Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology

A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.     

A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.     

Characterization of the bacterial and archaeal communities in rice field soils subjected to long-term fertilization practices

The bacterial and archaeal communities in rice field soils subjected to different fertilization regimes for 57 years were investigated in two different seasons, a non-planted, drained season (April) and a rice-growing, flooded season (August), by performing soil dehydrogenase assay, real-time PCR assay and pyrosequencing analysis. All fertilization regimes increased the soil dehydrogenase activity while the abundances of bacteria and archaea increased in the plots receiving inorganic fertilizers plus compost and not in those receiving inorganic fertilizers only. Rice-growing and flooding decreased the soil dehydrogenase activity while they increased the bacterial diversity in rice field soils. The bacterial communities were dominated by Chloroflexi, Proteobacteria, and Actinobacteria and the archaeal communities by Crenarchaeota at the phylum level. In principal coordinates analysis based on the weighted Fast UniFrac metric, the bacterial and archaeal communities were separated primarily by season, and generally distributed along with soil pH, the variation of which had been caused by long-term fertilization. Variations in the relative abundance according to the season or soil pH were observed for many bacterial and archaeal groups. In conclusion, the microbial activity, prokaryotic abundance and diversity, and prokaryotic community structure in the rice field soils were changed by season and long-term fertilization.     

Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities

The microbial world has been shown to hold an unimaginable diversity. The use of rRNA genes and PCR amplification to assess microbial community structure and diversity present biases that need to be analyzed in order to understand the risks involved in those estimates. Herein, we show that PCR amplification of specific sequence targets within a community depends on the fractions that those sequences represent to the total DNA template. Using quantitative, real-time, multiplex PCR and specific Taqman probes, the amplification of 16S rRNA genes from four bacterial species within a laboratory community were monitored. Results indicate that the relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template. Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the 'rare biosphere'. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa which are required to decipher the complete extent of microbial diversity in nature.     

Microbial communities in iron-silica-rich microbial mats at deep-sea hydrothermal fields of the Southern Mariana Trough

The abundance, diversity and composition of bacterial and archaeal communities in the microbial mats at deep-sea hydrothermal fields were investigated, using culture-independent 16S rRNA and functional gene analyses combined with mineralogical analysis. Microbial mats were collected at two hydrothermal areas on the ridge of the back-arc spreading centre in the Southern Mariana Trough. Scanning electron microscope and energy dispersive X-ray spectroscopic (SEM-EDS) analyses revealed that the mats were mainly composed of amorphous silica and contained numerous filamentous structures of iron hydroxides. Direct cell counting with SYBR Green I staining showed that the prokaryotic cell densities were more than 10⁸ cells g⁻¹. Quantitative polymerase chain reaction (Q-PCR) analysis revealed that Bacteria are more abundant than Archaea in the microbial communities. Furthermore, zetaproteobacterial cells accounted for 6% and 22% of the prokaryotic cells in each mat estimated by Q-PCR with newly designed primers and TaqMan probe. Phylotypes related to iron-oxidizers, methanotrophs/methylotrophs, ammonia-oxidizers and sulfate-reducers were found in the 16S rRNA gene clone libraries constructed from each mat sample. A variety of unique archaeal 16S rRNA gene phylotypes, several pmoA , dsrAB and archaeal amoA gene phylotypes were also recovered from the microbial mats. Our results provide insights into the diversity and abundance of microbial communities within microbial mats in deep-sea hydrothermal fields.     

In Situ Polymerase Chain Reaction Visualization of Vibrio halioticoli Using Alginate Lyase Gene AlyVG2

A prokaryotic in situ polymerase chain reaction (PI-PCR) technique was applied to visualize Vibrio halioticoli cells using alginate lyase gene alyVG2 as a target gene. Prior to PI-PCR, a primer set, VG2-OS3, for specific amplification of an approximately 1.0-kb fragment from V. halioticoli genomic DNA was developed with amplified fragments from V. pelagius and V. fischeri DNAs as reference strains. One-stage PI-PCR using the primer set, digoxigenin-labeled dUTP, and indirect alkaline-phosphatase-linked fluorescence detection technique (HNPP/Fast Red TR as a substrate) failed to differentiate V. halioticoli IAM14596^T cells from ATCC25916^T cells of the closely related species V. pelagius. However, two-stage PI-PCR adding the extension and digoxigenin-labeling step of the amplified fragment into the first amplification stage allowed us to differentiate V. halioticoli cells from V. pelagius cells. Received May 7, 1999; accepted September 4, 1999.     

Approaches to investigating the ecology of plasmids in marine bacterial communities

To better understand prokaryotic gene flux in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of plasmid populations in marine bacterial communities, information on the distribution, diversity, and ecological traits of marine plasmids is necessary. This mini-review highlights recent insights gained into the molecular diversity and ecology of plasmids occurring in marine microbial communities.     

Assessing the impact of the biological control agent Bacillus thuringiensis on the indigenous microbial community within the pepper plant phyllosphere

Although biological control agents (BCAs) have been used extensively for controlling insects and pathogens of plants, little is known regarding the effects of such agents on the indigenous microbial communities within the plant phyllosphere. We assessed the effect of the BCA Bacillus thuringiensis (Bt) on the microbial communities within the pepper plant phyllosphere using culture-independent methodologies. Phospholipid fatty acid (PLFA) analysis suggested that the bacterial and fungal biomass were not significantly affected following Bt application. However, principal component analysis of PLFA data indicated that Bt did change the phyllosphere microbial community structure significantly. 16S rRNA gene-directed PCR with denaturing gradient gel electrophoresis (DGGE) also suggested a significant change in the phyllosphere bacterial community structure following Bt inoculation. Phylogenetic analysis of excised DGGE bands suggested a change in bacterial phyla; bands from untreated samples predominantly belonged to the Firmicutes, while Gammaproteobacteria abounded in the treated samples.     

Colony structure of a consortium of nitrogen-fixing bacteria

Colonies produced by a consortium of nitrifying bacteria were studied using light and electron microscopy. The colonies were obtained by direct plating of inoculum from a two-stage nonsterile chemostat fermentor and by repeatedly passing the microbial community of the fermentor through selective media containing ammonium or nitrite. The colonies studied can be characterized by a specific combination of six types of cells differing in their ultrastructure and spatial location within the colony. The types of cells occurring within a given colony were found to depend on the nitrogen compound present in the medium. As a result of our study, morphological features of colonial bacterial communities were preliminarily identified. The proposed approach can be viewed as a method to describe microbial associations and communities.     

Colony Structure of a Consortium of Nitrifying Bacteria

Colonies produced by a consortium of nitrifying bacteria were studied using light and electron microscopy. The colonies were obtained by direct plating of inoculum from a two-stage nonsterile chemostat fermentor and by repeatedly passing the microbial community of the fermentor through selective media containing ammonium or nitrite. The colonies studied can be characterized by a specific combination of six types of cells differing in their ultrastructure and spatial location within the colony. The types of cells occurring within a given colony were found to depend on the nitrogen compound present in the medium. As a result of our study, morphological features of colonial bacterial communities were identified. The proposed approach can be viewed as a method to describe microbial associations and communities.     

PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

Bacterial community dynamics following antibiotic exposure in a trematode parasite

Parasites harbour rich microbial communities that may play a role in host-parasite interactions, from influencing the parasite’s infectivity to modulating its virulence. Experimental manipulation of a parasite’s microbes would be essential, however, in order to establish their causal role. Here, we tested whether indirect exposure of a trematode parasite within its snail intermediate host to a variety of antibiotics could alter its bacterial community. Based on sequencing the prokaryotic 16S ssrRNA gene, we characterised and compared the bacterial community of the trematode Philophthalmus attenuatus before, shortly after, and weeks after exposure to different antibiotics (penicillin, colistin, gentamicin) with distinct activity spectra. Our findings revealed that indirectly treating the parasites by exposing their snail host to antibiotics resulted in changes to their bacterial communities, measured as their diversity, taxonomic composition, and/or the relative abundance of certain taxa. However, alterations to the parasite’s bacterial community were not always as predicted from the activity spectrum of the antibiotic used. Furthermore, the bacterial communities of the parasites followed significantly divergent trajectories in the days post-exposure to antibiotics, but later converged toward a new state, i.e. a new bacterial community structure different from that pre-exposure. Our results confirm that a trematode’s microbial community can be experimentally altered by antibiotic exposure while within its snail host, with the dynamic nature of the bacterial assemblage driving it to a new state over time after the perturbation. This research opens new possibilities for future experimental investigations of the functional roles of microbes in host-parasite interactions.     

Different biogeographic patterns of prokaryotes and microbial eukaryotes in epilithic biofilms

Microbial biogeography studies expend much effort in determining whether environmental selection or stochastic processes related to dispersal are more important in shaping community composition. While both types of factors are possibly influential, it is tacitly assumed that protists, or microbial eukaryotes in general, behave biogeographically as prokaryotes because of their small physical size. However, direct evidence for this in exactly the same environment and at the same phylogenetic depth is lacking. In this study, we compared the structure of both prokaryotic and eukaryotic components of microbial communities forming biofilms on mineral substrates in different geographic locations at the level of small-subunit (SSU) rRNA-based operational taxonomic units (OTUs). These microbial communities are subjected to strong environmental selection and contain significant proportions of extremophilic microorganisms adapted to desiccation and UV radiation. We find that the nature of the substrate as well as climatic variables and geography influences microbial community structure. However, constrained correspondence analyses and distance-decay curves showed that, whereas the substrate type was the most significant factor structuring bacterial communities, geographic location was the most influential factor for microbial eukaryote communities. Biological explanations implying a higher dispersal success for bacteria combined with more mobile lifestyles for predatory protists may underlie these different prokaryote versus microbial eukaryote biogeographic patterns.     

Rapid quantitative profiling of complex microbial populations

Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities—a ‘census’—is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10 462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples—the current ‘gold standard’ method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.     

Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.     

Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.     

Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.