Molecular Evolutionary Genetics Analysis version 5.2 (MEGA5.2). Topological evaluations show common origin for native strains with other known strains with available sequences at GenBank database. Phylogenetic affiliation of different Pseudomonas sp based on 16S rRNA gene shows that molecular divergence contributes to the genetic diversity of Pseudomonas sp. Result indicate direct dynamic interactions with the rhizospheric pathogenic microbial community. The selection pressure acting on 16S rRNA gene was related to the nucleotide diversity of Pseudomonas sp in soil rhizosphere community among different agricultural crops. Besides, nucleotide diversity among the whole population was very low and tajima test statistic value (D) was also slightly positive (Tajima׳s test statistics D value 0.351). This data indicated increasing trends of infection of soil-borne pathogens under gangetic-alluvial regions of West Bengal due to high degree of nucleotide diversity with decreased population of plant growth promoting rhizobacteria like fluorescent Pseudomonads in soil.     

Improved Assessment of Denitrifying, N2-Fixing, and Total-Community Bacteria by Terminal Restriction Fragment Length Polymorphism Analysis Using Multiple Restriction Enzymes

A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N₂ fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.     

Effects of Soil pH on the Biodegradation of Chlorpyrifos and Isolation of a Chlorpyrifos-Degrading Bacterium

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of ≥6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of Pseudomonas. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (opd) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.     

Culture-independent analysis of Pseudomonas community structures in fertilized and unfertilized agricultural soils

Pseudomonas community structures were investigated by analyzing 16S rRNA clone libraries derived from fertilized and unfertilized soil plots under corn–alfalfa rotation in a long-term experiment. Amplified 16S rRNA fragments derived by polymerase chain reaction (PCR) were cloned and sequenced. A total of 729 clone sequences were analyzed, of which 51 were possible chimeras and discarded. The remaining clone sequences (678) belonged to γ-proteobacteria with 61.8 % (419) classified to the genus Pseudomonas. Unclassified Gammaproteobacteria accounted for 23.4 % of total clones sequences. Rarefaction analyses showed a more diverse community structure of both Gammaproteobacteria and Pseudomonas in unfertilized than fertilized field soils irrespective of plant types under cultivation. Bacterial or Pseudomonas community structures differed significantly between fertilized and unfertilized soil plots. Clone sequences that are affiliated to Pseudomonas putida and P. oryzihabitans were more prominent in libraries from fertilized plots, while those that clustered with Pseudomonas frederiksbergensis were more often retrieved from unfertilized soil plots. A strong influence of fertilizer applications on community structure was supported by principal component analysis. We conclude that long-term use of mineral fertilizers could influence Pseudomonas community structure.     

Bacterial biodiversity from Roopkund Glacier, Himalayan mountain ranges, India

The bacterial diversity of two soil samples collected from the periphery of the Roopkund glacial lake and one soil sample from the surface of the Roopkund Glacier in the Himalayan ranges was determined by constructing three 16S rRNA gene clone libraries. The three clone libraries yielded a total of 798 clones belonging to 25 classes. Actinobacteria was the most predominant class (>10% of the clones) in the three libraries. In the library from the glacial soil, class Betaproteobacteria (24.2%) was the most predominant. The rarefaction analysis indicated coverage of 43.4 and 41.2% in the samples collected from the periphery of the lake thus indicating a limited bacterial diversity covered; at the same time, the coverage of 98.4% in the glacier sample indicated most of the diversity was covered. Further, the bacterial diversity in the Roopkund glacier soil was low, but was comparable with the bacterial diversity of a few other glaciers. The results of principal component analysis based on the 16S rRNA gene clone library data, percentages of OTUs and biogeochemical data revealed that the lake soil samples were different from the glacier soil sample and the biogeochemical properties affected the diversity of microbial communities in the soil samples.