Direct detection of nif-gene sequences of Enterobacter agglomerans in soil

Abstract: Specific nif sequences of Enterobacter agglomerans plasmid pEA9 were detected in total DNA recovered from soil 70 days after its inoculation with the bacteria, when these were no longer culturable on agar medium. For this, a modified method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was necessary.     

Performance of the COX1 gene as a marker for the study of metabolically active Pezizomycotina and Agaricomycetes fungal communities from the analysis of soil RNA

In temperate forest soils, filamentous ectomycorrhizal and saprotrophic fungi affiliated to the Agaricomycetes and Pezizomycotina contribute to key biological processes. The diversity of soil fungal communities is usually estimated by studying molecular markers such as nuclear ribosomal gene regions amplified from soil-extracted DNA. However, this approach only reveals the presence of the corresponding genomic DNA in the soil sample and may not reflect the diversity of the metabolically active species. To circumvent this problem, we investigated the performance of the mitochondrial cytochrome c oxidase 1 (COX1)-encoding gene as a fungal molecular marker for environmental RNA-based studies. We designed PCR primers to specifically amplify Agaricomycetes and Pezizomycotina COX1 partial sequences and amplified them from both soil DNA and reverse-transcribed soil RNA. As a control, we also amplified the nuclear internal transcribed spacer ribosomal region from soil DNA. Fungal COX1 sequences were readily amplified from soil-extracted nucleic acids and were not significantly contaminated by nontarget sequences. We show that the relative abundance of fungal taxonomic groups differed between the different sequence data sets, with for example ascomycete COX1 sequences being more abundant among sequences amplified from soil DNA than from soil cDNAs.     

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Molecular identification of ectomycorrhizal mycelium in soil horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (> or = 99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had > or = 98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Direct characterization of Frankia and of close phyletic neighbors from an Alnus viridis rhizosphere

To analyze the population structure of Frankia soil populations, DNA was extracted from an Alnus viridis rhizosphere, PCR-amplified with a primer designed to be specific, and cloned. A PCR-RFLP analysis of the resulting clones showed that none had a pattern close to those expected from an analysis made on Frankia collection strains, on which Hha I, Hpa II and Hae III were found to be the most discriminant restriction enzymes. Sequencing was done on a subset of soil clones and these were compared to DNA data banks. Some sequences were typical of lowGC Firmicutes ( Bacillus and relatives), whereas others were typical of highGC Firmicutes (Actinomycetes and relatives). Two of these sequences appear to be in the same line of descent as Frankia and Geodermatophilus and represent as yet uncharacterized soil microorganisms. This shows that the design of specific or generic primers must be refined constantly to take into account the deposition of such new sequences in DNA data banks.     

Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd(1)-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.     

红树林土壤细菌群落16S rDNA V3片段PCR产物的DGGE分析

从土壤中抽提微生物总DNA ,直接扩增 16SrDNAV3片段 ,应用变性梯度凝胶电泳 (DGGE)和分子克隆技术分析 16SrDNAV3片段的多态性 ,发现地域因素和红树品种都是影响土壤细菌群落结构的因素。通过对杯萼海桑土壤 16SrDNAV3片段PCR产物两个DGGE条带进行分子克隆、序列测定和Blast分析 ,发现每个DGGE条带包含着许多不同的 16SrDNAV3片段 ,并且其中多数为NCBI未收录的序列。这表明DGGE和克隆技术相结合的方法是研究土壤微生物群落结构的一种可行方法。     

Development of a primer system to study abundance and diversity of the gene coding for alanine aminopeptidase pepN gene in Gram-negative soil bacteria

A new set of primers was developed allowing the specific detection of the pepN gene (coding for alanine aminopeptidase) from Gram-negative bacteria. The primers were designed in silico by sequence alignments based on available DNA sequence data. The PCR assay was validated using DNA from selected pure cultures. The analysis of gene libraries from extracted DNA from different soil samples revealed a high diversity of pepN related sequences mainly related to α-Proteobacteria. Most sequences obtained from clone libraries were closely related to already published sequences (<80% homology on amino acid level), which may be related to the conserved character of the amplified region of pepN. By linking the diversity data obtained by the clone library studies to potential enzymatic activities of alanine aminopeptidase, lowest diversity of pepN was found in those soil samples which displayed lowest activity levels, which confirms the importance of diversity for the ecosystem function mainly when transformation processes of complex molecules are studied.     

Effect of Incubation Conditions on the Enrichment of Pyrene-degrading Bacteria Identified by Stable-isotope Probing in an Aged,PAH-contaminated Soil

To determine whether the diversity of pyrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon-contaminated soil is affected by the addition of inorganic nutrients or by slurrying the soil, various incubation conditions (all including phosphate buffer) were examined by mineralization studies and stable-isotope probing (SIP). The addition of nitrogen to either continuously mixed slurry or static field-wet soil incubations increased the rate and extent of mineralization of [(14)C]pyrene, with the most rapid mineralization observed in slurried, nitrogen-amended soil. Microcosms of slurry and static field-wet soil amended with nitrogen were also examined by SIP with [U-(13)C]pyrene. Recovered (13)C-enriched deoxyribonucleic acid (DNA) was analyzed by denaturing-gradient gel electrophoresis (DGGE) and 16S ribosomal ribonucleic acid (rRNA) gene clone libraries. DGGE profiles of (13)C-enriched DNA fractions from both incubation conditions were similar, suggesting that pyrene-degrading bacterial community diversity may be independent of treatment method. The vast majority (67 of 71) of the partial sequences recovered from clone libraries were greater than or equal to 97% similar to one another, 98% similar to sequences of pyrene-degrading bacteria previously detected by SIP with pyrene in different soil, and only 89% similar to the closest cultivated genus. All of the sequences recovered from the field-wet incubation and most of the sequences recovered from the slurry incubation were in this clade. Of the four sequences from slurry incubations not within this clade, three possessed greater than 99% similarity to the 16S rRNA gene sequences of phylogenetically dissimilar Caulobacter spp.     

Distribution of a genetically-engineered Escherichia coli population introduced into soil

The spatial localization of the cells and the DNA of a genetically-engineered Escherichia coli population introduced into soil was investigated. Inoculated soils were size fractioned and bacterial numbers and E. coli EL 1003 specific chromosomal DNA target sequences were enumerated in each fraction using plate-counting and MPN-PCR, respectively. Different numbers of either indigenous or introduced bacteria were found in each fraction indicating that their distribution in the soil was non-uniform. The distributions of the indigenous bacteria and the E. coli cells within the size fractions were significantly different: the E. coli population was mainly associated with the dispersible clay fraction (79·0%) from which only 10·7% of the indigenous bacteria were recovered. The distribution of the E. coli target DNA sequences was in agreement with the location of the cells. The different distribution of the two populations is likely to restrict genetic interactions. These results are relevant to potential interactions between native soil microflora and populations introduced into soil for competitive purposes.     

DNA from soil mirrors plant taxonomic and growth form diversity

Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches.     

Pathways for lactose/galactose catabolism by Streptococcus salivarius

Using oligonucleotides derived from the N-terminal sequence of a triazine hydrolase from Nocardioides sp. strain C190, two DNA fragments containing trzN were cloned into Escherichia coli and their nucleotide sequences were determined. The 456-amino acid polypeptide predicted from the 1356-bp trzN ORF displayed significant similarity to triazine hydrolases from Pseudomonas and Rhodococcus isolates and belonged to the same amidohydrolase family. The trzN gene was flanked by two DNA sequences possessing 57 and 69% identity, respectively, at the protein level to Rhodococcus erythropolis sequences for a transposase and a transposase helper protein. Amplification primers specific to trzN were tested in soils inoculated with strain C190. The results demonstrated that the primers were specific to trzN, and could detect populations at 10(8) cfu g(-1) soil using 250-mg soil samples.     

Phylogenetic community ecology of soil biodiversity using mitochondrial metagenomics

High‐throughput DNA methods hold great promise for the study of taxonomically intractable mesofauna of the soil. Here, we assess species diversity and community structure in a phylogenetic framework, by sequencing total DNA from bulk specimen samples and assembly of mitochondrial genomes. The combination of mitochondrial metagenomics and DNA barcode sequencing of 1494 specimens in 69 soil samples from three geographic regions in southern Iberia revealed >300 species of soil Coleoptera (beetles) from a broad spectrum of phylogenetic lineages. A set of 214 mitochondrial sequences longer than 3000 bp was generated and used to estimate a well‐supported phylogenetic tree of the order Coleoptera. Shorter sequences, including cox1 barcodes, were placed on this mitogenomic tree. Raw Illumina reads were mapped against all available sequences to test for species present in local samples. This approach simultaneously established the species richness, phylogenetic composition and community turnover at species and phylogenetic levels. We find a strong signature of vertical structuring in soil fauna that shows high local community differentiation between deep soil and superficial horizons at phylogenetic levels. Within the two vertical layers, turnover among regions was primarily at the tip (species) level and was stronger in the deep soil than leaf litter communities, pointing to layer‐mediated drivers determining species diversification, spatial structure and evolutionary assembly of soil communities. This integrated phylogenetic framework opens the application of phylogenetic community ecology to the mesofauna of the soil, among the most diverse and least well‐understood ecosystems, and will propel both theoretical and applied soil science.     

Nematode 18S rRNA gene is a reliable tool for environmental biosafety assessment of transgenic banana in confined field trials

Information on relatedness in nematodes is commonly obtained by DNA sequencing of the ribosomal internal transcribed spacer region. However, the level of diversity at this locus is often insufficient for reliable species differentiation. Recent findings suggest that the sequences of a fragment of the small subunit nuclear ribosomal DNA (18S rRNA or SSU), identify genera of soil nematodes and can also distinguish between species in some cases. A database of soil nematode genera in a Ugandan soil was developed using 18S rRNA sequences of individual nematodes from a GM banana confined field trial site at the National Agricultural Research Laboratories, Kawanda in Uganda. The trial was planted to evaluate transgenic bananas for resistance to black Sigatoka disease. Search for relatedness of the sequences gained with entries in a public genomic database identified a range of 20 different genera and sometimes distinguished species. Molecular markers were designed from the sequence information to underpin nematode faunal analysis. This approach provides bio-indicators for disturbance of the soil environment and the condition of the soil food web. It is being developed to support environmental biosafety analysis by detecting any perturbance by transgenic banana or other GM crops on the soil environment.     

Molecular microbial diversity of an agricultural soil in Wisconsin.

A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.     

四种红树植物根际土壤微生物Ⅰ型和Ⅱ型PKS基因的检测与多样性分析

[目的]探究红树林土壤中聚酮合酶(Polyketide synthase,PKS)基因的多样性和新颖性.[方法]用Ⅰ型和Ⅱ型PKS基因酮基合成酶(Ketosynthase,KS)域的简并引物对海南清澜港红树林海莲、黄槿、银叶、老鼠簕4种红树根际土壤样品中DNA进行PCR扩增,之后利用PCR-限制性酶切片段多样性(PCR-RFLP)和测序分析法对Ⅰ型和Ⅱ型PKS基因的多样性进行探讨.[结果]对得到的72条Ⅰ型PKS基因的酮基合成酶(Ketosynthase,KS)域DNA序列进行PCR-RFLP分析,共得到51个可操作分类单元(Operational taxonomic unit,OTUs),其中37个OTUs为单克隆产生,没有明显的优势OTU.选取了26个代表不同OTU的克隆进行测序分析,这些序列与GenBank中已知序列的最大相似率均未超过85％. KS域氨基酸序列的系统发育分析显示,所得KS域来源广泛,包括蓝细菌门(Cyanobacteria)、变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和一些未可培养细菌;对55条PKSⅡ基因KS域DNA序列的PCR-RFLP分析后共得到25个OTUs,有两个明显的优势OTUs,代表的克隆子数所占比例超过10％.[结论]PCR-RFLP分析表明红树林根际土壤中存在着丰富多样的Ⅰ型和Ⅱ型PKS基因,且前者多样性更高;低的序列相似度表明所获得的PKSⅠ基因KS域序列独特;系统发育分析表明得到的PKSⅠ基因来源广泛.     

Detection in soil of a deletion in an engineered DNA sequence by using DNA probes

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain.     

Detection in soil of a deletion in an engineered DNA sequence by using DNA probes.

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain.     

Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay.

A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.