Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway

The viable and non-viable fractions of the bacterial community in a 2347-year-old permafrost soil from Spitsbergen were subjected to a comprehensive investigation using culture-independent and culture-dependent methods. LIVE/DEAD BacLight staining revealed that 26% of the total number of bacterial cells were viable. Quantitatively, aerobic microcolonies, aerobic colony-forming units and culturable anaerobic bacteria comprised a minor fraction of the total number of viable bacteria, which underlines the necessity for alternative cultivation approaches in bacterial cryobiology. Sulfate reduction was detected at temperatures between -2 degrees C and 29 degrees C while methanogenesis was not detected. Bacterial diversity was high with 162 operational taxonomic units observed from 800 16S rDNA clone sequences. The 158 pure cultures isolated from the permafrost soil affiliated with 29 different bacterial genera, the majority of which have not previously been isolated from permafrost habitats. Most of the strains isolated were affiliated to the genera Cellulomonas and Arthrobacter and several of the pure cultures were closely related to bacteria reported from other cryohabitats. Characterization of viable bacterial communities in permafrost soils is important as it will enable identification of functionally important groups together with the as yet undescribed adaptations that bacteria have evolved for surviving subzero temperatures for millennia.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

Gut-Associated Bacteria Throughout the Life Cycle of the Bark Beetle Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) and Their Cellulolytic Activities

Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) is an endemic economically important insect of the Sierra Madre Occidental in Mexico. This bark beetle has an atypical behavior within the genus because just one beetle couple colonizes and kills seedlings and young trees of 11 pine species. In this work, the bacteria associated with the Dendroctonus rhizophagus gut were analyzed by culture-dependent and culture-independent methods. Analysis of 16S rRNA sequences amplified directly from isolates of gut bacteria suggests that the bacterial community associated with Dendroctonus rhizophagus, like that of other Dendroctonus spp. and Ips pini, is limited in number. Nine bacterial genera of γ-Proteobacteria and Actinobacteria classes were detected in the gut of Dendroctonus rhizophagus. Stenotrophomonas and Rahnella genera were the most frequently found bacteria from Dendroctonus rhizophagus gut throughout their life cycle. Stenotrophomonas maltophilia, Ponticoccus gilvus, and Kocuria marina showed cellulolytic activity in vitro. Stenotrophomonas maltophilia, Rahnella aquatilis, Raoultella terrigena, Ponticoccus gilvus, and Kocuria marina associated with larvae or adults of Dendroctonus rhizophagus could be implicated in nitrogen fixation and cellulose breakdown, important roles associated to insect development and fitness, especially under the particularly difficult life conditions of this beetle.     

传统分离培养结合DGGE法检测榨菜腌制过程的细菌多样性

采用传统分离培养和基于16S rRNA 作为分子标记的变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)的方法, 分析榨菜腌制过程中不同时期的可培养细菌数量、多样性及其群落结构。结果表明, 用传统分离与分子鉴定方法获得7个属的细菌类群, 其中乳杆菌属(Acidobacterium)是优势菌群, 明串珠菌属(Leuconostoc)是次优势菌群。对通过DGGE方法得到的11条16S rRNA优势条带序列进行了比对, 结果表明明串珠菌属(Leucon     

Deciphering the role of <Emphasis Type="Italic">Paenibacillus</Emphasis> strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès

Background  

The recycling of the organic matter is a crucial function in any environment, especially in oligotrophic environments such as Acid Mine Drainages (AMDs). Polymer-degrading bacteria might play an important role in such ecosystem, at least by releasing by-products useful for the rest of the community. In this study, physiological, molecular and biochemical experiments were performed to decipher the role of a Paenibacillus strain isolated from the sediment of Carnoulès AMD.     

In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator,Euglena mutabilis

Euglena mutabilis is a photosynthetic protist found in acidic aquatic environments such as peat bogs, volcanic lakes and acid mine drainages (AMDs). Through its photosynthetic metabolism, this protist is supposed to have an important role in primary production in such oligotrophic ecosystems. Nevertheless, the exact contribution of E. mutabilis in organic matter synthesis remains unclear and no evidence of metabolite secretion by this protist has been established so far. Here we combined in situ proteo-metabolomic approaches to determine the nature of the metabolites accumulated by this protist or potentially secreted into an AMD. Our results revealed that the secreted metabolites are represented by a large number of amino acids, polyamine compounds, urea and some sugars but no fatty acids, suggesting a selective organic matter contribution in this ecosystem. Such a production may have a crucial impact on the bacterial community present on the study site, as it has been suggested previously that prokaryotes transport and recycle in situ most of the metabolites secreted by E. mutabilis. Consequently, this protist may have an indirect but important role in AMD ecosystems but also in other ecological niches often described as nitrogen-limited.     

包头泉山金矿浸矿酸性微生物群落优势度变化的比较研究

【目的】研究不同矿石组成对微生物群落结构的影响。【方法】选取包头泉山金矿的酸性矿坑水(Acid mine drainage,AMD)对4种不同组成的矿石样品进行浸矿,采用16S rRNA-PCR和RFLP相结合的方法,研究浸矿前后浸矿微生物种群优势度的变化情况。【结果】所选取的3个酸性矿坑水考查点之间浸矿微生物的种类及数量相似度较大,浸矿微生物结构相对变化度不大。H71和F11相对其他两个样品的群落结构差异较大,而J72和S71几乎一样,说明在钾长型和石英型矿石的选择压力下,浸矿微生物的被选择趋势是大致相同的。【结论】浸矿前后样品中浸矿微生物的系统发育分析结果表明,所考查的克隆子的序列可分为两大分支:Acidithiobacillus菌属和一个相对独立的菌属,且这两个分支内部菌株之间的发育距离较近。     

Microbial diversity in tropical marine sediments assessed using culture-dependent and culture-independent techniques

The microbial communities associated with marine sediments are critical for ecosystem function yet remain poorly characterized. While culture-independent (CI) techniques capture the broadest perspective on community composition, culture-dependent (CD) methods can select for low abundance taxa that are missed using CI approaches. This study aimed to assess microbial diversity in tropical marine sediments at five shallow-water sites in Belize using both CD and CI techniques. The CD methods captured approximately 3% of the >800 genera detected across all sites using the CI approach. Additionally, 39 genera were only detected in culture, revealing rare taxa that were missed with the CI approach. Significantly different communities were detected across sites, with rare taxa playing an important role in distinguishing among communities. This study provides important baseline data describing shallow-water sediment microbial communities, evidence that standard cultivation techniques may be more effective than previously recognized, and the first steps towards identifying new taxa that are amenable to agar plate cultivation.     

Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Microbial community dynamics and effect of environmental microbial reservoirs on red-backed salamanders (Plethodon cinereus)

Beneficial cutaneous bacteria on amphibians can protect against the lethal disease chytridiomycosis, which has devastated many amphibian species and is caused by the fungus Batrachochytrium dendrobatidis. We describe the diversity of bacteria on red-backed salamanders (Plethodon cinereus) in the wild and the stability of these communities through time in captivity using culture-independent Illumina 16S rRNA gene sequencing. After field sampling, salamanders were housed with soil from the field or sterile media. The captive conditions led to different trajectories of bacterial communities. Eight OTUs present on >90% of salamanders in the field, through time, and in both treatments were defined as the core community, suggesting that some bacteria are closely associated with the host and are independent of an environmental reservoir. One of these taxa, a Pseudomonas sp., was previously cultured from amphibians and found to be antifungal. As all host-associated bacteria were found in the soil reservoir, environmental microbes strongly influence host–microbial diversity and likely regulate the core community. Using PICRUSt, an exploratory bioinformatics tool to predict gene functions, we found that core skin bacteria provided similar gene functions to the entire community. We suggest that future experiments focus on testing whether core bacteria on salamander skin contribute to the observed resistance to chytridiomycosis in this species even under hygenic captive conditions. For disease-susceptible hosts, providing an environmental reservoir with defensive bacteria in captive-rearing programs may improve outcomes by increasing bacterial diversity on threatened amphibians or increasing the likelihood that defensive bacteria are available for colonization.     

Microbial diversity in bighorn sheep revealed by culture-independent methods

We investigated the effectiveness of culture-independent molecular methods for determining host-associated microbial diversity in bighorn sheep (Ovis canadensis). Results from bacterial culture attempts have been the primary source of information on host-associated bacteria, but studies have shown that culture-based results significantly underestimate bacterial diversity in biological samples. To test the effectiveness of culture-independent methods, we extracted DNA from nasal and oropharyngeal swab samples collected from bighorn sheep in four different populations. From these samples, we amplified, cloned, and sequenced small subunit (16S) ribosomal DNA (rDNA) to identify the scope of microbial diversity in bighorn respiratory tracts. Phylogenetic analysis of these rDNA gene sequences revealed organismal diversity an order of magnitude higher than was determined by culture methods. Pasteurellaceae bacteria were the most diverse phylogenetic group in live bighorn sheep, and members of bacterial genera often associated with respiratory disease were found in all the samples. Culture-independent methods were also able to directly detect leukotoxin (lktA) gene sequences in swab and lung tissue samples. Overall, our results show the power of culture-independent molecular methods for identifying microbial diversity in bighorn sheep and the potential for these methods to detect the presence of virulence genes in biological samples.     

Culture-dependent and culture-independent diversity surveys target different bacteria: a case study in a freshwater sample

Compared with culture-independent approaches, traditionally used culture-dependent methods have a limited capacity to characterize water microbiota. Nevertheless, for almost a century the latter have been optimized to detect and quantify relevant bacteria. A pertinent question is if culture-independent diversity surveys give merely an extended perspective of the bacterial diversity or if, even with a higher coverage, focus on a different set of organisms. We compared the diversity and phylogeny of bacteria in a freshwater sample recovered by currently used culture-dependent and culture-independent methods (DGGE and 454 pyrosequencing). The culture-dependent diversity survey presented lower coverage than the other methods. However, it allowed bacterial identifications to the species level, in contrast with the other procedures that rarely produced identifications below the order. Although the predominant bacterial phyla detected by both approaches were the same (Proteobacteria, Actinobacteria, Bacteroidetes), sequence similarity analysis showed that, in general, different operational taxonomical units were targeted by each method. The observation that culture-dependent and independent approaches target different organisms has implications for the use of the latter for studies in which taxonomic identification has a predictive value. In comparison to DGGE, 454 pyrosequencing method had a higher capacity to explore the bacterial richness and to detect cultured organisms, being also less laborious.     

Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential

The Cerrado is the second largest Brazilian biome, yet little is known about its wild fauna, flora and microbiota. This work aimed to identify epiphytic bacteria present in fruits native to three different regions of the Cerrado and to select cellulase-producing bacteria. Culture-dependent and culture-independent (PCR-DGGE) methods were used to characterize the microbiota from 32 native Cerrado fruits, and the selection of cellulase-producing bacteria was performed by a semi-quantitative test on carboxymethylcellulose agar medium. Analysis of the 16S rRNA gene sequences of 69 profile representatives showed that the isolates belonged to 29 bacterial genera (Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Streptomyces, Enterobacter, Microbacterium, Aerococcus, Bradyrhizobium, Methylobacterium, Erwinia, Pantoea, Acidithiobacillus, Ochrobactrum, Stenotrophomonas, Curtobacterium, Clostridium, Lactobacillus, Xanthomonas, Delftia, Klebsiella, Enterococcus, Burkholderia, Escherichia, Streptococcus, Citrobacter and Achromobacter). Species in the genera Methylobacterium, Stenotrophomonas, Clostridium, Pantoea and Enterobacter were detected by both culture-dependent and culture-independent methods. The species Lactobacillus fermentum, Acinetobacter sp. and Methylomonas methanica were detected only by PCR-DGGE. Additionally, 30 % (178 isolates) of the bacteria tested were able to produce cellulase. The best producers belonged to the genera Bacillus, Streptomyces, Paenibacillus, Enterobacter and Burkholderia, indicating that this ecosystem could be an attractive source for the study of novel enzymes.     

Litter and soil biodiversity jointly drive ecosystem functions

The decomposition of litter and the supply of nutrients into and from the soil are two fundamental processes through which the above- and belowground world interact. Microbial biodiversity, and especially that of decomposers, plays a key role in these processes by helping litter decomposition. Yet the relative contribution of litter diversity and soil biodiversity in supporting multiple ecosystem services remains virtually unknown. Here we conducted a mesocosm experiment where leaf litter and soil biodiversity were manipulated to investigate their influence on plant productivity, litter decomposition, soil respiration, and enzymatic activity in the littersphere. We showed that both leaf litter diversity and soil microbial diversity (richness and community composition) independently contributed to explain multiple ecosystem functions. Fungal saprobes community composition was especially important for supporting ecosystem multifunctionality (EMF), plant production, litter decomposition, and activity of soil phosphatase when compared with bacteria or other fungal functional groups and litter species richness. Moreover, leaf litter diversity and soil microbial diversity exerted previously undescribed and significantly interactive effects on EMF and multiple individual ecosystem functions, such as litter decomposition and plant production. Together, our work provides experimental evidence supporting the independent and interactive roles of litter and belowground soil biodiversity to maintain ecosystem functions and multiple services.     

植物叶际固氮菌研究进展

固氮菌广泛存在于植物叶际,能固定空气中的氮气,满足自身或植物的部分氮素需求。基于纯种分离和培养的传统生物学方法已经研究了部分叶际固氮菌的特性,但对叶际固氮菌的物种组成、群落结构及生态功能等方面的认识还非常有限。随着分子生物学技术的发展、微生物分子生态学研究方法的逐步成熟,人们对叶际微生物的多样性和生态功能的研究越来越深入。叶际固氮菌具有丰富的多样性,温度、湿度、光照等环境因素及植物种类和微生物互作均会影响叶际固氮菌的组成。不同于根际固氮菌,叶际固氮菌具有非专一性,且不易受化肥的影响,其在农业生产上已经表现出潜在的应用价值。为此,本文综述了农作物、森林、海域生态系统中叶际固氮菌的群落结构组成及生态功能,以及外界因素对叶际固氮系统的影响。     

Differences in Bacterial Community Structure in Two Color Morphs of the Hawaiian Reef Coral Montipora capitata

Corals harbor diverse bacterial associations that contribute to the health of the host. Using 16S rRNA pyrosequencing, we compared the bacterial communities of red and orange morphs of the Hawaiian coral Montipora capitata. Although both color morphs shared dominant bacterial genera, weighted and unweighted UniFrac analyses showed distinct bacterial communities. A single operational taxonomic unit (OTU), classified as Vibrio, represented the largest driver of differences between the color morphs. This OTU comprised 35.4% (±5.5%) of the orange morph bacterial community yet comprised 1.1% (±0.6%) of the red morph bacterial community. Cultivable bacteria from the two color morphs were also compared and tested for antibacterial activity. Cultured isolates represented 14 genera (7% of the total genera identified from sequencing data), and all but two cultured isolates had a matching OTU from the sequencing data. Half of the isolates tested (8 out of 16) displayed antibacterial activity against other cultured isolates but not against two known bacterial pathogens of M. capitata. The results from this study demonstrate that the specificity of coral-bacterial associations extends beyond the level of coral species. In addition, culture-dependent methods captured bacterial diversity that was representative of both rare and abundant members of the associated bacterial community, as characterized by culture-independent methods.     

Characterization of Epiphytic Bacterial Communities from Grapes,Leaves, Bark and Soil of Grapevine Plants Grown,and Their Relations

Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plant parts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, Curtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by T-RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.     

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.     

Diversity and structure of bacterial communities in Fildes Peninsula, King George Island

To examine the bacterial community structure in the Fildes Peninsula, King George Island, Antarctica, we examined the bacterial diversity and community composition of samples collected from lacustrine sediment, marine sediment, penguin ornithogenic sediments, and soils using culture-dependent and culture-independent methods. The 70 strains fell into five groups: Actinobacteria, Bacteroidetes, Firmicutes, Gammaproteobacteria, and Betaproteobacteria. Bacterial diversity at the phylum level detected in Denaturing Gradient Gel Electrophoresis (DGGE) profiles comprised Proteobacteria (including the subphyla Alpha-, Beta-, Gamma-, Deltaproteobacteria), Bacteroidetes, Firmicutes, Chlorobi, and Deinococcus-Thermus. Gammaproteobacteria was identified to be the dominant bacterial subphylum by cultivation and DGGE method. By cluster analysis, the overall structure and composition of bacterial communities in the soil and lacustrine sediment were similar to one another but significantly different from bacterial communities in penguin ornithogenic sediment and marine sediment, which were similar to one another. The majority of 16S rDNA sequences from cultured bacteria were closely related to sequences found in cold environments. In contrast, a minority of 16S rDNA sequences from the DGGE approach were closely related to sequences found in cold environments.