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.

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.

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.

An RND transporter in the monoterpene metabolism of Castellaniella defragrans.

The betaproteobacterium Castellaniella defragrans 65Phen grows on monoterpenes at concentrations toxic to many bacteria. Tolerance mechanisms include modifications of the membrane fatty acid composition and the mineralization of monoterpenes. In this study, we characterized an efflux transporter associated to the monoterpene metabolism. The inner-membrane transporter AmeD (apolar monoterpene efflux) affiliated to the HAE3 (hydrophobe/amphiphile efflux) family within the Resistance-Nodulation-Division (RND) superfamily. RND pumps of the HAE3 family are known for transporting substrates into the periplasm. AmeD is co-expressed with the outer membrane protein AmeA and the periplasmic proteins AmeB and AmeC, suggesting an export channel into the environment similar to HAE1-type RND exporters. Proteins AmeABCD are encoded within a genetic island involved in the metabolism of acyclic and cyclic monoterpenes. The deletion of ameABCD translated into a decrease in tolerance to monoterpenes in liquid cultures. The addition of acetate as cosubstrate in limonene-containing cultures partially alleviated monoterpene toxicity in the deletion mutant. Accumulation of Nile Red in cells of C. defragrans required dissipation of the proton motive force with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells lacking AmeABCD accumulated more Nile Red, suggesting an export function of the proteins. Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans.

Planctomycetes attached to algal surfaces: Insight into their genomes.

Planctomycetes are bacteria with complex molecular and cellular biology. They have large genomes, some over 7Mb, and complex life cycles that include motile cells and sessile cells. Some live on the complex biofilm of macroalgae. Factors governing their life in this environment were investigated at the genomic level. We analyzed the genomes of three planctomycetes isolated from algal surfaces. The genomes were 6.6Mbp to 8.1Mbp large. Genes for outer-membrane proteins, peptidoglycan and lipopolysaccharide biosynthesis were present. Rubripirellula obstinata LF1T, Roseimaritima ulvae UC8T and Mariniblastus fucicola FC18T shared with Rhodopirellula baltica and R. rubra SWK7 unique proteins related to metal binding systems, phosphate metabolism, chemotaxis, and stress response. These functions may contribute to their ecological success in such a complex environment. Exceptionally huge proteins (6000 to 10,000 amino-acids) with extracellular, periplasmic or membrane-associated locations were found which may be involved in biofilm formation or cell adhesion.

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.

Environmental and hydroclimatic factors influencing Vibrio populations in the estuarine zone of the Bengal delta.

The objective of this study was to determine environmental parameters driving Vibrio populations in the estuarine zone of the Bengal delta. Spatio-temporal data were collected at river estuary, mangrove, beach, pond, and canal sites. Effects of salinity, tidal amplitude, and a cyclone and tsunami were included in the study. Vibrio population shifts were found to be correlated with tide-driven salinity and suspended particulate matter (SPM). Increased abundance of Vibrio spp. in surface water was observed after a cyclone, attributed to re-suspension of benthic particulate organic carbon (POC), and increased availability of chitin and dissolved organic carbon (DOC). Approximately a two log10 increase in the (p < 0.05) number of Vibrio spp. was observed in < 20 µm particulates, compared with microphytoplankton (20-60 µm) and zooplankton > 60 µm fractions. Benthic and suspended sediment comprised a major reservoir of Vibrio spp. Results of microcosm experiments showed enhanced growth of vibrios was related to concentration of organic matter in SPM. It is concluded that SPM, POC, chitin, and salinity significantly influence abundance and distribution of vibrios in the Bengal delta estuarine zone.

Genomic and physiological analyses of 'Reinekea forsetii' reveal a versatile opportunistic lifestyle during spring algae blooms.

Gammaproteobacterial Reinekea spp. were detected during North Sea spring algae blooms in the years 2009-2012, with relative abundances of up to 16% in the bacterioplankton. Here, we explore the ecophysiology of 'R. forsetii' strain Hel1_31_D35 that was isolated during the 2010 spring bloom using (i) its manually annotated, high-quality closed genome, (ii) re-analysis of in situ data from the 2009-2012 blooms and (iii) physiological tests. High resolution analysis of 16S rRNA gene sequences suggested that 'R. forsetii' dominated Reinekea populations during these blooms. This was corroborated by retrieval of almost complete Hel1_31_D35 genomes from 2009 and 2010 bacterioplankton metagenomes. Strain Hel1_31_D35 can use numerous low-molecular weight substrates including diverse sugar monomers, and few but relevant algal polysaccharides such as mannan, α-glucans, and likely bacterial peptidoglycan. It oxidizes thiosulfate to sulfate, and ferments under anoxic conditions. The strain can attach to algae and thrives at low phosphate concentrations as they occur during blooms. Its genome encodes RTX toxin and secretion proteins, and in cultivation experiments Hel1_31_D35 crude cell extracts inhibited growth of a North Sea Polaribacter strain. Our data suggest that the combination of these traits make strain Hel1_31_D35 a versatile opportunist that is particularly competitive during spring phytoplankton blooms.

Description of Gramella forsetii sp. nov., a marine Flavobacteriaceae isolated from North Sea water, and emended description of Gramella gaetbulicola Cho et al. 2011.

Strain KT0803T was isolated from coastal eutrophic surface waters of Helgoland Roads near the island of Helgoland, North Sea, Germany. The taxonomic position of the strain, previously known as 'Gramella forsetii' KT0803, was investigated by using a polyphasic approach. The strain was Gram-stain-negative, chemo-organotrophic, heterotrophic, strictly aerobic, oxidase- and catalase-positive, rod-shaped, motile by gliding and had orange-yellow carotenoid pigments, but was negative for flexirubin-type pigments. It grew optimally at 22-25 °C, at pH 7.5 and at a salinity between 2-3 %. Strain KT0803T hydrolysed the polysaccharides laminarin, alginate, pachyman and starch. The respiratory quinone was MK-6. Polar lipids comprised phosphatidylethanolamine, six unidentified lipids and two unidentified aminolipids. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω7c and iso-C17 : 1ω7c, with smaller amounts of iso-C15 : 0 2-OH, C15 : 0, anteiso-C15 : 0 and C17 : 1ω6c. The G+C content of the genomic DNA was 36.6 mol%. The 16S rRNA gene sequence identities were 98.6 % with Gramella echinicola DSM 19838T, 98.3 % with Gramella gaetbulicola DSM 23082T, 98.1 % with Gramella aestuariivivens BG-MY13T and Gramella aquimixticola HJM-19T, 98.0 % with Gramella lutea YJ019T, 97.9 % with Gramella portivictoriae DSM 23547T and 96.9 % with Gramella marina KMM 6048T. The DNA-DNA relatedness values were <35 % between strain KT0803T and type strains with >98.2 % 16S rRNA gene sequence identity. Based on the chemotaxonomic, phenotypic and genomic characteristics, strain KT0803T has been assigned to the genus Gramella, as Gramella forsetii sp. nov. The type strain is KT0803T (=DSM 17595T=CGMCC 1.15422T). An emended description of Gramella gaetbulicolaCho et al. 2011 is also proposed.

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.

Use of carbon monoxide and hydrogen by a bacteria-animal symbiosis from seagrass sediments.

The gutless marine worm Olavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO2 ) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O. algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H2 ) might also be used as energy sources. We provide direct evidence that the O. algarvensis symbiosis consumes CO and H2 . Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3-symbiont, uses the energy from CO oxidation to fix CO2 . Pore water analysis revealed considerable in-situ concentrations of CO and H2 in the O. algarvensis environment, Mediterranean seagrass sediments. Pore water H2 concentrations (89-2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36-fold higher than previously known from shallow-water marine sediments. Pore water CO concentrations (17-51 nM) were twice as high as in the overlying seawater (no literature data from other shallow-water sediments are available for comparison). Ex-situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.

Dilution cultivation of marine heterotrophic bacteria abundant after a spring phytoplankton bloom in the North Sea.

The roles of individual bacterioplankton species in the re-mineralization of algal biomass are poorly understood. Evidence from molecular data had indicated that a spring diatom bloom in the German Bight of the North Sea in 2009 was followed by a rapid succession of uncultivated bacterioplankton species, including members of the genera Ulvibacter, Formosa, Polaribacter (class Flavobacteria) and Reinekea (class Gammaproteobacteria). We isolated strains from the same site during the diatom bloom in spring 2010 using dilution cultivation in an artificial seawater medium with micromolar substrate and nutrient concentrations. Flow cytometry demonstrated growth from single cells to densities of 10(4) -10(6) cells ml(-1) and a culturability of 35%. Novel Formosa, Polaribacter and Reinekea strains were isolated and had 16S rRNA gene sequence identities of > 99.8% with bacterioplankton in spring or summer 2009. Genomes of selected isolates were draft sequenced and used for read recruitment of metagenomes from bacterioplankton in 2009. Metagenome reads covered 93% of a Formosa clade B, 91% of a Reinekea and 74% of a Formosa clade A genome, applying a ≥ 94.5% nucleotide identity threshold. These novel strains represent abundant bacterioplankton species thriving on coastal phytoplankton blooms in the North Sea.

Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters.

The wide distribution of diverse nitrogenase (nifH) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N2 -fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2 -fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2 -fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9 × 10(4) and 4.7 × 10(4) nifH copies l(-1) respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.

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.

Proteomic insight into arabinogalactan utilization by particle-associated Maribacter sp. MAR_2009_72.

Arabinose and galactose are major, rapidly metabolized components of marine particulate and dissolved organic matter. In this study, we observed for the first time large microbiomes for the degradation of arabinogalactan and report a detailed investigation of arabinogalactan utilization by the flavobacterium Maribacter sp. MAR_2009_72. Cellular extracts hydrolysed arabinogalactan in vitro. Comparative proteomic analyses of cells grown on arabinogalactan, arabinose, galactose, and glucose revealed the expression of specific proteins in the presence of arabinogalactan, mainly glycoside hydrolases (GH). Extracellular glycan hydrolysis involved five alpha-l-arabinofuranosidases affiliating with glycoside hydrolase families 43 and 51, four unsaturated rhamnogalacturonylhydrolases (GH105) and a protein with a glycoside hydrolase family-like domain. We detected expression of three induced TonB-dependent SusC/D transporter systems, one SusC, and nine glycoside hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78, and GH115. The genes are located outside of and within canonical polysaccharide utilization loci classified as specific for arabinogalactan, for galactose-containing glycans, and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from the terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

The genome of the alga-associated marine flavobacterium Formosa agariphila KMM 3901T reveals a broad potential for degradation of algal polysaccharides.

In recent years, representatives of the Bacteroidetes have been increasingly recognized as specialists for the degradation of macromolecules. Formosa constitutes a Bacteroidetes genus within the class Flavobacteria, and the members of this genus have been found in marine habitats with high levels of organic matter, such as in association with algae, invertebrates, and fecal pellets. Here we report on the generation and analysis of the genome of the type strain of Formosa agariphila (KMM 3901(T)), an isolate from the green alga Acrosiphonia sonderi. F. agariphila is a facultative anaerobe with the capacity for mixed acid fermentation and denitrification. Its genome harbors 129 proteases and 88 glycoside hydrolases, indicating a pronounced specialization for the degradation of proteins, polysaccharides, and glycoproteins. Sixty-five of the glycoside hydrolases are organized in at least 13 distinct polysaccharide utilization loci, where they are clustered with TonB-dependent receptors, SusD-like proteins, sensors/transcription factors, transporters, and often sulfatases. These loci play a pivotal role in bacteroidetal polysaccharide biodegradation and in the case of F. agariphila revealed the capacity to degrade a wide range of algal polysaccharides from green, red, and brown algae and thus a strong specialization of toward an alga-associated lifestyle. This was corroborated by growth experiments, which confirmed usage particularly of those monosaccharides that constitute the building blocks of abundant algal polysaccharides, as well as distinct algal polysaccharides, such as laminarins, xylans, and κ-carrageenans.

Taxonomy and evolution of bacteriochlorophyll a-containing members of the OM60/NOR5 clade of marine gammaproteobacteria: description of Luminiphilus syltensis gen. nov., sp. nov., reclassification of Haliea rubra as Pseudohaliea rubra gen. nov., comb. nov., and emendation of Chromatocurvus halotolerans.

BACKGROUND: Aerobic gammaproteobacteria affiliated to the OM60/NOR5 clade are widespread in saline environments and of ecological importance in several marine ecosystems, especially the euphotic zone of coastal areas. Within this group a close relationship between aerobic anoxygenic photoheterotrophs and non-phototrophic members has been found. RESULTS: Several strains of aerobic red-pigmented bacteria affiliated to the OM60/NOR5 clade were obtained from tidal flat sediment samples at the island of Sylt (North Sea, Germany). Two of the novel isolates, Rap1red and Ivo14(T), were chosen for an analysis in detail. Strain Rap1red shared a 16S rRNA sequence identity of 99% with the type strain of Congregibacter litoralis and was genome-sequenced to reveal the extent of genetic microheterogeneity among closely related strains within this clade. In addition, a draft genome sequence was obtained from the isolate Ivo14(T), which belongs to the environmental important NOR5-1 lineage that contains so far no cultured representative with a comprehensive description. Strain Ivo14(T) was characterized using a polyphasic approach and compared with other red-pigmented members of the OM60/NOR5 clade, including Congregibacter litoralis DSM 17192(T), Haliea rubra DSM 19751(T) and Chromatocurvus halotolerans DSM 23344(T). All analyzed strains contained bacteriochlorophyll a and spirilloxanthin as photosynthetic pigments. Besides a detailed phenotypic characterization including physiological and chemotaxonomic traits, sequence information based on protein-coding genes and a comparison of draft genome data sets were used to identify possible features characteristic for distinct taxa within this clade. CONCLUSIONS: Comparative sequence analyses of the pufLM genes of genome-sequenced representatives of the OM60/NOR5 clade indicated that the photosynthetic apparatus of these species was derived from a common ancestor and not acquired by multiple horizontal gene transfer from phylogenetically distant species. An affiliation of the characterized bacteriochlorophyll a-containing strains to different genera was indicated by significant phenotypic differences and pufLM nucleotide sequence identity values below 82%. The revealed high genotypic and phenotypic diversity of closely related strains within this phylogenetic group reflects a rapid evolution and frequent niche separation in the OM60/NOR5 clade, which is possibly driven by the necessities of an adaptation to oligotrophic marine habitats.

rpoB gene as a novel molecular marker to infer phylogeny in Planctomycetales.

The 16S rRNA gene has been used in the last decades as a gold standard for determining the phylogenetic position of bacteria and their taxonomy. It is a well conserved gene, with some variations, present in all bacteria and allows the reconstruction of genealogies of microorganisms. Nevertheless, this gene has its limitations when inferring phylogenetic relationships between closely related isolates. To overcome this problem, DNA-DNA hybridization appeared as a solution to clarify interspecies relationships when the sequence similarity of the 16S rRNA gene is above 97 %. However, this technique is time consuming, expensive and laborious and so, researchers developed other molecular markers such as sequencing of housekeeping or functional genes for accurate determination of bacterial phylogeny. One of these genes that have been used successfully, particularly in clinical microbiology, codes for the beta subunit of the RNA polymerase (rpoB). The rpoB gene is sufficiently conserved to be used as a molecular clock, it is present in all bacteria and it is a mono-copy gene. In this study, rpoB gene sequencing was applied to the phylum Planctomycetes. Based on the genomes of 19 planctomycetes it was possible to determine the correlation between the rpoB gene sequence and the phylogenetic position of the organisms at a 95-96 % sequence similarity threshold for a novel species. A 1200-bp fragment of the rpoB gene was amplified from several new planctomycetal isolates and their intra and inter-species relationships to other members of this group were determined based on a 96.3 % species border and 98.2 % for intraspecies resolution.

Genetic diversity of Rhodopirellula strains.

Rhodopirellula baltica SH1(T) is a marine planctomycete with 7,325 genes in its genome. Ten strains of the genus Rhodopirellula were studied in whole genome microarray experiments to assess the extent of their genetic relatedness to R. baltica SH1(T). DNA of strains which were previously affiliated with the species R. baltica (OTU A) hybridized with 3,645-5,728 genes of the type strain on the microarray. Strains SH398 and 6C (OTU B), representing a closely related species with an average nucleotide identity of 88 %, showed less hybridization signals: 1,816 and 3,302 genes gave a hybridization signal, respectively. Comparative genomics of eight permanent draft genomes revealed the presence of over 4,000 proteins common in R. baltica SH1(T) and strains of OTU A or B. The genus Rhodopirellula is characterized by large genomes, with over 7,000 genes per genome and a core genome of around 3000 genes. Individual Rhodopirellula strains have a large portion of strain-specific genes.