Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.     

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_T1038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge.     

Site-specific variation in Antarctic marine biofilms established on artificial surfaces

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.     

Sulphide oxidation to elemental sulphur in a membrane bioreactor: performance and characterization of the selected microbial sulphur-oxidizing community

In leather tanning industrial areas sulphide management represents a major problem. However, biological sulphide oxidation to sulphur represents a convenient solution to this problem. Elemental sulphur is easy to separate and the process is highly efficient in terms of energy consumption and effluent quality. As the oxidation process is performed by specialized bacteria, selection of an appropriate microbial community is fundamental for obtaining a good yield. Sulphur oxidizing bacteria (SOB) represent a wide-ranging and highly diversified group of microorganisms with the capability of oxidizing reduced sulphur compounds. Therefore, it is useful to select new microbes that are able to perform this process efficiently. For this purpose, an experimental membrane bioreactor for sulphide oxidation was set up, and the selected microbial community was characterized by constructing 16S rRNA gene libraries and subsequent screening of clones. Fluorescence in situ hybridization (FISH) was then used to assess the relative abundance of different bacterial groups. Sulphide oxidation to elemental sulphur proceeded in an efficient (up to 79% conversion) and stable way in the bioreactor. Both analysis of clone libraries and FISH experiments revealed that the dominant operational taxonomic unit (OTU) in the bioreactor was constituted by Gammaproteobacteria belonging to the Halothiobacillaceae family. FISH performed with the specifically designed probe tios_434 demonstrated that this OTU constituted 90.6+/-1.3% of the bacterial community. Smaller fractions were represented by bacteria belonging to the classes Betaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Clostridia, Mollicutes, Sphingobacteria, Bacteroidetes and Chlorobia. Phylogenetic analysis revealed that clone sequences from the dominant OTU formed a stable clade (here called the TIOS44 cluster), within the Halothiobacillaceae family, with sequences from many organisms that have not yet been validly described. The data indicated that bacteria belonging to the TIOS44 cluster were responsible for the oxidation process.     

Gut microbiome of the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus)

The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current 'critically endangered' status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈ throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.     

Molecular- and cultivation-based analyses of microbial communities in oil field water and in microcosms amended with nitrate to control H2S production

Nitrate injection into oil fields is an alternative to biocide addition for controlling sulfide production (‘souring’) caused by sulfate-reducing bacteria (SRB). This study examined the suitability of several cultivation-dependent and cultivation-independent methods to assess potential microbial activities (sulfidogenesis and nitrate reduction) and the impact of nitrate amendment on oil field microbiota. Microcosms containing produced waters from two Western Canadian oil fields exhibited sulfidogenesis that was inhibited by nitrate amendment. Most probable number (MPN) and fluorescent in situ hybridization (FISH) analyses of uncultivated produced waters showed low cell numbers (≤10³ MPN/ml) dominated by SRB (>95% relative abundance). MPN analysis also detected nitrate-reducing sulfide-oxidizing bacteria (NRSOB) and heterotrophic nitrate-reducing bacteria (HNRB) at numbers too low to be detected by FISH or denaturing gradient gel electrophoresis (DGGE). In microcosms containing produced water fortified with sulfate, near-stoichiometric concentrations of sulfide were produced. FISH analyses of the microcosms after 55 days of incubation revealed that Gammaproteobacteria increased from undetectable levels to 5–20% abundance, resulting in a decreased proportion of Deltaproteobacteria (50–60% abundance). DGGE analysis confirmed the presence of Delta- and Gammaproteobacteria and also detected Bacteroidetes. When sulfate-fortified produced waters were amended with nitrate, sulfidogenesis was inhibited and Deltaproteobacteria decreased to levels undetectable by FISH, with a concomitant increase in Gammaproteobacteria from below detection to 50–60% abundance. DGGE analysis of these microcosms yielded sequences of Gamma- and Epsilonproteobacteria related to presumptive HNRB and NRSOB (Halomonas, Marinobacterium, Marinobacter, Pseudomonas and Arcobacter), thus supporting chemical data indicating that nitrate-reducing bacteria out-compete SRB when nitrate is added.     

Characterization of bacterial communities associated with deep-sea corals on Gulf of Alaska seamounts

Although microbes associated with shallow-water corals have been reported, deepwater coral microbes are poorly characterized. A cultivation-independent analysis of Alaskan seamount octocoral microflora showed that Proteobacteria (classes Alphaproteobacteria and Gammaproteobacteria), Firmicutes, Bacteroidetes, and Acidobacteria dominate and vary in abundance. More sampling is needed to understand the basis and significance of this variation.     

Microbial composition of biofilms in a brewery investigated by fatty acid analysis,fluorescence in situ hybridisation and isolation techniques

Biofilms associated with brewery plants can harbour spoiling microorganisms that potentially damage the final product. Most beer-spoiling microorganisms are thought to depend on numerous interactions with the accompanying microbiota. However, there is no information on the microbial community structure of biofilms from bottling plants. The conveyors that transport the bottles to and from the plant are known as potential sources of microbial contamination of beer. Consequently, the material buildup from two conveyors was analysed using a cultivation/isolation approach, and the culture-independent techniques of whole cell fatty acid analysis and fluorescence in situ hybridisation (FISH). Heterogeneous communities were present at both conveyors. Although characteristic fatty acids for Eukarya were present, FISH-signals for Eukarya were extremely low. The Proteobacteria, in particular the Gammaproteobacteria, were abundant at both sample sites. Bacterial isolates were obtained for every dominating group detected by FISH: the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, the Xanthomonadaceae, the Actinobacteria, the Bacteroidetes and the Firmicutes.     

Attached bacterial populations shared by four species of aquatic angiosperms

Symbiotic relationships between microbes and plants are common and well studied in terrestrial ecosystems, but little is known about such relationships in aquatic environments. We compared the phylogenetic diversities of leaf- and root-attached bacteria from four species of aquatic angiosperms using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of PCR-amplified 16S rRNA genes. Plants were collected from three beds in Chesapeake Bay at sites characterized as freshwater (Vallisneria americana), brackish (Potomogeton perfoliatus and Stuckenia pectinata), and marine (Zostera marina). DGGE analyses showed that bacterial communities were very similar for replicate samples of leaves from canopy-forming plants S. pectinata and P. perfoliatus and less similar for replicate samples of leaves from meadow-forming plants Z. marina and V. americana and of roots of all species. In contrast, bacterial communities differed greatly among plant species and between leaves and roots. DNA sequencing identified 154 bacterial phylotypes, most of which were restricted to single plant species. However, 12 phylotypes were found on more than one plant species, and several of these phylotypes were abundant in clone libraries and represented the darkest bands in DGGE banding patterns. Root-attached phylotypes included relatives of sulfur-oxidizing Gammaproteobacteria and sulfate-reducing Deltaproteobacteria. Leaf-attached phylotypes included relatives of polymer-degrading Bacteroidetes and phototrophic Alphaproteobacteria. Also, leaves and roots of three plant species hosted relatives of methylotrophic Betaproteobacteria belonging to the family Methylophilaceae. These results suggest that aquatic angiosperms host specialized communities of bacteria on their surfaces, including several broadly distributed and potentially mutualistic bacterial populations.     

非培养生物方法在堆肥微生物生态中的应用展望

微生物是堆肥化处理研究中被重点关注的因素,但大部分堆肥微生物都处于存活不可培养状态,使得了解堆肥过程中微生物实际群落结构比较困难。非培养生物方法的快速发展,为解决这一难题提供了技术依据。分别就生物化学,分子生物学,生理学中不依赖于传统培养技术的代表方法PLFA谱图分析法、FISH技术、Biolog微量板分析法进行了介绍,综述了3类方法的组合应用以及在堆肥微生物生态中的应用展望。     

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.     

Phylogeny and in situ identification of a novel gammaproteobacterium in activated sludge

Failure of a continuously aerated sequencing batch reactor (SBR) pilot plant-enhanced biological phosphorus removal (EBPR) process, designed to remove phosphorus from the clarified effluent from a conventional non-EBPR wastewater treatment plant, was associated with the dominance ( c . 50% of the biovolume) of gammaproteobacterial coccobacilli. Flow cytometry and subsequent clone library generation from an enriched population of these Gammaproteobacteria showed that their 16S rRNA genes were most similar to partial clone sequences obtained from an actively denitrifying SBR community, and from anaerobic : aerobic EBPR communities. Under the SBR operating conditions used here, these cells stained for poly-β-hydroxyalkanoates, but never polyphosphate. Applying FISH probes designed against them in combination with microautoradiography showed that they could also assimilate acetate 'aerobically'. FISH analyses of biomass samples from the full-scale treatment plant providing the pilot plant feed showed that they were present there in high numbers. However, they were not detected by FISH in laboratory-scale communities of the same aerated laboratory-scale EBPR process even when EBPR had failed, or from several full-scale EBPR plants or other activated sludge processes.     

河口细菌群落多样性及其控制因素:以切萨皮克湾为例

咸淡水的混合和重要营养盐与有机物的再循环,使得河口成为地球上生产力较高而动态变化明显的水生生态系统.一个典型的河口区断面中,细菌群落包含了一些从淡水到海洋的过渡类型:例如α-变形菌(Alphaproteobacteria)、p-变形菌(Betaproteobacteria)、γ-变形菌(Gammaproteobacteria)、蓝细菌(Cyanobactenia)[聚球藻(Synechococcus)]、拟杆菌(Bacteroidetes)、放线细菌(Actinobacteria)和疣微菌(Verrucomicrobia)等.此外,河口也包含其独特的细菌群落:SAR11组、玫瑰杆菌属(Roseobacter)、SAR86和放线细菌(Actinobacteria)的一些进化亚枝(subclades),表明海湾或者大型温带河口区细菌类群具有区域生态适应性.以研究较多的美国切萨皮克湾(Chesapeake Bay)为例,其细菌群落呈现出显著的季节性变化和周期性的年际变化特征;这些变化除了受水的滞留时间和细菌生长速度影响外,还可能受其他许多环境因子的影响.其中叶绿素a和水温变化的影响最大,其他环境因子如溶解氧、铵态氮、亚硝酸盐和硝酸盐以及病毒的丰度也有影响.近年来,基于群落水平的基因组学(genomics)和后基因组学(postgenomics)(转录组学和蛋白质组学)技术应用于研究自然条件下微生物群落错综复杂的基因多样性和表达,提供了揭示水环境中微生物群落组成和新功能基因的途径.     

A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge

Bathymodiolus azoricus and Bathymodiolus puteoserpentis are symbiont-bearing mussels that dominate hydrothermal vent sites along the northern Mid-Atlantic Ridge (MAR). Both species live in symbiosis with two physiologically and phylogenetically distinct Gammaproteobacteria: a sulfur-oxidizing chemoautotroph and a methane-oxidizer. A detailed analysis of mussels collected from four MAR vent sites (Menez Gwen, Lucky Strike, Rainbow, and Logatchev) using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH) showed that the two mussel species share highly similar to identical symbiont phylotypes. FISH observations of symbiont distribution and relative abundances showed no obvious differences between the two host species. In contrast, distinct differences in relative symbiont abundances were observed between mussels from different sites, indicating that vent chemistry may influence the relative abundance of thiotrophs and methanotrophs in these dual symbioses.     

Fate of heterotrophic microbes in pelagic habitats: focus on populations.

Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a"Gleasonian" perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed.     

Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations

Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a “Gleasonian” perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed.     

Ultrastructural and molecular characterization of a bacterial symbiosis in the ecologically important scale insect family Coelostomidiidae

Scale insects are important ecologically and as agricultural pests. The majority of scale insect taxa feed exclusively on plant phloem sap, which is carbon rich but deficient in essential amino acids. This suggests that, as seen in the related aphids and psyllids, scale insect nutrition might also depend upon bacterial symbionts, yet very little is known about scale insect-bacteria symbioses. We report here the first identification and molecular characterization of symbiotic bacteria associated with the New Zealand giant scale Coelostomidia wairoensis, using fluorescence in situ hybridization (FISH), transmission electron microscopy (TEM) and 16S rRNA gene-based analysis. Dissection and FISH confirmed the location of the bacteria in large, paired, multilobate organs in the abdominal region of the insect. TEM indicated that the dominant pleomorphic bacteria were confined to bacteriocytes in the sheath-enclosed bacteriome. Phylogenetic analysis revealed the presence of three distinct bacterial types, the bacteriome-associated B-symbiont (Bacteroidetes), an Erwinia-related symbiont (Gammaproteobacteria) and Wolbachia sp. (Alphaproteobacteria). This study extends the current knowledge of scale insect symbionts and is the first microbiological investigation of the ecologically important coelostomidiid scales.     

Bacterial Community Analyses of Two Red Sea Sponges

Red Sea sponges offer potential as sources of novel drugs and bioactive compounds. Sponges harbor diverse and abundant prokaryotic communities. The diversity of Egyptian sponge-associated bacterial communities has not yet been explored. Our study is the first culture-based and culture-independent investigation of the total bacterial assemblages associated with two Red Sea Demosponges, Hyrtios erectus and Amphimedon sp. Denaturing gradient gel electrophoresis fingerprint-based analysis revealed statistically different banding patterns of the bacterial communities of the studied sponges with H. erectus having the greater diversity. 16S rRNA clone libraries of both sponges revealed diverse and complex bacterial assemblages represented by ten phyla for H. erectus and five phyla for Amphimedon sp. The bacterial community associated with H. erectus was dominated by Deltaproteobacteria. Clones affiliated with Gammaproteobacteria were the major component of the clone library of Amphimedon sp. About a third of the 16S rRNA gene sequences in these communities were derived from bacteria that are novel at least at the species level. Although the overall bacterial communities were significantly different, some bacterial groups, including members of Alphaproteobacteria, Gammaproteobacteria, Acidobacteria, and Actinobacteria, were found in both sponge species. The culture-based component of this study targeted Actinobacteria and resulted in the isolation of 35 sponge-associated microbes. The current study lays the groundwork for future studies of the role of these diverse microbes in the ecology, evolution, and development of marine sponges. In addition, our work provides an excellent resource of several candidate bacteria for production of novel pharmaceutically important compounds.     

Diversity of the Bacterial Communities Associated with the Azooxanthellate Deep Water Octocorals <Emphasis Type="Italic">Leptogorgia minimata</Emphasis>, <Emphasis Type="Italic">Iciligorgia schrammi</Emphasis>, and <Emphasis Type="Italic">Swiftia exertia</Emphasis>

This study examined the microbiota associated with the marine azooxanthellate octocorals Leptogorgia minimata, Swiftia exertia, and Iciligorgia schrammi collected from moderate depths (45 m). Traditional aerobic plate culture, fluorescence in situ hybridization (FISH), and molecular identification of the 16S rDNA region were used for this purpose. In general, cultures were found to be selective for Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Interestingly, FISH counts for Firmicutes in the whole coral (holobiont) were near the detection limit of this assay, representing less than 6% of the total detectable microbiota in all counts. Proteobacteria, especially Alpha- and Gammaproteobacteria, made up the majority of the total microbiota in the holobionts. In addition, the absence of zooxanthellae in these three corals was confirmed by the use of polymerase chain reaction (PCR) and dinoflagellate-specific primers, and spectrophotometric chlorophyll pigment measurements. No evidence of zooxanthellae could be found in any of the corals by either of these techniques. This is the first study examining the microbiota marine octocorals, which grow at moderate depth (40 to 100 m) in the absence of direct sunlight. Thomas B. Brück and Wolfram M. Brück have made equal contributions to this publication.