Search

In tropical and subtropical oceanic surface waters phosphate scarcity can limit microbial productivity. However, these environments also have bioavailable forms of phosphorus incorporated into dissolved organic matter (DOM) that microbes with the necessary transport and hydrolysis metabolic pathways can access to supplement their phosphorus requirements. In this study we evaluated how the environment shapes the abundance and taxonomic distribution of the bacterial carbon-phosphorus (C-P) lyase pathway, an enzyme complex evolved to extract phosphate from phosphonates. Phosphonates are organophosphorus compounds characterized by a highly stable C-P bond and are enriched in marine DOM. Similar to other known bacterial adaptions to low phosphate environments, C-P lyase was found to become more prevalent as phosphate concentrations decreased. C-P lyase was particularly enriched in the Mediterranean Sea and North Atlantic Ocean, two regions that feature sustained periods of phosphate depletion. In these regions, C-P lyase was prevalent in several lineages of Alphaproteobacteria (Pelagibacter, SAR116, Roseobacter and Rhodospirillales), Gammaproteobacteria, and Actinobacteria. The global scope of this analysis supports previous studies that infer phosphonate catabolism via C-P lyase is an important adaptive strategy implemented by bacteria to alleviate phosphate limitation and expands the known geographic extent and taxonomic affiliation of this metabolic pathway in the ocean., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Nitrogen (N) is frequently a limiting nutrient in soil; its availability can govern ecosystem functions such as primary production and decomposition. Assimilation of N by microorganisms impacts the availability of N in soil. Despite its established ecological significance, the contributions of microbial taxa to N assimilation are unknown. Here we measure N uptake and use by microbial phylotypes and taxonomic groups within a diverse assemblage of soil microbes through quantitative stable isotope probing (qSIP) with 15 N. Following incubation with 15 NH4+, distinct patterns of 15 N assimilation among taxonomic groups were observed. For instance, glucose addition stimulated 15 N assimilation in most members of Actinobacteria and Proteobacteria but generally decreased 15 N use by Firmicutes and Bacteriodetes. While NH4+ is considered a preferred and universal source of N to prokaryotes, the majority (> 80%) of N assimilation in our soils could be attributed to a handful of active orders. Characterizing N assimilation of taxonomic groups with 15 N qSIP may provide a basis for understanding how microbial community composition influences N availability in the environment., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The diversity and composition of lake bacterial communities are driven by the interplay between local contemporary environmental conditions and dispersal of cells from the surroundings, i.e. the metacommunity. Still, a conceptual understanding of the relative importance of the two types of factors is lacking. For instance, it is unknown which sources of dispersal are most important and under which circumstances. Here, we investigated the seasonal variation in the importance of dispersal from different sources (mixing, precipitation, surface runoff and sediment resuspension) for lake bacterioplankton community and population dynamics. For that purpose, two small forest lakes and their dispersal sources were sampled over a period of 10 months. The influence of dispersal on communities and populations was determined by 454 sequencing of the 16S rRNA gene and SourceTracker analysis. On the community level direct effects of dispersal were questionable from all sources. Instead we found that the community of the preceding sampling occasion, representing growth of resident bacteria, was of great importance. On the population level, however, dispersal of individual taxa from the inlet could be occasionally important even under low water flow. The effect of sediment resuspension and precipitation appeared small., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Rhizodeposits play a key role in shaping rhizosphere microbial communities. In soybean, isoflavonoids are a key rhizodeposit component that aid in plant defense and enable symbiotic associations with rhizobia. However, it is uncertain if and how they influence rhizosphere microbial communities. Isoflavonoid biosynthesis was silenced via RNA interference of isoflavone synthase in soybean hairy root composite plants. Rhizosphere soil fractions tightly associated with roots were isolated, and PCR amplicons from 16S rRNA gene variable regions V1-V3 and V3-V5 from these fractions were sequenced using 454. The resulting data was resolved using MOTHUR and vegan to identify bacterial taxa and evaluate changes in rhizosphere bacterial communities. The soybean rhizosphere was enriched in Proteobacteria and Bacteroidetes, and had relatively lower levels of Actinobacteria and Acidobacteria compared with bulk soil. Isoflavonoids had a small effect on bacterial community structure, and in particular on the abundance of Xanthomonads and Comamonads. The effect of hairy root transformation on rhizosphere bacterial communities was largely similar to untransformed plant roots with approximately 74% of the bacterial families displaying similar colonization underscoring the suitability of this technique to evaluate the influence of plant roots on rhizosphere bacterial communities. However, hairy root transformation had notable influence on Sphingomonads and Acidobacteria., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Magnetotactic bacteria (MTB) are a group of phylogenetically and physiologically diverse Gram-negative bacteria that synthesize intracellular magnetic crystals named magnetosomes. MTB are affiliated with three classes of Proteobacteria phylum, Nitrospirae phylum, Omnitrophica phylum and probably with the candidate phylum Latescibacteria. The evolutionary origin and physiological diversity of MTB compared with other bacterial taxonomic groups remain to be illustrated. Here, we analysed the genome of the marine magneto-ovoid strain MO-1 and found that it is closely related to Magnetococcus marinus MC-1. Detailed analyses of the ribosomal proteins and whole proteomes of 390 genomes reveal that, among the Proteobacteria analysed, only MO-1 and MC-1 have coding sequences (CDSs) with a similarly high proportion of origins from Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria and Gammaproteobacteria. Interestingly, a comparative metabolic network analysis with anoxic network enzymes from sequenced MTB and non-MTB successfully allows the eventual prediction of an organism with a metabolic profile compatible for magnetosome production. Altogether, our genomic analysis reveals multiple origins of MO-1 and M. marinus MC-1 genomes and suggests a metabolism-restriction model for explaining whether a bacterium could become an MTB upon acquisition of magnetosome encoding genes., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Thirty kilometers south of the island of Hawai'i lies the Lō'ihi Seamount, an active submarine volcano that hosts a network of low-temperature hydrothermal vents enriched in ferrous iron that supports extensive microbial mats. These mats, which can be a half a meter deep, are composed of ferric iron bound to organic polymers - the metabolic byproduct of iron-oxidizing Zetaproteobacteria. Though the role of Zetaproteobacteria in mat formation is well established, we have a limited understanding of how differences in diversity are related to mat morphology. We used Minimum Entropy Decomposition and ZetaOtu classification to demonstrate cryptic diversity between closely related Zetaproteobacteria while showing habitat and geographic specificity. Veiled mats, common structures at Lō'ihi, exhibit distinct community composition and contain diversity not detected in other mat types, including specific Zetaproteobacteria and an unclassified Gammaproteobacteria. Our analyses also indicate that diversity can change dramatically across small spatial transects from points of active venting, yet we found comparatively few differences between major sampling sites. This study provides a better picture of the microbiome responsible for iron mat production at Lō'ihi and has broad implications for our understanding of these globally distributed communities., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Deep-sea hydrothermal vents are highly dynamic habitats characterized by steep temperature and chemical gradients. The oxidation of reduced compounds dissolved in the venting fluids fuels primary production providing the basis for extensive life. Until recently studies of microbial vent communities have focused primarily on chemolithoautotrophic organisms. In our study, we targeted the change of microbial community compositions along mixing gradients, focusing on distribution and capabilities of heterotrophic microorganisms. Samples were retrieved from different venting areas within the Menez Gwen hydrothermal field, taken along mixing gradients, including diffuse fluid discharge points, their immediate surroundings and the buoyant parts of hydrothermal plumes. High throughput 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization, and targeted metagenome analysis were combined with geochemical analyses. Close to diffuse venting orifices dominated by chemolithoautotrophic Epsilonproteobacteria, in areas where environmental conditions still supported chemolithoautotrophic processes, we detected microbial communities enriched for versatile heterotrophic Alpha- and Gammaproteobacteria. The potential for alkane degradation could be shown for several genera and yet uncultured clades. We propose that hotspots of chemolithoautotrophic life support a 'belt' of heterotrophic bacteria significantly different from the dominating oligotrophic microbiota of the deep sea., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Marine fish larvae are immature upon hatching, and share their environment with high numbers of bacteria. The microbial communities associated with developing fish larvae might be structured by other factors than those important in developing terrestrial animals. Here, we analysed the beta (β)-diversity of the microbiota associated with developing cod larvae and compared it with the bacterial communities in water and live feed by applying pyrosequencing of bar coded v4 16S rDNA amplicons. A total of 15 phyla were observed in the cod larval microbiota. Proteobacteria was the most abundant, followed by Firmicutes, Bacteroidetes and Actinobacteria. The composition and diversity of the cod larval microbiota changed considerably with age. The temporal and spatial patterns of β-diversity could not be explained by stochastic processes, and did not coincide with changes in the rearing conditions. Furthermore, the larval microbiota was highly distinct from the water and the live feed microbiota, particularly at early developmental stages. However, the similarity between larval and water microbiota increased with age. This study suggests that strong selection in the host structures the cod larval microbiota. The changes in community structure observed with increasing age can be explained by altered selection pressure due to development of the intestinal system., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Microbes colonize human oral surfaces within hours after delivery. During postnatal development, physiological changes, such as the eruption of primary teeth and replacement of the primary dentition with permanent dentition, greatly alter the microbial habitats, which, in return, may lead to community composition shifts at different phases in people's lives. By profiling saliva, supragingival and mucosal plaque samples from healthy volunteers at different ages and dentition stages, we observed that the oral cavity is a highly heterogeneous ecological system containing distinct niches with significantly different microbial communities. More importantly, the phylogenetic microbial structure varies with ageing. In addition, only a few taxa were present across the whole populations, indicating a core oral microbiome should be defined based on age and oral niches., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Diazotrophic bacteria potentially play an important functional role in supplying fixed nitrogen to the coral holobiont, but the value of such a partnership depends on the stability of the association. Here we evaluate the composition of diazotroph assemblages associated with the coral Acropora millepora throughout four seasons and at two reefs, an inshore and an offshore (mid-shelf) reef on the Great Barrier Reef, Australia. Amplicon pyrosequencing of the nifH gene revealed that diazotrophs are ubiquitous members of the bacterial community associated with A. millepora. Rhizobia (65% of the overall nifH sequences retrieved) and particularly Bradyrhizobia sp.-affiliated sequences (> 50% of rhizobia sequences) dominated diazotrophic assemblages across all coral samples from the two sites throughout the year. In contrast to this consistency in the spatial and temporal patterns of occurrence of diazotroph assemblages, the overall coral-associated bacterial community, assessed through amplicon sequencing of the general bacterial 16S ribosomal RNA gene, differed between inshore and mid-shelf reef locations. Sequences associated with the Oceanospirillales family, particularly with Endozoicomonas sp., dominated bacterial communities associated with inshore corals. Although rhizobia represented a variable and generally small fraction of the overall bacterial community associated with A. millepora, consistency in the structure of these diazotrophic assemblages suggests that they have a functional role in the coral holobiont., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A dataset of 87 020 nifH reads and 16 782 unique nifH protein sequences obtained over 2 years from four locations across a gradient of agricultural soil types in Argentina were analysed to provide a detailed and comprehensive picture of the diversity, abundance and responses of the N2 -fixing community in relation to differences in soil chemistry and agricultural practices. Phylogenetic analysis revealed an expected high proportion of Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria, mainly relatives to Bradyrhizobium and Methylosinus/Methylocystis, but a surprising paucity of Gammaproteobacteria. Analysis of variance and stepwise regression modelling suggested location and treatment-specific influences of soil type on diazotrophic community composition and organic carbon concentrations on nifH diversity. nifH gene abundance, determined by quantitative real-time polymerase chain reaction, was higher in agricultural soils than in non-agricultural soils, and was influenced by soil chemistry under intensive crop rotation but not under monoculture. At some locations, sustainable increased crop yields might be possible through the management of soil chemistry to improve the abundance and diversity of N2 -fixing bacteria., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The objective of this study was to evaluate the contribution of evolutionary history to variation in the biomass stoichiometry and underlying biochemical allocation patterns of heterotrophic marine bacteria. We hypothesized that phylogeny significantly constrains biochemical allocation strategy and elemental composition among taxa of heterotrophic marine bacteria. Using a 'common-garden' experimental design, we detected significant interspecific variation in stoichiometry, macromolecule allocation and growth rate among 13 strains of marine Proteobacteria. However, this variation was not well explained by 16S rRNA phylogenetic relationships or differences in growth rate. Heterotrophic bacteria likely experience C-limitation when consuming resources in Redfield proportions, which consequently decouples growth rate from allocation to rRNA and biomass P content. Accordingly, overall bacterial C : nutrient ratios (C : P = 77, C : N = 4.9) were lower than Redfield proportions, whereas the average N : P ratio of 17 was consistent with the Redfield ratio. Our results suggest that strain-level diversity is an important driver of variation in the C : N : P ratios of heterotrophic bacterial biomass and that the potential importance of non-nucleic acid pools of P warrants further investigation. Continued work clarifying the range and controls on the stoichiometry of heterotrophic marine bacteria will help improve understanding and predictions of global ocean C, N and P dynamics., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur-containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5'-diphosphate-sulphoquinovose (UDP-SQ) synthase involved in the SQDG biosynthetic pathway to assess potential microbial sources of SQDGs in the marine environment. The phylogeny of the sqdB-coding protein reveals two distinct clusters: one including green algae, higher plants and cyanobacteria, and another one comprising mainly non-photosynthetic bacteria, as well as other cyanobacteria and algal groups. Evolutionary analysis suggests that the appearance of UDP-SQ synthase occurred twice in cyanobacterial evolution, and one of those branches led to the diversification of the protein in members of the phylum Proteobacteria. A search of homologues of sqdB-proteins in marine metagenomes strongly suggested the presence of heterotrophic bacteria potential SQDG producers. Application of newly developed sqdB gene primers in the marine environment revealed a high diversity of sequences affiliated to cyanobacteria and Proteobacteria in microbial mats, while in North Sea surface water, most of the detected sqdB genes were attributed to the cyanobacterium Synechococcus sp. Lipid analysis revealed that specific SQDGs were characteristic of microbial mat depth, suggesting that SQDG lipids are associated with specific producers., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Sulphur is an essential element in the metabolism. The sulphur-containing amino acid methionine is a metabolic precursor for S-adenosylmethionine (SAM), which serves as a coenzyme for ubiquitous methyltrtansferases. Recycling of organic sulphur compounds, e.g. via the SAM cycle, is an important metabolic process that needs to be tightly regulated. Knowledge about transcriptional regulation of these processes is still limited for many free-living bacteria. We identified a novel transcription factor SahR from the ArsR family that controls the SAM cycle genes in diverse microorganisms from soil and aquatic ecosystems. By using comparative genomics, we predicted SahR-binding DNA motifs and reconstructed SahR regulons in the genomes of 62 Proteobacteria. The conserved core of SahR regulons includes all enzymes required for the SAM cycle: the SAH hydrolase AhcY, the methionine biosynthesis enzymes MetE/MetH and MetF, and the SAM synthetase MetK. By using a combination of experimental techniques, we validated the SahR regulon in the sulphate-reducing Deltaproteobacterium Desulfovibrio alaskensis. SahR functions as a negative regulator that responds to the S-adenosylhomocysteine (SAH). The elevated SAH level in the cell dissociates SahR from its DNA operators and induces the expression of SAM cycle genes. The effector-sensing domain in SahR is related to SAM-dependent methylases that are able to tightly bind SAH. SahR represents a novel type of transcriptional regulators for the control of sulphur amino acid metabolism., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Horizontal gene transfer (HGT), the transfer of genetic material other than by descent, is thought to have played significant roles in the evolution and distribution of genes in prokaryotes. These include those responsible for the ability of motile, aquatic magnetotactic bacteria (MTB) to align and swim along magnetic field lines and the biomineralization of magnetosomes that are responsible for this behaviour. There is some genomic evidence that HGT might be responsible for the distribution of magnetosome genes in different phylogenetic groups of bacteria. For example, in the genomes of a number of MTB, magnetosome genes are present as clusters within a larger structure known as the magnetosome genomic island surrounded by mobile elements such as insertion sequences and transposases as well as tRNA genes. Despite this, there is no strong direct proof of HGT between these organisms. Here we show that a phylogenetic tree based on magnetosome protein amino acid sequences from a number of MTB was congruent with the tree based on the organisms' 16S rRNA gene sequences. This shows that evolution and divergence of these proteins and the 16S rRNA gene occurred similarly. This suggests that magnetotaxis originated monophyletically in the Proteobacteria phylum and implies that the common ancestor of all Proteobacteria was magnetotactic., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Knowledge of intraspecific variation in symbioses may aid in understanding the ecology of widespread insects in different parts of their range. We investigated bacterial symbionts of Adelges tsugae, a pest of hemlocks in eastern North America introduced from Asia. Amplification, cloning, and sequencing of bacterial 16S rDNA, in situ hybridizations, and electron microscopy revealed that A. tsugae harbours up to five bacterial phylotypes, according to population. Three Gammaproteobacteria species are maternally transmitted. The first, designated 'Ca. Pseudomonas adelgestsugas' resides in the haemocoel, and was detected in all populations except Taiwan. The second phylotype, 'Ca. Serratia symbiotica', resides in bacteriocytes of populations on Tsuga sieboldii in Japan and in E. North America. The third phylotype, designated 'Ca. Annandia adelgestsuga', clustered within a lineage of several insect endosymbionts that included Buchnera aphidicola. It was detected in bacteriocytes in all populations, and in salivary glands of first instars. Two Betaproteobacteria phylotypes were detected in some Japanese T. sieboldii and eastern North America populations, and were observed only in salivary glands with no evidence of maternal transmission. Our results support the ideas that symbiont gain and loss has been volatile in adelgids, and that symbionts may help to trace the source of invasive species., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Submerged metal surfaces in marine waters undergo rapid microbial colonization and biocorrosion, causing huge damage to marine engineering facilities and significant financial losses. In coastal areas, an accelerated and particularly severe form of biocorrosion termed accelerated low water corrosion (ALWC) is widespread globally. While identification of biocorroding microorganisms and the dynamics of their community structures is the key for understanding the processes and mechanisms leading to ALWC, neither one is presently understood. In this study, analysis of constructed clone libraries and qPCR assays targeting group-specific 16S rRNA or functional marker genes were used to determine the identity and abundance of putative early carbon steel surface-colonizing and biocorroding microbes in coastal seawater. Diverse microbial groups including 10 bacterial phyla, archaea and algae were found to putatively participate in the surface-colonizing process. Analysis of the community structure of carbon steel surface microbiota revealed a temporal succession leading to ALWC. By extending the current state of knowledge, our work demonstrates the global importance of Alphaproteobacteria (mainly Rhodobacterales), Gammaproteobacteria (mainly Alteromonadales and Oceanospirillales), Bacteroidetes (mainly Flavobacteriales) and microalgae as the pioneer and sustaining surface colonizers that contribute to initial formation and development of surface biofilms. We also discovered Epsilonproteobacteria and the recently described Zetaproteobacteria as putative corrosion-causing microorganisms during early steps of the ALWC process. Hence, our study reports that Zetaproteobacteria may be ubiquitous also in non-hydrothermal coastal seawaters and that ALWC of submerged carbon steel surfaces in coastal waters may involve a highly diverse, complex and dynamic microbial consortium. Our finding that Epsilon- and Zetaproteobacteria may play pivotal roles in ALWC provides a new starting point for future investigation of the ALWC process and mechanism in marine environments. Further studies of Epsilon- and Zetaproteobacteria in particular may thus help with the design of effective corrosion prevention and control strategies., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The ultramafic-hosted Logatchev hydrothermal field (LHF) is characterized by vent fluids, which are enriched in dissolved hydrogen and methane compared with fluids from basalt-hosted systems. Thick sediment layers in LHF are partly covered by characteristic white mats. In this study, these sediments were investigated in order to determine biogeochemical processes and key organisms relevant for primary production. Temperature profiling at two mat-covered sites showed a conductive heating of the sediments. Elemental sulfur was detected in the overlying mat and metal-sulfides in the upper sediment layer. Microprofiles revealed an intensive hydrogen sulfide flux from deeper sediment layers. Fluorescence in situ hybridization showed that filamentous and vibrioid, Arcobacter-related Epsilonproteobacteria dominated the overlying mats. This is in contrast to sulfidic sediments in basalt-hosted fields where mats of similar appearance are composed of large sulfur-oxidizing Gammaproteobacteria. Epsilonproteobacteria (7-21%) and Deltaproteobacteria (20-21%) were highly abundant in the surface sediment layer. The physiology of the closest cultivated relatives, revealed by comparative 16S rRNA sequence analysis, was characterized by the capability to metabolize sulfur components. High sulfate reduction rates as well as sulfide depleted in (34)S further confirmed the importance of the biogeochemical sulfur cycle. In contrast, methane was found to be of minor relevance for microbial life in mat-covered surface sediments. Our data indicate that in conductively heated surface sediments microbial sulfur cycling is the driving force for bacterial biomass production although ultramafic-hosted systems are characterized by fluids with high levels of dissolved methane and hydrogen., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Culture-based and culture-independent methods were used to explore the diversity of phototrophic purple bacteria in Soap Lake, a small meromictic soda lake in the western USA. Among soda lakes, Soap Lake is unusual because it consists of distinct upper and lower water bodies of vastly different salinities, and its deep waters contain up to 175 mM sulfide. From Soap Lake water new alkaliphilic purple sulfur bacteria of the families Chromatiaceae and Ectothiorhodospiraceae were cultured, and one purple non-sulfur bacterium was isolated. Comparative sequence analysis of pufM, a gene that encodes a key photosynthetic reaction centre protein universally found in purple bacteria, was used to measure the diversity of purple bacteria in Soap Lake. Denaturing gradient gel electrophoresis and subsequent phylogenetic analyses of pufMs amplified from Soap Lake water revealed that a significant diversity of purple bacteria inhabit this soda lake. Although close relatives of several of the pufM phylotypes obtained from cultured species could also be detected in Soap Lake water, several other more divergent pufM phylotypes were also detected. It is possible that Soap Lake purple bacteria are major contributors of organic matter into the ecosystem of this lake, especially in its extensive anoxic and sulfidic deep waters., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Bacterial communities in the surface layer of the oceans consist of a few abundant phylotypes and many rare ones, most with unknown ecological functions and unclear roles in biogeochemical processes. To test hypotheses about relationships between abundant and rare phylotypes, we examined bacterial communities in the western Arctic Ocean using pyrosequence data of the V6 region of the 16S rRNA gene. Samples were collected from various locations in the Chukchi Sea, the Beaufort Sea and Franklin Bay in summer and winter. We found that bacterial communities differed between summer and winter at a few locations, but overall there was no significant difference between the two seasons in spite of large differences in biogeochemical properties. The sequence data suggested that abundant phylotypes remained abundant while rare phylotypes remained rare between the two seasons and among the Arctic regions examined here, arguing against the 'seed bank' hypothesis. Phylotype richness was calculated for various bacterial groups defined by sequence similarity or by phylogeny (phyla and proteobacterial classes). Abundant bacterial groups had higher within-group diversity than rare groups, suggesting that the ecological success of a bacterial lineage depends on diversity rather than on the dominance of a few phylotypes. In these Arctic waters, in spite of dramatic variation in several biogeochemical properties, bacterial community structure was remarkably stable over time and among regions, and any variation was due to the abundant phylotypes rather than rare ones.

Very few marine microbial communities are well characterized even with the weight of research effort presently devoted to it. Only a small proportion of this effort has been aimed at investigating temporal community structure. Here we present the first report of the application of high-throughput pyrosequencing to investigate intra-annual bacterial community structure. Microbial diversity was determined for 12 time points at the surface of the L4 sampling site in the Western English Channel. This was performed over 11 months during 2007. A total of 182 560 sequences from the V6 hyper-variable region of the small-subunit ribosomal RNA gene (16S rRNA) were obtained; there were between 11 327 and 17 339 reads per sample. Approximately 7000 genera were identified, with one in every 25 reads being attributed to a new genus; yet this level of sampling far from exhausted the total diversity present at any one time point. The total data set contained 17 673 unique sequences. Only 93 (0.5%) were found at all time points, yet these few lineages comprised 50% of the total reads sequenced. The most abundant phylum was Proteobacteria (50% of all sequenced reads), while the SAR11 clade comprised 21% of the ubiquitous reads and approximately 12% of the total sequenced reads. In contrast, 78% of all operational taxonomic units were only found at one time point and 67% were only found once, evidence of a large and transient rare assemblage. This time series shows evidence of seasonally structured community diversity. There is also evidence for seasonal succession, primarily reflecting changes among dominant taxa. These changes in structure were significantly correlated to a combination of temperature, phosphate and silicate concentrations.

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.

The widespread magnetotactic bacteria have the peculiar capacity of navigation along the geomagnetic field. Despite their ubiquitous distribution, only few axenic cultures have been obtained worldwide. In this study, we reported the first axenic culture of magnetotactic bacteria isolated from the Mediterranean Sea. This magneto-ovoid strain MO-1 grew in chemically defined O(2) gradient minimal media at the oxic-anoxic transition zone. It is phylogenetically related to Magnetococcus sp. MC-1 but might represent a novel genus of Proteobacteria. Pulsed-field gel electrophoresis analysis indicated that the genome size of the MO-1 strain is 5 ± 0.5 Mb, with four rRNA operons. Each cell synthesizes about 17 magnetosomes within a single chain, two phosphorous-oxygen-rich globules and one to seven lipid storage granules. The magnetosomes chain seems to divide in the centre during cell division giving rise to two daughter cells with an approximately equal number of magnetosomes. The MO-1 cell possesses two bundles of seven individual flagella that were enveloped in a unique sheath. They swam towards the north pole with a velocity up to 300 μm per second with frequent change from right-hand to left-hand helical trajectory. Using a magneto-spectrophotometry assay we showed that MO-1 flagella were powered by both proton-motive force and sodium ion gradient, which is a rare feature among bacteria.

Marine sponges contain complex assemblages of bacterial symbionts, the roles of which remain largely unknown. We identified diverse bacterial nifH genes within sponges and found that nifH genes are expressed in sponges. This is the first demonstration of the expression of any protein-coding bacterial gene within a sponge. Two sponges Ircinia strobilina and Mycale laxissima were collected from Key Largo, Florida and had delta(15)N values of c. 0-1 per thousand and 3-4 per thousand respectively. The potential for nitrogen fixation by symbionts was assessed by amplification of nifH genes. Diverse nifH genes affiliated with Proteobacteria and Cyanobacteria were detected, and expression of nifH genes affiliated with those from cyanobacteria was detected. The nifH genes from surrounding seawater were similar to those of Trichodesmium and clearly different from the cyanobacterial nifH genes detected in the two sponges. This study advances understanding of the role of bacterial symbionts in sponges and suggests that provision of fixed nitrogen is a means whereby symbionts benefit sponges in nutrient-limited reef environments. Nitrogen fixation by sponge symbionts is possibly an important source of new nitrogen to the reef environment that heretofore has been neglected and warrants further investigation.

OM43 is a clade of uncultured beta-proteobacteria that is commonly found in environmental nucleic acid sequences from productive coastal ocean ecosystems, and some freshwater environments, but is rarely detected in ocean gyres. Ecological studies associate OM43 with phytoplankton blooms, and evolutionary relationships indicate that they might be methylotrophs. Here we report on the genome sequence and metabolic properties of the first axenic isolate of the OM43 clade, strain HTCC2181, which was obtained using new procedures for culturing cells in natural seawater. We found that this strain is an obligate methylotroph that cannot oxidize methane but can use the oxidized C1 compounds methanol and formaldehyde as sources of carbon and energy. Its complete genome is 1304 428 bp in length, the smallest yet reported for a free-living cell. The HTCC2181 genome includes genes for xanthorhodopsin and retinal biosynthesis, an auxiliary system for producing transmembrane electrochemical potentials from light. The discovery that HTCC2181 is an extremely simple specialist in C1 metabolism suggests an unanticipated, important role for oxidized C1 compounds as substrates for bacterioplankton productivity in coastal ecosystems.

The marine environment represents a rich source of bio- and geogenically produced organohalogens, including the common pollutant perchloroethene (PCE). However, diversity and function of marine chloroethene-dechlorinating microorganisms are largely unknown. Here, we have studied the activity and composition of a tidal flat sediment bacterial and archaeal community from the North Sea exposed to low concentrations of PCE. After 2 weeks of incubation, PCE was rapidly dechlorinated via trichloroethene to dichloroethene (DCE). Unexpectedly, these microcosms produced 3.5-fold more trans-DCE than cis-DCE. The actively dechlorinating microbial populations were traced by stable isotope probing of rRNA with (13)C-labelled acetate for 4 days. Terminal restriction fragment length polymorphism fingerprinting and clone libraries of isotopically enriched, 'heavy'(13)C-labelled bacterial 16S rRNA revealed the populations potentially involved in reductive dechlorination. Major clone groups belonged to the Proteobacteria (50.0%; 22.4% delta-, 12.1% gamma-, 6.9% alpha-, 6.9% beta- and 1.7% epsilon-subgroup) and Chloroflexi (29.3%). Populations represented by the two dominant terminal restriction fragments were affiliated with the Dehalococcoidetes (subphylum II of the Chloroflexi), and were exclusively detected in the heavy fraction of the PCE-dechlorinating incubation. The phylogenetically novel, larger population, designated Tidal Flat Chloroflexi Cluster, was closely related to the recently discovered PCE-dechlorinating Lahn Cluster bacteria from anoxic river sediment but more distantly related to canonical Dehalococcoides spp. (92-94% sequence identity). The second population was closely related to 'Dehalobium chlorocoercia DF-1'. Both populations appear to be responsible for reductive dechlorination of highly chlorinated ethenes to predominantly trans-DCE in tidal flat sediment incubations.

The active methanotroph community was investigated for the first time in heather (Calluna)-covered moorlands and Sphagnum/Eriophorum-covered UK peatlands. Direct extraction of mRNA from these soils facilitated detection of expression of methane monooxygenase genes, which revealed that particulate methane monooxygenase and not soluble methane monooxygenase was probably responsible for CH(4) oxidation in situ, because only pmoA transcripts (encoding a subunit of particulate methane monooxygenase) were readily detectable. Differences in methanotroph community structures were observed between the Calluna-covered moorland and Sphagnum/Eriophorum-covered gully habitats. As with many other Sphagnum-covered peatlands, the Sphagnum/Eriophorum-covered gullies were dominated by Methylocystis. Methylocella and Methylocapsa-related species were also present. Methylobacter-related species were found as demonstrated by the use of a pmoA-based diagnostic microarray. In Calluna-covered moorlands, in addition to Methylocella and Methylocystis, a unique group of peat-associated type I methanotrophs (Gammaproteobacteria) and a group of uncultivated type II methanotrophs (Alphaproteobacteria) were also found. The pmoA sequences of the latter were only distantly related to Methylocapsa and also to the RA-14 group of methanotrophs, which are believed to be involved in oxidation of atmospheric concentrations of CH(4). Soil samples were also labelled with (13)CH(4), and subsequent analysis of the (13)C-labelled phospholipid fatty acids (PLFAs) showed that 16:1 omega 7, 18:1 omega 7 and 18:1 omega 9 were the major labelled PLFAs. The presence of (13)C-labelled 18:1 omega 9, which was not a major PLFA of any extant methanotrophs, indicated the presence of novel methanotrophs in this peatland.

Methane oxidation is a key process controlling methane emission from anoxic habitats into the atmosphere. Methanotrophs, responsible for aerobic methane oxidation, do not only oxidize but also assimilate methane. Once assimilated, methane carbon may be utilized by other organisms. Here we report on a microbial food web in a rice field soil driven by methane. A thin layer of water-saturated rice field soil was incubated under opposing gradients of oxygen and (13)C-labelled methane. Bacterial and eukaryotic communities incorporating methane carbon were analysed by RNA-stable isotope probing (SIP). Terminal restriction fragment length polymorphism (T-RFLP) and cloning showed that methanotrophs were the most prominent group of bacteria incorporating methane carbon. In addition, a few Myxobacteria-related sequences were obtained from the 'heavy' rRNA fraction. Denaturing gradient gel electrophoresis (DGGE) targeting 18S rRNA detected various groups of protists in the 'heavy' rRNA fraction including naked amoeba (Lobosea and Heterolobosea), ciliates (Colpodea) and flagellates (Cercozoa). Incubation of soil under different methane concentrations in air resulted in the development of distinct protozoan communities. These results suggest that methane carbon is incorporated into non-methanotrophic pro- and microeukaryotes probably via grazing, and that methane oxidation is a shaping force of the microeukaryotic community depending on methane availability.

Active methanotrophs in a landfill soil were revealed by detecting the 16S rRNA of methanotrophs and the mRNA transcripts of key genes involved in methane oxidation. New 16S rRNA primers targeting type I and type II methanotrophs were designed and optimized for analysis by denaturing gradient gel electrophoresis. Direct extraction of RNA from soil enabled the analysis of the expression of the functional genes: mmoX, pmoA and mxaF, which encode subunits of soluble methane monooxygenase, particulate methane monooxygenase and methanol dehydrogenase respectively. The 16S rRNA polymerase chain reaction (PCR) primers for type I methanotrophs detected Methylomonas, Methylosarcina and Methylobacter sequences from both soil DNA and cDNA which was generated from RNA extracted directly from the landfill cover soil. The 16S rRNA primers for type II methanotrophs detected primarily Methylocella and some Methylocystis 16S rRNA genes. Phylogenetic analysis of mRNA recovered from the soil indicated that Methylobacter, Methylosarcina, Methylomonas, Methylocystis and Methylocella were actively expressing genes involved in methane and methanol oxidation. Transcripts of pmoA but not mmoX were readily detected by reverse transcription polymerase chain reaction (RT-PCR), indicating that particulate methane monooxygenase may be largely responsible for methane oxidation in situ.

Tuber magnatum, an ascomycetous fungus and obligate ectomycorrhizal symbiont, forms hypogeous fruit bodies, commonly called Italian white truffles. The diversity of bacterial communities associated with T. magnatum truffles was investigated using culture-independent and -dependent 16S rRNA gene-based approaches. Eighteen truffles were classified in three groups, representing different degrees of ascocarp maturation, based on the percentage of asci containing mature spores. The culturable bacterial fraction was (4.17 +/- 1.61) x 10(7), (2.60 +/- 1.22) x 10(7) and (1.86 +/- 1.32) x 10(6) cfu g(-1) for immature, intermediate and mature ascocarps respectively. The total of bacteria count was two orders of magnitude higher than the cfu g(-1) count. Sequencing results from the clone library showed a significant presence of alpha-Proteobacteria (634 of the 771 total clones screened, c. 82%) affiliated with Sinorhizobium, Rhizobium and Bradyrhizobium spp. The bacterial culturable fraction was generally represented by gamma-Proteobacteria (210 of the 384 total strains isolated, c. 55%), which were mostly fluorescent pseudomonads. Fluorescent in situ hybridization confirmed that alpha-Proteobacteria (85.8%) were the predominant components of truffle bacterial communities with beta-Proteobacteria (1.5%), gamma-Proteobacteria (1.9%), Bacteroidetes (2.1%), Firmicutes (2.4%) and Actinobacteria (3%) only poorly represented. Molecular approaches made it possible to identify alpha-Proteobacteria as major constituents of a bacterial component associated with T. magnatum ascoma, independently from the degree of maturation.

Lilliput was discovered in 2005 as the southernmost known hydrothermal field along the Mid-Atlantic Ridge. It is exceptional in that it lacks high-temperature venting probably because of a thickened crust. The absence of thermophilic and hyperthermophilic prokaryotes in emissions supports the argument against the presence of a hot subsurface at Lilliput, as is typically suggested for diffuse emissions from areas of high-temperature venting. The high phylogenetic diversity and novelty of bacteria observed could be because of the low-temperature influence, the distinct location of the hydrothermal field or the Bathymodiolus assemblages covering the sites of discharge. The low-temperature fluids at the Lilliput are characterized by lowered pH and slightly elevated hydrogen (16 nM) and methane ( approximately 2.6 microM) contents compared with ambient seawater. No typical hydrogen and methane oxidizing prokaryotes were detected. The higher diversity of reverse tricarboxylic acid genes and the form II RubisCO genes of the Calvin Benson-Bassham (CBB) cycle compared with the form I RubisCO genes of the CBB cycle suggests that the chemoautotrophic community is better adapted to low oxygen concentrations. Thiomicrospira spp. and Epsilonproteobacteria dominated the autotrophic community. Sulfide is the most abundant inorganic energy source (0.5 mM). Diverse bacteria were associated with sulfur cycling, including Gamma-, Delta- and Epsilonproteobacteria, with the latter being the most abundant bacteria according to fluorescence in situ hybridization. With members of various Candidate Divisions constituting for 25% of clone library sequences we suggest that their role in vent ecosystems might be more important than previously assumed and propose potential mechanisms they might be involved in at the Lilliput hydrothermal field.

The high incidence of coral disease in shallow coastal marine environments suggests seawater depth and coastal pollution have an impact on the microbial communities inhabiting healthy coral tissues. A study was undertaken to determine how bacterial communities inhabiting tissues of the coral Montastraea annularis change at 5 m, 10 m and 20 m water depth in varying proximity to the urban centre and seaport of Willemstad, Curaçao, Netherlands Antilles. Analyses of terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA gene sequences show significant differences in bacterial communities of polluted and control localities only at the shallowest seawater depth. Furthermore, distinct differences in bacterial communities were found with increasing water depth. Comparisons of TRFLP peaks with sequenced clone libraries indicate the black band disease cyanobacterium clone CD1C11 is common and most abundant on healthy corals in less than 10 m water depth. Similarly, sequences belonging to a previously unrecognized group of likely phototrophic bacteria, herein referred to as CAB-I, were also more common in shallow water. To assess the influence of environmental and physiologic factors on bacterial community structure, canonical correspondence analysis was performed using explanatory variables associated with: (i) light availability; (ii) seawater pollution; (iii) coral mucus composition; (iv) the community structure of symbiotic algae; and (v) the photosynthetic activity of symbiotic algae. Eleven per cent of the variation in bacterial communities was accounted for by covariation with these variables; the most important being photosynthetically active radiation (sunlight) and the coral uptake of sewage-derived compounds as recorded by the delta(15)N of coral tissue.

Resin acids are tricyclic diterpenoids naturally synthesized by trees that are released from wood during pulping processes. Using a newly designed primer set, genes similar to that encoding the DitA1 catalytic alpha-subunit of the diterpenoid dioxygenase, a key enzyme in abietane resin acid degradation by Pseudomonas abietaniphila BKME-9, could be amplified from different Pseudomonas strains, whereas ditA1 gene sequence types representing distinct branches in the evolutionary tree were amplified from Burkholderia and Cupriavidus isolates. All isolates harbouring a ditA1-homologue were capable of growth on dehydroabietic acid as the sole source of carbon and energy and reverse transcription polymerase chain reaction analysis in three strains confirmed that ditA1 was expressed constitutively or in response to DhA, demonstrating its involvement in DhA-degradation. Evolutionary analyses indicate that gyrB (as a phylogenetic marker) and ditA1 genes have coevolved under purifying selection from their ancestral variants present in the most recent common ancestor of the genera Pseudomonas, Cupriavidus and Burkholderia. A polymerase chain reaction-single-strand conformation poylmorphism fingerprinting method was established to monitor the diversity of ditA1 genes in environmental samples. The molecular fingerprints indicated the presence ofa broad, previously unrecognized diversity of diterpenoid dioxygenase genes in soils, and suggest that other bacterial phyla may also harbour the genetic potential for DhA-degradation.

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.

Crude oil is a complex mixture of different hydrocarbons. While diverse bacterial communities can degrade oil, the specific roles of individual members within such communities remain unclear. To identify the key bacterial taxa involved in aerobic degradation of specific hydrocarbons, microcosm experiments were established using seawater from Stanford le Hope, Thames estuary, UK, adjacent to a major oil refinery. In all microcosms, hydrocarbon degradation was significant within 10 weeks, ranging from > 99% of low-molecular-weight alkanes (C(10)-C(18)), 41-84% of high-molecular-weight alkanes (C(20)-C(32)) and pristane, and 32-88% of polycyclic aromatic hydrocarbons (PAHs). Analysis of 16S rRNA sequences from clone libraries and denaturing gradient gel electrophoresis (DGGE) indicated that, except when incubated with fluorene, PAH-degrading communities were dominated by Cycloclasticus. Moreover, PAH-degrading communities were distinct from those in microcosms containing alkanes. Degradation of the branched alkane, pristane, was carried out almost exclusively by Alcanivorax. Bacteria related to Thalassolituus oleivorans (99-100% identity) were the dominant known alkane degraders in n-alkane (C(12)-C(32)) microcosms, while Roseobacter-related bacteria were also consistently found in these microcosms. However, in contrast to previous studies, Thalassolituus, rather than Alcanivorax, was dominant in crude oil-enriched microcosms. The communities in n-decane microcosms differed from those in microcosms supplemented with less volatile alkanes, with a phylogenetically distinct species of Thalassolituus out-competing T. oleivorans. These data suggest that the diversity and importance of the genus Thalassolituus is greater than previously established. Overall, these experiments demonstrate how degradation of different petroleum hydrocarbons is partitioned between different bacterial taxa, which together as a community can remediate petroleum hydrocarbon-impacted estuarine environments.

Microbial consumption is one of the main processes, along with photolysis and ventilation, that remove the biogenic trace gas dimethylsulfide (DMS) from the surface ocean. Although a few isolates of marine bacteria have been studied for their ability to utilize DMS, little is known about the characteristics or phylogenetic affiliation of DMS consumers in seawater. We enriched coastal and open-ocean waters with different carbon sources to stimulate different bacterial communities (glucose-consuming bacteria, methyl group-consuming bacteria and DMS consumers) in order to test how this affected DMS consumption and to examine which organisms might be involved. Dimethylsulfide consumption was greatly stimulated in the DMS addition treatments whereas there was no stimulation in the other treatments. Analysis of microbial DNA by two different techniques (sequenced bands from DGGE gels and clone libraries) showed that bacteria grown specifically with the presence of DMS were closely related to the genus Methylophaga. We also followed the fate of consumed DMS in some of the enrichments. Dimethylsulfide was converted mostly to DMSO in glucose or methanol enrichments, whereas it was converted mostly to sulfate in DMS enrichments, the latter suggesting use of DMS as a carbon and energy source. Our results indicate that unlike the biochemical precursor of DMS, dimethylsulfoniopropionate (DMSP), which is consumed by a broad spectrum of marine microorganisms, DMS seems to be utilized as a carbon and electron source by specialists. This is consistent with the usual observation that DMSP turns over at much higher rates than DMS.

Phototrophic anoxygenic purple bacteria play a key role in many aquatic ecosystems by oxidizing sulfur compounds and low-molecular-weight organic compounds using light as energy source. In this study, molecular methods based upon pufM gene (photosynthetic unit forming gene) were compared with culture-dependent methods to investigate anoxygenic purple phototrophic communities in sediments of an eutrophic brackish lagoon. Thirteen strains, belonging to eight different genera of purple phototrophic bacteria were isolated with a large dominance of the metabolically versatile purple non-sulfur bacteria (eight strains), some purple sulfur bacteria (three strains) and two strains belonging to the Roseobacter clade (aerobic phototrophs). The pufM genes amplified from the isolated strains were not detected by the molecular methods [terminal-restriction fragment length polymorphism (T-RFLP)] applied on in situ communities. An environmental clone library of the pufM gene was thus constructed from sediment samples. The results showed that most of the clones probably corresponded to aerobic phototrophic bacteria. Our results demonstrate that the culture-dependent techniques remain the best experimental approach for determining the diversity of phototrophic purple non-sulfur bacteria whereas the molecular approach clearly illustrated the abundance of organisms related to the Roseobacter clade in these eutrophic sediments.

Autotrophic microorganisms have been isolated that are able to derive energy from the oxidation of arsenite [As(III)] to arsenate [As(V)] under aerobic conditions. Based on chemical energetics, microbial oxidation of As(III) can occur in the absence of oxygen, and may be relevant in some environments. Enrichment cultures were established from an arsenic contaminated industrial soil amended with As(III) as the electron donor, inorganic C as the carbon source and nitrate as the electron acceptor. In the active enrichment cultures, oxidation of As(III) was stoichiometrically coupled to the reduction of NO(3) (-). Two autotrophic As(III)-oxidizing strains were isolated that completely oxidized 5 mM As(III) within 7 days under denitrifying conditions. Based on 16S rRNA gene sequencing results, strain DAO1 was 99% related to Azoarcus and strain DAO10 was most closely related to a Sinorhizobium. The nitrous oxide reductase (nosZ) and the RuBisCO Type II (cbbM) genes were successfully amplified from both isolates underscoring their ability to denitrify and fix CO(2) while coupled to As(III) oxidation. Although limited work has been done to examine the diversity of anaerobic autotrophic oxidizers of As(III), this process may be an important component in the biological cycling of arsenic within the environment.

A putative carbon monoxide dehydrogenase large subunit gene (BMS putative coxL) was amplified from genomic DNA extracts of four recent (42-300 year old) Hawaiian volcanic deposits by polymerase chain reaction (PCR). Sequence databases derived from clone libraries constructed using PCR products were analysed phylogenetically and statistically. These analyses indicated that each of the deposits supported distinct BMS putative coxL gene assemblages. Statistical analyses also showed that the youngest deposit (42 years old) contained the least diverse sequences (P<0.05), but that diversity did not vary significantly among three older deposits with ages from about 108-300 years. Although diversity indices did not vary among the older deposits, mismatch analyses suggested population structures increased in complexity with increasing deposit age. At each of the sites, most of the clone sequences appeared to originate from Proteobacteria not currently represented in culture or recognized as CO oxidizers.

We outline an approach to simultaneously assess multilevel microbial diversity patterns utilizing 16S-ITS rDNA clone libraries coupled with automated ribosomal intergenic spacer analysis (ARISA). Sequence data from 512 clones allowed estimation of ARISA fragment lengths associated with bacteria in a coastal marine environment. We matched 92% of ARISA peaks (each comprising >1% total amplified product) with corresponding lengths from clone libraries. These peaks with putative identification accounted for an average of 83% of total amplified community DNA. At 16S rDNA similarities <98%, most taxa displayed differences in ARISA fragment lengths >10 bp, readily detectable and suggesting ARISA resolution is near the 'species' level. Prochlorococcus abundance profiles from ARISA were strongly correlated (r2=0.86) to Prochlorococcus cell counts, indicating ARISA data are roughly proportional to actual cell abundance within a defined taxon. Analysis of ARISA profiles for 42 months elucidated patterns of microbial presence and abundance providing insights into community shifts and ecological niches for specific organisms, including a coupling of ecological patterns for taxa within the Prochlorococcus, the Gamma Proteobacteria and Actinobacteria. Clade-specific ARISA protocols were developed for the SAR11 and marine cyanobacteria to resolve ambiguous identifications and to perform focused studies. 16S-ITS data allowed high-resolution identification of organisms by ITS sequence analysis, and examination of microdiversity.

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.

The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 microg CH4 m(-2) h(-1)) of atmospheric CH4 at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real-time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia-oxidizing bacteria. As yet uncultured methanotrophs of a group previously named 'forest soil cluster' or 'USC alpha' were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group 'Cluster I' were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10(6) pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia-oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH4 uptake rates due to their low cell-specific CH4 oxidation activity. Based on CH4 flux and bacterial abundance data, estimated cell-specific CH4 oxidation rates of the detected methanotrophic bacteria were 540-800 x 10(-18) mol cell(-1) h(-1), which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell-specific CH4 oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH4 alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USC alpha methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA transcripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USC alpha and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell-specific CH4 oxidation activities.

Patterns of aerobic methane (CH4) oxidation and associated methanotroph community composition were investigated during the development of seasonal stratification in Mono Lake, California (USA). CH4 oxidation rates were measured using a tritiated CH4 radiotracer technique. Fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequence analysis were used to characterize methanotroph community composition. A temporally shifting zone of elevated CH4 oxidation (59-123 nM day(-1)) was consistently associated with a suboxycline, microaerophilic zone that migrated upwards in the water column as stratification progressed. FISH analysis revealed stable numbers of type I (4.1-9.3 x 10(5) cells ml(-1)) and type II (1.4-3.4 x 10(5) cells ml(-1)) methanotrophs over depth and over time. Denaturing gradient gel electrophoresis and sequence analysis indicated slight shifts in methanotroph community composition despite stable absolute cell numbers. Variable CH4 oxidation rates in the presence of a relatively stable methanotroph population suggested that zones of high CH4 oxidation resulted from an increase in activity of a subset of the existing methanotroph population. These results challenge existing paradigms suggesting that zones of elevated CH4 oxidation activity result from the accumulation of methanotrophic biomass and illustrate that type II methanotrophs may be an important component of the methanotroph population in saline and/or alkaline pelagic environments.

The microbial community associated with the reef building coral Pocillopora damicornis located on the Great Barrier Reef was investigated using culture-independent molecular microbial techniques. The microbial communities of three separate coral colonies were assessed using clone library construction alongside restriction fragment length polymorphism and phylogenetic analysis. Diversity was also investigated spatially across six replicate samples within each single coral colony using 16S rDNA and rpoB-DGGE analysis. Clone libraries demonstrated that the majority of retrieved sequences from coral tissue slurry libraries affiliated with gamma-Proteobacteria. This contrasted with clone libraries of seawater and coral mucus, which were dominated by alpha-Proteobacteria. A number of retrieved clone sequences were conserved between coral colonies; a result consistent with previous studies suggesting a specific microbe-coral association. rpoB-DGGE patterns of replicate tissue slurry samples underestimated microbial diversity, but demonstrated that fingerprints were identical within the same coral. These fingerprints were also conserved across coral colonies. The 16S rDNA-DGGE patterns of replicate tissue slurry samples were more complex, although non-metric multidimensional scaling (nMDS) analysis showed groupings of these banding patterns indicating that some bacterial diversity was uniform within a coral colony. Sequence data retrieved from DGGE analysis support clone library data in that the majority of affiliations were within the gamma-Proteobacteria. Many sequences retrieved also affiliated closely with sequences derived from previous studies of microbial diversity of healthy corals in the Caribbean. Clones showing high 16S rDNA sequence identity to both Vibrio shiloi and Vibrio coralliilyticus were retrieved, suggesting that these may be opportunist pathogens. Comparisons of retrieved microbial diversity between two different sampling methods, a syringe extracted coral mucus sample and an airbrushed coral tissue slurry sample were also investigated. Non-metric multidimensional scaling of clone library data highlighted that clone diversity retrieved from a coral mucus library more closely reflected the diversity of surrounding seawater than a corresponding coral tissue clone library.

Marine bacteria that oxidized methyl bromide and methyl chloride were enriched and isolated from seawater samples. Six methyl halide-oxidizing enrichments were established from which 13 isolates that grew on methyl bromide and methyl chloride as sole sources of carbon and energy were isolated and maintained. All isolates belonged to three different clades in the Roseobacter group of the alpha subdivision of the Proteobacteria and were distinct from Leisingera methylohalidivorans, the only other identified marine bacterium that grows on methyl bromide as sole source of carbon and energy. Genes encoding the methyltransferase/corrinoid-binding protein CmuA, which is responsible for the initial step of methyl chloride oxidation in terrestrial methyl halide-oxidizing bacteria, were detected in enrichments and some of the novel marine strains. Gene clusters containing cmuA and other genes implicated in the metabolism of methyl halides were cloned from two of the isolates. Expression of CmuA during growth on methyl halides was demonstrated by analysis of polypeptides expressed during growth on methyl halides by SDS-PAGE and mass spectrometry in two isolates representing two of the three clades. These findings indicate that certain marine methyl halide degrading bacteria from the Roseobacter group contain a methyltransferase pathway for oxidation of methyl bromide that may be similar to that responsible for methyl chloride oxidation in Methylobacterium chloromethanicum. This pathway therefore potentially contributes to cycling of methyl halides in both terrestrial and marine environments.

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.

In order to understand the effect of the maize rhizosphere on denitrification, the diversity and the activity of the denitrifying community were studied in soil amended with maize mucilage. Diversity of the denitrifying community was investigated by polymerase chain reaction (PCR) amplification of total community DNA extracted from soils using gene fragments, encoding the nitrate reductase (narG) and the nitrous oxide reductase (nosZ), as molecular markers. To assess the underlying diversity, PCR products were cloned and 10 gene libraries were obtained for each targeted gene. Libraries containing 738 and 713 narG and nosZ clones, respectively, were screened by restriction fragment analysis, and grouped based on their RFLP (restriction fragment length polymorphism) patterns. In all, 117 and 171 different clone families have been identified for narG and nosZ and representatives of RFLP families containing at least two clones were sequenced. Rarefaction curves of both genes did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity. Recovered NarG sequences were related to NarG from Actinomycetales and from Proteobacteria but most of them are not related to NarG from known bacteria. In contrast, most of the NosZ sequences were related to NosZ from alpha, beta, and gammaProteobacteria. Denitrifying activity was monitored by incubating the control and amended soils anaerobically in presence of acetylene. The N2O production rates revealed denitrifying activity to be greater in amended soil than in control soil. Altogether, our results revealed that mucilage addition to the soil results in a strong impact on the activity of the denitrifying community and minor changes on its diversity.