Sarah Hahnke

The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker's guide to life in the sea.

Dinoroseobacter shibae DFL12T, a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12T is able to synthesize the vitamins B1 and B12 for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B12 are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B12 was confirmed to be functional, and D. shibae DFL12T was shown to provide the growth-limiting vitamins B1 and B12 to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12T is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12T has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12T shows the most complex viral defense system of all Rhodobacterales sequenced to date.

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.

Complete genome sequence of the novel Porphyromonadaceae bacterium strain ING2-E5B isolated from a mesophilic lab-scale biogas reactor

In this study, the whole genome sequence of the mesophilic, anaerobic Porphyromonadaceae bacterium strain ING2-E5B (LMG 28429, DSM 28696) is reported. The new isolate belongs to the phylum Bacteroidetes and was obtained from a biogas-producing lab-scale completely stirred tank reactor (CSTR) optimized for anaerobic digestion of maize silage in co-fermentation with pig and cattle manure. The genome of strain ING2-E5B contains numerous genes encoding proteins and enzymes involved in the degradation of complex carbohydrates and proteinaceous compounds. Moreover, it possesses genes catalyzing the production of volatile fatty acids. Hence, this bacterium was predicted to be involved in hydrolysis and acidogenesis during anaerobic digestion and biomethanation.

Physiological diversity of Roseobacter clade bacteria co-occurring during a phytoplankton bloom in the North Sea.

Organisms of the Roseobacter clade are an important component in marine ecosystems, partially due to their metabolic variety. Not much is known, however, about the physiological diversity of different roseobacters present within one habitat. By using serial dilution cultures with low-nutrient media seven roseobacter strains, co-occurring during a phytoplankton bloom in the southern North Sea, were obtained in this study. Physiological characterization exhibited distinct substrate spectra of the isolates. Although no isolate showed growth on algal osmolyte dimethylsulfoniopropionate (DMSP), feeding experiments revealed that all new strains converted [²H₆]DMSP into a variety of volatile compounds. Six strains mainly decomposed DMSP via the demethylation pathway, but four strains were also capable of cleaving DMSP to DMS and acrylate. It is hypothesized that the great physiological diversity of the roseobacters reflects their ability to inhabit different ecological niches and enables the organisms to cope differently with changing substrate supplies during phytoplankton blooms. Denaturing gradient gel electrophoresis and sequencing of excised bands resulted in detection of five additional roseobacters. Three of these sequences showed affiliation with three of the four major clusters of the Roseobacter clade, consisting predominantly of uncultured organisms (i.e. the Roseobacter clade-affiliated (RCA)), the NAC11-7 and the CHAB-I-5 clusters.

Erratum to Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov., isolated from mesophilic laboratory-scale biogas reactors, and emended description of the genus Proteiniphilum.

Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic laboratory-scale biogas reactors. The strains were Gram-negative rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1-7.8. The main fermentation products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA-DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3-45.6 and 23.8-25.7 % relatedness, respectively, which supports the conclusion that both isolates represent novel species. Phylogenetic analysis and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiological, genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addition, an emended description of the genus Proteiniphilum is provided.

Clostridium bornimense sp. nov., isolated from a mesophilic, two-phase, laboratory-scale biogas reactor

A novel anaerobic, mesophilic, hydrogen-producing bacterium, designated strain M2/40(T), was isolated from a mesophilic, two-phase, laboratory-scale biogas reactor fed continuously with maize silage supplemented with 5% wheat straw. 16S rRNA gene sequence comparison revealed an affiliation to the genus Clostridium sensu stricto (cluster I of the clostridia), with Clostridium cellulovorans as the closest characterized species, showing 93.8% sequence similarity to the type strain. Cells of strain M2/40(T) were rods to elongated filamentous rods that showed variable Gram staining. Optimal growth occurred at 35 °C and at pH 7. Grown on glucose, the main fermentation products were H2, CO2, formate, lactate and propionate. The DNA G+C content was 29.6 mol%. The major fatty acids (>10 %) were C(16 : 0), summed feature 10 (C(18 : 1)ω11c/ω9t/ω6t and/or unknown ECL 17.834) and C(18 : 1)ω11c dimethylacetal. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain M2/40(T) represents a novel species within the genus Clostridium, for which we propose the name Clostridium bornimense sp. nov. The type strain is M2/40(T) ( = DSM 25664(T) = CECT 8097(T)).

Planktotalea frisia gen. nov., sp. nov., isolated from the southern North Sea

A heterotrophic, aerobic bacterium, designated strain SH6-1(T), was obtained from a seawater sample collected from the open North Sea during a phytoplankton bloom. Strain SH6-1(T) was isolated from a 10(-6) dilution culture, which indicated a high abundance of this organism in the environmental sample. 16S rRNA gene sequence comparison revealed that strain SH6-1(T) belonged to the marine Roseobacter clade (order Rhodobacterales) within the class Alphaproteobacteria. Pelagicola litoralis CL-ES2(T) was the closest phylogenetic neighbour (96.4% 16S rRNA gene sequence similarity). Cells of strain SH6-1(T) were small or elongated irregular rods. Optimal growth occurred between 20 and 25 °C and between pH 7.5 and 9.0 with peptone and yeast extract. On marine agar, the isolate formed non-pigmented, small, circular, convex colonies. For growth, cells required sodium ions and the vitamins pantothenic acid and nicotinic acid amide. The DNA G+C content was 53.8 mol%. The fatty acids (>1%) were C(10:0) 3-OH, C(16:0), C(12:1), C(12:1) 3-OH, C(18:0), C(18:1)ω7c, C(18:2) and 11-methyl C(18:1)ω7c. The polar lipid pattern indicated the presence of phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and one unidentified phospholipid. The major respiratory lipoquinone was ubiquinone Q-10. Strain SH6-1(T) contained the genes pufLM, which code for the bacterial photosynthesis reaction centre; however, no bacteriochlorophyll a could be detected. Physiological, genotypic and phenotypic differences from P. litoralis support the description of a novel genus and species, for which we suggest the name Planktotalea frisia gen. nov., sp. nov; the type strain of the type species is SH6-1(T) (=DSM 23709(T)=LMG 25294(T)).

Influence of light and anoxia on chemiosmotic energy conservation in Dinoroseobacter shibae

In the present study we have investigated the influence of light and anoxia on the energetic state of the aerobic anoxygenic phototroph (AAP) Dinoroseobacter shibae. Respiration, chemiosmotic proton translocation and the adenylate energy charge (AEC) of the cells were measured comparing light versus dark and oxic versus anoxic conditions. Light caused a decrease of the respiration rates of washed cells. This might be a substitution rather than a direct inhibitory effect, because both photosynthesis and respiration contribute to the proton-motive force. As known from other AAPs, light alone did not induce proton translocation if applied to anoxic cell suspensions. However, additions of small oxygen pulses to anoxic cell suspensions caused two times more proton translocation in the light than in the dark. The AEC of the cells was measured by means of a modified luciferin-luciferase method. Growing cells of D. shibae kept an AEC of 0.93, indicating that the adenylate pool was highly phosphorylated. After harvesting and storing the cells under anoxic conditions for 2 h, the AEC dropped to 0.12. However, the cells remained reactive. Upon addition of oxygen, the AEC increased to its original value within 40 s by the formation of about 12 mM of intracellular ATP. There were no differences whether this recovery experiment was carried out in the dark or in the light. We conclude that D. shibae is able to change its energetic state not only in response to the light regime but also during oxic–anoxic transitions. Both responses appear suited to save in situ organic substrates and endogenous electron donors, thus enhancing the role of photosynthetic energy conservation.

Pelagimonas varians gen. nov., sp. nov., isolated from the southern North Sea

A heterotrophic, Gram-stain-negative, aerobic bacterium, designated strain SH4-1(T), was obtained from a seawater sample collected from the southern North Sea during a phytoplankton bloom. The 16S rRNA gene sequence comparison revealed affiliation to the Roseobacter clade (class Alphaproteobacteria) with Sulfitobacter marinus SW-265(T) as the most closely related characterized strain, showing 97.2 % 16S rRNA gene sequence similarity. Calculation of phylogenetic trees based on 16S rRNA gene sequences indicated, however, that members of the genus Roseobacter, Roseobacter denitrificans Och 114(T) and Roseobacter litoralis Och 149(T) (95 % and 96 % sequence similarity, respectively) fall between strain SH4-1(T) and the Sulfitobacter cluster including Oceanibulbus indolifex HEL-45(T) (≥95.4 % sequence similarity). Cells of strain SH4-1(T) are irregular rods with at least one flagellum. Optimal growth occurred between 28 and 32 °C and at a pH between 7.0 and 8.5. Cells require the vitamin nicotinic acid amide as well as sodium ions for growth. The DNA G+C content was 55.1 mol%. The fatty acids (>1 %) comprised C10 : 0 3-OH, C12 : 1, C14 : 1 3-OH, C16 : 0, C18 : 0, C18 : 2, C18 : 1ω7c and 11-methyl C18 : 1ω7c. The polar lipid pattern indicated the presence of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, an unidentified aminolipid, one unidentified phospholipid and one other unidentified lipid. On the basis of phenotypic, chemotaxonomic and phylogenetic differences, strain SH4-1(T) represents a novel species in a new genus within the family Rhodobacteraceae, for which we propose the name Pelagimonas varians gen. nov., sp. nov. The type strain of the type species is SH4-1(T) ( = DSM 23678(T) = LMG 26343(T) = CIP 110297(T)).

Complete genome analysis of Clostridium bornimense strain M2/40T: A new acidogenic Clostridium species isolated from a mesophilic two-phase laboratory-scale biogas reactor

Taxonomic and functional profiling based on metagenome analyses frequently revealed that members of the class Clostridia dominate biogas reactor communities and perform different essential metabolic pathways in the biogas fermentation process. Clostridium bornimense strain M2/40(T) was recently isolated from a mesophilic two-phase lab-scale biogas reactor continuously fed with maize silage and wheat straw. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding carbohydrate active enzyme production and fermentation of organic compounds for consolidated biofuel production from biomass. The C. bornimense M2/40(T) genome consists of a chromosome (2,917,864bp in size) containing 2613 protein coding sequences, and a 699,161bp chromid (secondary replicon) harboring 680 coding sequences. Both replicons feature very similar GC-contents of approximately 29%. The complex genome comprises three prophage regions, two CRISPR-cas systems and a putative cellulosomal gene cluster that is located on the second replicon (chromid) of the strain. The overexpressed glycosyl hydrolases (GH) CelK (GH9) and CelA (GH48) encoded in the cellulosomal gene cluster were shown to be active on the substrates xylan and xyloglucan whereas XghA (GH74) is highly active on xyloglucan. Reconstruction of fermentation pathways from genome sequence data revealed that strain M2/40(T) encodes all enzymes for hydrogen, acetate, formate, lactate, butyrate, and ethanol production, leading to the classification of the isolate as acidogenic bacterium. Phylogenetic analyses uncovered that the closest characterized relative of C. bornimense is C. cellulovorans. Comparative analyses of the C. bornimense and C. cellulovorans genomes revealed considerable rearrangements within their chromosomes suggesting that both species evolved separately for a relatively long period of time and adapted to specific tasks within microbial consortia responsible for anaerobic digestion.