Influence of diet on the structure and function of the bacterial hindgut community of crickets

The effect of diets varying in carbohydrate and protein content on the structure and function of the hindgut microbiota of crickets was evaluated by determining bacterial densities, fermentation activity, and guanine plus cytosine (G + C) profiles of the DNA extracted from the microbial hindgut community. DNA isolated from the gut community was fractionated and quantified according to G + C content as a comprehensive, coarse-level measure of the composition and structure of the community. The bacterial densities measured by direct counts were not significantly different among the four diets. The crickets were initially reared in the laboratory on cricket chow, which resulted in a hindgut community dominated by bacteria with a G + C content between 32% and 57%. Crickets shifted to an alfalfa diet showed a similar hindgut community G + C profile, although microbial populations with DNA between 35% and 45% G + C were more abundant in alfalfa- than chow-fed crickets. The apparent complexity of the gut community was reduced in crickets fed beet-pulp and protein-based diets compared to those fed chow and alfalfa, and was dominated by populations with a low percentage G + C content. Hindgut communities in crickets fed pulp and protein diets also showed a decrease in hydrogen and carbon dioxide production, suggesting that these diets affected the biochemical activity of the hindgut community. The protein-based diet resulted in a decrease in the rate of evolution of volatile fatty acids, while the ratio of butyrate production to acetate and propionate production was significantly higher in these crickets. Our results show the emergence of a new microbial community structure concomitant with changes in microbial biochemical activity due to shifts in the cricket's dietary regime.     

Molecular analysis of deep-subsurface bacteria.

Bacterial isolates from deep-sediment samples from three sites at the Savannah River site, near Aiken, S.C., were studied to determine their microbial community composition and DNA structure by using total DNA hybridization and moles percent G + C. Standard phenotypic identification underestimated the bacterial diversity at the three sites, since isolates with the same phenotype had different DNA structures in terms of moles percent G + C and DNA homology. The G + C content of deep-subsurface bacteria ranged from 20 to 77 mol%. More than 60% of the isolates tested had G + C values similar to those of Pseudomonas spp., and 12% had values similar to those of Acinetobacter spp. No isolates from deeper formations showed the same DNA composition as isolates from upper formations. Total-DNA hybridization and DNA base composition analysis provided a better resolution than phenotypic tests for the understanding of the diversity and structure of deep-subsurface bacterial communities. On the basis of the moles percent G + C values, deep-subsurface isolates tested seemed to belong to the families Pseudomonadaceae and Neisseriaceae, which might reflect a long period of adaptation to the environmental conditions of the deep subsurface.     

Molecular analysis of deep-subsurface bacteria

Bacterial isolates from deep-sediment samples from three sites at the Savannah River site, near Aiken, S.C., were studied to determine their microbial community composition and DNA structure by using total DNA hybridization and moles percent G + C. Standard phenotypic identification underestimated the bacterial diversity at the three sites, since isolates with the same phenotype had different DNA structures in terms of moles percent G + C and DNA homology. The G + C content of deep-subsurface bacteria ranged from 20 to 77 mol%. More than 60% of the isolates tested had G + C values similar to those of Pseudomonas spp., and 12% had values similar to those of Acinetobacter spp. No isolates from deeper formations showed the same DNA composition as isolates from upper formations. Total-DNA hybridization and DNA base composition analysis provided a better resolution than phenotypic tests for the understanding of the diversity and structure of deep-subsurface bacterial communities. On the basis of the moles percent G + C values, deep-subsurface isolates tested seemed to belong to the families Pseudomonadaceae and Neisseriaceae, which might reflect a long period of adaptation to the environmental conditions of the deep subsurface.     

New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples.     

New Threshold and Confidence Estimates for Terminal Restriction Fragment Length Polymorphism Analysis of Complex Bacterial Communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples.     

Broad-scale approaches to the determination of soil microbial community structure: Application of the community DNA hybridization technique

Broad-scale approaches seek to integrate information on whole microbial communities. It is widely recognized that culture techniques are too selective and unrepresentative to allow a realistic assessment of the overall structure of microbial communities. Techniques based on fatty acid or metabolic profiles determine the phenotypic composition of the community. Complementary information about the genotypic structure of soil microbial communities necessitates analysis of community DNA. To determine broad-scale differences in soil microbial community structure (i.e., differences at the whole community level, rather than specific differences in species composition), we have applied a community hybridization technique to determine the similarity and relative diversity of two samples by cross hybridization. In previous studies this assay failed with whole-soil community DNA. Usable hybridization signals were obtained using whole-soil DNA, in this study, by digesting the DNA with restriction enzymes before the labeling with a random-primer reaction. The community hybridization technique was tested using a graded series of microbial fractions, increasing in complexity, all isolated from the same soil sample. This demonstrated that single bacterial species and a mixture of cultivable bacteria were less complex and only 5% similar to whole-community DNA or bacteria directly extracted from the soil. Extracted bacterial and whole-community DNA were 75% similar to each other and equally complex. When DNA was extracted from four different agricultural soils, their similarities ranged from 35 to 75%. The potential usefulness of community hybridization applied to soil microbial communities is discussed.     

Assessment of bacterial community structure in a long-term copper-polluted ex-vineyard soil

The influence of long-term copper contamination on the diversity of bacterial communities was investigated in an ex-vineyard soil. Two sites of the same area but exhibiting different 3-fold exchangeable copper (Ex-Cu) concentrations were analysed. Culturable bacterial community structure was assessed using a variety of approaches: determination of culturable bacteria number, analyses of 132 isolates, and denaturing gradient gel lectrophoresis (DGGE) patterns of bacterial biomass grown on agar plates and of soil DNA. There was no significant difference in the number of total heterotrophs at the two sites, whereas the percentage of fast-growing bacteria growing in 1 day, was lower at the site with the higher Ex-Cu content. A high percentage of Cu-tolerant bacteria was found in both sites (63-70%) and it was relatively independent of the Cu content. Shifts in species composition of the culturable bacterial community were detected by analysing isolates from the two soils, Gram-positive bacteria prevailed in the less-polluted soil while Gram-negative bacteria in the more-polluted soil. Each sample site had a community with a different metal resistance pattern. Our study seems to indicate that in this soil ecosystem, copper influenced the culturable bacterial communities, affecting the structural diversity and altering some of the metal resistance of the microorganisms. The Sorensen similarity index calculated on DGGE profiles of 16S rDNA of total and culturable bacterial communities indicated a different species composition at the two sites, although both sites had the same biodiversity degree and different dominance.     

Composition influences the pathway but not the outcome of the metabolic response of bacterioplankton to resource shifts

Bacterioplankton community metabolism is central to the functioning of aquatic ecosystems, and strongly reactive to changes in the environment, yet the processes underlying this response remain unclear. Here we explore the role that community composition plays in shaping the bacterial metabolic response to resource gradients that occur along aquatic ecotones in a complex watershed in Québec. Our results show that the response is mediated by complex shifts in community structure, and structural equation analysis confirmed two main pathways, one involving adjustments in the level of activity of existing phylotypes, and the other the replacement of the dominant phylotypes. These contrasting response pathways were not determined by the type or the intensity of the gradients involved, as we had hypothesized, but rather it would appear that some compositional configurations may be intrinsically more plastic than others. Our results suggest that community composition determines this overall level of community plasticity, but that composition itself may be driven by factors independent of the environmental gradients themselves, such that the response of bacterial communities to a given type of gradient may alternate between the adjustment and replacement pathways. We conclude that community composition influences the pathways of response in these bacterial communities, but not the metabolic outcome itself, which is driven by the environment, and which can be attained through multiple alternative configurations.     

Comparing bacterial community fingerprints from white colonizations in Altamira Cave (Spain)

Monitoring bacterial communities is critical for assessing biodeterioration among other processes. This study presents a strategy and an example of comparative analysis of bacterial communities developing in a cave environment, Altamira Cave which contains unique paleolithic paintings. The analyzed question was whether white colonizations discovered throughout the cave corresponded to similar or different bacterial communities. Molecular fingerprints were obtained by PCR–DGGE from DNA and RNA and statistically compared. Results based on DNA analysis showed that a similar bacterial community was present in white colonizations throughout the cave. Fingerprints based on RNA confirmed the similarity of the major metabolically active components of these communities. The proposed procedure confirmed that white colonizations in Altamira Cave were a consequence of the development of a single complex bacterial community, and the method proves to be highly useful for comparative analysis of microbial communities, including biodeteriorating processes and any other comparative analysis of bacterial communities.     

Bacterial Community Affects Toxin Production by Gymnodinium catenatum

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell⁻¹) was similar to the parent cultures (169–206 fmol STX cell⁻¹), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell⁻¹) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell⁻¹). Specific toxin production rate (fmol STX day⁻¹) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell⁻¹ day⁻¹) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.     

Community structural differences shape microbial responses to high molecular weight organic matter

The extent to which differences in microbial community structure result in variations in organic matter (OM) degradation is not well understood. Here, we tested the hypothesis that distinct marine microbial communities from North Atlantic surface and bottom waters would exhibit varying compositional succession and functional shifts in response to the same pool of complex high molecular weight (HMW-OM). We also hypothesized that microbial communities would produce a broader spectrum of enzymes upon exposure to HMW-OM, indicating a greater potential to degrade these compounds than reflected by initial enzymatic activities. Our results show that community succession in amended mesocosms was congruent with cell growth, increased bacterial production and most notably, with substantial shifts in enzymatic activities. In all amended mesocosms, closely related taxa that were initially rare became dominant at time frames during which a broader spectrum of active enzymes were detected compared to initial timepoints, indicating a similar response among different communities. However, succession on the whole-community level, and the rates, spectra and progression of enzymatic activities, reveal robust differences among distinct communities from discrete water masses. These results underscore the crucial role of rare bacterial taxa in ocean carbon cycling and the importance of bacterial community structure for HMW-OM degradation.     

Protein Extraction and Fingerprinting Optimization of Bacterial Communities in Natural Environment

Recent development in molecular approaches allows access to genetic structure and diversity of indigenous microbial communities. In contrast, the functional analysis of microorganisms in their environment is still hampered by methodological limitations. Analysis of total proteins expressed at the whole community level (metaproteome) has been proposed to characterize the functional structure of microbial communities in their environment. However, developments are still required to perform such analysis. Our aim was to optimize methods to extract and characterize metaproteome of indigenous microbial communities. Experiments were first conducted in monoxenic bacterial cultures, and various methods were examined to define a procedure of protein extraction ensuring an efficient recovery regardless of the taxonomic affiliation of the cells. These developments were next applied to characterize the metaproteome from indigenous bacterial communities in freshwater samples. Bacterial cells were recovered from water using a high-speed density gradient centrifugation method before protein extraction and fingerprinting. The reactivity and sensitivity of this metaproteomic approach were tested by analyzing the variations of protein fingerprints according to perturbations (cadmium or mercury contamination). The genetic structure of the corresponding communities was also characterized by automated ribosomal spacer analysis (ARISA) DNA fingerprinting. Both protein and DNA fingerprints were statistically analyzed. Results obtained showed that the method developed for protein recovery and fingerprinting was efficient, sensitive, and reproducible. Both the functional and genetic structures of the freshwater bacterial community were complex and varied with perturbations. These variations occurred at both population and protein expression levels and were specific to the perturbation applied.     

Temporal,regional and geochemical drivers of microbial community variation in the melt ponds of the Ross Sea region,Antarctica

To expand the understanding of the poorly described planktonic bacterial communities inhabiting Antarctic meltwater ponds, this study characterized the community composition and identified environmental drivers influencing community structure from a total of 41 meltwater ponds: 37 from the McMurdo Ice Shelf (Bratina Island) and four from a terrestrial locale (Miers Valley) during three austral summers. DNA fingerprinting coupled with in situ pH and conductivity was utilized to select ponds for in-depth nutrient and chemical analysis and high-throughput sequencing of the bacterial 16S rRNA gene V5–V6 hypervariable region. Conductivity was the strongest driver of community structure across all ponds and for all time points; however, other influential factors (pH, climatological, Hg, Fe, and PO₄) were also identified. Unique members of communities (sequences absent in at least one pond) represented a small percentage of total reads but also represented a large proportion of pond biodiversity that was strongly driven by differing environmental variables (Si, B and S). Significant temporal variation in community structure was also identified within the same ponds although major taxa remained present. Miers Valley ponds exhibit greater similarity to Bratina Island ponds rather than between each other, thereby suggesting regional movement of microorganisms. In summary, these data provide the first in-depth investigation of the intra-seasonal and regional variation of the microbial communities inhabiting these ponds and proved that a total of ten cosmopolitan OTUs were the dominant components of ponds throughout all sampling times and locations, their variable relative abundances driving the major dissimilarities in community structure.     

Comparison of DNA‐ and RNA‐based bacterial community structures in soil exposed to 2,4‐dichlorophenol

Aims: To examine the effect of the pollutant 2,4‐dichlorophenol on DNA‐ and RNA‐based bacterial communities in soil. Methods and Results: Soil was exposed to 100 mg kg^?1 of 2,4‐dichlorophenol (2,4‐DCP), and degradation was monitored over 35 days. DNA and RNA were coextracted, and terminal restriction fragment length polymorphism (T‐RFLP) was used to report changes in bacterial communities in response to the presence of the chlorophenol. The phylogenetic composition of the soil during degradation was determined by creating a clone library of amplified 16S rRNA sequences from both DNA and reverse‐transcribed RNA from exposed soil. Resulting clones were sequenced, and putative identities were assigned. Conclusions: A significant difference between active (RNA‐based) and total (DNA‐based) bacterial community structure was observed for both T‐RFLP and phylogenetic analyses in response to 2,4‐DCP, with more pronounced changes seen in RNA‐based communities. Phylogenetic analysis indicated the dominance of Proteobacteria in both profiles. Significance and Impact of the Study: This study describes the response of soil bacterial communities to the addition of the xenobiotic compound 2,4‐DCP, and highlights the importance of including RNA‐based 16S rRNA analysis to complement any molecular study in a perturbed soil.     

Impact of protozoan grazing on bacterial community structure in soil microcosms

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.     

Soil Mineral Composition Matters: Response of Microbial Communities to Phenanthrene and Plant Litter Addition in Long-Term Matured Artificial Soils

The fate of polycyclic aromatic hydrocarbons (PAHs) in soil is determined by a suite of biotic and abiotic factors, and disentangling their role in the complex soil interaction network remains challenging. Here, we investigate the influence of soil composition on the microbial community structure and its response to the spiked model PAH compound phenanthrene and plant litter. We used long-term matured artificial soils differing in type of clay mineral (illite, montmorillonite) and presence of charcoal or ferrihydrite. The soils received an identical soil microbial fraction and were incubated for more than two years with two sterile manure additions. The matured artificial soils and a natural soil were subjected to the following spiking treatments: (I) phenanthrene, (II) litter, (III) litter + phenanthrene, (IV) unspiked control. Total community DNA was extracted from soil sampled on the day of spiking, 7, 21, and 63 days after spiking. Bacterial 16S rRNA gene and fungal internal transcribed spacer amplicons were quantified by qPCR and subjected to denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed that the bacterial community composition, which was strongly shaped by clay minerals after more than two years of incubation, changed in response to spiked phenanthrene and added litter. DGGE and qPCR showed that soil composition significantly influenced the microbial response to spiking. While fungal communities responded only in presence of litter to phenanthrene spiking, the response of the bacterial communities to phenanthrene was less pronounced when litter was present. Interestingly, microbial communities in all artificial soils were more strongly affected by spiking than in the natural soil, which might indicate the importance of higher microbial diversity to compensate perturbations. This study showed the influence of soil composition on the microbiota and their response to phenanthrene and litter, which may increase our understanding of complex interactions in soils for bioremediation applications.     

Effects of plant species richness and evenness on soil microbial community diversity and function

Understanding the links between plant diversity and soil communities is critical to disentangling the mechanisms by which plant communities modulate ecosystem function. Experimental plant communities varying in species richness, evenness, and density were established using a response surface design and soil community properties including bacterial and archaeal abundance, richness, and evenness were measured. The potential to perform a representative soil ecosystem function, oxidation of ammonium to nitrite, was measured via archaeal and bacterial amoA genes. Structural equation modeling was used to explore the direct and indirect effects of the plant community on soil diversity and potential function. Plant communities influenced archaea and bacteria via different pathways. Species richness and evenness had significant direct effects on soil microbial community structure, but the mechanisms driving these effects did not include either root biomass or the pools of carbon and nitrogen available to the soil microbial community. Species richness had direct positive effects on archaeal amoA prevalence, but only indirect impacts on bacterial communities through modulation of plant evenness. Increased plant evenness increased bacterial abundance which in turn increased bacterial amoA abundance. These results suggest that plant community evenness may have a strong impact on some aspects of soil ecosystem function. We show that a more even plant community increased bacterial abundance, which then increased the potential for bacterial nitrification. A more even plant community also increased total dissolved nitrogen in the soil, which decreased the potential for archaeal nitrification. The role of plant evenness in structuring the soil community suggests mechanisms including complementarity in root exudate profiles or root foraging patterns.     

Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H'), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from beta-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

Effect of ferritin overexpression in tobacco on the structure of bacterial and pseudomonad communities associated with the roots

The genetic structures of total bacterial and pseudomonad communities were characterized in rhizosphere soil and rhizoplane+root tissues of tobacco wild type and a ferritin overexpressor transgenic line (P6) by a cultivation-independent method using directly extracted DNA at the end of three consecutive plant cultures. The structure of total bacterial communities was characterized by automated ribosomal intergenic spacer analysis (A-RISA), and that of pseudomonad communities was characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) from DNA amplified with specific primers. The structure of total bacterial communities was significantly modified in the rhizosphere soil by the overaccumulation of iron in the tobacco transgenic P6 line at the first culture, to a lesser extent at the second culture, and not at all at the third culture. No significant difference was recorded between the total communities associated with the roots (rhizoplane+root tissues) of the two plant genotypes in any of the cultures. In contrast, the difference in pseudomonad structure between the two plant genotypes increased with successive culture at the root level, but was not detected at a significant level in the rhizosphere soil. The impact of iron overaccumulation by the tobacco transgenic P6 line on pseudomonads supports previous findings on the importance of iron competition among fluorescent pseudomonads.