Environmental Microbiology

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process.

Publication Date: 2012 Feb 1 PMID: 22296107
Authors: van der Zaan, B. M. - Talarico Saia, F. - Stams, A. J. - Plugge, C. M. - de Vos, W. M. - Smidt, H. - Langenhoff, A. A. - Gerritse, J.
Journal: Environ Microbiol

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.

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Common bacterial responses in six ecosystems exposed to 10 years of elevated atmospheric carbon dioxide.

Publication Date: 2012 Jan 20 PMID: 22264231
Authors: Dunbar, J. - Eichorst, S. A. - Gallegos-Graves, L. V. - Silva, S. - Xie, G. - Hengartner, N. W. - Evans, R. D. - Hungate, B. A. - Jackson, R. B. - Megonigal, J. P. - Schadt, C. W. - Vilgalys, R. - Zak, D. R. - Kuske, C. R.
Journal: Environ Microbiol

Six terrestrial ecosystems in the USA were exposed to elevated atmospheric CO(2) in single or multifactorial experiments for more than a decade to assess potential impacts. We retrospectively assessed soil bacterial community responses in all six-field experiments and found ecosystem-specific and common patterns of soil bacterial community response to elevated CO(2) . Soil bacterial composition differed greatly across the six ecosystems. No common effect of elevated atmospheric CO(2) on bacterial biomass, richness and community composition across all of the ecosystems was identified, although significant responses were detected in individual ecosystems. The most striking common trend across the sites was a decrease of up to 3.5-fold in the relative abundance of Acidobacteria Group 1 bacteria in soils exposed to elevated CO(2) or other climate factors. The Acidobacteria Group 1 response observed in exploratory 16S rRNA gene clone library surveys was validated in one ecosystem by 100-fold deeper sequencing and semi-quantitative PCR assays. Collectively, the 16S rRNA gene sequencing approach revealed influences of elevated CO(2) on multiple ecosystems. Although few common trends across the ecosystems were detected in the small surveys, the trends may be harbingers of more substantive changes in less abundant, more sensitive taxa that can only be detected by deeper surveys.

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Anaerobic degradation of 4-methylbenzoate via a specific 4-methylbenzoyl-CoA pathway.

Publication Date: 2012 Jan 20 PMID: 22264224
Authors: Lahme, S. - Eberlein, C. - Jarling, R. - Kube, M. - Boll, M. - Wilkes, H. - Reinhardt, R. - Rabus, R.
Journal: Environ Microbiol

The pathway for anaerobic degradation of 4-methylbenzoate was studied in the denitrifying alphaproteobacterium Magnetospirillum sp. strain pMbN1. Adaptation studies with whole cells indicated substrate-dependent induction of the capacity to degrade 4-methylbenzoate. Differential protein profiling (2D-DIGE) of 4-methylbenzoate- in comparison with benzoate- or succinate-adapted cells revealed the specific abundance increase of substrate-specific protein sets. Their coding genes form distinct clusters on the genome, two of which were assigned to 4-methylbenzoate and one to benzoate degradation. The predicted functions of the gene products agree with a specific 4-methylbenzoyl-CoA degradation pathway in addition to and analogous to the known anaerobic benzoyl-CoA degradation pathway. In vitro benzoyl-CoA and 4-methylbenzoyl-CoA reductase activities revealed the electron donor and ATP-dependent formation of the corresponding conjugated cyclic dienoyl-CoA/4-methyl-dienoyl-CoA products. The 4-methylbenzoyl-CoA reductase activity was induced in the presence of 4-methylbenzoate. In accordance, metabolite analysis of cultures grown with 4-methylbenzoate tentatively identified 4-methylcyclohex-1,5-diene-1-carboxylate. The 4-methylbenzoate induced genes were assigned to code for the putative 4-methylbenzoyl-CoA reductase; their products display pronounced sequence disparity from the conventional class I benzoyl-CoA reductase, which does not accept substituents at the para-position. Identification of 3-methylglutarate together with the formation of specific proteins for ring cleavage and beta-oxidation in 4-methylbenzoate-adapted cells suggest conservation of the methyl group along the specific 4-methylbenzoyl-CoA degradation pathway.

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Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

Publication Date: 2012 Jan 18 PMID: 22252016
Authors: Sorensen, J. L. - Hansen, F. T. - Sondergaard, T. E. - Staerk, D. - Lee, T. V. - Wimmer, R. - Klitgaard, L. G. - Purup, S. - Giese, H. - Frandsen, R. J.
Journal: Environ Microbiol

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

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Basin-scale patterns in the abundance of SAR11 subclades, marine Actinobacteria (OM1), members of the Roseobacter clade and OCS116 in the South Atlantic.

Publication Date: 2012 Jan 9 PMID: 22225975
Authors: Morris, R. M. - Frazar, C. D. - Carlson, C. A.
Journal: Environ Microbiol

Bacterioplankton are major biogeochemical agents responsible for mediating the flux of dissolved organic matter (DOM) and subsequent cycling of nutrients in the oceans. Most information about the composition of bacterioplankton communities has come from studies along well-defined biogeochemical gradients in the northern hemisphere. This study extends observations of spatial and temporal dynamics for SAR11, Actinobacteria and OCS116 in the North Atlantic by demonstrating distinct spatial variability in the abundance and distribution of these and other lineages across the South Atlantic gyre and in the Benguela upwelling system. We identified shifts in SAR11, Actinobacteria, OCS116, SAR86, SAR116 and members of the Roseobacter clade along basin-scale gradients in nutrients, chlorophyll and dissolved organic carbon (DOC). Distinct SAR11 subclades dominated the western and eastern regions of the gyre, and Actinobacteria, OCS116 and members of the Roseobacter lineages were most abundant at the deep chlorophyll maxima. SAR86 and SAR116 accounted for a significant fraction of coastal and open ocean communities, respectively, and members of the gamma sulfur oxidizer (GSO) clade persisted in the Benguela upwelling system. These data suggest that distinct communities are partitioned along basin-scale biogeochemical gradients, that SAR11 community structure varies across the gyre and that Actinobacteria, OCS116, and members of the Roseobacter clade are closely associated with phytoplankton in the gyre.

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Variation in transport explains polymorphism of histidine and urocanate utilization in a natural Pseudomonas population.

Publication Date: 2012 Jan 9 PMID: 22225938
Authors: Zhang, X. X. - Chang, H. - Tran, S. L. - Gauntlett, J. C. - Cook, G. M. - Rainey, P. B.
Journal: Environ Microbiol

Phenotypic variation is a fundamental requirement for evolution by natural selection. While evidence of phenotypic variation in natural populations abounds, its genetic basis is rarely understood. Here we report variation in the ability of plant-colonizing Pseudomonas to utilize histidine, and its derivative, urocanate, as sole sources of carbon and nitrogen. From a population of 164 phyllosphere-colonizing Pseudomonas strains, 77% were able to utilize both histidine and urocanate (His(+) , Uro(+) ) as growth substrates, whereas the remainder could utilize histidine, but not urocanate (His(+) , Uro(-) ), or vice versa (His(-) , Uro(+) ). An in silico analysis of the hut locus, which determines capacity to utilize both histidine and urocanate, from genome-sequenced Pseudomonas strains, showed significant variation in the number of putative transporters. To identify transporter genes specific for histidine and urocanate, we focused on a single genotype of Pseudomonas fluorescens, strain SBW25, which is capable of utilizing both substrates. Site-directed mutagenesis, combined with [(3) H]histidine transport assays, shows that hutT(u) encodes a urocanate-specific transporter; hutT(h) encodes the major high-affinity histidine transporter; and hutXWV encodes an ABC-type transporter that plays a minor role in histidine uptake. Introduction of cloned copies of hutT(h) and hutT(u) from SBW25 into strains incapable of utilizing either histidine, or urocanate, complemented the defect, demonstrating a lack of functional transporters in these strains. Taken together our data show that variation in transport systems, and not in metabolic genes, explains a naturally occurring phenotypic polymorphism.

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The origin and ecological significance of multiple branches for histidine utilization in Pseudomonas aeruginosa PAO1.

Publication Date: 2012 Jan 9 PMID: 22225844
Authors: Gerth, M. L. - Ferla, M. P. - Rainey, P. B.
Journal: Environ Microbiol

Pseudomonas proliferate in a wide spectrum of harsh and variable environments. In many of these environments, amino acids, such as histidine, are a valuable source of carbon, nitrogen and energy. Here, we demonstrate that the histidine uptake and utilization (hut) pathway of Pseudomonas aeruginosa PAO1 contains two branches from the intermediate formiminoglutamate to the product glutamate. Genetic analysis revealed that the four-step route is dispensable as long as the five-step route is present (and vice versa). Mutants with deletions of either the four-step (HutE) or five-step (HutFG) branches were competed against each other and the wild-type strain to test the hypothesis of ecological redundancy; that is, that the presence of two pathways confers no benefit beyond that delivered by the individual pathways. Fitness assays performed under several environmental conditions led us to reject this hypothesis; the four-step pathway can provide an advantage when histidine is the sole carbon source. An IclR-type regulator (HutR) was identified that regulates the four-step pathway. Comparison of sequenced genomes revealed that P. aeruginosa strains and P. fluorescens Pf-5 have branched hut pathways. Phylogenetic analyses suggests that the gene encoding formiminoglutamase (hutE) was acquired by horizontal gene transfer from a Ralstonia-like ancestor. Potential barriers to inter-species transfer of the hutRE module were explored by transferring it from P. aeruginosa PAO1 to P. fluorescens SBW25. Transfer of the operon conferred the ability to utilize histidine via the four-step pathway in a single step, but the fitness cost of acquiring this new operon was found to be environment dependent.

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The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters.

Publication Date: 2012 Jan 9 PMID: 22225785
Authors: McDonald, J. E. - Houghton, J. N. - Rooks, D. J. - Allison, H. E. - McCarthy, A. J.
Journal: Environ Microbiol

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR). Fibrobacter spp. and Clostridium clusters III, IV and XIV were detected in almost all leachate samples and cluster III and XIV clostridia were the most abundant (1-6% and 1-17% of total bacterial 16S rRNA gene copies respectively). Two landfill leachate microcosms were constructed to specifically assess those microbial communities that colonize and degrade cellulose substrates in situ. Scanning electron microscopy (SEM) of colonized cotton revealed extensive cellulose degradation in one microcosm, and Fibrobacter spp. and Clostridium cluster III represented 29% and 17%, respectively, of total bacterial 16S rRNA gene copies in the biofilm. Visible cellulose degradation was not observed in the second microcosm, and this correlated with negligible relative abundances of Clostridium cluster III and Fibrobacter spp. (
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Genomic and functional analysis of Vibrio phage SIO-2 reveals novel insights into ecology and evolution of marine siphoviruses.

Publication Date: 2012 Jan 9 PMID: 22225728
Authors: Baudoux, A. C. - Hendrix, R. W. - Lander, G. C. - Bailly, X. - Podell, S. - Paillard, C. - Johnson, J. E. - Potter, C. S. - Carragher, B. - Azam, F.
Journal: Environ Microbiol

We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species of great ecological interest including the broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V. harveyi ATTC BAA-1116 and V. campbellii ATCC 25920). Vibrio phage SIO-2 has a circularly permuted genome of 80 598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO-2 capsid, which assembles as a large (80 nm) shell with a novel T = 12 symmetry. These atypical structural features confer on SIO-2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO-2 emerges as a model of considerable interest in ecological and evolutionary studies.

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The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida.

Publication Date: 2012 Jan 9 PMID: 22225632
Authors: Escapa, I. F. - Garcia, J. L. - Buhler, B. - Blank, L. M. - Prieto, M. A.
Journal: Environ Microbiol

The synthesis and degradation of polyhydroxyalkanoates (PHAs), the storage polymer of many bacteria, is linked to the operation of central carbon metabolism. To rationalize the impact of PHA accumulation on central carbon metabolism of the prototype bacterium Pseudomonas putida, we have revisited PHA production in quantitative physiology experiments in the wild-type strain vs. a PHA negative mutant growing under low nitrogen conditions. When octanoic acid was used as PHA precursor and as carbon and energy source, we have detected higher intracellular flux via acetyl-CoA in the mutant strain than in the wild type, which correlates with the stimulation of the TCA cycle and glyoxylate shunt observed on the transcriptional level. The mutant defective in carbon and energy storage spills the additional resources, releasing CO(2) instead of generating biomass. Hence, P. putida operates the metabolic network to optimally exploit available resources and channels excess carbon and energy to storage via PHA, without compromising growth. These findings demonstrate that the PHA metabolism plays a critical role in synchronizing global metabolism to availability of resources in PHA-producing microorganisms.

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Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska.

Publication Date: 2012 Jan 9 PMID: 22225623
Authors: Petersen, D. G. - Blazewicz, S. J. - Firestone, M. - Herman, D. J. - Turetsky, M. - Waldrop, M.
Journal: Environ Microbiol

Nitrification and denitrification processes are crucial to plant nutrient availability, eutrophication and greenhouse gas production both locally and globally. Unravelling the major environmental predictors for nitrification and denitrification is thus pivotal in order to understand and model environmental nitrogen (N) cycling. Here, we sampled five plant community types characteristic of interior Alaska, including black spruce, bog birch, tussock grass and two fens. We assessed abundance of functional genes affiliated with nitrification (bacterial and archaeal amoA) and denitrification (nirK/S and nosZ) using qPCR, soil characteristics, potential nitrification and denitrification rates (PNR and PDR) and gross mineralization rates. The main chemical and biological predictors for PNR and PDR were assigned through path analysis. The potential N cycling rates varied dramatically between sites, from some of the highest (in fens) to some of the lowest (in black spruce) measured globally. Based on path analysis, functional gene abundances were the most important variables to predict potential rates. PNR was best explained by bacterial amoA gene abundance followed by ammonium content, whereas PDR was best explained directly by nosZ gene abundance and indirectly by nirK/S gene abundance and nitrate. Hence, functional gene abundance is a valuable index that integrates recent environmental history and recent process activity, and therefore is a good predictor of potential rates. The results of this study contribute to our understanding of the relative importance of different biological and chemical factors in driving the potential for nitrification and denitrification across terrestrial ecosystems.

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Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression.

Publication Date: 2012 Jan 6 PMID: 22222000
Authors: Blokesch, M.
Journal: Environ Microbiol

Although Vibrio cholerae is a human pathogen its primary habitat are aquatic environments. In this environment, V. cholerae takes advantage of the abundance of zooplankton, whose chitinous exoskeletons provide a nutritious surface. Chitin also induces the developmental programme of natural competence in several species of the genus Vibrio. Because the chitin surface can serve as the sole carbon source for V. cholerae, the link between carbon catabolite repression and chitin-induced natural competence for transformation was investigated in this study. Provision of competing phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS)-dependent carbon sources in addition to chitin significantly lowered natural transformability. These sugars are known to interfere with the accumulation of 3',5'-cyclic AMP (cAMP); therefore, the contributions of the cAMP-producing enzyme, adenylate cyclase and the cAMP receptor protein (CRP) to chitin surface colonization, chitin degradation and natural transformation were also analysed. The results provided here indicate that cAMP and CRP are important in at least three interlinked areas of the chitin-induced natural competence programme. First, cAMP and CRP are required for the efficient colonization of the chitin surface; second both contribute to chitin degradation and utilization, and third, cAMP plus CRP play a role in increasing competence gene expression. These findings highlight the complex regulatory circuit of chitin-induced natural competence in V. cholerae.

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N and C control of ABC-type bicarbonate transporter Cmp and its LysR-type transcriptional regulator CmpR in a heterocyst-forming cyanobacterium, Anabaena sp.

Publication Date: 2012 Jan 6 PMID: 22221957
Authors: Lopez-Igual, R. - Picossi, S. - Lopez-Garrido, J. - Flores, E. - Herrero, A.
Journal: Environ Microbiol

In the model, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, gene cluster alr2877-alr2880, which encodes an ABC-type transport system, was induced under conditions of carbon limitation and its inactivation impaired the uptake of bicarbonate. Thus, this gene cluster encodes a Cmp bicarbonate transporter. ORF all0862, encoding a LysR-type transcriptional regulator, was expressed under carbon limitation and at higher levels in the absence than in the presence of combined nitrogen, with a positive effect of the N-control transcription factor NtcA. all0862 was expressed from two putative transcription start sites located 164 and 64 bp upstream from the gene respectively. The latter was induced under carbon limitation and was dependent on positive autoregulation by All0862. All0862 was required for the induction of the Cmp bicarbonate transporter, thus representing a CmpR regulator of Anabaena sp. These results show a novel mode of co-regulation by C and N availability through the concerted action of N- and C-responsive transcription factors.

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Effect of oxygen on the anaerobic methanotroph 'Candidatus Methylomirabilis oxyfera': kinetic and transcriptional analysis.

Publication Date: 2012 Jan 6 PMID: 22221911
Authors: Luesken, F. A. - Wu, M. L. - Op den Camp, H. J. - Keltjens, J. T. - Stunnenberg, H. - Francoijs, K. J. - Strous, M. - Jetten, M. S.
Journal: Environ Microbiol

'Candidatus Methylomirabilis oxyfera' is a denitrifying methanotroph that performs nitrite-dependent anaerobic methane oxidation through a newly discovered intra-aerobic pathway. In this study, we investigated the response of a M. oxyfera enrichment culture to oxygen. Addition of either 2% or 8% oxygen resulted in an instant decrease of methane and nitrite conversion rates. Oxygen exposure also led to a deviation in the nitrite to methane oxidation stoichiometry. Oxygen-uptake and inhibition studies with cell-free extracts displayed a change from cytochrome c to quinol as electron donor after exposure to oxygen. The change in global gene expression was monitored by deep sequencing of cDNA using Illumina technology. After 24 h of oxygen exposure, transcription levels of 1109 (out of 2303) genes changed significantly when compared with the anoxic period. Most of the genes encoding enzymes of the methane oxidation pathway were constitutively expressed. Genes from the denitrification pathway, with exception of one of the putative nitric oxide reductases, norZ2, were severely downregulated. The majority of known genes involved in the vital cellular functions, such as nucleic acid and protein biosynthesis and cell division processes, were downregulated. The alkyl hydroperoxide reductase, ahpC, and genes involved in the synthesis/repair of the iron-sulfur clusters were among the few upregulated genes. Further, transcription of the pmoCAB genes of aerobic methanotrophs present in the non-M. oxyfera community were triggered by the presence of oxygen. Our results show that oxygen-exposed cells of M. oxyfera were under oxidative stress and that in spite of its oxygenic capacity, exposure to microoxic conditions has an overall detrimental effect.

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Look@NanoSIMS - a tool for the analysis of nanoSIMS data in environmental microbiology.

Publication Date: 2012 Jan 6 PMID: 22221878
Authors: Polerecky, L. - Adam, B. - Milucka, J. - Musat, N. - Vagner, T. - Kuypers, M. M.
Journal: Environ Microbiol

We describe an open-source freeware programme for high throughput analysis of nanoSIMS (nanometre-scale secondary ion mass spectrometry) data. The programme implements basic data processing and analytical functions, including display and drift-corrected accumulation of scanned planes, interactive and semi-automated definition of regions of interest (ROIs), and export of the ROIs' elemental and isotopic composition in graphical and text-based formats. Additionally, the programme offers new functions that were custom-designed to address the needs of environmental microbiologists. Specifically, it allows manual and automated classification of ROIs based on the information that is derived either from the nanoSIMS dataset itself (e.g. from labelling achieved by halogen in situ hybridization) or is provided externally (e.g. as a fluorescence in situ hybridization image). Moreover, by implementing post-processing routines coupled to built-in statistical tools, the programme allows rapid synthesis and comparative analysis of results from many different datasets. After validation of the programme, we illustrate how these new processing and analytical functions increase flexibility, efficiency and depth of the nanoSIMS data analysis. Through its custom-made and open-source design, the programme provides an efficient, reliable and easily expandable tool that can help a growing community of environmental microbiologists and researchers from other disciplines process and analyse their nanoSIMS data.

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Genome sequences of siphoviruses infecting marine Synechococcus unveil a diverse cyanophage group and extensive phage-host genetic exchanges.

Publication Date: 2012 Feb PMID: 22188618
Authors: Huang, S. - Wang, K. - Jiao, N. - Chen, F.
Journal: Environ Microbiol

Investigating the interactions between marine cyanobacteria and their viruses (phages) is important towards understanding the dynamic of ocean's primary productivity. Genome sequencing of marine cyanophages has greatly advanced our understanding about their ecology and evolution. Among 24 reported genomes of cyanophages that infect marine picocyanobacteria, 17 are from cyanomyoviruses and six from cyanopodoviruses, and only one from cyanosiphovirus (Prochlorococcus phage P-SS2). Here we present four complete genome sequences of siphoviruses (S-CBS1, S-CBS2, S-CBS3 and S-CBS4) that infect four different marine Synechococcus strains. Three distinct subtypes were recognized among the five known marine siphoviruses (including P-SS2) in terms of morphology, genome architecture, gene content and sequence similarity. Our study revealed that cyanosiphoviruses are genetically diverse with polyphyletic origin. No core genes were found across these five cyanosiphovirus genomes, and this is in contrast to the fact that many core genes have been found in cyanomyovirus or cyanopodovirus genomes. Interestingly, genes encoding three structural proteins and a lysozyme of S-CBS1 and S-CBS3 showed homology to a prophage-like genetic element in two freshwater Synechococcus elongatus genomes. Re-annotation of the prophage-like genomic region suggests that S. elongatus may contain an intact prophage. Cyanosiphovirus genes involved in DNA metabolism and replication share high sequence homology with those in cyanobacteria, and further phylogenetic analysis based on these genes suggests that ancient and selective genetic exchanges occurred, possibly due to past prophage integration. Metagenomic analysis based on the Global Ocean Sampling database showed that cyanosiphoviruses are present in relatively low abundance in the ocean surface water compared to cyanomyoviruses and cyanopodoviruses.

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Bacterial species may exist, metagenomics reveal.

Publication Date: 2012 Feb PMID: 22151572
Authors: Caro-Quintero, A. - Konstantinidis, K. T.
Journal: Environ Microbiol

Whether or not bacterial species exist remains an unresolved issue of paramount theoretical as well as practical consequences. Here we review and synthesize the findings emerging from metagenomic surveys of natural microbial populations and argue that microbial communities are predominantly organized in genetically and ecologically discernible populations, which possess the attributes expected for species. These sequence-discrete populations represent a major foundation for beginning high-resolution investigations on how populations are organized, interact, and evolve within communities. We also attempt to reconcile these findings with those of previous studies that reported indiscrete species and a genetic continuum within bacterial taxa and discuss the implications for the current bacterial species definition.

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Sponge-specific clusters revisited: a comprehensive phylogeny of sponge-associated microorganisms.

Publication Date: 2012 Feb PMID: 22151434
Authors: Simister, R. L. - Deines, P. - Botte, E. S. - Webster, N. S. - Taylor, M. W.
Journal: Environ Microbiol

Marine sponges often contain diverse and abundant communities of microorganisms including bacteria, archaea and eukaryotic microbes. Numerous 16S rRNA-based studies have identified putative 'sponge-specific' microbes that are apparently absent from seawater and other (non-sponge) marine habitats. With more than 7500 sponge-derived rRNA sequences (from clone, isolate and denaturing gradient gel electrophoresis data) now publicly available, we sought to determine whether the current notion of sponge-specific sequence clusters remains valid. Comprehensive phylogenetic analyses were performed on the 7546 sponge-derived 16S and 18S rRNA sequences that were publicly available in early 2010. Overall, 27% of all sequences fell into monophyletic, sponge-specific sequence clusters. Such clusters were particularly well represented among the Chloroflexi, Cyanobacteria, 'Poribacteria', Betaproteobacteria and Acidobacteria, and in total were identified in at least 14 bacterial phyla, as well as the Archaea and fungi. The largest sponge-specific cluster, representing the cyanobacterium 'Synechococcus spongiarum', contained 245 sequences from 40 sponge species. These results strongly support the existence of sponge-specific microbes and provide a suitable framework for future studies of rare and abundant sponge symbionts, both of which can now be studied using next-generation sequencing technologies.

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TPV1, the first virus isolated from the hyperthermophilic genus Thermococcus.

Publication Date: 2012 Feb PMID: 22151304
Authors: Gorlas, A. - Koonin, E. V. - Bienvenu, N. - Prieur, D. - Geslin, C.
Journal: Environ Microbiol

We describe a novel virus, TPV1 (Thermococcus prieurii virus 1), which was discovered in a hyperthermophilic euryarchaeote isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2700 m at the East Pacific Rise. TPV1 is the first virus isolated and characterized from the hyperthermophilic euryarchaeal genus Thermococcus. TPV1 particles have a lemon-shaped morphology (140 nm x 80 nm) similar to the structures previously reported for Fuselloviruses and for the unclassified virus-like particle PAV1 (Pyrococcus abyssi virus 1). The infection with TPV1 does not cause host lysis and viral replication can be induced by UV irradiation. TPV1 contains a double-stranded circular DNA of 21.5 kb, which is also present in high copy number in a free form in the host cell. The TPV1 genome encompasses 28 predicted genes; the protein sequences encoded in 16 of these genes show no significant similarity to proteins in public databases. Proteins predicted to be involved in genome replication were identified as well as transcriptional regulators. TPV1 encodes also a predicted integrase of the tyrosine recombinase family. The only two genes that are homologous between TPV1 and PAV1 are TPV1-22 and TPV1-23, which encode proteins containing a concanavalin A-like lectin/glucanase domain that might be involved in virus-host recognition.

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amoA-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions.

Publication Date: 2012 Feb PMID: 22141924
Authors: Pester, M. - Rattei, T. - Flechl, S. - Grongroft, A. - Richter, A. - Overmann, J. - Reinhold-Hurek, B. - Loy, A. - Wagner, M.
Journal: Environ Microbiol

Ammonia-oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species-level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species-level and represented 30-37 additional species-level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8-83 co-existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.

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Vertical stratification of subsurface microbial community composition across geological formations at the Hanford Site.

Publication Date: 2012 Feb PMID: 22122741
Authors: Lin, X. - Kennedy, D. - Fredrickson, J. - Bjornstad, B. - Konopka, A.
Journal: Environ Microbiol

Microbial diversity in subsurface sediments at the Hanford Site 300 Area near Richland, Washington state (USA) was investigated by analysing 21 samples recovered from depths of 9-52 m. Approximately 8000 near full-length 16S rRNA gene sequences were analysed across geological strata that include a natural redox transition zone. These strata included the oxic coarse-grained Hanford formation, fine-grained oxic and anoxic Ringold Formation sediments, and the weathered basalt group. We detected 1233 and 120 unique bacterial and archaeal OTUs (operational taxonomic units at the 97% identity level) respectively. Microbial community structure and richness varied substantially across the different geological strata. Bacterial OTU richness (Chao1 estimator) was highest (> 700) in the upper Hanford formation, and declined to about 120 at the bottom of the Hanford formation. Just above the Ringold oxic-anoxic interface, richness was about 325 and declined to less than 50 in the deeper reduced zones. The deeper Ringold strata were characterized by a preponderance (c. 90%) of Proteobacteria. The bacterial community in the oxic sediments contained not only members of nine well-recognized phyla but also an unusually high proportion of three candidate divisions (GAL15, NC10 and SPAM). Additionally, 13 novel phylogenetic orders were identified within the Deltaproteobacteria, a clade rich in microbes that carry out redox transformations of metals that are important contaminants on the Hanford Site.

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Human norovirus occurrence and diversity in the Llobregat river catchment, Spain.

Publication Date: 2012 Feb PMID: 22118046
Authors: Perez-Sautu, U. - Sano, D. - Guix, S. - Kasimir, G. - Pinto, R. M. - Bosch, A.
Journal: Environ Microbiol

Human noroviruses (NoV) were quantified and characterized in an 18 month survey conducted along the Llobregat river catchment in Spain. Sample types included freshwater, untreated and treated wastewater and drinking water. High NoV genome copy numbers were reported, reaching up to 10(6) l(-1) and 10(9) l(-1) in freshwater and raw sewage respectively. In both types of samples, GII NoV genome copies outnumbered those of GI, although without significance. All samples of semi-treated and treated drinking water were negative for NoV. A clear seasonality of NoV occurrence was observed both in river water and sewage samples, with significantly higher genome copy numbers in the cold than in the warm months period. Mean NoV log reduction rates after biological treatment of sewage were 2.2 and 3.1 for GI and GII respectively. A total of 77 NoV strains isolated in the Llobregat river catchment could be phylogenetically characterized, 44 belonging to GI and 33 to GII. The most prevalent genotype was GI.4, followed by GII.4 and GII.21. Several variants of the pandemic GII.4 strain were detected in the environment, corroborating their circulation among the population.

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Microdiversity and evidence for high dispersal rates in the marine actinomycete 'Salinispora pacifica'.

Publication Date: 2012 Feb PMID: 22117917
Authors: Freel, K. C. - Edlund, A. - Jensen, P. R.
Journal: Environ Microbiol

In July of 2006 and January of 2008, a total of 671 marine sediment samples were collected at depths from 5 to 2012 m throughout the Fijian islands and selectively processed for the cultivation of marine actinomycetes belonging to the genus Salinispora. The primary objectives were to assess the diversity, distribution and phylogeny of 'S. pacifica', the least well studied of the three species in the genus. Employing a sequential screening method based on antibiotic sensitivity, RFLP patterns, and 16S rRNA and ITS sequence analyses, 42 of 750 isolates with Salinispora-like features were identified as 'S. pacifica'. These strains represent the first report of 'S. pacifica' from Fiji and include 15 representatives of 4 new 'S. pacifica' 16S rRNA sequence types. Among the 'S. pacifica' strains isolated, little evidence for geographical isolation emerged based on 16S, ITS or secondary metabolite biosynthetic gene fingerprinting. The inclusion of isolates from additional collection sites and other Salinispora spp. revealed a high degree of dispersal among 'S. pacifica' populations and phylogenetic support for the delineation of this lineage as a third species.

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Genetic structure of three fosmid-fragments encoding 16S rRNA genes of the Miscellaneous Crenarchaeotic Group (MCG): implications for physiology and evolution of marine sedimentary archaea.

Publication Date: 2012 Feb PMID: 22117845
Authors: Li, P. Y. - Xie, B. B. - Zhang, X. Y. - Qin, Q. L. - Dang, H. Y. - Wang, X. M. - Chen, X. L. - Yu, J. - Zhang, Y. Z.
Journal: Environ Microbiol

Archaea of the Miscellaneous Crenarchaeotic Group (MCG) exist widely in soil, freshwater and marine sediments of both surface and subsurface. However, current knowledge about this group is limited to its phylogenetic diversity. An archaeal 16S library was constructed from a sediment sample from the South China Sea, which was dominated by MCG and Marine Group I (MG-I). A metagenomic library was constructed from the same sediment sample, and three MCG fosmids (E6-3G, E37-7F and E48-1C) containing 16S rRNA genes were screened. Annotation showed that the three genomic fragments encode a variety of open reading frames (ORFs) that are potentially homologous to important functional genes related to lipid biosynthesis, energy metabolism, and resistance to oxidants. No colinear regions were found between MCG fosmids and reported archaeal genomic fragments or genomes, suggesting that the MCG archaea are quite different from the sequenced archaea in gene arrangement. Analyses of both the phylogenies of 16S rRNA genes and several informational processing genes and nucleotide frequencies showed that MCG archaea are distinct from MG-I plus relatives. In addition, tetranucleotide frequency analysis in combination with phylogenetic analysis suggested that some fragments in the MCG fosmids are probably derived from non-MCG or non-archaeal genomes.

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Analysis of two marine metagenomes reveals the diversity of plasmids in oceanic environments.

Publication Date: 2012 Feb PMID: 22059529
Authors: Ma, Y. - Paulsen, I. T. - Palenik, B.
Journal: Environ Microbiol

Plasmid diversity is still poorly understood in pelagic marine environments. Metagenomic approaches have the potential to reveal the genetic diversity of microbes actually present in an environment and the contribution of mobile genetic elements such as plasmids. By searching metagenomic datasets from flow cytometry-sorted coastal California seawater samples dominated by cyanobacteria (SynMeta) and from the Global Ocean Survey (GOS) putative marine plasmid sequences were identified as well as their possible hosts in the same samples. Based on conserved plasmid replication protein sequences predicted from the SynMeta metagenomes, PCR primers were designed for amplification of one plasmid family and used to confirm that metagenomic contigs of this family were derived from plasmids. These results suggest that the majority of plasmids in SynMeta metagenomes were small and cryptic, encoding mostly their own replication proteins. In contrast, probable plasmid sequences identified in the GOS dataset showed more complexity, consistent with a much more diverse microbial population, and included genes involved in plasmid transfer, mobilization, stability and partitioning. Phylogenetic trees were constructed based on common replication protein functional domains and, even within one replication domain family, substantial diversity was found within and between different samples. However, some replication protein domain families appear to be rare in the marine environment.

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Metagenomic analysis of DNA viruses in a wastewater treatment plant in tropical climate.

Publication Date: 2012 Feb PMID: 22040222
Authors: Tamaki, H. - Zhang, R. - Angly, F. E. - Nakamura, S. - Hong, P. Y. - Yasunaga, T. - Kamagata, Y. - Liu, W. T.
Journal: Environ Microbiol

Viruses have been detected in the different stages of wastewater treatment plants (WWTPs) at concentrations of 10(8) -10(10) ml(-1) of virus-like particles (VLPs), 10-1000 times higher than in natural aquatic environments, suggesting that WWTPs can be considered as an important reservoir and source of viruses. This study revealed novel diversity and function with the DNA viral communities in the influent, activated sludge, anaerobic digester, and effluent of a domestic WWTP using metagenomics. WWTP was a very specific environment, with less than 5% of the > 936 000 metagenomic sequences obtained ( approximately 70-119 Mbp per sample) similar to sequences present in other environmental viromes. Many viruses found in the WWTP were novel, resulting in only < 5-20% of the reads being phylogenetically or functionally assigned. DNA metabolism was observed as the most abundant function with DNA methylase detected at levels 4.2-fold higher than other published viromes, while carbohydrate and amino acids metabolisms were 3.7- and 4.2-fold less abundant respectively. These specific aspects of the WWTP community functions are likely due to high biomass concentration, turnover rate and microbial activity in WWTPs, and likely include mechanisms that help viruses increase their infectivity. Among approximately 500 genotypes estimated in individual WWTP viromes, > 82% were shared. These data suggested that VLPs of most viral types could be present between 1 and 30 days in the process before they were discharged. Viruses in WWTP and the discharged ones can have potential impacts on the functioning of the wastewater treatment system and on the dynamics of microbial community in the surrounding aquatic environments respectively.

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Prokaryotic taxonomy in the sequencing era - the polyphasic approach revisited.

Publication Date: 2012 Feb PMID: 22040009
Authors: Kampfer, P. - Glaeser, S. P.
Journal: Environ Microbiol

The ultimate goal of taxonomy is to establish a system that mirrors the 'order in nature'. In prokaryote microbiology, almost all taxonomic concepts try to mirror the whole evolutionary order back to the origin of life with the cell as basic unit. The introduction of the 16S rRNA gene as molecular marker allowed for the first time the creation of a hierarchical taxonomic system based on one practical molecular marker. With the development of new and rapid sequencing technologies a wealth of new data can and will be used for critical evaluation of the taxonomic system. Comprehensive analyses of other molecular markers as well as total or partial genome comparisons confirmed the 16S rRNA based hierarchical system as 'backbone of prokaryote taxonomy' at least at the genus level and above. A tendency is visible to classify novel taxa more and more based on the genotype, i.e. comparative analyses of 16S rRNA and/or other gene sequence data (in multilocus sequence analysis, MLSA) at the genus and the species level, sometimes contrary to the indications of other (often phenotypic) data. The understanding of all the information behind these data is lagging far behind their accumulation. Genes and genomes do not function on its own and can only display their potential within the cell as the basic unit of evolution (and hence taxonomy). It is the phenotype and the natural selection that 'drive' evolution in a given environment. In this context, the 'polyphasic taxonomic approach' should be revisited again, taking into account the novel insights into genomes and other 'omic' sciences in a more strict and detailed context with the phenotype. This approach allows a more holistic view and provides a sound basis for describing the diversity of prokaryotes and has the potential to become the foundation of a more stable, in-depth taxonomy of the prokaryotes.

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Global network of specific virus-host interactions in hypersaline environments.

Publication Date: 2012 Feb PMID: 22003883
Authors: Atanasova, N. S. - Roine, E. - Oren, A. - Bamford, D. H. - Oksanen, H. M.
Journal: Environ Microbiol

Hypersaline environments are dominated by archaea and bacteria and are almost entirely devoid of eukaryotic organisms. In addition, hypersaline environments contain considerable numbers of viruses. Currently, there is only a limited amount of information about these haloviruses. The ones described in detail mostly resemble head-tail bacteriophages, whereas observations based on direct microscopy of the hypersaline environmental samples highlight the abundance of non-tailed virus-like particles. Here we studied nine spatially distant hypersaline environments for the isolation of new halophilic archaea (61 isolates), halophilic bacteria (24 isolates) and their viruses (49 isolates) using a culture-dependent approach. The obtained virus isolates approximately double the number of currently described archaeal viruses. The new isolates could be divided into three tailed and two non-tailed virus morphotypes, suggesting that both types of viruses are widely distributed and characteristic for haloarchaeal viruses. We determined the sensitivity of the hosts against all isolated viruses. It appeared that the host ranges of numerous viruses extend to hosts in distant locations, supporting the idea that there is a global exchange of microbes and their viruses. It suggests that hypersaline environments worldwide function like a single habitat.

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A novel genus of multicellular magnetotactic prokaryotes from the Yellow Sea.

Publication Date: 2012 Feb PMID: 21978297
Authors: Zhou, K. - Zhang, W. Y. - Yu-Zhang, K. - Pan, H. M. - Zhang, S. D. - Zhang, W. J. - Yue, H. D. - Li, Y. - Xiao, T. - Wu, L. F.
Journal: Environ Microbiol

Multicellular magnetotactic prokaryotes (MMPs) are a group of magnetotactic microorganisms composed of 10-40 Gram-negative cells. Currently, all the identified MMPs show a spherical morphology and synthesize mainly iron sulfide magnetosomes. In this study, we report a novel genus of MMPs with peculiar ellipsoidal morphology and iron oxide magnetosomes, which were discovered in intertidal sediment of the Yellow Sea in China. Optical and fluorescence microscopy revealed that this organism was approximately 10 x 8 microm in size and composed of approximately 40 cells enveloped by an outer layer. Scanning electron microscopy showed that the cells were arranged in 4-6 interlaced circles. Bullet-shaped magnetite magnetosomes were organized in chains roughly parallel to the long axis of the ellipsoidal MMPs when analysed by transmission electron microscopy. These MMPs displayed special escape motility, i.e. swimming rapidly from the edge to the centre of the droplet and then slowly back to the edge. In addition, they exhibited negative phototaxis. Light microscopy observations showed that the ellipsoidal MMPs reproduced by division along the body long axis. Both analysis of 16S rRNA gene sequence and fluorescence in situ hybridization revealed the ellipsoidal MMPs as a new genus of the Deltaproteobacteria. In summary, this novel genus of MMPs exhibit unique morphology, peculiar division process and distinct phylogenetic affiliation compared with the other MMPs.

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Towards a taxonomy of Bacteria and Archaea based on interactive and cumulative data repositories.

Publication Date: 2012 Feb PMID: 21958017
Authors: Rossello-Mora, R.
Journal: Environ Microbiol

Taxonomy in the second decade of the 21st century is benefiting from technological advances in molecular microbiology, especially those related to genomics. Gene and genome databases are significantly increasing due to intense research activities in the field of molecular ecology and genomics. Taxa, and especially species, are tailored by means of the recognition of a phylogenetic, genomic and phenotypic coherence that reveal their uniqueness in the classification schema. Phylogenetic coherence is mainly revealed by means of 16S rRNA gene analyses for which curated databases such as EzTaxon and LTP provide a valuable tool for tree reconstruction to taxonomy users. On the other hand, in silico full or partial genomic sequence comparisons are called on to substitute cumbersome techniques such as DNA-DNA hybridization (DDH) to genomically circumscribe species. DDH similarity values around 70% would be equivalent to ANI values of 96%. Finally, finding an exclusive phenotypic property for the taxa to be classified is of paramount relevance to producing an operative and predictive classification system. The current methods used for taxonomic classification require significant laboratory experimentation, and generally will not produce interactive databases. The new high-throughput metabolomic technologies, such as ICR-FT and MALDI-TOF mass spectrometry methods, open the door to the construction of metabolic databases for taxonomic purposes. It is to be foreseen that, in the future, taxonomists will benefit significantly from public databases speeding up the classification process. However, serious effort will be needed to harmonize them and to prevent inaccurate material.

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Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes.

Publication Date: 2012 Feb PMID: 21923700
Authors: Gihring, T. M. - Green, S. J. - Schadt, C. W.
Journal: Environ Microbiol

Technologies for massively parallel sequencing are revolutionizing microbial ecology and are vastly increasing the scale of ribosomal RNA (rRNA) gene studies. Although pyrosequencing has increased the breadth and depth of possible rRNA gene sampling, one drawback is that the number of reads obtained per sample is difficult to control. Pyrosequencing libraries typically vary widely in the number of sequences per sample, even within individual studies, and there is a need to revisit the behaviour of richness estimators and diversity indices with variable gene sequence library sizes. Multiple reports and review papers have demonstrated the bias in non-parametric richness estimators (e.g. Chao1 and ACE) and diversity indices when using clone libraries. However, we found that biased community comparisons are accumulating in the literature. Here we demonstrate the effects of sample size on Chao1, ACE, CatchAll, Shannon, Chao-Shen and Simpson's estimations specifically using pyrosequencing libraries. The need to equalize the number of reads being compared across libraries is reiterated, and investigators are directed towards available tools for making unbiased diversity comparisons.

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Detecting unknown sequences with DNA microarrays: explorative probe design strategies.

Publication Date: 2012 Feb PMID: 21895914
Authors: Dugat-Bony, E. - Peyretaillade, E. - Parisot, N. - Biderre-Petit, C. - Jaziri, F. - Hill, D. - Rimour, S. - Peyret, P.
Journal: Environ Microbiol

Designing environmental DNA microarrays that can be used to survey the extreme diversity of microorganisms existing in nature, represents a stimulating challenge in the field of molecular ecology. Indeed, recent efforts in metagenomics have produced a substantial amount of sequence information from various ecosystems, and will continue to accumulate large amounts of sequence data given the qualitative and quantitative improvements in the next-generation sequencing methods. It is now possible to take advantage of these data to develop comprehensive microarrays by using explorative probe design strategies. Such strategies anticipate genetic variations and thus are able to detect known and unknown sequences in environmental samples. In this review, we provide a detailed overview of the probe design strategies currently available to construct both phylogenetic and functional DNA microarrays, with emphasis on those permitting the selection of such explorative probes. Furthermore, exploration of complex environments requires particular attention on probe sensitivity and specificity criteria. Finally, these innovative probe design approaches require exploiting newly available high-density microarray formats.

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Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil.

Publication Date: 2012 Feb PMID: 21824242
Authors: Hubert, C. R. - Oldenburg, T. B. - Fustic, M. - Gray, N. D. - Larter, S. R. - Penn, K. - Rowan, A. K. - Seshadri, R. - Sherry, A. - Swainsbury, R. - Voordouw, G. - Voordouw, J. K. - Head, I. M.
Journal: Environ Microbiol

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, > 99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n = 382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n = 96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.

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Multi-locus sequence analysis, taxonomic resolution and biogeography of marine Synechococcus.

Publication Date: 2012 Feb PMID: 21651684
Authors: Mazard, S. - Ostrowski, M. - Partensky, F. - Scanlan, D. J.
Journal: Environ Microbiol

Conserved markers such as the 16S rRNA gene do not provide sufficient molecular resolution to identify spatially structured populations of marine Synechococcus, or 'ecotypes' adapted to distinct ecological niches. Multi-locus sequence analysis targeting seven 'core' genes was employed to taxonomically resolve Synechococcus isolates and correlate previous phylogenetic analyses encompassing a range of markers. Despite the recognized importance of lateral gene transfer in shaping the genomes of marine cyanobacteria, multi-locus sequence analysis of more than 120 isolates reflects a clonal population structure of major lineages and subgroups. A single core genome locus, petB, encoding the cytochrome b(6) subunit of the cytochrome b(6) f complex, was selected to expand our understanding of the diversity and ecology of marine Synechococcus populations. Environmental petB sequences cloned from contrasting sites highlight numerous genetically and ecologically distinct clusters, some of which represent novel, environmentally abundant clades without cultured representatives. With a view to scaling ecological analyses, the short sequence, taxonomic resolution and accurate automated alignment of petB is ideally suited to high-throughput and high-resolution sequencing projects to explore links between the ecology, evolution and biology of marine Synechococcus.

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Marine sponges and their microbial symbionts: love and other relationships.

Publication Date: 2012 Feb PMID: 21443739
Authors: Webster, N. S. - Taylor, M. W.
Journal: Environ Microbiol

Many marine sponges harbour dense and diverse microbial communities of considerable ecological and biotechnological importance. While the past decade has seen tremendous advances in our understanding of the phylogenetic diversity of sponge-associated microorganisms (more than 25 bacterial phyla have now been reported from sponges), it is only in the past 3-4 years that the in situ activity and function of these microbes has become a major research focus. Already the rewards of this new emphasis are evident, with genomics and experimental approaches yielding novel insights into symbiont function. Key steps in the nitrogen cycle [denitrification, anaerobic ammonium oxidation (Anammox)] have recently been demonstrated in sponges for the first time, with diverse bacteria - including the sponge-associated candidate phylum 'Poribacteria'- being implicated in these processes. In this minireview we examine recent major developments in the microbiology of sponges, and identify several research areas (e.g. biology of viruses in sponges, effects of environmental stress) that we believe are deserving of increased attention.

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