In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Phylogenetic Resolution and Quantifying the Phylogenetic Diversity and Dispersion of Communities

Conservation biologists and community ecologists have increasingly begun to quantify the phylogenetic diversity and phylogenetic dispersion in species assemblages. In some instances, the phylogenetic trees used for such analyses are fully bifurcating, but in many cases the phylogenies being used contain unresolved nodes (i.e. polytomies). The lack of phylogenetic resolution in such studies, while certainly not preferred, is likely to continue particularly for those analyzing diverse communities and datasets with hundreds to thousands of taxa. Thus it is imperative that we quantify potential biases and losses of statistical power in studies that use phylogenetic trees that are not completely resolved. The present study is designed to meet both of these goals by quantifying the phylogenetic diversity and dispersion of simulated communities using resolved and gradually ‘unresolved’ phylogenies. The results show that: (i) measures of community phylogenetic diversity and dispersion are generally more sensitive to loss of resolution basally in the phylogeny and less sensitive to loss of resolution terminally; and (ii) the loss of phylogenetic resolution generally causes false negative results rather than false positives.     

Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities

Abstract In this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.     

Phylogenetic Diversity of Microbial Communities from the Surface of Polyethylene Terephthalate Materials Exposed to Different Water Environments

Microbiology - The goal of the present work was to investigate the composition of microbial communities developing on polyethylene terephthalate (PET) samples immersed in aqueous media of various...     

Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin,Guaymas Basin

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.     

Aspects of Diversity Measurement for Microbial Communities

A useful measure of diversity was calculated for microbial communities collected from lake water and sediment samples using the Shannon index (H′) and rarefaction [E(S)]. Isolates were clustered by a numerical taxonomy approach in which limited (<20) tests were used so that the groups obtained represented a level of resolution other than species. The numerical value of diversity for each sample was affected by the number of tests used; however, the relative diversity compared among several sampling locations was the same whether 11 or 19 characters were examined. The number of isolates (i.e., sample size) strongly influenced the value of H′ so that unequal sized samples could not be compared. Rarefaction accounts for differences in sample size inherently so that such comparisons are made simple. Due to the type of sampling carried out by microbiologists, H′ is estimated and not determined and therefore requires a statement of error associated with it. Failure to report error provided potentially misleading results. Calculation of the variance of H′ is not a simple matter and may be impossible when handling a large number of samples. With rarefaction, the variance of E(S) is readily determined, facilitating the comparison of many samples.     

矿山废水中微生物生态多样性研究

从湖北铜绿山铜矿和山西中条山铜矿两地采集了矿山废水样品, 用RFLP方法(限制性片段长度多态性方法)分析不同矿山废水中微生物多样性及其群落结构组成变化。结果发现：通过16S rDNA基因序列的系统发育树分析, 在5个矿山废水样品中检测到的细菌主要分为4大类：即Proteobacteria纲、Nitrospira纲、Firmicutes纲和Bacteroidetes纲。通过对古菌16S rDNA 的PCR分析发现, 取自湖北铜绿山铜矿的样品中检测到古菌的存在, 而取自山西中条山铜矿的样品中没有检测到古菌的存在。检测到的古菌主要是属于Ferroplasma属和Thermoplasma属。结合5个矿山废水样品的化学元素分析结果和细菌群落结构数据, 进行PCA分析(主成分分析), 发现不同矿山废水样品的生物地球化学性质及其微生物组成存在很大差异, pH值、温度、不同浓度的元素如S、Cu、Ni和Co可能是形成细菌种群结构差异的关键因素。     

DGGE技术在森林土壤微生物多样性研究中的应用

微生物在森林土壤物质转化中扮演着重要角色,与森林的林型、土壤理化性质存在着密切关系.森林土壤微生物多样性及其变化在一定程度上反映了土壤环境的生产力和稳定性,对表征森林演替,土壤生态修复等有重要意义.变性梯度凝胶电泳(DGGE)技术测定微生物多样性具有快捷、高效和可重复性高等优点.简要介绍DGGE技术的原理,分析这一技术的局限性和优化方法.重点以实例说明该技术在森林土壤微生物多样性研究中的应用现状,展望该技术的发展前景,以期能为今后这一领域的研究提供科学依据.     

Oxygen and the Spatial Structure of Microbial Communities

Oxygen has two faces. On one side it is the terminal electron acceptor of aerobic respiration – the most efficient engine of energy metabolism. On the other hand, oxygen is toxic because the reduction of molecular O₂ creates reactive oxygen species such as the superoxide anion, peroxide, and the hydroxyl radical. Probably most prokaryotes, and virtually all eukaryotes, depend on oxygen respiration, and we show that the ambiguous relation to oxygen is both an evolutionary force and a dominating factor driving functional interactions and the spatial structure of microbial communities.We focus on microbial communities that are specialised for life in concentration gradients of oxygen, where they acquire the full panoply of specific requirements from limited ranges of PO₂ , which also support the spatial organisation of microbial communities. Marine and lake sediments provide examples of steep O₂ gradients, which arise because consumption or production of oxygen exceeds transport rates of molecular diffusion. Deep lakes undergo thermal stratification in warm waters, resulting in seasonal anaerobiosis below the thermocline, and lakes with a permanent pycnocline often have permanent anoxic deep water. The oxycline is here biologically similar to sediments, and it harbours similar microbial biota, the main difference being the spatial scale. In sediments, transport is dominated by molecular diffusion, and in the water column, turbulent mixing dominates vertical transport.Cell size determines the minimum requirement of aerobic organisms. For bacteria (and mitochondria), the half‐saturation constant for oxygen uptake ranges within 0.05 – 0.1% atmospheric saturation; for the amoeba Acanthamoeba castellanii it is 0.2%, and for two ciliate species measuring around 150 μm, it is 1‐2 % atmospheric saturation. Protection against O₂ toxicity has an energetic cost that increases with increasing ambient O₂ tension. Oxygen sensing seems universal in aquatic organisms. Many aspects of oxygen sensing are incompletely understood, but the mechanisms seem to be evolutionarily conserved. A simple method of studying oxygen preference in microbes is to identify the preferred oxygen tension accumulating in O₂ gradients. Microorganisms cannot sense the direction of a chemical gradient directly, so they use other devices to orient themselves. Different mechanisms in different prokaryotic and eukaryotic microbes are described. In O₂ gradients, many bacteria and protozoa are vertically distributed according to oxygen tension and they show a very limited range of preferred PO₂. In some pigmented protists the required PO₂ is contingent on light due to photochemically generated reactive oxygen species. In protists that harbour endosymbiotic phototrophs, orientation towards light is mediated through the oxygen production of their photosynthetic symbionts. Oxygen plays a similar role for the distribution of small metazoans (meiofauna) in sediments, but there is little experimental evidence for this. Thus the oxygenated sediments surrounding ventilated animal burrows provide a special habitat for metazoan meiofauna as well as unicellular organisms.     

UniFrac: a New Phylogenetic Method for Comparing Microbial Communities

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.     

Effects of Abiotic Factors on the Phylogenetic Diversity of Bacterial Communities in Acidic Thermal Springs

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E_h of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.     

The Influence of Tropical Plant Diversity and Composition on Soil Microbial Communities

There is growing interest in understanding the linkages between above- and belowground communities, and very little is known about these linkages in tropical systems. Using an experimental site at La Selva Biological Station, Costa Rica, we examined whether plant diversity, plant community composition, and season influenced microbial communities. We also determined whether soil characteristics were related to differences in microbial communities. Phospholipid fatty acid (PLFA) composition revealed that microbial community composition differed across a plant diversity gradient (plots contained 1, 3, 5, or over 25 species). Plant species identity also was a factor influencing microbial community composition; PLFA composition significantly varied among monocultures, and among three-species combinations that differed in plant species composition. Differences among treatments within each of these comparisons were apparent in all four sampling dates of the study. There was no consistent shift in microbial community composition between wet and dry seasons, although we did see significant changes over time. Of all measured soil characteristics, soil C/N was most often associated with changes in microbial community composition across treatment groups. Our findings provide evidence for human alteration of soil microbial communities via the alteration of plant community composition and diversity and that such changes are mediated in part by changes in soil carbon quality.     

Impact of Carbon and Flooding on the Metabolic Diversity of Microbial Communities in Soils

The assumption that carbon and soil water content are major determinants of microbial community structure and function is rarely questioned because of substantial evidence of the impacts of these variables on specific populations and functions. The significance of carbon and water for metabolic diversity at the microbial community level was tested on the field scale in agricultural plots varying in carbon inputs and in whether they were flooded. Surface soils in which rice straw was incorporated or burned and which were flooded or unflooded were sampled at monthly intervals three times during the flooded winter period (January to March) and again 1 month postdraining. Biomass carbon and nitrogen were not affected by treatments, active bacterial counts showed slight increases, and respiration rates were increased by carbon inputs and flooding. Biolog microplates were inoculated with soil extracts to quantify the metabolic diversity of the soil microbial community. Canonical correspondence analysis and the Monte Carlo permutation testing showed that differences in substrate utilization patterns were significantly related (P < 0.001) to carbon and flooding treatments. Biolog substrates whose metabolism was altered by the treatments were consistent across dates and tended to be positively related (utilization enhancement) to carbon inputs and negatively related to winter flooding. The importance of carbon as an environmental variable increased over time after straw treatment, whereas the importance of water became evident after flooding and decreased after drainage. The effect of long-term rice straw incorporation on substrate utilization patterns at another field site was consistent with these results despite the dissimilarities of the two soils.     

Microbial Diversity and Community Structure on Corroding Concretes

The objective of this study was to analyze bacterial diversity in two different concrete samples to understand the dominant types of bacteria that may contribute to concrete corrosion. Two concrete samples, HN-1 from the sunny side and HN-2 from dark and damp side, were collected from Zijin Mountain in Nanjing and genomic DNA was extracted. The partial bacterial 16S rRNA gene fragment was PCR amplified and two clone libraries were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). Phylogenetic trees of bacterial 16S rDNA nucleotide sequences were constructed. Sample HN-1 and HN-2 contained 21 OTUs and 26 OTUs, respectively. Proteobacteria and Planctomycetes were the predominant bacteria in both samples, and they are distributed among Herbaspirillum, Archangium, Phyllobacteriaceae and Planctomycetaceae. Cyanobacteria and Rubrobacter sp. are dominant in HN-1; while Acidobacteriaceae, Adhaeribacter sp. and Nitrospira sp. are predominant in HN-2. This distribution pattern was consistent with local environmental conditions of these two samples. The inferred physiological characteristics of these bacteria, based on relatedness of the DNA clone sequences to cultivated species, revealed different mechanisms of concrete corrosion depending on the local environmental conditions.