CiteULike: Group: microbiology_nijmegen - library [475 articles]

Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase.

N Hasegawa — 2008-08-06T14:28:52-00:00

Biochemical and biophysical research communications, Vol. 288, No. 5. (16 November 2001), pp. 1223-1230.

Three c-type cytochromes, NirM, NirC, and NirN, are encoded in the nirSMCFDLGHJEN gene cluster for cytochrome cd(1)-type nitrite reductase (NIR) of Pseudomonas aeruginosa. nirS is the structural gene for NIR. NirM (cytochrome c(551)) is reported to be a physiological electron donor for nitrite reductase. The respective functions of NirC and NirN have remained unclear. In this study, we produced recombinant NirC and NirN in P. aeruginosa, and purified them from the periplasmic fraction. N-terminal amino acid sequences of the purified proteins showed that the N-terminal 31 and 18 residues of NirC and NirN precursors were cleaved, respectively, indicating that cleaved peptides act as signals for membrane translocation. In addition, the ability of NirC for electron donation to nitrite reductase was investigated. NirC, as well as NirM, was able to mediate the electron donation from the membrane electron pathway to NIR, suggesting that the structural gene for NIR is followed by the genes for two electron donors for NIR.

RibAlign: a software tool and database for eubacterial phylogeny based on concatenated ribosomal protein subunits.

H Teeling — 2008-02-19T21:24:17-00:00

BMC Bioinformatics, Vol. 7 (2006)

BACKGROUND: Until today, analysis of 16S ribosomal RNA (rRNA) sequences has been the de-facto gold standard for the assessment of phylogenetic relationships among prokaryotes. However, the branching order of the individual phlya is not well-resolved in 16S rRNA-based trees. In search of an improvement, new phylogenetic methods have been developed alongside with the growing availability of complete genome sequences. Unfortunately, only a few genes in prokaryotic genomes qualify as universal phylogenetic markers and almost all of them have a lower information content than the 16S rRNA gene. Therefore, emphasis has been placed on methods that are based on multiple genes or even entire genomes. The concatenation of ribosomal protein sequences is one method which has been ascribed an improved resolution. Since there is neither a comprehensive database for ribosomal protein sequences nor a tool that assists in sequence retrieval and generation of respective input files for phylogenetic reconstruction programs, RibAlign has been developed to fill this gap. RESULTS: RibAlign serves two purposes: First, it provides a fast and scalable database that has been specifically adapted to eubacterial ribosomal protein sequences and second, it provides sophisticated import and export capabilities. This includes semi-automatic extraction of ribosomal protein sequences from whole-genome GenBank and FASTA files as well as exporting aligned, concatenated and filtered sequence files that can directly be used in conjunction with the PHYLIP and MrBayes phylogenetic reconstruction programs. CONCLUSION: Up to now, phylogeny based on concatenated ribosomal protein sequences is hampered by the limited set of sequenced genomes and high computational requirements. However, hundreds of full and draft genome sequencing projects are on the way, and advances in cluster-computing and algorithms make phylogenetic reconstructions feasible even with large alignments of concatenated marker genes. RibAlign is a first step in this direction and may be particularly interesting to scientists involved in whole genome sequencing of representatives of new or sparsely studied eubacterial phyla. RibAlign is available at http://www.megx.net/ribalign.

A bacterial metapopulation adapts locally to phage predation despite global dispersal

Victor Kunin — 2008-04-12T02:43:33-00:00

Genome Res., Vol. 18, No. 2. (1 February 2008), pp. 293-297.

Using a combination of bacterial and phage-targeted metagenomics, we analyzed two geographically remote sludge bioreactors enriched in a single bacterial species Candidatus Accumulibacter phosphatis (CAP). We inferred unrestricted global movement of this species and identified aquatic ecosystems as the primary environmental reservoirs facilitating dispersal. Highly related and geographically remote CAP strains differed principally in genomic regions encoding phage defense mechanisms. We found that CAP populations were high density, clonal, and nonrecombining, providing natural targets for "kill-the-winner" phage predation. Community expression analysis demonstrated that phages were consistently active in the bioreactor community. Genomic signatures linking CAP to past phage exposures were observed mostly between local phage and host. We conclude that CAP strains disperse globally but must adapt to phage predation pressure locally. 10.1101/gr.6835308

The Crystal Structure of [Fe]-Hydrogenase Reveals the Geometry of the Active Site

Seigo Shima — 2008-07-28T01:31:43-00:00

Science, Vol. 321, No. 5888. (25 July 2008), pp. 572-575.

Biological formation and consumption of molecular hydrogen (H2) are catalyzed by hydrogenases, of which three phylogenetically unrelated types are known: [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenase. We present a crystal structure of [Fe]-hydrogenase at 1.75 angstrom resolution, showing a mononuclear iron coordinated by the sulfur of cysteine 176, two carbon monoxide (CO) molecules, and the sp2-hybridized nitrogen of a 2-pyridinol compound with back-bonding properties similar to those of cyanide. The three-dimensional arrangement of the ligands is similar to that of thiolate, CO, and cyanide ligated to the low-spin iron in binuclear [NiFe]- and [FeFe]-hydrogenases, although the enzymes have evolved independently and the CO and cyanide ligands are not found in any other metalloenzyme. The related iron ligation pattern of hydrogenases exemplifies convergent evolution and presumably plays an essential role in H2 activation. This finding may stimulate the ongoing synthesis of catalysts that could substitute for platinum in applications such as fuel cells. 10.1126/science.1158978

Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2 — PNAS

Mohamed Baani — 2008-07-17T11:10:26-00:00

Proceedings of the National Academy of Sciences of the USA, Vol. 105, No. 28. (July 2008), pp. 10203-10208.

Methane-oxidizing bacteria (methanotrophs) attenuate methane emission from major sources, such as wetlands, rice paddies, and landfills, and constitute the only biological sink for atmospheric methane in upland soils. Their key enzyme is particulate methane monooxygenase (pMMO), which converts methane to methanol. It has long been believed that methane at the trace atmospheric mixing ratio of 1.75 parts per million by volume (ppmv) is not oxidized by the methanotrophs cultured to date, but rather only by some uncultured methanotrophs, and that type I and type II methanotrophs contain a single type of pMMO. Here, we show that the type II methanotroph Methylocystis sp. strain SC2 possesses two pMMO isozymes with different methane oxidation kinetics. The pmoCAB1 genes encoding the known type of pMMO (pMMO1) are expressed and pMMO1 oxidizes methane only at mixing ratios >600 ppmv. The pmoCAB2 genes encoding pMMO2, in contrast, are constitutively expressed, and pMMO2 oxidizes methane at lower mixing ratios, even at the trace level of atmospheric methane. Wild-type strain SC2 and mutants expressing pmoCAB2 but defective in pmoCAB1 consumed atmospheric methane for >3 months. Growth occurred at 10–100 ppmv methane. Most type II but no type I methanotrophs possess the pmoCAB2 genes. The apparent K m of pMMO2 (0.11 μM) in strain SC2 corresponds well with the K m(app) values for methane oxidation measured in soils that consume atmospheric methane, thereby explaining why these soils are dominated by type II methanotrophs, and some by Methylocystis spp., in particular. These findings change our concept of methanotroph ecology.

Stable carbon isotope fractionation between substrates and products of Methanosarcina barkeri

Kathleen Londry — 2008-07-15T17:47:23-00:00

Organic Geochemistry, Vol. 39, No. 5. (May 2008), pp. 608-621.

Stable carbon isotope ratios are an important tool for understanding methanogenesis in the environment. When applied to biological methanogenesis, interpretation of carbon isotope ratios requires a thorough understanding of how the availability of different substrates affects the eventual [delta]13C of methane, biomass and lipids. Methanosarcina barkeri was grown on four substrates: methanol, trimethylamine (TMA), acetate, and H2/CO2, under variable conditions in which the substrate was either present in excess or limited in availability. The extent of isotopic fractionation between the carbon substrate and the products of M. barkeri was dependent on the substrate type and availability. Growth on unlimited substrate resulted in a range of observed isotope fractionation, with growth on methanol yielding methane, biomass and lipids most depleted in 13C relative to substrate, and growth on acetate yielding the least depleted products. Autotrophic fractionations were intermediate. Substrate-limited growth afforded smaller depletions in 13C on all substrates. There were large differences in the [delta]13C among the M. barkeri lipids produced within each experiment, with the notable exception of growth on acetate. The 13C content of lipids was generally well correlated with that of biomass, with archaeol showing the strongest relationship. The 13C content of individual lipids varied with substrate availability in some cases, but did not show patterns that could be used to identify the growth substrate of methanogens in natural environments.

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification

Christopher Jones — 2008-07-14T08:26:50-00:00

Mol Biol Evol (8 July 2008), msn146.

Denitrification is a facultative respiratory pathway in which nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N2), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility, however these results remain speculative, as they are based on visual comparisons of phylogenies from partial sequences. We re-analyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and Maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. While these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ, makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the dataset of the nirK-harboring genomes. However, inspection of codon usage and G+C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. While HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene. 10.1093/molbev/msn146

Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

Gregory Crowther — 2008-07-13T19:16:09-00:00

J. Bacteriol., Vol. 190, No. 14. (15 July 2008), pp. 5057-5062.

In serine cycle methylotrophs, methylene tetrahydrofolate (H4F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H4F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H4F to generate methylene H4F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H4F via H4F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H4F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H4F and formate are unable to grow on methanol, suggesting that this route for methylene H4F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H4F synthesis pathway through formate. These results suggested that the methylene H4F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1. 10.1128/JB.00228-08

Preferential Use of an Anode as an Electron Acceptor by an Acidophilic Bacterium in the Presence of Oxygen

Moustafa Malki — 2008-07-13T19:10:36-00:00

Appl. Environ. Microbiol., Vol. 74, No. 14. (15 July 2008), pp. 4472-4476.

Several anaerobic metal-reducing bacteria have been shown to be able to donate electrons directly to an electrode. This property is of great interest for microbial fuel cell development. To date, microbial fuel cell design requires avoiding O2 diffusion from the cathodic compartment to the sensitive anodic compartment. Here, we show that Acidiphilium sp. strain 3.2 Sup 5 cells that were isolated from an extreme acidic environment are able to colonize graphite felt electrodes. These bacterial electrodes were able to produce high-density electrocatalytic currents, up to 3 A/m2 at a poised potential of +0.15 V (compared to the value for the reference standard calomel electrode) in the absence of redox mediators, by oxidizing glucose even at saturating air concentrations and very low pHs. 10.1128/AEM.00209-08

Genetic Determinants of Self Identity and Social Recognition in Bacteria

Karine Gibbs — 2008-07-11T20:20:47-00:00

Science, Vol. 321, No. 5886. (11 July 2008), pp. 256-259.

The bacterium Proteus mirabilis is capable of movement on solid surfaces by a type of motility called swarming. Boundaries form between swarming colonies of different P. mirabilis strains but not between colonies of a single strain. A fundamental requirement for boundary formation is the ability to discriminate between self and nonself. We have isolated mutants that form boundaries with their parent. The mutations map within a six-gene locus that we term ids for identification of self. Five of the genes in the ids locus are required for recognition of the parent strain as self. Three of the ids genes are interchangeable between strains, and two encode specific molecular identifiers. 10.1126/science.1160033

Calling on a million minds for community annotation in WikiProteins

Barend Mons — 2008-05-28T10:54:00-00:00

Genome Biology, Vol. 9, No. 5. (2008)

WikiProteins enables Community Annotation in a Wiki-based system. Extracts of major data sources have been fused into an editable environment with a link out to the original sources. Data from Community edits create automatic copies of the original data. Semantic technology captures concepts co-occurring in one sentence and thus potential factual statements. In addition, indirect associations between concepts have been calculated. We call on a 'million minds' to annotate a 'million concepts' and to collect facts from the literature with the reward of collaborative knowledge discovery.

Reactive Oxygen Species Special Feature: Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling

Pablo Castello — 2008-06-18T08:59:29-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 24. (17 June 2008), pp. 8203-8208.

Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (NO2-)-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the NO2--dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on NO2--dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells. 10.1073/pnas.0709461105

Selectivity of bacterial proteome fractionation based on differential solubility: A mass spectrometry evaluation

Yassel Ramos — 2008-06-13T07:02:27-00:00

Analytical Biochemistry, Vol. 377, No. 2. (15 June 2008), pp. 134-140.

We investigate the selectivity achieved after differential solubilization of bacterial proteomes following two procedures, both based on successive extraction of proteins in solutions of increasing solubilizing power. Recently, these procedures have gained notable popularity and several commercial kits are now available. A total of 225 proteins in one case and 227 proteins in the other were identified by LC MSMS analysis; 146 of them were identified in both procedures. The proportions of proteins identified as present in only one fraction were 64 and 57%, respectively. The distribution of cytosolic, membrane, and ribosomal proteins among the successive extracts was analyzed in detail. The effect of (1) replacement of low-speed with high-speed centrifugation, (2) omission of detergents in urea solutions, (3) successive washes of pellets, and (4) reproducibility was evaluated. Proteins with positive grand averages of hydropathicity values and membrane proteins were found in all fractions. This study highlights the benefits and limitations of differential solubilization methods, focusing on practical aspects that may strongly influence their selectivity.

Characterization and expression analysis of the cytochrome bd oxidase operon from Desulfovibrio gigas.

P Machado — 2008-06-03T18:22:48-00:00

Current microbiology, Vol. 52, No. 4. (April 2006), pp. 274-281.

Although classified as anaerobic, Desulfovibrio gigas contains a functional canonical membrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In the present study, we report the identification of the operon cydAB encoding the two subunits of cytochrome bd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptional regulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity to cytochrome bd from other organisms, presenting the conserved residues typical from these proteins. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed the operon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in different media and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in the presence of 150 microM: NO. However, in the presence of 10 microM: NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 microM: O2 or 10 microM: NO.

Desulfovibrio gigas flavodiiron protein affords protection against nitrosative stress in vivo.

R Rodrigues — 2008-06-03T18:21:10-00:00

Journal of bacteriology, Vol. 188, No. 8. (April 2006), pp. 2745-2751.

Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O(2). Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas Deltaroo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O(2) reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.

Cholest-4-en-3-one-delta 1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism.

YR Chiang — 2008-06-03T18:19:38-00:00

Applied and environmental microbiology, Vol. 74, No. 1. (January 2008), pp. 107-113.

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Delta(1)-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Delta(1)-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Delta(1)-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.

Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans.

YR Chiang — 2008-06-03T18:18:48-00:00

Journal of bacteriology, Vol. 190, No. 3. (February 2008), pp. 905-914.

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1S(T). The second enzyme of the proposed pathway, cholest-4-en-3-one-Delta1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

Initial steps in the anoxic metabolism of cholesterol by the denitrifying Sterolibacterium denitrificans.

YR Chiang — 2008-06-03T18:17:51-00:00

The Journal of biological chemistry, Vol. 282, No. 18. (4 May 2007), pp. 13240-13249.

The anoxic metabolism of the ubiquitous triterpene cholesterol is challenging because of its complex chemical structure, low solubility in water, low number of active functional groups, and the presence of four alicyclic rings and two quaternary carbon atoms. Consequently, the aerobic metabolism depends on oxygenase catalyzed reactions requiring molecular oxygen as co-substrate. Sterolibacterium denitrificans is shown to metabolize cholesterol anoxically via the oxidation of ring A, followed by an oxygen-independent hydroxylation of the terminal C-25 of the side chain. The anaerobic hydroxylation of a tertiary carbon using water as oxygen donor is unprecedented and may be catalyzed by a novel molybdenum containing enzyme.

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase.

A Alfieri — 2008-06-03T18:16:29-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 4. (23 January 2007), pp. 1177-1182.

p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-A radius and a 29-A3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. The shape and position of this cavity are perfectly fit for housing the oxygen atoms of the flavin C4a-hydroperoxide intermediate that is formed upon reaction of the C2-bound reduced flavin with molecular oxygen. The side chain of His-396 is predicted to act as a hydrogen-bond donor to the oxygen atoms of the intermediate. This architecture promotes the nucleophilic attack of the substrate onto the terminal oxygen of the hydroperoxyflavin. Comparative analysis with the structures of other flavoenzymes indicates that a distinctive feature of monooxygenases is the presence of specific cavities that encapsulate and stabilize the crucial hydroperoxyflavin intermediate.

Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase.

L Li — 2008-06-03T18:15:35-00:00

Journal of molecular biology, Vol. 376, No. 2. (15 February 2008), pp. 453-465.

LadA, a long-chain alkane monooxygenase, utilizes a terminal oxidation pathway for the conversion of long-chain alkanes (up to at least C(36)) to corresponding primary alcohols in thermophilic bacillus Geobacillus thermodenitrificans NG80-2. Here, we report the first structure of the long-chain alkane hydroxylase, LadA, and its complex with the flavin mononucleotide (FMN) coenzyme. LadA is characterized as a new member of the SsuD subfamily of the bacterial luciferase family via a surprising structural relationship. The LadA:FMN binary complex structure and a LadA:FMN:alkane model reveal a hydrophobic cavity that has dual roles: to provide a hydrogen-bond donor (His138) for catalysis and to create a solvent-free environment in which to stabilize the C4a-hydroperoxyflavin intermediate. Consequently, LadA should catalyze the conversion of long-chain alkanes via the acknowledged flavoprotein monooxygenase mechanism. This finding suggests that the ability of LadA to catalyze the degradation of long-chain alkanes is determined by the binding mode of the long-chain alkane substrates. The LadA structure opens a rational perspective to explore and alter the substrate binding site of LadA, with potential biotechnological applications in areas such as petroleum exploration and treatment of environmental oil pollution.

Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir.

L Feng — 2008-06-03T18:15:04-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 13. (27 March 2007), pp. 5602-5607.

The complete genome sequence of Geobacillus thermodenitrificans NG80-2, a thermophilic bacillus isolated from a deep oil reservoir in Northern China, consists of a 3,550,319-bp chromosome and a 57,693-bp plasmid. The genome reveals that NG80-2 is well equipped for adaptation into a wide variety of environmental niches, including oil reservoirs, by possessing genes for utilization of a broad range of energy sources, genes encoding various transporters for efficient nutrient uptake and detoxification, and genes for a flexible respiration system including an aerobic branch comprising five terminal oxidases and an anaerobic branch comprising a complete denitrification pathway for quick response to dissolved oxygen fluctuation. The identification of a nitrous oxide reductase gene has not been previously described in Gram-positive bacteria. The proteome further reveals the presence of a long-chain alkane degradation pathway; and the function of the key enzyme in the pathway, the long-chain alkane monooxygenase LadA, is confirmed by in vivo and in vitro experiments. The thermophilic soluble monomeric LadA is an ideal candidate for treatment of environmental oil pollutions and biosynthesis of complex molecules.

TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7.

D Han — 2008-06-03T18:13:11-00:00

Proteins, Vol. 70, No. 3. (15 February 2008), pp. 900-914.

Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.

Temporal evolution of methane cycling and phylogenetic diversity of archaea in sediments from a deep-sea whale-fall in Monterey Canyon, California.

Shana K Goffredi — 2008-01-27T22:06:30-00:00

ISME J (24 January 2008)

Whale-falls represent localized areas of extreme organic enrichment in an otherwise oligotrophic deep-sea environment. Anaerobic remineralization within these habitats is typically portrayed as sulfidogenic; however, we demonstrate that these systems are also favorable for diverse methane-producing archaeal assemblages, representing up to 40% of total cell counts. Chemical analyses revealed elevated methane and depleted sulfate concentrations in sediments under the whale-fall, as compared to surrounding sediments. Carbon was enriched (up to 3.5%) in whale-fall sediments, as well as the surrounding sea floor to at least 10 m, forming a 'bulls eye' of elevated carbon. The diversity of sedimentary archaea associated with the 2893 m whale-fall in Monterey Canyon (California) varied both spatially and temporally. 16S rRNA diversity, determined by both sequencing and terminal restriction fragment length polymorphism analysis, as well as quantitative PCR of the methyl-coenzyme M reductase gene, revealed that methanogens, including members of the Methanomicrobiales and Methanosarcinales, were the dominant archaea (up to 98%) in sediments immediately beneath the whale-fall. Temporal changes in this archaeal community included the early establishment of methylotrophic methanogens followed by development of methanogens thought to be hydrogenotrophic, as well as members related to the newly described methanotrophic lineage, ANME-3. In comparison, archaeal assemblages in 'reference' sediments collected 10 m from the whale-fall primarily consisted of Crenarchaeota affiliated with marine group I and marine benthic group B. Overall, these results indicate that whale-falls can favor the establishment of metabolically and phylogenetically diverse methanogen assemblages, resulting in an active near-seafloor methane cycle in the deep sea.The ISME Journal advance online publication, 24 January 2008; doi:10.1038/ismej.2007.103.

Analysis of the respiratory chain in Ethanologenic Zymomonas mobilis with a cyanide-resistant bd-type ubiquinol oxidase as the only terminal oxidase and its possible physiological roles.

K Sootsuwan — 2008-06-03T18:11:45-00:00

Journal of molecular microbiology and biotechnology, Vol. 14, No. 4. (2008), pp. 163-175.

The respiratory chain of the ethanologenic bacterium Zymomonas mobilis was investigated, in which the pyruvate-to-ethanol pathway has been demonstrated to be mainly responsible for NADH oxidation and the tricarboxylic acid cycle is incomplete. Membranes from cells cultivated under aerobic or anaerobic growth conditions showed dehydrogenase and oxidase activities for NADH, D-lactate and D-glucose and ubiquinol oxidase activity. Intriguingly, the NADH oxidase activity level of membrane fractions from cells grown aerobically was found to be higher than that of membrane fractions from Escherichia coli or Pseudomonas putida grown aerobically, indicating a crucial role of the respiratory chain in NADH oxidation in the organism. Cyanide-resistant terminal oxidase activity was observed and appeared to be due to a bd-type ubiquinol oxidase as the only terminal oxidase encoded by the entire genome. The terminal oxidase with a relatively strong ubiquinol oxidase activity exhibited remarkably weak signals of cytochrome d. Considering these findings and the presence of a type-II NADH dehydrogenase but not a type-I, a simple respiratory chain that generates less energymay have evolved in Z. mobilis.

Cytochrome bd oxidase, oxidative stress, and dioxygen tolerance of the strictly anaerobic bacterium Moorella thermoacetica.

A Das — 2008-06-03T18:09:23-00:00

Journal of bacteriology, Vol. 187, No. 6. (March 2005), pp. 2020-2029.

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (approximately 70%) inhibited both the endogenous and CO/methanol-dependent O2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-beta-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b561, cytochrome b595, and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine + H2S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.

Presence and expression of terminal oxygen reductases in strictly anaerobic sulfate-reducing bacteria isolated from salt-marsh sediments.

Margarida Santana — 2008-06-03T18:08:08-00:00

Anaerobe (25 March 2008)

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough genes were found encoding membrane terminal oxygen reductases of two types: a cytochrome c oxidase and a cytochrome bd oxidase, both enzymes are terminal oxidases typical of facultative or aerobic microorganisms (Heidelberg JF, et al., The genome sequence of the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough. Nat Biotechnol 2004; 22: 554-9). To apprehend the presence of both oxidases in other sulfate-reducing bacteria (SRB), several assays were performed on isolates recovered from salt-marsh sediments in Portugal, representative of the different phylogenetic groups identified. Hybridization and PCR experiments for DNA sequencing were performed on the chosen isolates. Primers were selected to amplify conserved regions of cytochrome c oxidases and cytochrome bd oxidases taking into consideration alignment of corresponding subunit I sequences. The results showed that both oxidase genes are present on the chromosome of several isolates characterized as Desulfovibrio. These genes were shown to be transcribed, as demonstrated by Reverse Transcriptase-PCR experiments on total RNA. In order to assess the relative contribution of each oxidase to oxygen consumption, oxygen uptake was measured for each isolate and further characterized by the effect of cyanide on oxygen consumption. It was concluded that cytochrome bd oxidase was the terminal membrane oxygen reductase allowing oxygen consumption. In addition, it was observed that isolates containing cytochrome bd oxidase had higher resistance to air exposure, suggesting an important role of this enzyme in survival to air exposure. The pattern for the presence of oxygen reductase genes was compared to the physiological pattern of substrate use, which was determined for each isolate. Salinity tolerance, pH and temperature growth of each isolate were also analyzed.

NOX family NADPH oxidases: not just in mammals.

K Bedard — 2008-06-03T18:06:27-00:00

Biochimie, Vol. 89, No. 9. (September 2007), pp. 1107-1112.

NOX family NADPH oxidases are enzymes whose biological function is electron transport and the generation of reactive oxygen species (ROS). NOX enzymes in mammalian organisms have received most attention. However, NOX enzymes are widely distributed in different kingdoms of life. While they are not found in prokaryotes and most unicellular eukaryotes, they are present in fungi, plants, and animals. The identity of the ancestral NOX is not known, but most likely it: (i) possessed the basic NOX structure consisting of 6 transmembrane domains (containing two assymmetrical hemes) and a long cytoplasmic C-terminal (containing the FAD and NADPH binding sites); and (ii) emerged before the divergence of life into fungi, plants, and animals. During evolution, acquisition of a Ca(2+)-binding EF hand domain by an ancestral NOX, led to NOX5-like isoforms. DUOX isoforms presumably developed from a NOX5-like isoform through the additional acquisition of a peroxidase homology domain. The expression pattern of NOX enzymes is specific to each kingdom of life. Fungi express only ancestral-type isoforms, and plants only NOX5-like isoforms. NOX expression patterns in animals are complex and ancestral NOXes, NOX5-like isoforms and DUOXes are generally found. But there are exceptions; for example rodents lack NOX5 and Caenorhabditis elegans expresses only DUOXes. Biological functions of NOX enzymes include, among others, host defense, post-translational modification of proteins, and regulation cell growth and differentiation. In summary, the invention of NOX enzymes early in the development of life was a success story: there is no evidence of multicellular life without NOX enzymes.

Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes

Tsukasa Kawahara — 2007-07-07T15:19:10-00:00

BMC Evolutionary Biology, Vol. 7 (06 July 2007), 109.

Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species.

Hideki Sumimoto — 2008-06-03T18:04:06-00:00

The FEBS journal (30 May 2008)

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc(1) complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca(2+)-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin-NADP(+) reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1-4 subfamily in animals form a stable heterodimer with the membrane protein p22(phox), which functions as a docking site for the SH3 domain-containing regulatory proteins p47(phox), p67(phox), and p40(phox); the small GTPase Rac binds to p67(phox) (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67(phox)-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation.

Metagenomic studies reveal the critical and wide-ranging ecological importance of uncultivated archaea: the role of ammonia oxidizers

Ricardo Cavicchioli — 2007-02-26T22:18:32-00:00

BioEssays, Vol. 29, No. 1. (2007), pp. 11-14.

Microbial genome sequencing has entered a new phase, where DNA sequence information is gathered from entire microbial communities (metagenomics or environmental genomics) rather than from individual microorganisms. By providing access to the genetic material of vast numbers of organisms, most of which are organisms that have never been isolated or cultivated, a new level of insight is being gained into the diversity and extent of the microbial processes that are presently occuring in environmental communities. By extending metagenomic-based approaches to the study of very complex and methodologically recalcitrant soil environments, a recent study has found that ammonia-oxidizing archaea are more abundant in many soils than bacteria. These findings not only highlight the undoubtedly critical yet unknown roles that archaea play in global nutrient cycles but illustrate the importance of genomic studies for informing us about the functional capacity of life on Earth. BioEssays 29: 11-14, 2007. © 2006 Wiley Periodicals, Inc.

Building on basic metagenomics with complementary technologies

Falk Warnecke — 2007-12-29T10:56:51-00:00

Genome Biology, Vol. 8 (28 December 2007), 231.

Tracking microbial biodiversity through molecular and genomic ecology.

P López-García — 2008-06-03T12:18:47-00:00

Research in microbiology, Vol. 159, No. 1. (b 2008), pp. 67-73.

Molecular ecology and metagenomics applied to the study of microbial biodiversity are changing our comprehension of the biosphere. An impressive diversity of archaea, bacteria and, more recently, protists has been uncovered by molecular tools. Efforts to couple function to the phylogenetic diversity observed in natural environments are leading to the discovery of novel metabolisms and to a re-evaluation of the global ecological impact of known ones. Interesting questions relating to mechanisms of speciation and evolutionary trends at the smallest and largest phylogenetic scales are emerging.

Bacterial carbon processing by generalist species in the coastal ocean

Xiaozhen Mou — 2008-01-31T11:58:30-00:00

Nature (27 January 2008)

Toward an Online Repository of Standard Operating Procedures (SOPs) for (Meta)genomic Annotation.

Samuel V Angiuoli — 2008-06-03T12:16:59-00:00

Omics : a journal of integrative biology (16 April 2008)

Abstract The methodologies used to generate genome and metagenome annotations are diverse and vary between groups and laboratories. Descriptions of the annotation process are helpful in interpreting genome annotation data. Some groups have produced Standard Operating Procedures (SOPs) that describe the annotation process, but standards are lacking for structure and content of these descriptions. In addition, there is no central repository to store and disseminate procedures and protocols for genome annotation. We highlight the importance of SOPs for genome annotation and endorse an online repository of SOPs.

MEGAN analysis of metagenomic data.

DH Huson — 2008-02-14T19:21:34-00:00

Genome Res, Vol. 17, No. 3. (March 2007), pp. 377-386.

Metagenomics is the study of the genomic content of a sample of organisms obtained from a common habitat using targeted or random sequencing. Goals include understanding the extent and role of microbial diversity. The taxonomical content of such a sample is usually estimated by comparison against sequence databases of known sequences. Most published studies use the analysis of paired-end reads, complete sequences of environmental fosmid and BAC clones, or environmental assemblies. Emerging sequencing-by-synthesis technologies with very high throughput are paving the way to low-cost random "shotgun" approaches. This paper introduces MEGAN, a new computer program that allows laptop analysis of large metagenomic data sets. In a preprocessing step, the set of DNA sequences is compared against databases of known sequences using BLAST or another comparison tool. MEGAN is then used to compute and explore the taxonomical content of the data set, employing the NCBI taxonomy to summarize and order the results. A simple lowest common ancestor algorithm assigns reads to taxa such that the taxonomical level of the assigned taxon reflects the level of conservation of the sequence. The software allows large data sets to be dissected without the need for assembly or the targeting of specific phylogenetic markers. It provides graphical and statistical output for comparing different data sets. The approach is applied to several data sets, including the Sargasso Sea data set, a recently published metagenomic data set sampled from a mammoth bone, and several complete microbial genomes. Also, simulations that evaluate the performance of the approach for different read lengths are presented.

Use of simulated data sets to evaluate the fidelity of metagenomic processing methods.

Konstantinos Mavromatis — 2007-05-17T17:08:06-00:00

Nat Methods (29 April 2007)

Metagenomics is a rapidly emerging field of research for studying microbial communities. To evaluate methods presently used to process metagenomic sequences, we constructed three simulated data sets of varying complexity by combining sequencing reads randomly selected from 113 isolate genomes. These data sets were designed to model real metagenomes in terms of complexity and phylogenetic composition. We assembled sampled reads using three commonly used genome assemblers (Phrap, Arachne and JAZZ), and predicted genes using two popular gene-finding pipelines (fgenesb and CRITICA/GLIMMER). The phylogenetic origins of the assembled contigs were predicted using one sequence similarity-based (blast hit distribution) and two sequence composition-based (PhyloPythia, oligonucleotide frequencies) binning methods. We explored the effects of the simulated community structure and method combinations on the fidelity of each processing step by comparison to the corresponding isolate genomes. The simulated data sets are available online to facilitate standardized benchmarking of tools for metagenomic analysis.

Binning sequences using very sparse labels within a metagenome

Chon-Kit Chan — 2008-04-29T09:23:48-00:00

BMC Bioinformatics, Vol. 9 (28 April 2008), 215.

Gene prediction in metagenomic fragments: a large scale machine learning approach

Katharina Hoff — 2008-04-29T09:23:48-00:00

BMC Bioinformatics, Vol. 9 (28 April 2008), 217.

Metagenomics is not enough.

J Handelsman — 2008-06-03T12:11:10-00:00

DNA and cell biology, Vol. 27, No. 5. (May 2008), pp. 219-221.

Community Genomics Among Stratified Microbial Assemblages in the Ocean's Interior

Edward Delong — 2006-01-27T15:51:55-00:00

Science, Vol. 311, No. 5760. (27 January 2006), pp. 496-503.

Microbial life predominates in the ocean, yet little is known about its genomic variability, especially along the depth continuum. We report here genomic analyses of planktonic microbial communities in the North Pacific Subtropical Gyre, from the ocean's surface to near-sea floor depths. Sequence variation in microbial community genes reflected vertical zonation of taxonomic groups, functional gene repertoires, and metabolic potential. The distributional patterns of microbial genes suggested depth-variable community trends in carbon and energy metabolism, attachment and motility, gene mobility, and host-viral interactions. Comparative genomic analyses of stratified microbial communities have the potential to provide significant insight into higher-order community organization and dynamics.

Recent advances in molecular techniques for the detection of phylogenetic markers and functional genes in microbial communities

Stanley Lau — 2008-04-18T18:53:15-00:00

FEMS Microbiology Letters, Vol. 275, No. 2. (2007), pp. 183-190.

Abstract The detection and analysis of nucleic acids extracted from microbial communities are the ultimate ways to determine the diversity and functional capability of microbial communities in the environments. However, it remains a challenge to use molecular techniques for unequivocal determination and quantification of microbial species composition and functional activities. Considerable efforts have been made to enhance the capability of molecular techniques. Here an update of the recent developments in molecular techniques for environmental microbiology is provided.

Quantitative Phylogenetic Assessment of Microbial Communities in Diverse Environments

C von Mering — 2007-02-26T09:46:57-00:00

Science, Vol. 315, No. 5815. (23 February 2007), pp. 1126-1130.

The taxonomic composition of environmental communities is an important indicator of their ecology and function. We used a set of protein-coding marker genes, extracted from large-scale environmental shotgun sequencing data, to provide a more direct, quantitative, and accurate picture of community composition than that provided by traditional ribosomal RNA-based approaches depending on the polymerase chain reaction. Mapping marker genes from four diverse environmental data sets onto a reference species phylogeny shows that certain communities evolve faster than others. The method also enables determination of preferred habitats for entire microbial clades and provides evidence that such habitat preferences are often remarkably stable over time. 10.1126/science.1133420

Comparative Metagenomics of Microbial Communities

Susannah Tringe — 2006-06-19T04:39:47-00:00

Science, Vol. 308, No. 5721. (22 April 2005), pp. 554-557.

The species complexity of microbial communities and challenges in culturing representative isolates make it difficult to obtain assembled genomes. Here we characterize and compare the metabolic capabilities of terrestrial and marine microbial communities using largely unassembled sequence data obtained by shotgun sequencing DNA isolated from the various environments. Quantitative gene content analysis reveals habitat-specific fingerprints that reflect known characteristics of the sampled environments. The identification of environment-specific genes through a gene-centric comparative analysis presents new opportunities for interpreting and diagnosing environments. 10.1126/science.1107851

Proteogenomic approaches for the molecular characterization of natural microbial communities.

JF Banfield — 2006-07-27T21:27:20-00:00

OMICS, Vol. 9, No. 4. (2005), pp. 301-333.

At the present time we know little about how microbial communities function in their natural habitats. For example, how do microorganisms interact with each other and their physical and chemical surroundings and respond to environmental perturbations? We might begin to answer these questions if we could monitor the ways in which metabolic roles are partitioned amongst members as microbial communities assemble, determine how resources such as carbon, nitrogen, and energy are allocated into metabolic pathways, and understand the mechanisms by which organisms and communities respond to changes in their surroundings. Because many organisms cannot be cultivated, and given that the metabolisms of those growing in monoculture are likely to differ from those of organisms growing as part of consortia, it is vital to develop methods to study microbial communities in situ. Chemoautotrophic biofilms growing in mine tunnels hundreds of meters underground drive pyrite (FeS(2)) dissolution and acid and metal release, creating habitats that select for a small number of organism types. The geochemical and microbial simplicity of these systems, the significant biomass, and clearly defined biological-inorganic feedbacks make these ecosystem microcosms ideal for development of methods for the study of uncultivated microbial consortia. Our approach begins with the acquisition of genomic data from biofilms that are sampled over time and in different growth conditions. We have demonstrated that it is possible to assemble shotgun sequence data to reveal the gene complement of the dominant community members and to use these data to confidently identify a significant fraction of proteins from the dominant organisms by mass spectrometry (MS)-based proteomics. However, there are technical obstacles currently restricting this type of "proteogenomic" analysis. Composite genomic sequences assembled from environmental data from natural microbial communities do not capture the full range of genetic potential of the associated populations. Thus, it is necessary to develop bioinformatics approaches to generate relatively comprehensive gene inventories for each organism type. These inventories are critical for expression and functional analyses. In proteomic studies, for example, peptides that differ from those predicted from gene sequences can be measured, but they generally cannot be identified by database matching, even if the difference is only a single amino acid residue. Furthermore, many of the identified proteins have no known function. We propose that these challenges can be addressed by development of proteogenomic, biochemical, and geochemical methods that will be initially deployed in a simple, natural model ecosystem. The resulting approach should be broadly applicable and will enhance the utility and significance of genomic data from isolates and consortia for study of organisms in many habitats. Solutions draining pyrite-rich deposits are referred to as acid mine drainage (AMD). AMD is a very prevalent, international environmental problem associated with energy and metal resources. The biological-mineralogical interactions that define these systems can be harnessed for energy-efficient metal recovery and removal of sulfur from coal. The detailed understanding of microbial ecology and ecosystem dynamics resulting from the proposed work will provide a scientific foundation for dealing with the environmental challenges and technological opportunities, and yield new methods for analysis of more complex natural communities.

Toward a census of bacteria in soil.

PD Schloss — 2008-03-25T13:50:10-00:00

PLoS Comput Biol, Vol. 2, No. 7. (21 July 2006)

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome.

A statistical toolbox for metagenomics: assessing functional diversity in microbial communities

Patrick Schloss — 2008-01-24T03:54:24-00:00

BMC Bioinformatics, Vol. 9 (23 January 2008), 34.

Opinion: Whither or wither geomicrobiology in the era of 'community metagenomics'

Ronald Oremland — 2005-07-01T19:24:26-00:00

Nature Reviews Microbiology, Vol. 3, No. 7. (01 July 2005), pp. 572-578.

Signature, a web server for taxonomic characterization of sequence samples using signature genes.

Bas E Dutilh — 2008-06-03T11:52:05-00:00

Nucleic acids research (23 May 2008)

Signature genes are genes that are unique to a taxonomic clade and are common within it. They contain a wealth of information about clade-specific processes and hold a strong evolutionary signal that can be used to phylogenetically characterize a set of sequences, such as a metagenomics sample. As signature genes are based on gene content, they provide a means to assess the taxonomic origin of a sequence sample that is complementary to sequence-based analyses. Here, we introduce Signature (http://www.cmbi.ru.nl/signature), a web server that identifies the signature genes in a set of query sequences, and therewith phylogenetically characterizes it. The server produces a list of taxonomic clades that share signature genes with the set of query sequences, along with an insightful image of the tree of life, in which the clades are color coded based on the number of signature genes present. This allows the user to quickly see from which part(s) of the taxonomy the query sequences likely originate.

Signature Genes as a Phylogenomic Tool.

Bas E Dutilh — 2008-06-03T11:51:43-00:00

Molecular biology and evolution (19 May 2008)

Gene content has been shown to contain a strong phylogenetic signal, yet its usage for phylogenetic questions is hampered by horizontal gene transfer and parallel gene loss, and until now required completely sequenced genomes. Here, we introduce an approach that allows the phylogenetic signal in gene content to be applied to any set of sequences, using signature genes for phylogenetic classification. The hundreds of publicly available genomes allow us to identify signature genes at various taxonomic depths, and we show how the presence of signature genes in an unspecified sample can be used to characterize its taxonomic composition. We identify 8,362 signature genes specific for 112 prokaryotic taxa. We show that these signature genes can be used to address phylogenetic questions on the basis of gene content in cases where classic gene content or sequence analyses provide an ambiguous answer, such as for Nanoarchaeum equitans, and even in cases where complete genomes are not available, such as for metagenomics data. Cross-validation experiments leaving out up to 30% of the species show that approximately 92% of the signature genes correctly place the species in a related clade. Analyses of metagenomics data sets with the signature gene approach are in good agreement with the previously reported species distributions based on phylogenetic analysis of marker genes. Summarising, signature genes can complement traditional sequence based methods in addressing taxonomic questions.

Metagenomics: Exploring unseen communities

Nathan Blow — 2008-05-29T14:31:11-00:00

Nature, Vol. 453, No. 7195. (28 May 2008), pp. 687-690.