Recording and Simulating Proton-Related Metabolism in Bacterial Cell Suspensions.

Proton release and uptake induced by metabolic activities were measured in non-buffered cell suspensions by means of a pH electrode. Recorded data were used for simulating substrate turnover rates by means of a new freeware app (proton.exe). The program applies Michaelis-Menten or first-order kinetics to the metabolic processes and allows for parametrization of simultaneously ongoing processes. The simulation includes changes of the transmembrane ΔpH, membrane potential and ATP gains, and demonstrates the principles of chemiosmotic energy conservation. In our experiments, the versatile sulfate-reducing bacterium Desulfovibrio desulfuricans CSN (DSM 9104) was used as model organism. We analysed sulfate uptake by proton-sulfate symport, scalar alkalinization by sulfate reduction to sulfide, as well as nitrate and nitrite reduction to ammonia, and electron transport-coupled proton translocation. Two types of experiments were performed: In oxidant pulse experiments, cells were kept under H2, and micromolar amounts of sulfate, nitrate or nitrite were added. For reductant pulse experiments, small amounts of H2-saturated KCl were added to cells incubated under N2 with an excess of one of the above-mentioned electron acceptors. To study electron-transport driven proton translocation, the membrane potential was neutralized by addition of KSCN (100 mM). H+/e- ratios of electron-transport driven proton translocation were calculated by simulation with proton.exe. This method gave lower but more realistic values than logarithmic extrapolation. We could verify the kinetic simulation parameters found with proton.exe using series of increasing additions of the reactants. Our approach allows for studying a broad variety of proton-related metabolic activities at micromolar concentrations and time scales of seconds to minutes.

Heterozygous, Polyploid, Giant Bacterium, Achromatium, Possesses an Identical Functional Inventory Worldwide across Drastically Different Ecosystems.

Achromatium is large, hyperpolyploid and the only known heterozygous bacterium. Single cells contain approximately 300 different chromosomes with allelic diversity far exceeding that typically harbored by single bacteria genera. Surveying all publicly available sediment sequence archives, we show that Achromatium is common worldwide, spanning temperature, salinity, pH, and depth ranges normally resulting in bacterial speciation. Although saline and freshwater Achromatium spp. appear phylogenetically separated, the genus Achromatium contains a globally identical, complete functional inventory regardless of habitat. Achromatium spp. cells from differing ecosystems (e.g., from freshwater to saline) are, unexpectedly, equally functionally equipped but differ in gene expression patterns by transcribing only relevant genes. We suggest that environmental adaptation occurs by increasing the copy number of relevant genes across the cell's hundreds of chromosomes, without losing irrelevant ones, thus maintaining the ability to survive in any ecosystem type. The functional versatility of Achromatium and its genomic features reveal alternative genetic and evolutionary mechanisms, expanding our understanding of the role and evolution of polyploidy in bacteria while challenging the bacterial species concept and drivers of bacterial speciation.

Cell Architecture of the Giant Sulfur Bacterium Achromatium oxaliferum: Extra-cytoplasmic Localization of Calcium Carbonate Bodies.

Achromatium oxaliferum is a large sulfur bacterium easily recognized by large intracellular calcium carbonate bodies. Although these bodies often fill major parts of the cells' volume, their role and specific intracellular location are unclear. In this study, we used various microscopy and staining techniques to identify the cell compartment harboring the calcium carbonate bodies. We observed that Achromatium cells often lost their calcium carbonate bodies, either naturally or induced by treatments with diluted acids, ethanol, sodium bicarbonate and UV radiation which did not visibly affect the overall shape and motility of the cells (except for UV radiation). The water-soluble fluorescent dye fluorescein easily diffused into empty cavities remaining after calcium carbonate loss. Membranes (stained with Nile Red) formed a network stretching throughout the cell and surrounding empty or filled calcium carbonate cavities. The cytoplasm (stained with FITC and SYBR Green for nucleic acids) appeared highly condensed and showed spots of dissolved Ca2+ (stained with Fura-2). From our observations, we conclude that the calcium carbonate bodies are located in the periplasm, in extra-cytoplasmic pockets of the cytoplasmic membrane and are thus kept separate from the cell's cytoplasm. This periplasmic localization of the carbonate bodies might explain their dynamic formation and release upon environmental changes.

Chemiosmotic Energy Conservation in Dinoroseobacter shibae: Proton Translocation Driven by Aerobic Respiration, Denitrification, and Photosynthetic Light Reaction.

Dinoroseobacter shibae is an aerobic anoxygenic phototroph and able to utilize light energy to support its aerobic energy metabolism. Since the cells can also grow anaerobically with nitrate and nitrite as terminal electron acceptor, we were interested in how the cells profit from photosynthesis during denitrification and what the steps of chemiosmotic energy conservation are. Therefore, we conducted proton translocation experiments and compared O2-, NO3-, and NO2- respiration during different light regimes and in the dark. We used wild type cells and transposon mutants with knocked-out nitrate- and nitrite- reductase genes (napA and nirS), as well as a mutant (ppsR) impaired in bacteriochlorophyll a synthesis. Light had a positive impact on proton translocation, independent of the type of terminal electron acceptor present. In the absence of an electron acceptor, however, light did not stimulate proton translocation. The light-driven add-on to proton translocation was about 1.4 H+/e- for O2 respiration and about 1.1 H+/e- for NO3- and NO2-. We could see that the chemiosmotic energy conservation during aerobic respiration involved proton translocation, mediated by the NADH dehydrogenase, the cytochrome bc1 complex, and the cytochrome c oxidase. During denitrification the last proton translocation step of the electron transport was missing, resulting in a lower H+/e- ratio during anoxia. Furthermore, we studied the type of light-harvesting and found that the cells were able to channel light from the green-blue spectrum most efficiently, while red light has only minor impact. This fits well with the depth profiles for D. shibae abundance in the ocean and the penetration depth of light with different wavelengths into the water column.

A Crispy Diet: Grazers of Achromatium oxaliferum in Lake Stechlin Sediments.

Achromatium is the largest freshwater bacterium known to date and easily recognised by conspicuous calcite bodies filling the cell volume. Members of this genus are highly abundant in diverse aquatic sediments and may account for up to 90% of the bacterial biovolume in the oxic-anoxic interfaces. The high abundance implies that Achromatium is either rapidly growing or hardly prone to predation. As Achromatium is still uncultivated and does not appear to grow fast, one could assume that the cells might escape predation by their unusual shape and composition. However, we observed various members of the meiofauna grazing or parasitizing on Achromatium. By microphotography, we documented amoebae, ciliates, oligochetes and plathelminthes having Achromatium cells ingested. Some Achromatium cells harboured structures resembling sporangia of parasitic fungi (chytrids) that could be stained with the chitin-specific dye Calcofluor White. Many Achromatia carried prokaryotic epibionts in the slime layer surrounding the cells. Their regular distribution over the cell might indicate that they are commensalistic rather than harming their hosts. In conclusion, we report on various interactions of Achromatium with the sediment community and show that although Achromatium cells are a crispy diet, full of calcite bodies, predators do not spare them.

Propidium ion enters viable cells with high membrane potential during live-dead staining.

Live-dead staining with propidium iodide can give erroneous results for bacteria showing high membrane potentials. We observed uptake of propidium ions across intact cell membranes for Dinoroseobacter shibae and Bacillus subtilis. Apparently, a high membrane potential facilitates breakthrough of the double-charged propidium ion and can mark viable cells as dead.

Community-like genome in single cells of the sulfur bacterium Achromatium oxaliferum.

Polyploid bacteria are common, but the genetic and functional diversity resulting from polyploidy is unknown. Here we use single-cell genomics, metagenomics, single-cell amplicon sequencing, and fluorescence in situ hybridization, to show that individual cells of Achromatium oxaliferum, the world's biggest known freshwater bacterium, harbor genetic diversity typical of whole bacterial communities. The cells contain tens of transposable elements, which likely cause the unprecedented diversity that we observe in the sequence and synteny of genes. Given the high within-cell diversity of the usually conserved 16S ribosomal RNA gene, we suggest that gene conversion occurs in multiple, separated genomic hotspots. The ribosomal RNA distribution inside the cells hints to spatially differential gene expression. We also suggest that intracellular gene transfer may lead to extensive gene reshuffling and increased diversity.The cells of Achromatium bacteria are remarkably large and contain multiple chromosome copies. Here, Ionescu et al. show that chromosome copies within individual cells display high diversity, similar to that of bacterial communities, and contain tens of transposable elements.

Draft Genome Sequence of Pseudoruegeria sp. SK021, a Representative of the Marine Roseobacter Group, Isolated from North Sea Sediment.

Pseudoruegeria sp. SK021 is a member of the Roseobacter group, isolated under aerobic conditions from North Sea sediment. The draft genome comprises 3.95 Mb and contains 3,747 protein-coding sequences. Although the strain is nonmotile under laboratory conditions, the entire set of genes for the formation of a flagellar apparatus was found.

Marinisporobacter balticus gen. nov., sp. nov., Desulfosporosinus nitroreducens sp. nov. and Desulfosporosinus fructosivorans sp. nov., new spore-forming bacteria isolated from subsurface sediments of the Baltic Sea.

Four novel Gram-stain-positive, endospore-forming bacteria of the order Clostridiales were isolated from subsurface sediments sampled during International Ocean Discovery Program Expedition 347 to the Baltic Sea. One strain (59.4MT) grew as an obligate heterotroph by aerobic respiration and anaerobically by fermentation. Optimum growth was observed with 0.5 % NaCl at 25 °C and pH 7.0-7.3. Analysis of 16S rRNA gene sequences of 59.4MT revealed Alkaliphilus transvaalensis (92.3 % identity), Candidatus Geosporobacter ferrireducens (92.2 %), Geosporobacter subterraneus (91.9 %) and Alkaliphilus peptidifermentans (91.7 %) to be the closest relatives. On the basis of the results of phenotypic and genotypic analyses, we propose that strain 59.4MT represents a novel species within a novel genus, Marinisporobacter balticus gen. nov., sp. nov., with the type strain 59.4MT (=DSM 102940T=JCM 31103T). Three other strains, 59.4F, 59.4BT and 63.6FT, were affiliated with the genus Desulfosporosinus and grew as strictly anaerobic sulfate reducers. These strains additionally used thiosulfate, elemental sulfur, sulfite and DMSO as electron acceptors and hydrogen as an electron donor. Strains 59.4F and 59.4BT had identical 16S rRNA gene sequences, which were most similar to those of Desulfosporosinus lacus (97.8 %), Desulfosporosinus hippei (97.3 %) and Desulfosporosinus orientis (97.3 %). Strain 63.6FT was closely related to D. lacus (97.7 %), Desulfosporosinus meridiei (96.6 %) and D. hippei (96.5 %). The similarity of 16S rRNA gene sequences of strains 59.4BT and 63.6FT was 96.6 %. We propose the new names Desulfosporosinus nitroreducens sp. nov., incorporating strain 59.4F (=DSM 101562=JCM 31104) and the type strain 59.4BT (=DSM 101608T=JCM 31105T), and Desulfosporosinus fructosivorans sp. nov., with the type strain 63.6FT (=DSM 101609T=JCM 31106T).

Boosted Membrane Potential as Bioenergetic Response to Anoxia in Dinoroseobacter shibae.

Dinoroseobacter shibae DFL 12T is a metabolically versatile member of the world-wide abundant Roseobacter clade. As an epibiont of dinoflagellates D. shibae is subjected to rigorous changes in oxygen availability. It has been shown that it loses up to 90% of its intracellular ATP when exposed to anoxic conditions. Yet, D. shibae regenerates its ATP level quickly when oxygen becomes available again. In the present study we focused on the bioenergetic aspects of the quick recovery and hypothesized that the proton-motive force decreases during anoxia and gets restored upon re-aeration. Therefore, we analyzed ΔpH and the membrane potential (ΔΨ) during the oxic-anoxic transitions. To visualize changes of ΔΨ we used fluorescence microscopy and the carbocyanine dyes DiOC2 (3; 3,3'-Diethyloxacarbocyanine Iodide) and JC-10. In control experiments the ΔΨ-decreasing effects of the chemiosmotic inhibitors CCCP (carbonyl cyanide m-chlorophenyl hydrazone), TCS (3,3',4',5-tetrachlorosalicylanilide) and gramicidin were tested on D. shibae and Gram-negative and -positive control bacteria (Escherichia coli and Micrococcus luteus). We found that ΔpH is not affected by short-term anoxia and does not contribute to the quick ATP regeneration in D. shibae. By contrast, ΔΨ was increased during anoxia, which was astonishing since none of the control organisms behaved that way. Our study shows physiological and bioenergetical aspects comparing to previous studies on transcriptomic responses to the transition from aerobic to nitrate respiration in D. shibae. For the lifestyle as an epibiont of a dinoflagellate, the ability to stand phases of temporary oxygen depletion is beneficial. With a boosted ΔΨ, the cells are able to give their ATP regeneration a flying start, once oxygen is available again.

Labilibaculum manganireducens gen. nov., sp. nov. and Labilibaculum filiforme sp. nov., Novel Bacteroidetes Isolated from Subsurface Sediments of the Baltic Sea.

Microbial communities in deep subsurface sediments are challenged by the decrease in amount and quality of organic substrates with depth. In sediments of the Baltic Sea, they might additionally have to cope with an increase in salinity from ions that have diffused downward from the overlying water during the last 9000 years. Here, we report the isolation and characterization of four novel bacteria of the Bacteroidetes from depths of 14-52 m below seafloor (mbsf) of Baltic Sea sediments sampled during International Ocean Discovery Program (IODP) Expedition 347. Based on physiological, chemotaxonomic and genotypic characterization, we propose that the four strains represent two new species within a new genus in the family Marinifilaceae, with the proposed names Labilibaculum manganireducens gen. nov., sp. nov. (type strain 59.10-2MT) and Labilibaculum filiforme sp. nov. (type strains 59.16BT) with additional strains of this species (59.10-1M and 60.6M). The draft genomes of the two type strains had sizes of 5.2 and 5.3 Mb and reflected the major physiological capabilities. The strains showed gliding motility, were psychrotolerant, neutrophilic and halotolerant. Growth by fermentation of mono- and disaccharides as well as pyruvate, lactate and glycerol was observed. During glucose fermentation, small amounts of electron equivalents were transferred to Fe(III) by all strains, while one of the strains also reduced Mn(IV). Thereby, the four strains broaden the phylogenetic range of prokaryotes known to reduce metals to the group of Bacteroidetes. Halotolerance and metal reduction might both be beneficial for survival in deep subsurface sediments of the Baltic Sea.

Modelling anaerobic digestion in an industrial biogas digester: Application of lactate-including ADM1 model (Part II).

A modified Anaerobic Digestion Model No. 1 (ADM1xp) including lactate was applied to a full-scale biogas plant. This model considers monosaccharides to degrade through lactic acid, which further degrades majorly into acetate followed by propionate and butyrate. Experimental data were derived from the previous works in the same laboratory, and the proposed parameters were validated against batch experiments. After successful validation, the biogas plant bearing a fermenter size of 7 dam3 and operated with food waste and cattle manure was simulated. The biogas production and methane content were reliably simulated, and a good fit could be obtained against the experimental data with an average difference of less than 1%. When compared to the original ADM1 model, the performance of the lactate-incorporated model was found to be improved. Inclusion of lactate as a parameter in the ADM1xp model is recommended for an increased sensitivity and enhanced prediction principally for systems dealing with high carbohydrate and lactate loads.

Modelling anaerobic digestion in a biogas reactor: ADM1 model development with lactate as an intermediate (Part I).

The Anaerobic Digestion Model No. 1 (ADM1) was extended to include lactate, a crucial metabolic product during sugar fermentation. This study tests the validity of the modified ADM1 model in improving the predictions of a standard biogas reactor. This reactor was prepared in the laboratory with simple organic substrates with an intention to represent an 'average biogas plant'. Kinetic parameters were determined from a lactic acid enriched steady-state reactor. The parameters were adjusted further in order to acquire satisfying simulation results systematically with the batch experiments and then against the standard biogas reactor. Arresting methanogenesis revealed that lactate degradation occurred majorly via acetate followed by propionate, and a non-negligible proportion of butyrate too was found, which were further updated in the model. The modified ADM1 provided a successful correlation with the experimental results for the batch and continuous experiments. We justified that inclusion of lactate in the model resulted in optimized simulation for both biogas and methane content in the standard biogas reactor.

Genome sequence of Shimia str. SK013, a representative of the Roseobacter group isolated from marine sediment.

Shimia strain SK013 is an aerobic, Gram-negative, rod shaped alphaproteobacterium affiliated with the Roseobacter group within the family Rhodobacteraceae. The strain was isolated from surface sediment (0-1 cm) of the Skagerrak at 114 m below sea level. The 4,049,808 bp genome of Shimia str. SK013 comprises 3,981 protein-coding genes and 47 RNA genes. It contains one chromosome and no extrachromosomal elements. The genome analysis revealed the presence of genes for a dimethylsulfoniopropionate lyase, demethylase and the trimethylamine methyltransferase (mttB) as well as genes for nitrate, nitrite and dimethyl sulfoxide reduction. This indicates that Shimia str. SK013 is able to switch from aerobic to anaerobic metabolism and thus is capable of aerobic and anaerobic sulfur cycling at the seafloor. Among the ability to convert other sulfur compounds it has the genetic capacity to produce climatically active dimethyl sulfide. Growth on glutamate as a sole carbon source results in formation of cell-connecting filaments, a putative phenotypic adaptation of the surface-associated strain to the environmental conditions at the seafloor. Genome analysis revealed the presence of a flagellum (fla1) and a type IV pilus biogenesis, which is speculated to be a prerequisite for biofilm formation. This is also related to genes responsible for signalling such as N-acyl homoserine lactones, as well as quip-genes responsible for quorum quenching and antibiotic biosynthesis. Pairwise similarities of 16S rRNA genes (98.56 % sequence similarity to the next relative S. haliotis) and the in silico DNA-DNA hybridization (21.20 % sequence similarity to S. haliotis) indicated Shimia str. SK013 to be considered as a new species. The genome analysis of Shimia str. SK013 offered first insights into specific physiological and phenotypic adaptation mechanisms of Roseobacter-affiliated bacteria to the benthic environment.

Different substrates and starter inocula govern microbial community structures in biogas reactors.

The influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.

Distinct compositions of free-living, particle-associated and benthic communities of the Roseobacter group in the North Sea.

The Roseobacter group is one of the predominant lineages in the marine environment. While most investigations focus on pelagic roseobacters, the distribution and metabolic potential of benthic representatives is less understood. In this study, the diversity of the Roseobacter group was characterized in sediment and water samples along the German/Scandinavian North Sea coast by 16S rRNA gene analysis and cultivation-based methods. Molecular analysis indicated an increasing diversity between communities of the Roseobacter group from the sea surface to the seafloor and revealed distinct compositions of free-living and attached fractions. Culture media containing dimethyl sulfide (DMS), dimethyl sulfonium propionate (DMSP) or dimethyl sulfoxide (DMSO) stimulated growth of roseobacters showing highest most probable numbers (MPN) in DMSO-containing dilutions of surface sediments (2.1 × 10(7) roseobacters cm(-3)). Twenty roseobacters (12 from sediments) were isolated from DMSP- and DMS-containing cultures. Sequences of the isolates represented 0.04% of all Bacteria and 4.7% of all roseobacters in the pyrosequencing dataset from sediments. Growth experiments with the isolate Shimia sp. SK013 indicated that benthic roseobacters are able to switch between aerobic and anaerobic utilization of organic sulfur compounds. This response to changing redox conditions might be an adaptation to specific environmental conditions on particles and in sediments.

Temperature and pressure adaptation of a sulfate reducer from the deep subsurface.

Microbial life in deep marine subsurface faces increasing temperatures and hydrostatic pressure with depth. In this study, we have examined growth characteristics and temperature-related adaptation of the Desulfovibrio indonesiensis strain P23 to the in situ pressure of 30 MPa. The strain originates from the deep subsurface of the eastern flank of the Juan de Fuca Ridge (IODP Site U1301). The organism was isolated at 20°C and atmospheric pressure from ~61°C-warm sediments approximately 5 m above the sediment-basement interface. In comparison to standard laboratory conditions (20°C and 0.1 MPa), faster growth was recorded when incubated at in situ pressure and high temperature (45°C), while cell filamentation was induced by further compression. The maximum growth temperature shifted from 48°C at atmospheric pressure to 50°C under high-pressure conditions. Complementary cellular lipid analyses revealed a two-step response of membrane viscosity to increasing temperature with an exchange of unsaturated by saturated fatty acids and subsequent change from branched to unbranched alkyl moieties. While temperature had a stronger effect on the degree of fatty acid saturation and restructuring of main phospholipids, pressure mainly affected branching and length of side chains. The simultaneous decrease of temperature and pressure to ambient laboratory conditions allowed the cultivation of our moderately thermophilic strain. This may in turn be one key to a successful isolation of microorganisms from the deep subsurface adapted to high temperature and pressure.

Oxidative stress and starvation in Dinoroseobacter shibae: the role of extrachromosomal elements.

Aerobic anoxygenic phototrophic bacteria (AAP) are abundant in the photic zone of the marine environment. Dinoroseobacter shibae, a representative of the Roseobacter group, converts light into additional energy that enhances its survival especially under starvation. However, light exposure results in the production of cytotoxic reactive oxygen species in AAPs. Here we investigated the response of D. shibae to starvation and oxidative stress, focusing on the role of extrachromosomal elements (ECRs). D. shibae possessing five ECRs (three plasmids and two chromids) was starved for 4 weeks either in the dark or under light/dark cycles and the survival was monitored. Transcriptomics showed that on the chromosome genes with a role in oxidative stress response and photosynthesis were differentially expressed during the light period. Most extrachromosomal genes in contrast showed a general loss of transcriptional activity, especially in dark-starved cells. The observed decrease of gene expression was not due to plasmid loss, as all five ECRs were maintained in the cells. Interestingly, the genes on the 72-kb chromid were the least downregulated, and one region with genes of the oxygen stress response and a light-dependent protochlorophyllide reductase of cyanobacterial origin was strongly activated under the light/dark cycle. A Δ72-kb curing mutant lost the ability to survive under starvation in a light/dark cycle demonstrating the essential role of this chromid for adaptation to starvation and oxidative stress. Our data moreover suggest that the other four ECRs of D. shibae have no vital function under the investigated conditions and therefore were transcriptionally silenced.

High virus-to-cell ratios indicate ongoing production of viruses in deep subsurface sediments.

Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (∼14 Ma old) and the most oligotrophic subsurface sediments of the world's oceans (South Pacific Gyre (SPG)). The numbers of viruses (10(4)-10(9) cm(-3), counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.

Genomics and physiology of a marine flavobacterium encoding a proteorhodopsin and a xanthorhodopsin-like protein.

Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete ß-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.