Metaproteogenomic analysis of a community of sponge symbionts

Sponges harbour complex communities of diverse microorganisms, which have been postulated to form intimate symbiotic relationships with their host. Here we unravel some of these interactions by characterising the functional features of the microbial community of the sponge Cymbastela concentrica through a combined metagenomic and metaproteomic approach. We discover the expression of specific transport functions for typical sponge metabolites (for example, halogenated aromatics, dipeptides), which indicates metabolic interactions between the community and the host. We also uncover the simultaneous performance of aerobic nitrification and anaerobic denitrification, which would aid to remove ammonium secreted by the sponge. Our analysis also highlights the requirement for the microbial community to respond to variable environmental conditions and hence express an array of stress protection proteins. Molecular interactions between symbionts and their host might also be mediated by a set of expressed eukaryotic-like proteins and cell–cell mediators. Finally, some sponge-associated bacteria (for example, a Phyllobacteriaceae phylotype) appear to undergo an evolutionary adaptation process to the sponge environment as evidenced by active mobile genetic elements. Our data clearly show that a combined metaproteogenomic approach can provide novel information on the activities, physiology and interactions of sponge-associated microbial communities.     

Life based on phosphite: a genome-guided analysis of Desulfotignum phosphitoxidans

Background

The Delta-Proteobacterium Desulfotignum phosphitoxidans is a type strain of the genus Desulfotignum, which comprises to date only three species together with D. balticum and D. toluenicum. D. phosphitoxidans oxidizes phosphite to phosphate as its only source of electrons, with either sulfate or CO₂ as electron acceptor to gain its metabolic energy, which is of exclusive interest. Sequencing of the genome of this bacterium was undertaken to elucidate the genomic basis of this so far unique type of energy metabolism.

Results

The genome contains 4,998,761 base pairs and 4646 genes of which 3609 were assigned to a function, and 1037 are without function prediction. Metabolic reconstruction revealed that most biosynthetic pathways of Gram negative, autotrophic sulfate reducers were present. Autotrophic CO₂ assimilation proceeds through the Wood-Ljungdahl pathway. Additionally, we have found and confirmed the ability of the strain to couple phosphite oxidation to dissimilatory nitrate reduction to ammonia, which in itself is a new type of energy metabolism. Surprisingly, only two pathways for uptake, assimilation and utilization of inorganic and organic phosphonates were found in the genome. The unique for D. phosphitoxidans Ptx-Ptd cluster is involved in inorganic phosphite oxidation and an atypical C-P lyase-coding cluster (Phn) is involved in utilization of organophosphonates.

Conclusions

We present the whole genome sequence of the first bacterium able to gain metabolic energy via phosphite oxidation. The data obtained provide initial information on the composition and architecture of the phosphite–utilizing and energy-transducing systems needed to live with phosphite as an unusual electron donor.     

Complete genome sequence of the hydrogenotrophic Archaeon Methanobacterium sp. Mb1 isolated from a production-scale biogas plant

Methanobacterium sp. Mb1, a hydrogenotrophic methanogenic Archaeon, was isolated from a rural biogas plant producing methane-rich biogas from maize silage and cattle manure in Germany. Here we report the complete genome sequence of the novel methanogenic isolate Methanobacterium sp. Mb1 harboring a 2,029,766 bp circular chromosome featuring a GC content of 39.74%. The genome encodes two rRNA operons, 41 tRNA genes and 2021 coding sequences and represents the smallest genome currently known within the genus Methanobacterium.     

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

Recent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.     

Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a Psychrotrophic Rhodococcus sp.

The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C₁₀ to C₂₁ alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5°C. Mineralization of hexadecane at 5°C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction–gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25°C.     

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with ¹³C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent β-oxidation and the reverse Wood–Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.     

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.     

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 ??mol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery.     

Genome-scale dynamic modeling of the competition between Rhodoferax and Geobacter in anoxic subsurface environments

The advent of rapid complete genome sequencing, and the potential to capture this information in genome-scale metabolic models, provide the possibility of comprehensively modeling microbial community interactions. For example, Rhodoferax and Geobacter species are acetate-oxidizing Fe(III)-reducers that compete in anoxic subsurface environments and this competition may have an influence on the in situ bioremediation of uranium-contaminated groundwater. Therefore, genome-scale models of Geobacter sulfurreducens and Rhodoferax ferrireducens were used to evaluate how Geobacter and Rhodoferax species might compete under diverse conditions found in a uranium-contaminated aquifer in Rifle, CO. The model predicted that at the low rates of acetate flux expected under natural conditions at the site, Rhodoferax will outcompete Geobacter as long as sufficient ammonium is available. The model also predicted that when high concentrations of acetate are added during in situ bioremediation, Geobacter species would predominate, consistent with field-scale observations. This can be attributed to the higher expected growth yields of Rhodoferax and the ability of Geobacter to fix nitrogen. The modeling predicted relative proportions of Geobacter and Rhodoferax in geochemically distinct zones of the Rifle site that were comparable to those that were previously documented with molecular techniques. The model also predicted that under nitrogen fixation, higher carbon and electron fluxes would be diverted toward respiration rather than biomass formation in Geobacter, providing a potential explanation for enhanced in situ U(VI) reduction in low-ammonium zones. These results show that genome-scale modeling can be a useful tool for predicting microbial interactions in subsurface environments and shows promise for designing bioremediation strategies.     

Single-cell genomics reveals the lifestyle of Poribacteria,a candidate phylum symbiotically associated with marine sponges

In this study, we present a single-cell genomics approach for the functional characterization of the candidate phylum Poribacteria, members of which are nearly exclusively found in marine sponges. The microbial consortia of the Mediterranean sponge Aplysina aerophoba were singularized by fluorescence-activated cell sorting, and individual microbial cells were subjected to phi29 polymerase-mediated ‘whole-genome amplification''. Pyrosequencing of a single amplified genome (SAG) derived from a member of the Poribacteria resulted in nearly 1.6 Mb of genomic information distributed among 554 contigs analyzed in this study. Approximately two-third of the poribacterial genome was sequenced. Our findings shed light on the functional properties and lifestyle of a possibly ancient bacterial symbiont of marine sponges. The Poribacteria are mixotrophic bacteria with autotrophic CO₂-fixation capacities through the Wood–Ljungdahl pathway. The cell wall is of Gram-negative origin. The Poribacteria produce at least two polyketide synthases (PKSs), one of which is the sponge-specific Sup-type PKS. Several putative symbiosis factors such as adhesins (bacterial Ig-like domains, lamininin G domain proteins), adhesin-related proteins (ankyrin, fibronectin type III) and tetratrico peptide repeat domain-encoding proteins were identified, which might be involved in mediating sponge–microbe interactions. The discovery of genes coding for 24-isopropyl steroids implies that certain fossil biomarkers used to date the origins of metazoan life on earth may possibly be of poribacterial origin. Single-cell genomic approaches, such as those shown herein, contribute to a better understanding of beneficial microbial consortia, of which most members are, because of the lack of cultivation, inaccessible by conventional techniques.     

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.     

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.     

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern

Background

Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome.

Results

PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y’s phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism.

Conclusions

In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-384) contains supplementary material, which is available to authorized users.     

Selenoproteins in Archaea and Gram-positive bacteria

Selenium is an essential trace element for many organisms by serving important catalytic roles in the form of the 21st co-translationally inserted amino acid selenocysteine. It is mostly found in redox-active proteins in members of all three domains of life and analysis of the ever-increasing number of genome sequences has facilitated identification of the encoded selenoproteins. Available data from biochemical, sequence, and structure analyses indicate that Gram-positive bacteria synthesize and incorporate selenocysteine via the same pathway as enterobacteria. However, recent in vivo studies indicate that selenocysteine-decoding is much less stringent in Gram-positive bacteria than in Escherichia coli. For years, knowledge about the pathway of selenocysteine synthesis in Archaea and Eukarya was only fragmentary, but genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has not only led to the characterization of the pathways but has also shown that they are principally identical. This review summarizes current knowledge about the metabolic pathways of Archaea and Gram-positive bacteria where selenium is involved, about the known selenoproteins, and about the respective pathways employed in selenoprotein synthesis.     

湿地松和马尾松人工林土壤甲烷代谢微生物群落的结构特征

土壤甲烷代谢微生物影响甲烷的产生和氧化,然而关于林型对土壤中甲烷代谢微生物群落结构影响的研究较少。采用基因芯片GeoChip 3.0研究了湿地松人工林和马尾松人工林土壤甲烷代谢微生物群落结构特征。结果如下,(1)两种林型的甲烷代谢微生物群落结构存在极显著差异(P=0.008),林型能解释其34.9%的变异;(2)产甲烷菌(包含甲基辅酶M还原α亚基基因mcrA的微生物)的优势菌群发生了变化,湿地松人工林的的优势菌为Methanocorpusculum labreanum Z,马尾松人工林的优势菌群除Methanocorpusculum labreanum Z外,还包括产甲烷古菌和Methanosarcina mazei Gol;(3)甲烷营养菌(包含甲烷单加氧酶基因pmoA基因的微生物)的优势菌为Ⅱ型,有3种不可培养细菌只在湿地松人工林检测到,在马尾松人工林中未检测到;(4)mcrA基因丰度或同源基因数量与土壤容重正相关,与土壤粘粒含量呈显著负相关;pmoA基因信号强度或多样性指数与土壤全碳含量、全磷含量和速效氮含量显著正相关。总之,相比本地种马尾松人工林,引进种湿地松人工林的土壤甲烷代谢微生物群落结构发生了显著变化。