Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

Three Microbial Musketeers of the Seas: Shewanella baltica, Aliivibrio fischeri and Vibrio harveyi, and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.

Genomic and proteomic profiles of biofilms on microplastics are decoupled from artificial surface properties.

Microplastics in marine ecosystems are colonized by diverse prokaryotic and eukaryotic communities. How these communities and their functional profiles are shaped by the artificial surfaces remains broadly unknown. In order to close this knowledge gap, we set up an in situ experiment with pellets of the polyolefin polymer polyethylene (PE), the aromatic hydrocarbon polymer polystyrene (PS), and wooden beads along a coastal to estuarine gradient in the Baltic Sea, Germany. We used an integrated metagenomics/metaproteomics approach to evaluate the genomic potential as well as protein expression levels of aquatic plastic biofilms. Our results suggest that material properties had a minor influence on the plastic-associated assemblages, as genomic and proteomic profiles of communities associated with the structurally different polymers PE and PS were highly similar, hence polymer-unspecific. Instead, it seemed that these communities were shaped by biogeographic factors. Wood, on the other hand, induced the formation of substrate-specific biofilms and served as nutrient source itself. Our study indicates that, while PE and PS microplastics may be relevant in the photic zone as opportunistic colonization grounds for phototrophic microorganisms, they appear not to be subject to biodegradation or serve as vectors for pathogenic microorganisms in marine habitats.

A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan.

Marine seaweeds increasingly grow into extensive algal blooms, which are detrimental to coastal ecosystems, tourism and aquaculture. However, algal biomass is also emerging as a sustainable raw material for the bioeconomy. The potential exploitation of algae is hindered by our limited knowledge of the microbial pathways-and hence the distinct biochemical functions of the enzymes involved-that convert algal polysaccharides into oligo- and monosaccharides. Understanding these processes would be essential, however, for applications such as the fermentation of algal biomass into bioethanol or other value-added compounds. Here, we describe the metabolic pathway that enables the marine flavobacterium Formosa agariphila to degrade ulvan, the main cell wall polysaccharide of bloom-forming Ulva species. The pathway involves 12 biochemically characterized carbohydrate-active enzymes, including two polysaccharide lyases, three sulfatases and seven glycoside hydrolases that sequentially break down ulvan into fermentable monosaccharides. This way, the enzymes turn a previously unexploited renewable into a valuable and ecologically sustainable bioresource.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprenes, and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here, we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a DTT:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently, the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism (ctm) in C. defragrans and are annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB, and CtmAB in Escherichia coli demonstrated that limonene dehydrogenase activity required both subunits, each carrying a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest placing in Enzyme Nomenclature as new entry EC 1.17.99.8.

Transcriptomic and proteomic insight into the mechanism of cyclooctasulfur- versus thiosulfate-oxidation by the chemolithoautotroph Sulfurimonas denitrificans.

Chemoautotrophic bacteria belonging to the genus Sulfurimonas (class Campylobacteria) were previously identified as key players in the turnover of zero-valence sulfur, a central intermediate in the marine sulfur cycle. S. denitrificans was further shown to be able to oxidize cyclooctasulfur (S8 ). However, at present the mechanism of activation and metabolism of cyclooctasulfur is not known. Here, we assessed the transcriptome and proteome of S. denitrificans grown with either thiosulfate or S8 as the electron donor. While the overall expression profiles under the two growth conditions were rather similar, distinct differences were observed that could be attributed to the utilization of S8 . This included a higher abundance of expressed genes related to surface attachment in the presence of S8 , and the differential regulation of the sulfur-oxidation multienzyme complex (SOX), which in S. denitrificans is encoded in two gene clusters: soxABXY 1 Z 1 and soxCDY 2 Z 2 . While the proteins of both clusters were present with thiosulfate, only proteins of the soxCDY 2 Z 2 were detected at significant levels with S8 . Based on these findings a model for the oxidation of S8 is proposed. Our results have implications for interpreting metatranscriptomic and -proteomic data and for the observed high level of diversification of soxY 2 Z 2 among sulfur-oxidizing Campylobacteria.

Microbial metal-sulfide oxidation in inactive hydrothermal vent chimneys suggested by metagenomic and metaproteomic analyses.

Metal-sulfides are wide-spread in marine benthic habitats. At deep-sea hydrothermal vents, they occur as massive sulfide chimneys formed by mineral precipitation upon mixing of reduced vent fluids with cold oxygenated sea water. Although microorganisms inhabiting actively venting chimneys and utilizing compounds supplied by the venting fluids are well studied, only little is known about microorganisms inhabiting inactive chimneys. In this study, we combined 16S rRNA gene-based community profiling of sulfide chimneys from the Manus Basin (SW Pacific) with radiometric dating, metagenome (n = 4) and metaproteome (n = 1) analyses. Our results shed light on potential lifestyles of yet poorly characterized bacterial clades colonizing inactive chimneys. These include sulfate-reducing Nitrospirae and sulfide-oxidizing Gammaproteobacteria dominating most of the inactive chimney communities. Our phylogenetic analysis attributed the gammaproteobacterial clades to the recently described Woeseiaceae family and the SSr-clade found in marine sediments around the world. Metaproteomic data identified these Gammaproteobacteria as autotrophic sulfide-oxidizers potentially facilitating metal-sulfide dissolution via extracellular electron transfer. Considering the wide distribution of these gammaproteobacterial clades in marine environments such as hydrothermal vents and sediments, microbially accelerated neutrophilic mineral oxidation might be a globally relevant process in benthic element cycling and a considerable energy source for carbon fixation in marine benthic habitats.

Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta.

Marine sponges represent one of the few eukaryotic groups that frequently harbour symbiotic members of the Thaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However, in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of ammonia-oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and isotope-based functional assays. 'Candidatus Nitrosospongia ianthellae' is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbour nitrite-oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system, represent important adaptations of AOA to life within these ancient filter-feeding animals.

Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium.

Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 µm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms.IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions.

Alpha- and beta-mannan utilization by marine Bacteroidetes.

Marine microscopic algae carry out about half of the global carbon dioxide fixation into organic matter. They provide organic substrates for marine microbes such as members of the Bacteroidetes that degrade algal polysaccharides using carbohydrate-active enzymes (CAZymes). In Bacteroidetes genomes CAZyme encoding genes are mostly grouped in distinct regions termed polysaccharide utilization loci (PULs). While some studies have shown involvement of PULs in the degradation of algal polysaccharides, the specific substrates are for the most part still unknown. We investigated four marine Bacteroidetes isolated from the southern North Sea that harbour putative mannan-specific PULs. These PULs are similarly organized as PULs in human gut Bacteroides that digest α- and ß-mannans from yeasts and plants respectively. Using proteomics and defined growth experiments with polysaccharides as sole carbon sources we could show that the investigated marine Bacteroidetes express the predicted functional proteins required for α- and ß-mannan degradation. Our data suggest that algal mannans play an as yet unknown important role in the marine carbon cycle, and that biochemical principles established for gut or terrestrial microbes also apply to marine bacteria, even though their PULs are evolutionarily distant.

The anaerobic linalool metabolism in Thauera linaloolentis 47 Lol.

BACKGROUND: The betaproteobacterium Thauera linaloolentis 47Lol(T) was isolated on the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. Growth experiments indicated the formation of geraniol and geranial. Thus, a 3,1-hydroxyl-Δ(1)-Δ(2)-mutase (linalool isomerase) activity may initiate the degradation, followed by enzymes of the acyclic terpene utilization (Atu) and leucine/isovalerate utilization (Liu) pathways that were extensively studied in Pseudomonas spp. growing on citronellol or geraniol. RESULTS: A transposon mutagenesis yielded 39 transconjugants that could not grow anaerobically on linalool and nitrate in liquid medium. The deficiencies were apparently based on gene functions required to overcome the toxicity of linalool, but not due to inactivation of genes in the degradation pathway. Growing cultures formed geraniol and geranial transiently, but also geranic acid. Analysis of expressed proteins detected several enzymes of the Atu and Liu pathways. The draft genome of T. linaloolentis 47Lol(T) had atu and liu genes with homology to those of Pseudomonas spp.. CONCLUSION: The in comparison to monoterpenes larger toxicity of monoterpene alcohols is defeated by several modifications of the cellular structure and metabolism in Thauera linaloolentis 47Lol(T). The acyclic terpene utilization pathway is used in T. linaloolentis 47Lol(T) during growth on (R,S)-linalool and nitrate under anoxic conditions. This is the first experimental verification of an active Atu pathway outside of the genus Pseudomonas.

Linalool isomerase, a membrane-anchored enzyme in the anaerobic monoterpene degradation in Thauera linaloolentis 47Lol.

BACKGROUND: Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ(1)-Δ(2)-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool. RESULTS: The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42 ± 0.28 nkat mg * protein(-1) and a kM value of 455 ± 124 µM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C. CONCLUSION: The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.

Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use.

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO(2). Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO(2) fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

The genome of the obligate intracellular parasite Trachipleistophora hominis: new insights into microsporidian genome dynamics and reductive evolution.

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

BACKGROUND: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic ß-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway. RESULTS: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate. CONCLUSIONS: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

Marine bacteroidetes use a conserved enzymatic cascade to digest diatom ß-mannan.

The polysaccharide ß-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a ß-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed ß-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for ß-mannan utilization. A conserved GH26 ß-mannanase with endo-activity depolymerized the ß-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial ß-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-ß-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of ß-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom ß-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade ß-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling.

Differential regulation of degradation and immune pathways underlies adaptation of the ectosymbiotic nematode Laxus oneistus to oxic-anoxic interfaces.

Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm's Toll-like innate immunity pathway and several immune effectors (e.g., bactericidal/permeability-increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires the oxidative phosphorylation and reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

Diverse events have transferred genes for edible seaweed digestion from marine to human gut bacteria.

Humans harbor numerous species of colonic bacteria that digest fiber polysaccharides in commonly consumed terrestrial plants. More recently in history, regional populations have consumed edible macroalgae seaweeds containing unique polysaccharides. It remains unclear how extensively gut bacteria have adapted to digest these nutrients. Here, we show that the ability of gut bacteria to digest seaweed polysaccharides is more pervasive than previously appreciated. Enrichment-cultured Bacteroides harbor previously discovered genes for seaweed degradation, which have mobilized into several members of this genus. Additionally, other examples of marine bacteria-derived genes, and their mobile DNA elements, are involved in gut microbial degradation of seaweed polysaccharides, including genes in gut-resident Firmicutes. Collectively, these results uncover multiple separate events that have mobilized the genes encoding seaweed-degrading-enzymes into gut bacteria. This work further underscores the metabolic plasticity of the human gut microbiome and global exchange of genes in the context of dietary selective pressures.