Spatial distribution of 16S rRNA levels from uncultured acidobacteria in soil

The activity of uncultured acidobacteria was monitored in Dutch grassland soils by quantifying their ribosomes. These bacteria were detectable by five different 16S rRNA RT-PCR products in temperature gradient gel electrophoresis fingerprints. The ribosomes in surface soil samples were quantified with multiple competitive RT-PCR along a 1.5-km transect through the grassland. In total, the five members of the acidobacteria were estimated to contribute 4 x 1010 to 1 x 1011 ribosomes g soil-1, representing 7-14% of all bacterial ribosomes. These results indicate that ribosomes from acidobacteria are continuously present and abundant in soil and might contribute significantly to microbial activity in soil.     

In Situ Detection of an Uncultured Predominant Bacillus in Dutch Grassland Soils

Uncultured predominant Bacillus ribotype DA001 in Dutch Drentse A grassland soils, as revealed by its 16S rRNA sequence, was detected in soil by fluorescent whole-cell in situ hybridization. A prominent rod-shaped cell type was identified in bacterial suspensions prepared from soil by a multiple 16S rRNA probing approach.     

呼伦贝尔草原不同退化梯度土壤细菌多样性季节变化

为了研究草地退化程度与土壤微生物多样性的关系,在呼伦贝尔草地上选取羊草草甸草原和贝加尔针茅草甸草原两个典型放牧点,按照轻度、中度和重度划分取样点,分别于6、8月份和10月份3个不同季节采集土壤样品。应用变性梯度凝胶电泳技术(PCR-DGGE)研究两个放牧地点不同退化程度、不同季节草地的细菌群落结构变化。结果表明,呼伦贝尔草地不同退化梯度的草地土壤中细菌种类较为丰富。从丰富度和Shannon-Winner指数的变化看,两个放牧点8月份丰富度和Shannon-Winner指数最高,8月份的丰富度平均为32.4,比6月和10月份分别高11%和7.4%;8月份Shannon-Winner指数平均为4.15,比6月和10月份分别高7.7%和5.4%。DGGE图谱聚类分析结果显示,随着季节变化和草地退化程度由轻至重的变化,土壤中的细菌优势种群没有受到明显的影响。回收DGGE图谱中10个条带进行测序分析,结果显示,所有序列与GenBank数据库中的相似度在87%100%之间。基于98%的相似度,可将其中的7个鉴定为Proteobacteria(变形菌门),将其中的1个鉴定为Actinobacteria(放线菌门)。另外2个同已知序列相似性较低,可能是未知的细菌。结果表明,Proteobacteria(变形菌门)为呼伦贝尔草原土壤中的优势细菌类群,尽管所选取样点草地植被有不同程度的退化,但土壤微生物优势种群并没有发生变化。     

Dynamics of bacterial community succession in a salt marsh chronosequence: evidences for temporal niche partitioning

The mechanisms underlying community assembly and promoting temporal succession are often overlooked in microbial ecology. Here, we studied an undisturbed salt marsh chronosequence, spanning over a century of ecosystem development, to understand bacterial succession in soil. We used 16S rRNA gene-based quantitative PCR to determine bacterial abundance and multitag 454 pyrosequencing for community composition and diversity analyses. Despite 10-fold lower 16S rRNA gene abundances, the initial stages of soil development held higher phylogenetic diversities than the soil at late succession. Temporal variations in phylogenetic β-diversity were greater at initial stages of soil development, possibly as a result of the great dynamism imposed by the daily influence of the tide, promoting high immigration rates. Allogenic succession of bacterial communities was mostly driven by shifts in the soil physical structure, as well as variations in pH and salinity, which collectively explained 84.5% of the variation concerning community assemblage. The community assembly data for each successional stage were integrated into a network co-occurrence analysis, revealing higher complexity at initial stages, coinciding with great dynamism in turnover and environmental variability. Contrary to a spatial niche-based perspective of bacterial community assembly, we suggest temporal niche partitioning as the dominant mechanism of assembly (promoting more phylotype co-occurrence) in the initial stages of succession, where continuous environmental change results in the existence of multiple niches over short periods of time.     

Changes in the community structure of ammonia-oxidizing bacteria during secondary succession of calcareous grasslands

The community structure of β-subclass Proteobacteria ammonia-oxidizing bacteria was determined in semi-natural chalk grassland soils at different stages of secondary succession. Both culture-mediated (most probable number; MPN) and direct nucleic acid-based approaches targeting genes encoding 16S rRNA and the AmoA subunit of ammonia monooxygenase were used. Similar shifts were detected in the composition of the ammonia oxidizer communities by both culture-dependent and independent approaches. A predominance of Nitrosospira sequence cluster 3 in early successional fields was replaced by Nitrosospira sequence cluster 4 in late successional fields. The rate of this shift differed between the two areas examined. This shift occurred in a background of relative stability in the dominant bacterial populations in the soil, as determined by domain-level polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). Molecular analysis of enrichment cultures obtained using different ammonia concentrations revealed biases towards Nitrosospira sequence cluster 3 or Nitrosospira sequence cluster 4 under high- or low-ammonia conditions respectively. High-ammonia MPNs suggested a decease in ammonia oxidizer numbers with succession, but low-ammonia MPNs and competitive PCR targeting amoA failed to support such a trend. Ammonia turnover rate, not specific changes in plant diversity and species composition, is implicated as the major determinant of ammonia oxidizer community structure in successional chalk grassland soils.     

Bacterial succession during 500?years of soil development under agricultural use

Soil bacterial succession under intensive anthropogenic disturbances is not well known. Using terminal restriction fragment length polymorphisms and 454 pyrosequencing of 16S rRNA genes, this study investigated how soil bacterial diversity and community structure changed under two agricultural land uses (paddy rice and upland cropping) in relation to soil development along a 500-year chronosequence created by intermittent reclamation of estuarine salt marshes. Multivariate analysis revealed orderly changes in soil physicochemical properties and bacterial community structure with time, confirming the occurrence of soil development and bacterial succession. Patterns of soil development and bacterial succession resembled each other, with recent land uses affecting their trajectories but not the overall direction. Succession of bacterial community structure was mainly associated with changes in ??-Proteobacteria and Verrucomicrobia. Two stages of bacterial succession were observed, a dramatic-succession stage during the first several decades when bacterial diversity increased evidently and bacterial community structure changed rapidly, and a long gradual-succession stage that lasted for centuries. Canonical correspondence analysis identified soil Na⁺, potentially mineralizable nitrogen, total phosphorous, and crystallinity of iron oxyhydrates as potential environmental drivers of bacterial succession. To conclude, orderly succession of soil bacterial communities occurred along with the long-term development of agroecosystems, which in turn was associated with soil physicochemical changes over time.     

Diversity of bacteria associated with grassland soil nematodes of different feeding groups

The bacterial diversity associated with soil nematodes and its relationship with their feeding habits are as yet poorly understood. In the present study the diversity and abundance of bacteria from nematodes and their surrounding soil were analysed and compared. The nematodes were collected from a grassland soil and sorted into bacterial, fungal, plant, predatory and omnivore feeding groups and assigned to taxonomic groups. Total DNA was extracted from the nematodes and partial bacterial 16S rRNA genes were PCR amplified, cloned and sequenced. The abundance and composition of bacterial taxa differed between and within feeding groups. The lowest bacterial diversity was found in the predatory nematodes Prionchulus sp., whereas the highest bacterial diversity was associated with the bacterial-feeding nematode Acrobeles sp. The soil had a more diverse bacterial community than the communities found in the nematode groups. The 16S rRNA gene sequences of bacteria associated with nematodes did not overlap with those detected in soil as determined using the cloning screening approach. However, bacterial sequences identified from nematodes could be detected in the soil with targeted PCR. Our data suggest that the nematodes do not feed on the most abundant bacteria present in soil. Furthermore, several nematodes contained suspected bacterial symbionts and parasites.     

Airborne bacterial emission fluxes from manure-fertilized agricultural soil

This is the first study to quantify the dependence on wind velocity of airborne bacterial emission fluxes from soil. It demonstrates that manure bacteria get aerosolized from fertilized soil more easily than soil bacteria, and it applies bacterial genomic sequencing for the first time to trace environmental faecal contamination back to its source in the chicken barn. We report quantitative, airborne emission fluxes of bacteria during and following the fertilization of agricultural soil with manure from broiler chickens. During the fertilization process, the concentration of airborne bacteria culturable on blood agar medium increased more than 600 000-fold, and 1 m³ of air carried 2.9 × 10⁵ viable enterococci, i.e. indicators of faecal contamination which had been undetectable in background air samples. Trajectory modelling suggested that atmospheric residence times and dispersion pathways were dependent on the time of day at which fertilization was performed. Measurements in a wind tunnel indicated that airborne bacterial emission fluxes from freshly fertilized soil under local climatic conditions on average were 100-fold higher than a previous estimate of average emissions from land. Faecal bacteria collected from soil and dust up to seven weeks after fertilization could be traced to their origins in the poultry barn by genomic sequencing. Comparative analyses of 16S rRNA gene sequences from manure, soil and dust showed that manure bacteria got aerosolized preferably, likely due to their attachment to low-density manure particles. Our data show that fertilization with manure may cause substantial increases of bacterial emissions from agricultural land. After mechanical incorporation of manure into soil, however, the associated risk of airborne infection is low.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Influence of Changing Plant Food Sources on the Gut Microbiota of Saltmarsh Detritivores

Changes in agricultural land-use of saltmarshes along the German North Sea coast have favoured the succession of the marsh grass Elytrigia atherica over the long-established Spartina anglica. Consequently, E. atherica represents a potential food source of increasing importance for plant-feeding soil detritivores. Considering the importance of this ecological guild for decomposition processes and nutrient cycling, we focussed on two sympatric saltmarsh soil macrodetritivores and their associated gut microbiota to investigate how the digestive processes of these species may be affected by changing plant food sources. Using genetic fingerprints of partial 16S rRNA gene sequences, we analysed composition and diversity of the bacterial gut community in a diplopod and an amphipod crustacean in relation to different feeding regimes representing the natural vegetation changes. Effects of syntopy on the host-specific gut microbiota were also taken into account by feeding the two detritivore species either independently or on the same plant sample. Bacterial community composition was influenced by both the host species and the available plant food sources, but the latter had a stronger effect on microbial community structure. Furthermore, bacterial diversity was highest after feeding on a mixture of both plant species, regardless of the host species. The gut microbiota of these two detritivores can thus be expected to change along with the on-going succession at the plant community level in this environment. Cloning and sequencing of bacterial 16S rRNA gene fragments further indicated a host-related effect since the two detritivores differed in terms of predominant bacterial taxa: diplopods harboured mainly representatives of the phyla Bacteroidetes and Gammaproteobacteria. In contrast, the genus Vibrio was found for the amphipod host across all feeding conditions.     

Molecular characterization of bacterial diversity from British Columbia forest soils subjected to disturbance

Bacteria from forest soils were characterized by DNA sequence analysis of cloned 16S rRNA gene fragments (16S clones). Surface organic matter and mineral soil samples from a British Columbia Ministry of Forests Long-Term Soil Productivity (LTSP) installation were collected during winter and summer from two disturbance treatments: whole-tree harvesting with no soil compaction (plot N) and whole-tree harvesting plus complete surface organic matter removal with heavy soil compaction (plot S). Phylogenetic analyses revealed that 87% of 580 16S clones were classified as Proteobacteria, Actinobacteria, Acidobacterium, Verrucomicrobia, Bacillus/Clostridium group, Cytophaga-Flexibacter-Bacteroides group, green nonsulfur bacteria, Planctomyces, and candidate divisions TM6 and OP10. Seventy-five 16S clones could not be classified into known bacterial divisions, and five 16S clones were related to chloroplast DNA. Members of Proteobacteria represented 46% of the clone library. A higher proportion of 16S clones affiliated with y-Proteobacteria were from plot N compared with plot S. 16S rRNA gene fragments amplified with Pseudomonas-specific primers and cloned (Ps clones) were examined from mineral-soil samples from plots N and S from three LTSP installations. A significantly greater proportion of sequenced Ps clones from plot N contained Pseudomonas 16S rRNA gene fragments compared with Ps clones from plot S.     

Succession of sulfur-oxidizing bacteria in the microbial community on corroding concrete in sewer systems

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O3(2-)). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.     

Assessment of the bacterial community structure in a Brazilian clay soil treated with atrazine

In the present paper, the bacterial communities in two soils, one from an agricultural sugarcane cropped field and the other from an unperturbed soil with similar geopedological characteristics, were characterized using the Fluorescence In Situ Hybridization (FISH) method. FISH consists of in situ identification of bacteria using fluorescent labeled 16S rRNA targeted oligonucleotide probes visualizable under epifluorescence microscope. In the cultivated soil, in line with agricultural practice, the pre-emergence herbicide atrazine had been regularly applied each year at a concentration of 5 L/ha. The Shannon Diversity and Evenness Indices were also calculated using the phylogenetic data obtained from the FISH analysis. Although, at the sampling time (6 months after soil atrazine treatment), no residual herbicide concentration was found, the overall bacterial community results show a lower diversity and evenness in the agricultural soil than in the unperturbed one, demonstrating how microbiological indicators are sensitive to anthropogenic disturbance. In the natural soil, the dominant groups were α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria (representing more than 50 % of the bacteria), but in the agricultural soil, their abundance decreased significantly and represented just 31 % of the bacteria domain.     

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.     

Reviewing the DA001-files: a 16S rRNA chase on suspect #X99967, a Bacillus and Dutch underground activist.

A variant of 'the rRNA approach' on uncultured soil bacteria is discussed, which is mainly based on 16S rRNA rather than on genomic 16S rDNA. While the rDNA only reflects the presence of bacteria, the rRNA indicates much more the activity of bacteria. Hence, the presented strategy can indicate the involvement of uncultured bacteria to the metabolic activity of the total microbial community. The potentials and limitations of the applied techniques will be discussed: isolation of ribosomes from soil, temperature gradient gel electrophoresis, cloning and sequencing, and the verification of these data by V6 Southern blot hybridization, dot blot hybridization and in situ hybridization. By this and another novel rRNA quantification approach, the multiple competitive RT-PCR, it could be found that an uncultured Bacillus, recognized as ribotype DA001, contributes approximately 5-10% to all bacterial ribosomes in Dutch Drentse A grassland soils. These bacteria should be major operators of biogeochemical processes in soil.     

长期施用化肥及秸秆还田对砂姜黑土细菌群落的影响

【目的】在施用化肥的基础上进行秸秆还田是提高砂姜黑土肥力的有效措施,以往的研究只注重秸秆还田对土壤结构、肥力等物理化学性状方面的研究,缺少施肥对砂姜黑土微生物群落影响的研究。本研究以安徽蒙城典型的砂姜黑土为研究对象,以期揭示长期施用化肥和秸秆还田对砂姜黑土细菌群落的影响。【方法】采用454高通量测序对砂姜黑土不同农业施肥措施下的细菌群落进行分析研究,并通过生物信息学的分析方法揭示影响砂姜黑土细菌群落的主要因素。【结果】通过对454高通量测序数据的分析,发现砂姜黑土主要的细菌门类为放线菌、变形菌、酸杆菌、绿弯菌和拟杆菌。长期施用化肥显著提高了砂姜黑土肥力和作物产量,但导致了细菌群落结构的显著变化和多样性的显著降低。秸秆还田有利于土壤肥力的进一步提高,但是并没有缓解长期施用化肥对土壤细菌群落产生的不利影响。分析发现土壤pH的变化是导致土壤细菌群落变异的主要因素。【结论】在施用化肥的基础上进行秸秆还田有利于砂姜黑土肥力的提升,然而并没有缓解由施肥导致的土壤酸化对土壤细菌群落组成和多样性产生的不利影响。这暗示秸秆还田可能并未对砂姜黑土微生物生态产生根本性的有益影响,对于秸秆农田的利用方式还需要进一步研究,以达到农业生产效益和生态效益的并重。     

The nonrandom microheterogeneity of 16S rRNA genes in Vibrio splendidus may reflect adaptation to versatile lifestyles

16S rRNA molecules in a microbial strain can differ due to nucleotide variation between their genes. This is a typical trait of fast-growing bacteria to cope with different niches. We investigated characteristics of 16S rRNA genes in Vibrio splendidus strain PB1-10, from the normal flora of Atlantic halibut. Sequencing of 16S rRNA gene clones detected 35 variable positions in a total of 13 different gene copies. More than two-thirds of the substitutions occurred in regions corresponding to helix H6 and helix H17 of the 16S rRNA molecule. Possible recombination between these helixes in related bacteria ( Vibrio, Photobacterium, Colwellia ) from similar environments impacts 16S rRNA-based phylogeny of V. splendidus . We argue that these nonrandom modifications are maintained to provide a fine-tuning of the ribosome function to optimize translation machinery performance and ultimately bacterial niche fitness.     

Long-term after effects of fertilization on above-ground phytomass and species diversity in calcareous grassland

Abstract. Long-term after effects on species number and productivity in calcareous grassland were analyzed after cessation of fertilization. Three series of permanent plots were monitored yearly from 1971 to 1993. These series differed in duration of fertilization and in fertilizer composition, notably the amount of nitrogen and phosphorus. Yearly above-ground production decreased in all series after fertilization had been stopped, however at different speeds. The grass/forb ratio also decreased, while species number and Shannon index of diversity (H') increased. In the series where fertilizer treatment was stopped in 1967 (1968-series), productivity and grass/forb ratio decreased equally as in the plots where fertilization with a high nitrogen content was stopped in 1979 (80/Npk-series). However, species number in the plots of the 80/Npk-series increased faster than in the 1968-series. This was probably the result of a higher number of species present in the seed rain from the surrounding vegetation in the period after 1980 than from after 1968. In the plots receiving a high amount of phosphorus (80/nPk series), the productivity decreased more slowly than in the high nitrogen plots (80/Npk-series). At approximately the same total production level, the grass/forb ratio remained higher for another five-year period in the 80/nPk-series. Species number and the Shannon index of diversity increased more slowly in the 80/nPk-series than in the 80/Npk-series. Over a given range of productivity, changes in species number and Shannon index correlated better with the grass/forb ratio than with total above-ground phytomass. Therefore, restoration of species-rich grassland should not only be focused on lowering the yearly production, but also on reducing the grass component of the vegetation.