Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem

Rumen microbial community comprising of bacteria, archaea, fungi, and protozoa is characterized not only by the high population density but also by the remarkable diversity and the most complex microecological interactions existing in the biological world. This unprecedented biodiversity is quite far from full elucidation as only about 15-20?% of the rumen microbes are identified and characterized till date using conventional culturing and microscopy. However, the last two decades have witnessed a paradigm shift from cumbersome and time-consuming classical methods to nucleic acid-based molecular approaches for deciphering the rumen microbial community. These techniques are rapid, reproducible and allow both the qualitative and quantitative assessment of microbial diversity. This review describes the different molecular methods and their applications in elucidating the rumen microbial community.     

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.     

Microbial diversity and community respiration in freshwater sediments influenced by artificial light at night

An increasing proportion of the Earth''s surface is illuminated at night. In aquatic ecosystems, artificial light at night (ALAN) may influence microbial communities living in the sediments. These communities are highly diverse and play an important role in the global carbon cycle. We combined field and laboratory experiments using sediments from an agricultural drainage system to examine how ALAN affects communities and alters carbon mineralization. Two identical light infrastructures were installed parallel to a drainage ditch before the start of the experiment. DNA metabarcoding indicated that both sediment communities were similar. After one was lit for five months (July–December 2012) we observed an increase in photoautotroph abundance (diatoms, Cyanobacteria) in ALAN-exposed sediments. In laboratory incubations mimicking summer and winter (six weeks each), communities in sediments that were exposed to ALAN for 1 year (July 2012–June 2013) showed less overall seasonal change compared with ALAN-naive sediments. Nocturnal community respiration was reduced in ALAN-exposed sediments. In long-term exposed summer-sediments, we observed a shift from negative to positive net ecosystem production. Our results indicate ALAN may alter sediment microbial communities over time, with implications for ecosystem-level functions. It may thus have the potential to transform inland waters to nocturnal carbon sinks.     

典型高原湖滨带底泥细菌群落结构及多样性特征

【背景】高原湖泊的富营养化日趋严重,而湖滨带作为湖泊的保护屏障对外源污染物具有拦截净化等作用,水环境变化则会对底泥细菌产生深刻影响。【目的】探究高原湖滨带底泥细菌群落结构特征及与水体富营养化之间的联系。【方法】基于16S rRNA基因高通量测序技术分析了阳宗海南岸湖滨带8个不同样点的底泥细菌群落结构及多样性,并结合样品水体环境因子,采用主成分分析(PCA)和冗余分析(redundancy analysis,RDA)探讨了水体富营养化对底泥细菌群落结构及丰富度的影响。【结果】湖滨带底泥细菌与水体富营养化程度存在响应关系,在水体富营养化程度高的区域(S3)细菌丰富度较高,操作分类单元(operationaltaxonomicunits,OTU)高达1473。反之,在富营养化程度低的区域(S1)细菌丰富度较低,OTU为730。阳宗海南岸湖滨带底泥中主要优势菌门为变形菌门(Proteobacteria)和绿弯菌门(Chloroflexi),含有少量的放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)和厚壁菌门(Firmicutes);绿弯菌门(Chloroflex...     

Molecular analyses of methanogen diversity associated with cattle dung

Methanogen communities were characterized in cattle dung of different ages by using a culture-independent approach. Community structures were determined by the phylogenetic analyses of methyl-coenzyme M reductase A (mcrA) clones of fresh, 8-month-old, and 24-month-old-dry dung samples. The clones in the mcrA libraries of fresh and 8-month old dung samples were identified as belonging to Methanomicrobiales, Methanobacteriales, and Methanosarcinales. However, clones in the library of 24-month-old dung were not affiliated to Methanomicrobiales. Anaerobic digestion of 2-month-old dung produced only 15% less methane compared to fresh dung which indicated the possibility of using dry dung to fuel the biogas plants in areas where unavailability of fresh dung hinders their continuous functioning. Our results first time showed the presence of viable methanogens in dry cattle dung stored for prolonged periods of time.     

Ciliate community structure, diversity and trophic role in offshore sediments from the Yellow Sea

We investigated the community structure, diversity and trophic role of ciliates in the sediments from 48 stations in the Yellow Sea using Ludox density centrifugation and quantitative protargol stain. The ciliate abundance ranged from 1 to 221cellscm(-3) and biomass from 0.0001 to 0.47μgCcm(-3) in the upper 8cm of the sediments. On average, 77% of ciliate abundance and 81% of biomass were distributed in the 0-2cm sediment layers, while the respective proportions were only about 6% and 3% in the 5-8-cm layers. Among the 198 morphospecies, Prostomatea was the most dominant group accounting for 45% of the total abundance and 58% of the total biomass. Carnivorous ciliates constituted the primary feeding type, occupying about 64% of the total biomass, followed by bacterivores (21%), algivores (12%) and omnivores (3%). The ciliate abundance and biomass in the upper 5cm of sediments were two orders of magnitude higher than those in the upper 10m of the Yellow Sea water column. The estimated ciliate bacterivory and herbivory indicate that ciliate ingestion had little direct influence on bacterial standing stock but possibly had an important impact on diatoms in the sediments from the Yellow Sea.     

Shear rate moderates community diversity in freshwater biofilms

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S(-1)) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S(-1)). The intermediate shear rate (122 S(-1)) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S(-1)), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.     

黄河内蒙古段表层沉积物细菌多样性及群落结构类型

为掌握黄河内蒙古段表层沉积物微生物多样性、群落结构类型及其影响因素,采用高通量测序技术分析了6个采样点表层沉积物中的微生物多样性及群落结构。研究结果表明,黄河内蒙古段沉积物细菌丰度大小排序为乌拉特前旗(H3)老牛湾(H6)临河(H2)包头(H4)托县(H5)乌海(H1),微生物多样性排序为H6 H2 H5 H4 H3 H1,乌海沉积物中细菌丰度和微生物多样性都是最低的一个采样点。黄河内蒙古段表层沉积物中三大优势菌群分别为变形菌门(Proteobacteria,相对丰度32.39%)、绿弯菌门(Chloroflexi,13.25%)和拟杆菌门(Bacteroidetes,12.16%)。细菌群落丰度与环境因子之间的冗余分析结果显示,沉积物中总有机碳(TOC)、离子交换容量(CEC)和总磷(TP)、总氮(TN)等环境因子对黄河内蒙古段沉积物细菌群落分布影响较大,负相关系数分别为82.5%、80.1%、85.5%和85.2%;微生物多样性与环境理化因子相关性分析结果表明,黄河沉积物微生物多样性格局与对氮磷等营养物质的损耗有直接关系。     

Microenvironments and microbial community structure in sediments

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.     

Molecular microbiology of gut bacteria: genetic diversity and community structure analysis

Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.     

Molecular tools for isolate and community studies of Pyrenomycete fungi

The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny established from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples.     

Molecular evidence for the broad distribution of anaerobic ammonium-oxidizing bacteria in freshwater and marine sediments

Previously available primer sets for detecting anaerobic ammonium-oxidizing (anammox) bacteria are inefficient, resulting in a very limited database of such sequences, which limits knowledge of their ecology. To overcome this limitation, we designed a new primer set that was 100% specific in the recovery of approximately 700-bp 16S rRNA gene sequences with >96% homology to the "Candidatus Scalindua" group of anammox bacteria, and we detected this group at all sites studied, including a variety of freshwater and marine sediments and permafrost soil. A second primer set was designed that exhibited greater efficiency than previous primers in recovering full-length (1,380-bp) sequences related to "Ca. Scalindua," "Candidatus Brocadia," and "Candidatus Kuenenia." This study provides evidence for the widespread distribution of anammox bacteria in that it detected closely related anammox 16S rRNA gene sequences in 11 geographically and biogeochemically diverse freshwater and marine sediments.     

Molecular tools and analytical approaches for the characterization of farm animal genetic diversity

Genetic studies of livestock populations focus on questions of domestication, within- and among-breed diversity, breed history and adaptive variation. In this review, we describe the use of different molecular markers and methods for data analysis used to address these questions. There is a clear trend towards the use of single nucleotide polymorphisms and whole-genome sequence information, the application of Bayesian or Approximate Bayesian analysis and the use of adaptive next to neutral diversity to support decisions on conservation.     

Quinone profiles in lake sediments: Implications for microbial diversity and community structures

Microbial quinone compositions of sediment mud samples from five different lakes in Japan were studied by spectrochromatography and mass spectrometry. The total quinone content of these samples ranged from 1.97 to 18.0 nmol/g dry weight of sediment, of which a combined fraction of ubiquinones and menaquinones accounted for 42 to 74%. The remaining fraction (26 to 58%) consisted of the photosynthetic quinones, plastoquinones and phylloquinone. The sediment samples produced PQ-9 or Q-8 as the most abundant quinone type regardless of their geographic locations and depths. These results indicate that oxygenic phototrophic microorganisms and Q-8-containing proteobacteria constituted major parts of microbial populations in the lake sediment. In the surface water of the same sampling sites, plastoquinones and phylloquinone occurred in much higher proportions. These findings suggested that the high abundance of oxygenic phototrophs in the sediment muds resulted from their constant movement or sedimentation from the surface water. Numerical analyses of the quinone profiles showed that the microbial communities of the sediment were diverse and different in different lakes but similar to each other in the diversity of bioenergetic modes. Three physiological groups of microbes showing ubiquinone-mediated aerobic respiration, oxygenic photosynthesis, and menaquinone-associated respiration were suggested to inhabit the lake sediments in balance.     

Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures

Bacterial community structure and diversity in rhizospheres in two types of grassland, distinguished by both plant species and fertilization regimen, were assessed by performing a 16S ribosomal DNA (rDNA) sequence analysis of DNAs extracted from triplicate soil plots. PCR products were cloned, and 45 to 48 clones from each of the six libraries were partially sequenced. Phylogenetic analysis of the resultant 275 clone sequences indicated that there was considerable variation in abundance in replicate unfertilized, unimproved soil samples and fertilized, improved soil samples but that there were no significant differences in the abundance of any phylogenetic group. Several clone sequences were identical in the 16S rDNA region analyzed, and the clones comprised eight pairs of duplicate clones and two sets of triplicate clones. Many clones were found to be most closely related to environmental clones obtained in other studies, although three clones were found to be identical to culturable species in databases. The clones were clustered into operational taxonomic units at a level of sequence similarity of >97% in order to quantify diversity. In all, 34 clusters containing two or more sequences were identified, and the largest group contained nine clones. A number of diversity, dominance, and evenness indices were calculated, and they all indicated that diversity was high, reflecting the low coverage of rDNA libraries achieved. Differences in diversity between sample types were not observed. Collector's curves, however, indicated that there were differences in the underlying community structures; in particular, there was reduced diversity of organisms of the alpha subdivision of the class Proteobacteria (alpha-proteobacteria) in improved soils.     

Heterotrophic flagellate biodiversity and community structure in freshwater streams

The species diversity and community structure of heterotrophic flagellates in watercourses from different geographical regions were investigated. Forty-one species have been identified. The species diversity of cercomonads, kinetoplastids, choanoflagellates, and flagellates is the highest. Bodo saltans, B. designis and Goniomonas truncata are the most common species. Planktonic communities in different streams are most similar in terms of species composition. The highest similarity within benthic cenoses was noted between geographically distant communities. Communities of heterotrophic flagellates seem to be highly heterogenous systems; species distribution depends mainly on the type of habitat and not on geographical factors.     

Biological diversity and the community structure of organisms

Biological diversity and the complexity of the community structure of benthic animals in different bodies of water are evaluated using the Shannon index. The literature data on the communities in Lake Krasnoye (Karelian Isthmus, Leningrad Oblast), Shchuchy Bay of Lake Ladoga, Neva Bay, and the eastern part of the Gulf of Finland have been analyzed. The extrema (maxima and minima) and the inflection point of the curve of functions are determined with the help of derivatives. The rate of the secondary succession is evaluated for the first time by the example of benthic communities. It is shown that the community structure of bottom animals in the Shchuchy Bay ecosystem formed in the process of succession proves to be more complex than in Lake Krasnoye and the Neva Bay communities that have been formed for a long time. Communities of bottom animals are less complex in polluted waters of the top of the eastern part of the Gulf of Finland. It has taken 12 years to reach the maximum diversity of the bottom animal communities in Shchuchy Bay, 16 years in Lake Krasnoye, and 7–10 years in Neva Bay. It is supposed that, under favorable conditions in the waterbodies of a moderate climate and in the absence of heavy pollution or eutrophication, it takes 12–14 years on average to form the most complex community structure of bottom animals (3–4 bit/spec.). The structure of communities in polluted waters remains simple.     

Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

Sialic acid diversity in the human gut: Molecular impacts and tools for future discovery

Sialic acids are a family of structurally related sugars that are prevalent in mucosal surfaces, including the human intestine. In the gut, sialic acids have diverse biological roles at the interface of the host epithelium and the microbiota. N-acetylneuraminic acid (Neu5Ac), the best studied sialic acid, is a nutrient source for bacteria and, when displayed on the cell surface, a binding site for host immune factors, viruses, and bacterial toxins. Neu5Ac is extensively modified by host and microbial enzymes, and the impacts of Neu5Ac derivatives on host–microbe interactions, and generally on human and microbial biology, remain underexplored. In this mini-review, we highlight recent reports describing how host and microbial proteins differentiate Neu5Ac and its derivatives, draw attention to gaps in knowledge related to sialic acid biology, and suggest cutting-edge methodologies that may expand our appreciation and understanding of Neu5Ac in health and disease.     

Microbial diversity and community structure in Fynbos soil

The Fynbos biome in South Africa is renowned for its high plant diversity and the conservation of this area is particularly important for the region. This is especially true in the case of endangered vegetation types on the lowlands such as Sand Fynbos, of which only small fragments remain. The question is thus whether the diversity of the above‐ground flora is mirrored in the below‐ground microbial communities. In order to determine the relationship of the above‐ and below‐ground communities, the soil community composition of both fungal and bacterial groups in Sand Fynbos was characterized over space and time. A molecular approach was used based on the isolation of total soil genomic DNA and automated ribosomal intergenic spacer analysis of bacterial and fungal communities. Soil from four different sites was compared to resolve the microbial diversity of eubacterial and fungal groups on a local (alpha diversity) scale as well as a landscape scale (beta diversity). The community structures from different sites were compared and found to exhibit strong spatial patterns which remained stable over time. The plant community data were compared with the fungal and the bacterial communities. We concluded that the microbial communities in the Sand Fynbos are highly diverse and closely linked to the above‐ground floral communities.