Multivariate Analysis of Complex DNA Sequence Electropherograms for High-Throughput Quantitative Analysis of Mixed Microbial Populations

High-throughput quantification of genetically coherent units (GCUs) is essential for deciphering population dynamics and species interactions within a community of microbes. Current techniques for microbial community analyses are, however, not suitable for this kind of high-throughput application. Here, we demonstrate the use of multivariate statistical analysis of complex DNA sequence electropherograms for the effective and accurate estimation of relative genotype abundance in cell samples from mixed microbial populations. The procedure is no more labor-intensive than standard automated DNA sequencing and provides a very effective means of quantitative data acquisition from experimental microbial communities. We present results with the Campylobacter jejuni strain-specific marker gene gltA, as well as the 16S rRNA gene, which is a universal marker across bacterial assemblages. The statistical models computed for these genes are applied to genetic data from two different experimental settings, namely, a chicken infection model and a multispecies anaerobic fermentation model, demonstrating collection of time series data from model bacterial communities. The method presented here is, however, applicable to any experimental scenario where the interest is quantification of GCUs in genetically heterogeneous DNA samples.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

High-Yield and Phylogenetically Robust Methods of DNA Recovery for Analysis of Microbial Biofilms Adherent to Plant Biomass in the Herbivore Gut

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the ??repeated bead beating and column (RBB+C) purification?? method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions.     

Method for Studying Microbial Biofilms in Flowing-Water Systems

A method for the study of microbial biofilms in flowing-water systems was developed with special reference to the flow conditions in electrochemical concentration cells. Seawater was circulated in a semiclosed flow system through biofilm reactors (3 cm s⁻¹) with microscope cover slips arranged in lamellar piles parallel with the flow. At fixed time intervals cover slips with their biofilm were removed from the pile, stained with crystal violet, and mounted on microscope slides. The absorbances of the slides were measured at 590 nm and plotted against time to give microbial biofilm development. From calibration experiments a staining time of 1 min and a rinse time of 10 min in a tap water flow (3 cm s⁻¹) were considered sufficient. When an analysis of variance was performed on biofilm development data, 78% of the total variance was found to be due to random natural effects; the rest could be explained by experimental effects. The absorbance values correlated well with protein N, dry weight, and organic weight in two biofilm experiments, one with a biofilm with a high (75%) and one with a low (~25%, normal) inorganic content. Comparisons of regression lines revealed that the absorbance of the stained biofilms was an estimate closely related to biofilm dry weight.     

Methods for Observing Microbial Biofilms Directly on Leaf Surfaces and Recovering Them for Isolation of Culturable Microorganisms

Epifluorescence microscopy, scanning electron microscopy, and confocal laser scanning microscopy were used to observe microbial biofilms directly on leaf surfaces. Biofilms were observed on leaves of all species sampled (spinach, lettuce, Chinese cabbage, celery, leeks, basil, parsley, and broad-leaved endive), although the epifluorescent images were clearest when pale green tissue or cuticle pieces were used. With these techniques, biofilms were observed that were about 20 (mu)m in depth and up to 1 mm in length and that contained copious exopolymeric matrices, diverse morphotypes of microorganisms, and debris. The epifluorescence techniques described here can be used to rapidly determine the abundance and localization of biofilms on leaves. An additional technique was developed to recover individual biofilms or portions of single biofilms from leaves and to disintegrate them for isolation of the culturable microorganisms they contained. Nineteen biofilms from broad-leaved endive, spinach, parsley, and olive leaves were thus isolated and characterized to illustrate the applications of this technique.     

Microbial Biofilms on the Sandstone Monuments of the Angkor Wat Complex,Cambodia

Discoloring biofilms from Cambodian temples Angkor Wat, Preah Khan, and the Bayon and West Prasat in Angkor Thom contained a microbial community dominated by coccoid cyanobacteria. Molecular analysis identified Chroococcidiopsis as major colonizer, but low similarity values (<95%) suggested a similar genus or species not present in the databases. In only two of the six sites sampled were filamentous cyanobacteria, Microcoleus, Leptolyngbya, and Scytonema, found; the first two detected by sequencing of 16S rRNA gene library clones from samples of a moist green biofilm on internal walls in Preah Khan, where Lyngbya (possibly synonymous with Microcoleus) was seen by direct microscopy as major colonizer. Scytonema was detected also by microscopy on an internal wall in the Bayon. This suggests that filamentous cyanobacteria are more prevalent in internal (high moisture) areas. Heterotrophic bacteria were found in all samples. DNA sequencing of bands from DGGE gels identified Proteobacteria (Stenotrophomonas maltophilia and Methylobacterium radiotolerans) and Firmicutes (Bacillus sp., Bacillus niacini, Bacillus sporothermodurans, Lysinibacillus fusiformis, Paenibacillus sp., Paenibacillus panacisoli, and Paenibacillus zanthoxyli). Some of these bacteria produce organic acids, potentially degrading stone. Actinobacteria, mainly streptomycetes, were present in most samples; algae and fungi were rare. A dark-pigmented filamentous fungus was detected in internal and external Preah Khan samples, while the alga Trentepohlia was found only in samples taken from external, pink-stained stone at Preah Khan. Results show that these microbial biofilms are mature communities whose major constituents are resistant to dehydration and high levels of irradiation and can be involved in deterioration of sandstone. Such analyses are important prerequisites to the application of control strategies.     

Spatial Autocorrelation Analysis of the Distribution of Genotypes within Populations of Lodgepole Pine

Spatial autocorrelation analyses of point samples within two populations of lodgepole pine (Pinus contorta ssp. latifolia) indicate that single-locus mature tree and pollen genotypes are distributed in a nearly random fashion for most of the allozyme loci assayed. This lack of structure in the distributions of most genotypes is consistent with outcrossing rates that are very nearly 1.0 and with estimates indicating that both pollen and seed are dispersed over long distances in lodgepole pine. However, spatial autocorrelation of genotypes for a few loci suggests that genotypes at these loci may be under natural selection.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

塔里木荒漠绿洲过渡带主要种群生态位与空间格局分析

于新疆塔里木盆地荒漠绿洲过渡带设置3条样带(长900 m×宽50 m)进行植物群落调查,运用生态位测度指标和点格局法对种群空间格局、空间关联性及生态位特征进行研究。结果显示,胡杨(Populus euphratica Oliv.)、多枝柽柳(Tamarix ramosissima Ledeb.)和甘草(Glycyrrhiza inflate Bat.)具有较大的重要值和生态位宽度,对其它生态位较窄的物种具有资源竞争和扩张优势,生态适应能力强,对群落结构和环境变化起决定性作用;生态过渡带植物的种间生态位重叠普遍较高,尤其是草本植物间更明显,此分配格局反映出荒漠植物长期适应旱化生境而发生趋同适应,生态位分化程度低,种间资源竞争激烈;优势种群空间格局呈明显的空间变异性,0~25 m尺度内多枝柽柳和甘草分别呈随机分布和聚集分布,胡杨种群在≤5 m尺度内呈聚集分布,并随尺度增大转为随机分布;胡杨与多枝柽柳、胡杨与甘草、多枝柽柳与甘草分别在4 m、≤16 m和≤25 m尺度内呈显著空间负关联,种间相互排斥。表明在匮乏环境资源条件下植物趋同适应与资源竞争是驱动荒漠植物群落演替和限制物种共存的主要原因。     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.     

枇杷园蜘蛛混合种群空间分布型的动态分析

枇杷园蜘蛛混合种群的空间分布型在不同季节存在差异,其空间分布型主要由枇杷园蜘蛛本身的生殖水平、游猎型和定居型数量的多寡,主要目标害虫的数量及分布,气候环境的变化及其越冬生态习性,枇杷园蜘蛛的种群密度及其种间竞争程度决定。     

Competition and Coexistence of Sulfate-Reducing and Methanogenic Populations in Anaerobic Biofilms

The microbial population structure and function of natural anaerobic communities maintained in laboratory fixed-bed biofilm reactors were tracked before and after a major perturbation, which involved the addition of sulfate to the influent of a reactor that had previously been fed only glucose (methanogenic), while sulfate was withheld from a reactor that had been fed both glucose and sulfate (sulfidogenic). The population structure, determined by using phylogenetically based oligonucleotide probes for methanogens and sulfate-reducing bacteria, was linked to the functional performance of the biofilm reactors. Before the perturbation, the methanogenic reactor contained up to 25% methanogens as well as 15% sulfate-reducing bacteria, even though sulfate was not present in the influent of this reactor. Methanobacteriales and Desulfovibrio spp. were the most abundant methanogens and sulfate-reducing bacteria, respectively. The presence of sulfate-reducing bacteria (primarily Desulfovibrio spp. and Desulfobacterium spp.) in the absence of sulfate may be explained by their ability to function as proton-reducing acetogens and/or fermenters. Sulfate reduction began immediately following the addition of sulfate consistent with the presence of significant levels of sulfate-reducing bacteria in the methanogenic reactor, and levels of sulfate-reducing bacteria increased to a new steady-state level of 30 to 40%; coincidentally, effluent acetate concentrations decreased. Notably, some sulfate-reducing bacteria (Desulfococcus/Desulfosarcina/Desulfobotulus group) were more competitive without sulfate. Methane production decreased immediately following the addition of sulfate; this was later followed by a decrease in the relative concentration of methanogens, which reached a new steady-state level of approximately 8%. The changeover to sulfate-free medium in the sulfidogenic reactor did not cause a rapid shift to methanogenesis. Methane production and a substantial increase in the levels of methanogens were observed only after approximately 50 days following the perturbation.     

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.     

Spatial Patterns of Carbonate Biomineralization in Biofilms

Microbially catalyzed precipitation of carbonate minerals is an important process in diverse biological, geological, and engineered systems. However, the processes that regulate carbonate biomineralization and their impacts on biofilms are largely unexplored, mainly because of the inability of current methods to directly observe biomineralization within biofilms. Here, we present a method for in situ, real-time imaging of biomineralization in biofilms and use it to show that Pseudomonas aeruginosa biofilms produce morphologically distinct carbonate deposits that substantially modify biofilm structures. The patterns of carbonate biomineralization produced in situ were substantially different from those caused by accumulation of particles produced by abiotic precipitation. Contrary to the common expectation that mineral precipitation should occur at the biofilm surface, we found that biomineralization started at the base of the biofilm. The carbonate deposits grew over time, detaching biofilm-resident cells and deforming the biofilm morphology. These findings indicate that biomineralization is a general regulator of biofilm architecture and properties.     

Microbial Community Analysis of Fresh and Old Microbial Biofilms on Bayon Temple Sandstone of Angkor Thom, Cambodia

The temples of Angkor monuments including Angkor Thom and Bayon in Cambodia and surrounding countries were exclusively constructed using sandstone. They are severely threatened by biodeterioration caused by active growth of different microorganisms on the sandstone surfaces, but knowledge on the microbial community and composition of the biofilms on the sandstone is not available from this region. This study investigated the microbial community diversity by examining the fresh and old biofilms of the biodeteriorated bas-relief wall surfaces of the Bayon Temple by analysis of 16S and 18S rRNA gene sequences. The results showed that the retrieved sequences were clustered in 11 bacterial, 11 eukaryotic and two archaeal divisions with disparate communities (Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria; Alveolata, Fungi, Metazoa, Viridiplantae; Crenarchaeote, and Euyarchaeota). A comparison of the microbial communities between the fresh and old biofilms revealed that the bacterial community of old biofilm was very similar to the newly formed fresh biofilm in terms of bacterial composition, but the eukaryotic communities were distinctly different between these two. This information has important implications for understanding the formation process and development of the microbial diversity on the sandstone surfaces, and furthermore to the relationship between the extent of biodeterioration and succession of microbial communities on sandstone in tropic region.     

Mapping the Centimeter-Scale Spatial Variability of PAHs and Microbial Populations in the Rhizosphere of Two Plants

Rhizoremediation uses root development and exudation to favor microbial activity. Thus it can enhance polycyclic aromatic hydrocarbon (PAH) biodegradation in contaminated soils. Spatial heterogeneity of rhizosphere processes, mainly linked to the root development stage and to the plant species, could explain the contrasted rhizoremediation efficiency levels reported in the literature. Aim of the present study was to test if spatial variability in the whole plant rhizosphere, explored at the centimetre-scale, would influence the abundance of microorganisms (bacteria and fungi), and the abundance and activity of PAH-degrading bacteria, leading to spatial variability in PAH concentrations. Two contrasted rhizospheres were compared after 37 days of alfalfa or ryegrass growth in independent rhizotron devices. Almost all spiked PAHs were degraded, and the density of the PAH-degrading bacterial populations increased in both rhizospheres during the incubation period. Mapping of multiparametric data through geostatistical estimation (kriging) revealed that although root biomass was spatially structured, PAH distribution was not. However a greater variability of the PAH content was observed in the rhizosphere of alfalfa. Yet, in the ryegrass-planted rhizotron, the Gram-positive PAH-degraders followed a reverse depth gradient to root biomass, but were positively correlated to the soil pH and carbohydrate concentrations. The two rhizospheres structured the microbial community differently: a fungus-to-bacterium depth gradient similar to the root biomass gradient only formed in the alfalfa rhizotron.     

Molecular Identification and Biofilm-Forming Ability of Culturable Aquatic Bacteria In Microbial Biofilms Formed in Drinking Water Distribution Networks

Drinking water distribution networks are known to harbor microbial biofilms. The aim of the present work is to (i) identify the culturable bacteria presented in the drinking-water distribution network, (ii) investigate the ability of isolated bacteria to form biofilm under some environmental stress conditions and some eliminating or removing treatments. To achieve it, 57 strains were isolated from biofilm (43 isolates) and water samples (14 isolates) collected from five stations in drinking-water distribution network in Taif city, Kingdom of Saudi Arabia (KSA). Partial sequences of 16S rRNA gene in the 57 isolates ensured the presence of only 22 different strains in biofilm samples. Among these strains, only 14 strains were also detected in water samples. Gram-negative Aeromonas hydrophila was the most occurred bacterium in the microbial biofilm obtained from the purified-water storage tanks followed by Gram-negative Pseudomonas sp. Gram-positive Bacillus subtilis was the most occurred bacterium in the microbial biofilm collected from the ends of the distribution pipes. Among the 22 isolated strains, 13 strains were strong biofilm producers at 30 and 37°C. The effects of environmental stresses including nutrient starvation (diluted TSB, 20:1), heating (100°C for 10 min), UV-treatment (240 nm for 10 min) and dynamic incubation (150 rpm min^?1) on the formation of biofilm were also investigated. These conditions affected the biofilm formation ability of the isolated strains at different levels. Nutrient starvation enhanced biofilm formation by most of the isolates. Among some biofilm deforming treatments, SDS and trypsin had considerable effects on preventing biofilm formation by most of the isolated strains. In conclusion, the results of the present work indicated that not all biofilm strains released from biofilm to the drinking water. Also, not all biofilm strains were able to form biofilm. Most of isolated bacteria had ability to form biofilm at suboptimum temperature of growth. These results may provide basic information on formation of microbial biofilms and overcome the problem of deteriorating of water quality in the drinking-water distribution networks.