Monday 20 August

PS02 – Light and Microbial Life

421A Differential regulation of proteorhodopsin gene expression in the marine bacterium Vibrio sp. AND4 Neelam Akram*1, Joakim Palovaara1, Jeremy Forsberg1, José M González2, Jarone Pinhassi1 1Linnaeus University, SE-39182 Kalmar, Sweden, 2University of La Laguna, ES-38206 La Laguna, Tenerife, Spain

Proteorhodopsin, a light driven proton pump, is a widely distributed and highly expressed photoprotein in marine environments. It is expected to be important to bacteria by supplying energy for cell metabolism. Indeed first experiments have shown that proteorhodopsin in different bacteria can sustain growth yields or assist improved survival during starvation. However, knowledge of factors that regulate proteorhodopsin gene expression in different bacteria remains strongly limited. In the present study, starvation experiments with Vibrio sp. strain AND4 from different growth phases showed that proteorhodopsin phototrophy mediated a light-stimulated survival response only in cells from stationary phase and not in actively growing cells. Real time quantitative PCR analyses showed that proteorhodopsin gene expression was tightly regulated through different phases of growth with very low values in exponential phase, a pronounced peak at the intersection between exponential and stationary phase and a marked decline in stationary phase. Thus, proteorhodopsin gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in this Vibrio species. Our experiments further showed that nutrient limitation, and not light, was the major regulating factor for the differential proteorhodopsin gene expression seen in this bacterium. In an ecological context, our findings suggest that some proteorhodopsin-containing bacteria adapted to the exploitation of nutrient-rich microenvironments, like organic particles or on/in , rely on a phase of relatively slowly declining resources to prepare for oligotrophic conditions dispersed in the sun-lit waters. Additional screenings of 24 isolated marine vibrios revealed one new vibrio isolate containing the proteorhodopsin gene. The proteorhodopsin gene is also found in three other recently genome sequenced vibrios and comparative genomics analysis among all five shows that the potential for proteorhodopsin phototrophy is found in three separate clusters in the genus Vibrio. It is noteworthy that the proteorhodopsins in these vibrios originate from horizontal gene transfer events, either from alpha or gamma proteobacteria, and that the genomic localization of the proteorhodopsin gene differs greatly among closely related bacteria. These findings emphasizse that proteorhodopsin phototrophy may be more widely distributed among marine vibrios than previously recognized, which in turn, signifies the importance of photoheterotrophic potential in vibrios and other potential pathogenic bacteria.

422A Genome mining revealed coding genes belonging to the photolyase-cryptochrome family in UV-resistant bacteria isolated from High-Altitude Andean Lakes Virginia Helena Albarracin*1, Daniel Kurth2, Gabriela Ferrer2, Omar Federico Ordoñez2, Santiago Revale3, Estefania Mancini3, Soledad Romero3, Martin Vazquez3, Wolfgang Gärtner1, Maria Eugenia Farias2 1Max-Planck Institute for Bioinorganic Chemistry, Germany, 2Lab of Microbial Research on Andean Lakes(LIMLA), PROIMI-CCT-CONICET, , 3Rosario Agrobiotechnological Institute (INDEAR) – CONICET, CCT-CONICET, Argentina

High-Altitude Andean Lakes (HAAL) are a disperse group of shallow lakes allocated in the Puna-High Andes ecoregion above 4,000 m, at Northwestern Argentina. Environmental conditions in the HAAL are stressful for life, but UV irradiation is giving the greatest pressure on the ecology of its outstanding microbial diversity. This work presents the analysis of recently made available genomes of three UV resistant bacterial strains, isolated from these extreme environments and highlights the presence of coding-genes for members of the photolyase-cryptochrome protein family (PL-Cry).

We used Acinetobacter sp. Ver3, Exiguobacterium sp. S17, and Nesterenkonia sp. Act20, isolated from shallow water of Laguna Verde (4,400 m asl), from modern and from the surrounding soil of Laguna (4,000 m asl), respectively. Maximal UV-B intensity registered in situ was 10.78 W m-2. Genome sequences were obtained using a whole-genome shotgun strategy with a 454 GS Titanium pyrosequencer at INDEAR, Argentina. Genomes were annotated and analyzed in the RAST annotation server. PSI-BLAST and ClustalW were used to compare and align sequences, and phylogenetic trees were built using Mega4.

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Genome sizes were 3,349 Mb for Ver3, 3,139 Mb for S17 and 2,941 Mb for Act20. A total of 3,213 Coding Sequences (CDS) and 66 structural RNAs were predicted in Ver3. Annotation covered 320 RAST subsystems (45%) with 1417 CDS and 55 hypothetical proteins; S17 displayed 3220 CDS and 49 structural RNAs. Only 43% CDS were annotated in RAST subsystems with 1381 CDS and 73 hypothetical proteins. In Act20, we found 2,666 CDS and 50 structural RNAs. Annotation covered 302 RAST subsystems (41%) with 1070 CDS and 34 hypothetical proteins.

PL-Cry proteins were found in all the studied genomes, and were classified phylogenetically. These photolyases fell within three groups: CPD-PLs, photolyase-related proteins (PRPs), and Cryptochromes. The first two groups were represented in the three organisms studied, while a Cry- DASH related protein with 72% identity with Exiguobacterium sibiricum was found only in S17. CPD- PLs from Ver3 and S17 cluster within the Class I-CPD PLs. Interestingly, Act20-PL1 does not cluster within the group of the so-called actinomycetes CPD-photolyases but as a sister branch of the plant cryptochromes and Class III CPD-photolyases. Finally, Ver3 PRPs is related to the newly described FeS bacterial cryptochromes and photolyases (FeS-BCPs). S17 and Act20 also bear PRPs in the same group, but more distantly related to the known characterized FeS-BCPs.

We have found a rich diversity and complex phylogenetic arrangement of the Cry-PL genes in different strains from HAAL. These gene products most probably enable them to sense and respond to the high UV light intensity they suffered in their original environment. Overexpression experiments are currently processed aiming to demonstrate that these “extreme” photoreceptors are functional and effective for these purposes.

423A Reactive oxygen production induced by visible and near-infrared radiation in chlorophyll-d containing Lars Behrendt*1, Marc Staal1, Simona M. Cristescu2, Frans J.M. Harren2, Martin Schliep3, Anthony W. D. Larkum3, Michael Kühl1 1Copenhagen University, Denmark, 2Radboud University Nijmegen, Netherlands, 3University of Technology Sydney,

Cyanobacteria in the genus Acaryochloris are the only known organisms using Chl d to harvest near- infrared-radiation (NIR, >700 nm) for oxygenic photosynthesis, a light-driven electron pathway that is particularly prone to generate reactive oxygen species (ROS). Here we present the first study of ROS production in Acaryochloris strains. We recorded ROS formation in one novel (Acaryochloris strain CRS) and two previously described strains (Acaryochloris strains MBIC11017 and HICR111A) as a function of irradiance and spectral light composition. ROS was quantified using a real-time laser-based ethylene detector in conjunction with the substrate 2-keto-4-thiomethylbutyric acid (KMBA) that is converted to ethylene in proportion to the amount of certain types of reactive oxygen in the sample. We tested the effect of light conditioning by adapting all strains to either NIR or visible light before experimental treatments. Adaptation to NIR resulted in higher ROS production in the strains MBIC11017 and HICR111A when experimentally exposed to visible light or NIR. This is in contrast to strain CRS, which was more susceptible to generating ROS when previously grown at visible light. Action spectra performed on strain MBIC11017 and HICR111A revealed that blue (470 nm) and cyan (495 nm) light is the strongest inducer of ROS, while amber (595 nm) light was the least effective. Strain HICR111A showed less ROS production than strain MBIC11017 when exposed to monochromatic light; we hypothesize that the preference of HICR111A to form cell-aggregates results in lower ROS production due to self-shading, possibly providing a selective advantage in high-light environments.

424A Oxygenic photosynthesis as a defense mechanism against iron-oxide encrustation Bettina Buchmann1, Lubos Polerecky1, Christine Heim2, Dirk de Beer1, Danny Lonescu*1 1Max Planck Institute for Marine Microbiology, Germany, 2University of Goettingen, Germany

At circumneutral pH, under oxic conditions, Fe2+ is rapidly oxidized to Fe3+ which precipitates as FeO(OH). This process is accelerated as the pH and O2 increase. We connected sets of 2 illuminated bioreactors to 2 groundwater aquifers with different Fe2+ concentrations in the Aspo Hard Rock Laboratory, Sweden. At each station one of the bioreactors had an air headspace. Cyanobacterial mats developed in each of the reactors and were shown to be phylogenetically different from each other. Interestingly, the cyanobacteria growing in the lowest Fe2+ concentration (1 µM) were encrusted

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in iron, whereas, those growing in high Fe2+ concentration (30 µM) were not. In-situ measurements using pH and O2 microelectrodes have shown that all the cyanobacteria from the reactors perform oxygenic photosynthesis with those continuously submerged in Fe2+ rich water (reactor with no headspace), at a much higher rate than the others. Lab measurements on cultures from each of the reactors showed different reactions to increasing Fe2+ concentrations. The cyanobacteria normally growing at low Fe2+ were inhibited when the Fe2+ concentration was increase above 5 µM. The cyanobacteria obtained from a floating mat in the reactor with headspace at the high Fe2+ aquifer, were inhibited as well above 5 µM Fe2+. In contrast, the cyanobacteria obtained from the reactor with no headspace at the high Fe2+ aquifer showed increasing photosynthesis with increasing iron concentrations (up to 50 µM). In a similar experiment at a pH above 8, where given the oxic conditions, the half life time of Fe2+ is <1 s, no inhibitory effects were detected. A diffusion model showed a photosynthetically induced decrease in Fe2+ concentrations in the diffusive boundary layer. 2+ Using the O2 and pH data obtained in-situ from the high-Fe , constantly submerged cyanobacteria, the model shows that no Fe2+ reaches the mat. Therefore, we suggest that the two cyanobacterial communities living under high Fe+2 concentrations use two different strategies: The cyanobacteria in the reactors with headspace form floating mats and therefore do not come in contact with the high Fe2+; The cyanobacteria constantly submerged in the reactor with no headspace use high rate 2+ photosynthesis, thus increasing the O2 and the pH in their microenvironment enough to prevent Fe to reach the cells. The submerged cyanobacteria in the low Fe2+ aquifer do not alter their environment sufficiently to prevent encrustation; however, they survive due to the low Fe2+ concentration.

425A Surface Plasmon Resonance assays indicate that NtcA binds several L-amino acids Laura Calvo, Taís Mayumi Kuniyoshi*, María del Mar Pascual, Andrés González, María Fillat, Maria Luisa Peleato Universidad de Zaragoza/Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Spain

In Microcystis aeruginosa, the mcy operon encodes the genes responsible for microcystin synthesis. The aim of this work is the study of the regulation of the expression of those genes. Using electrophoretic band-shift assay (EMSA), we described that the master nitrogen transcriptional regulator NtcA exhibits affinity for two fragments of the bidirectional mcyDA promoter, as well as for other internal promoter regions such as mcyE and mcyH. The presence of 2-oxoglutarate increased by 2.5 fold the affinity of NtcA for the mcyA promoter region. This suggests that the 2-oxoglutarate levels, as signal of the C to N balance of the cells, regulate the microcystin gene cluster.

Kinetic analysis of the interaction between NtcA and 2-oxoglutarate and several amin oacids or metabolites has been studied by using SPR (surface plasmon resonance).

We used a sensor chip CM5, with high capture capacity, to inmobilize anti-histidine tag antibodies with amino coupling (EDC/NHS). After that, we captured the his-tagged NtcA each cycle, and performed the measure of the binding of the compounds tested at different concentrations.

2-oxoglutarate binds NtcA with a KD in the nanomolar range, and unexpectedly other L-amino acids such as glutamic acid, aspartic acid and asparagine binds the regulator. Lysine, arginine, alanine, glutamine and other L-amino acids tested do not bind NtcA.

The results obtained suggest that other molecules than 2-oxoglutarate can act as signalling of the C:N metabolic balance in cyanobacterial cells, with interesting consequences in the mcy operon regulation.

426A Response of Prochlorococcus to extended darkness Allison Coe*, Stephen Biller, Sara Roggensack, Sallie Chisholm MIT, United States

Prochlorococcus is the smallest (<1µm) and most abundant (sometimes over 105 cells/mL) oxygenic phototroph in the ocean euphotic zone (~0-200m) between 40˚N and 40˚S. The ecology of of high light (HL) and low light (LL)-adapted Prochlorococcus ecotypes have been studied extensively over the diel cycle in the lab and in the field, but not much is known about the ability of Prochlorococcus to survive extended darkness. There is good reason to believe that Prochlorococcus could get mixed below the euphotic zone for extended periods, and the ability to withstand this would influence its fitness. We set

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out to examine this by subjecting of a number of axenic HL and LL strains to varying periods of darkness, and then returning the cells to their normal growth conditions to examine their ability to recover as a function of dark exposure time. Some strains can survive extended darkness for short periods of time (up to 35 hours), while others cannot. LL-adapted ecotypes are not more resistant to extended darkness than the high light HL-adapted strains, and thus far there appears to be no relationship between dark survival ability and rRNA phylogeny. There is, however, a difference between phylogeny in the response time of recovery after extended darkness, with members of the LL clade responding quicker. Whether they recover or not, cells exhibit little change in flow cytometric properties after months in the dark.

Recent studies have suggested that co-culture with heterotrophic bacteria reduces oxidative stress in Prochlorococcus, and increases its fitness. We have found that the addition of the heterotroph Alteromonas to cultures of Prochlorococcus, with no added organics to the media, increased Prochlorococcus' ability to tolerate extended darkness. To determine if this was related to oxidative stress, we tested multiple pure strains of Prochlorococcus with additions of reactive oxygen species (ROS) scavenging compounds sodium pyruvate and sodium thiosulfate. Only under very high concentrations, well above the levels needed to reduce hydrogen peroxide in the media, were the compounds able to prolong the ability of Prochlorococcus to survive extended darkness. This suggests that it is not likely hydrogen peroxide toxicity that would be preventing the cells from recovering from extended darkness, but rather potential uptake of these organic carbon compounds by Prochlorococcus. The role or function Alteromonas performs remains a mystery, but we have been able to eliminate reduction of oxidative stress as the sole explanation. We are in the process of studying the transcriptional response to dark exposure with and without Alteromonas to begin to understand how cells reshape their metabolism when they cannot carry out photosynthesis for extended periods.

427A The phyllosphere: a phototrophic niche? Omri Finkel*1, Nof Atamna-Ismaeel2, Fabian Glaser2, Itai Sharon3, Ron Schneider1, Anton F. Post4, John L. Spudich5, Christian von Mering6, Julia A. Vofholt7, David Iluz8, Oded Béjà2, Shimshon Belkin1 1Hebrew University of Jerusalem, Israel, 2Technion – Israel Institute of Technology, Israel, 3University of California, United States, 4Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biology Laboratory, United States, 5University of Texas-Houston Medical School, United States, 6Institute of Molecular Life Sciences and Swiss Institute of Bioinformatics, University of Zurich, Switzerland, 7Institute of Microbiology, Eidgenössische Technische Hochschule, Switzerland, 8Bar Ilan University, Israel

Solar radiation is the main source of energy for both marine and terrestrial organisms, with terrestrial plants and aquatic performing an equivalent ecological function as chlorophyll-based photosynthetic primary producers. Marine surface waters are now known to harbor additional types of phototrophy; many marine microbes utilize rhodopsins, retinal-containing membrane-embedded proteins, as light-driven proton pumps. Microbial rhodopsins have been found in diverse aquatic habitats including freshwater, sea ice, hypersaline and brackish environments.

As light is an abundant resource on land, we tested the hypothesis that microbial rhodopsins also exist and play an important role in terrestrial niches. The leaf surface of terrestrial plants covers a surface area of an estimated 6.4X108 km2 and comprises the main interface between terrestrial biomass and solar photon flux. This habitat harbors diverse microbial communities of up to 106–107 cells per cm2 leaf surface. A mode of phototrophy that is compatible with the plant’s photosynthesis would offer a significant ecological advantage to microbes inhabiting this environment.

Based on 454-pyrosequencing-generated data, later corroborated by PCR, we report on the existence of genes encoding microbial rhodopsins in all five phyllosphere metagenomes that were tested: tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Most sequences were found to represent novel and distinct clades of both sensory and proton pumping rhodopsins. Interestingly, all reads containing position 105 of the translated sequence, encoded a Leucine residue, suggesting that these rhodopsins absorb in green, thus avoiding competition with the plant’s chlorophyll.

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Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.

428A The Roseobacter clade affiliated (RCA) cluster: Do these microbes conduct aerobic anoxygenic photosynthesis? Helge-Ansgar Giebel*1, Bernd Wemheuer2, Sonja Voget3, Meinhard Simon4 1University Oldenburg, Institute for Chemistry and Biology of the Marine Environment (ICBM), Germany, 2Georg-August University of Goettingen, Department of Genomic and Applied Microbiology, Institute of Microbiology and Genetics, Germany, 3Georg-August University of Goettingen, Goettingen Genomics Laboratory, Institute of Microbiology and Genetics, Germany, 4University Oldenburg / Institute for Chemistry and Biology of the Marine Environment (ICBM), Germany

Recent studies showed that among the main bacterioplankton groups photoheterotrophy is a surprisingly common feature. Within Alphaproteobacteria quite a few representatives of the Roseobacter clade are known to carry genes encoding bacteriochlorophyll a (BChla) and thus are capable of aerobic anoxygenic photosynthesis (AAnP). However, still little is known about the genomic organisation and expression of these genes in the major bacterioplankton groups including the Roseobacter Clade Affiliated (RCA) cluster.

Genomic analyses of RCA isolates revealed the presence of the entire BChla operon. The operon organisation and structure of these isolates differ from that of several type strains of the Roseobacter clade and are more related to those of the Rhodobacter clade. Main sections of the photosynthetic operon of strain RCA23 are analog to that of the genera of Loktanella and Thallassobium of the Roseobacter clade but differ in the pufX gene relative to Rhodobacter species. Physiological growth tests of RCA isolates failed to express the photosynthetic operon such that no BChla containing RCA cells have been detected. Signatures of BChla genes of the RCA cluster were identified in samples of the Global Ocean Sampling (GOS) data set in temperate regions complying with the known biogeographic distribution of this cluster. Expression of the photosynthetic operon of the RCA cluster in environmental samples was detected in the German Bight (North Sea) during a metatranscriptomic study. The regulation of AAnP of the RCA bacteria is still unknown, but a new specific transcriptional qPCR targeting the RCA photosynthetic genes will gain new insights into the significance of AAnP. Because RCA clones constitute the largest cluster within the Roseobacter clade and because of the high abundance of these organisms in several oceanic regions it is of utmost importance to understand the stimulation factors and regulation of the RCA BChla expression in bacterioplankton communities.

429A Inhibition of heterotrophic bacteria by solar radiation in a humic lake Jens Glaeser*1, Stefanie Glaeser1, Franziska Leunert2, Ivette Salka2, Hans-Peter Grossart2 1Justus-Liebig-University, Germany, 2Leibniz-Institut of Freshwater Ecology and Inland Fisheries, Germany

Solar radiation causes photochemical reactions that lead to the breakdown of dissolved organic matter in aquatic ecosystems. This process enhances the availability of low molecular weight substrates for heterotrophic bacteria. Simultaneously reactive oxygen species are generated. Humic substances act as photosensitizers and therefore mediate the photochemical formation of reactive oxygen species and therefore bacteria inhabiting humic rich lakes are exposed to increased oxidative stress. The formation of reactive oxygen species by humic substances has been known for decades, but detailed experiments on their impact on bacterial activity and community composition were lacking. In order to shed light on the question if reactive oxygen species affect bacterial activity and community structure we i) monitored diurnal cycles of bacterial activity in the humic south-west basin of Lake Grosse Fuchskuhle and ii) assessed differences in the sensitivity of dominating bacterioplankton lineages to selected reactive oxygen species.

High solar radiation caused strong inhibition of bacterial 14C-leucine and 14C-acetate uptake in surface waters and increased the fraction of membrane-damaged cells assessed by life/dead staining. The observed inhibition was paralleled by the formation of reactive oxygen species as singlet oxygen and hydrogen peroxide. The impact of increased oxidative stress on bacterial community composition was

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assessed by the generation of 16S rRNA gene clone libraries and 16S rRNA targeting RT-PCR DGGE analysis using Bacteria and group-specific primer-systems. Alphaproteobacteria (Novosphingobium acidiphilum) and Betaproteobacteria (Polynucleobacter necessarius and Limnohabitans related species) increased in relative abundance after singlet oxygen but not after hydrogen peroxide exposure. In contrast AcI-B Actinobacteria were sensitive to singlet oxygen exposure. Dominating bacterial groups responded differently to singlet oxygen and hydrogen peroxide exposure. Therefore the impact of oxidative stress on the bacterial communities strongly depends on the nature and toxicity of individual reactive oxygen species.

In order to verify our data, isolates representing predominant bacterial phylotypes were incubated in the surface water layer by using dialysis bags. N. acidiphilum represents a persistent species of the south-west basin and was not hampered in the assimilation of leucine and acetate by solar radiation. In contrast, leucine and acetate uptake by P. necessarius - a predominant Betaproteobacteria representative - was strongly inhibited during solar radiation exposure. Cultures of both strains showed a high fraction of life cells that did not decrease during daytime hours and therefore efficient mechanisms to cope with inhibitory products of photochemical reactions may be present in both species. In conclusion, different patterns of metabolic activity of dominating bacterioplankton phylotypes are strongly affected by solar radiation and subsequent photochemical reactions generating reactive oxygen species. As a consequence the presence of reactive oxygen species may strongly affect the activity and subsequently the structure of bacterial communities inhabiting lakes containing humic substances.

430A Diel cycles of dissolved B-Vitamins in the coastal ocean Laura Gómez-Consarnau*1, Lynda Cutter1, Jacob A. Cram1, David Needham1, Guillem Salazar2, Jed A. Fuhrman1, Josep M. Gasol2, Sergio A. Sañudo-Wilhelmy1 1University of Southern California, USA, 2Institut de Ciències del Mar-CSIC, Spain

Although vitamins play a crucial role in cellular metabolism, most phytoplankton species are unable to synthesize them de novo. It has recently been shown that large areas of the world’s ocean contain undetectable levels of B-vitamins (< 1pM), suggesting that they are important limiting growth factors for different members of the planktonic community. Because phytoplankton metabolism is highly regulated by diel cycles, fluctuations on vitamin concentrations throughout the day should be expected. We measured diel patterns in dissolved B-vitamins (B1, B2, B6, B7, and B12) concentrations in 200 liter mesocosm incubations carried out with surface water collected from the Blanes Bay Observatory in the Mediterranean sea under simulated in situ light conditions. While bacterial community composition analyzed with ARISA remained constant throughout the light-dark cycle, B-vitamin concentrations oscillated from day to night and appeared to be independent from each other. For example, while the highest concentrations of B2 were detected at night, the highest concentrations of B6 were measured in the light. These trends are consistent with the vitamin diel patterns observed in situ off Catalina Island in the Pacific Ocean, as well as with gene expression data showing a diel cycle in the up-regulation of some vitamin transporters. Our data suggest that the synthesis and uptake of B-vitamins is a very dynamic process that occurs similarly in different oceanic regimes of the world’s ocean. Furthermore, changes in the microbial diversity appear to be no good predictors of the hourly changes in vitamin concentrations. Measurements of different metabolic processes that dominate in light and dark will be required to better understand the processes controlling B-vitamin concentrations in the marine environment.

431A Comparative proteomics of a green and a purple sulfur bacterium through the light gradient in the water column of Lake Cadagno, Switzerland Kirsten S. Habicht*, Lasse G. Falkenby Univeristy of Southern Denmark, Denmark

The meromictic Lake Cadagno is dominated by anoxygenic phototrophs oxidizing sulfide to elemental sulfur and sulfate with light. In the lake anoxygenic photosynthesis can count for more than 60% of the total carbon fixation. The high concentration of purple and green sulfur bacteria makes this environment a good model system for understanding the physiology of anoxygenic phototrophs in their natural environment. We present a proteomic study of the green sulfur bacterium Chlorobium clathratiforme and the purple sulfur bacterium Thiodictyon Cad16 with the goal to understand how these organisms adapt to the natural physicochemical gradient of the lake with a special focus on how

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these organisms survive in the dark. Although metagenomic data is not available for Lake Cadagno, a proteomic analysis was possible as the complete genome sequences of the two species were available. In Lake Cadagno C. clathratiforme is the most common carbon-fixing organism with maximum cell densities of more than 107 cells ml-1 whereas Thiodictyon CAD16 is the most common purple sulfur bacterium although at a cell density more than 100 times lower than for C. clathratiforme. Both species are found at the photic chemocline and far down into the dark part of the lake. Cells from four depths in the water column were used in a comparative proteomic analysis. Using a gel-free, filter-aided sample preparation (FASP) protocol together with an in-solution isotope labeling strategy and with LC-MS/MS analysis we were able to indentify about 1200 proteins from C. clathratiforme and 400 from Thiodictyon CAD16 and to quantify ¼ of these proteins. Comparing the protein expression found at the oxic/anoxic interface with the expression through the anoxic water column we find that the purple sulfur bacteria more than double their expression of key enzymes involved in carbon metabolism and photosynthesis at their maximum density in the photic zone and then down-regulate the expression in the dark. In contrast, similar enzymes in the green sulfur bacteria had the same expression in both light and dark. Proteins participating in nitrogen and sulfur metabolism were for both organisms half as abundant in the dark compared to light. Both organisms furthermore showed high expression of N2-fixing enzymes in light but not in the dark. From the enzyme expression we suggest that C. clathratiforme in the dark part of the water column obtains energy for maintenance from the fermentation of organic compounds. Our comparative proteomic study gives unique insight into the physiology of phototrophic bacteria in their natural environment.

432A Light dependent oxygen consumption in a green sulfur bacteria biofilm from the Dead Sea Stefan Haeusler*1, Danny Ionescu1, Christian Lott2, Miriam Weber2, Dirk de Beer1 1Max Planck Institute for Marine Microbiology, Germany, 2HYDRA Institute for Marine Sciences, Italy

The Dead Sea is hypersaline terminal lake with up to 350 g/l total dissolved salts. The high Mg (2 M) and Ca (0.5 M) concentrations make it a hostile environment for most organisms. A recently discovered underwater freshwater system in the Dead Sea is an oasis of life in an otherwise desert environment. Within this system biofilms were described for the first time in the Dead Sea. We studied a green biofilm commonly found on cobble in some of these springs.

Microscopy showed the presence of small unicellular autofluorescent cyanobacteria, , and small green cells which lack autofluorescence in the visible spectrum. Single cell pigment analysis using hyperspectral imaging showed specific absorption maxima at 740 nm indicating bacteriochlorophyll c, a specific pigment of green sulfur bacteria. Cyanobacterial cells could be identified by specific absorption of chlorophyll a (640 nm) in close association. The presence of both pigments was confirmed by high-performance liquid chromatography analysis. We used oxygen microsensors to analyze the photosynthetic properties of this system. While oxygen production was never measured in a green biofilm it was occasionally detected in adjunct white biofilms. Applying the light-dark shift method using an oxygen microsensor we detected a light-dependent oxygen consumption. This could be shown for light intensities ranging from 20 to 1500 µE with increasing consumption with higher light intensities. Furthermore, the community was able to perform light- dependent oxygen consumption in a gradient from 10% to 100% Dead Sea water without loss of response. Maximum rates were measured at a 50% or 60% Dead Sea water depending on light intensity. Light-dependent oxygen consumption was previously described in microbial mats and attributed to the Mehler reaction in oxygenic phototrophs or to the activity of type A flavoproteins in anoxygenic phototrophs. We propose that light-dependent oxygen consumption is a survival strategy used by the strictly anaerobic green sulfur bacteria to coexsist in a non stratified mat with oxygenic phototrophs. The large salinity range in which the biofilm maintains its activity suggests the organisms are well adapted to the fluctuating conditions in the underwater springs.

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433A Summer Solar Radiation effects on autotrophic activity in a high anathalassohaline evaporitic basin: Salar de Huasco-Chilean Altiplano Klaudia Hernandez*1, Lasse Olsen2, Verónica Molina3, Martha Hengst4, Chris Harrod5, Cristina Dorador6 1Universidad Austral de /CIEN Austral, Chile, 2Norwegian University of Science and Technology, Norway, 3Universidad Andrés Bello, Chile, 4Centro de Bioinovación/Facultad de Recursos del Mar/Universidad de Antofagasta, Chile, 5Queen's University Belfast, United Kingdom, 6Facultad de Recursos del Mar/Universidad de Antofagasta, Chile

Solar radiation is a key-driving factor for microbial diversity and function in ecosystems. Even under natural levels, solar radiation might impair organisms and cause DNA damage, photoinhibition of photosynthesis, and cell death. Salar de Huasco (a high-altitude saline located at 3800m in the Chilean Altiplano) represents an extreme and dynamic environment, with a high photoautotrophic microbial diversity, ranging from picoplankton to micro-phytoplankton. Here we assessed the effects of natural sunlight on autotrophic activity (as a proxy of photosynthesis) in Salar de Huasco. Our aim was to investigate the short-term response of autotrophs to variable irradiance as either photo-acclimation or photo-inhibition. Two short-term (24h) experiments were developed under 'Bolivian winter' weather conditions (February 2012, mid Austral summer) in a shallow pond (Site-H0), historically described as the far north freshwater stream within the natural salinity gradient of Salar de Huasco. Water from H0 was incubated under ambient irradiance for 4, 7 and 9 h and exposed to 3 spectral radiation treatments: photosynthetically active radiation (PAR, 400 to 700 nm), PAR+UVA (PA, 320 to 700 nm), and PAR+UVR (PAB, 280 to 700 nm), plus a dark treatment (without sunlight). Maximum and operational quantum yield of photosystem 2 (Qy) was measured by changes in variable fluorescence. Qy fluorometer was used as a proxy of photosynthetic performance at excitation wavelength 450 nm for eukaryotic phytoplankton, and at 620 nm for cyanobacteria. The results show low stratospheric events (275 DU) that were predominant during the summer season 2012, with one registered "ozone hole" event (250 DU). Maximum total solar radiation registered for the period was 2000 W/m2. Regarding daily doses during experiments Total Radiation registered was ~30 MJ/m2, PAR 28 MJ/m2, UVA 3,7MJ/m2, and UVB 0,3 MJ/m2. The experiments showed strong solar radiation effects on Qy, mainly for phytoplankton and in a lower extent for cyanobacteria. Lowest phytoplankton QY values were observed under PA and PAB treatment (QY ~0.03) compared to PAR (QY ~0.23) during and 3 hours following solar noon. Maximum phytoplankton QY values were observed during dark treatment at sunset (QY ~0.6). In the case of cyanobacteria, the QY trend was similar but strong inhibition occurred in PA treatment (QY ~0.03) mainly at noon. Ammonium concentration (0.3-0.35 µM) showed a variable response in all irradiated treatments, but decreased overall after 9 hours of exposure (0.19 µM). No clear trend was observed for in situ chlorophyll-a concentrations, which ranged between 0.38 and 1.14 mg Chla/m3. Phytoplankton abundance showed high numbers (~9.5x106 cells/ml) and cyanobacteria were mainly dominated by Oscillatoria sp. (~1.82x106 cells/ml). Our results suggest that in this environment with extreme radiation at noon, strong photoinhibition is present during the day with photorepair processes starting during the late afternoon, influencing also nitrogen cycle.

434A Summer community structure of aerobic anoxygenic phototrophic bacteria in the western Arctic Ocean Christian Jeanthon*1, Dominique Boeuf1, Matthew Cottrell2, David Kirchman2, Philippe Lebaron3 1Station Biologique, France, 2University of Delaware, USA, 3Observatoire de Banyuls, France

Photoheterotrophy, which is the ability to utilize organic substrates and harvest light energy, occurs in a broad range of microbes. Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that require oxygen for their growth and for bacteriochlorophyll a synthesis. In the Arctic Ocean, we actually observe an increase in solar radiation due to a decrease in summer ice cover and increase the export of organic carbon to the ocean due to permafrost thawing and increased river-runoff. Therefore, the hybrid metabolism of photoheterotrophs (respiration and phototrophy) may likely make them key players in the response of coastal arctic ecosystems to ongoing change.

Using infrared epifluorescence microscopy, cultural and molecular approaches, we analyzed the AAP bacterial abundance and diversity in the Mackenzie river/Beaufort Sea system in samples collected during the MALINA cruise (July-August 2009). Very low relative abundances (

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A collection of 92 bacteriochlorophyll a–containing isolates were isolated from arctic surface waters. Based on their 16S rRNA gene sequences, all were Alphaproteobacteria affiliated to the family Rhodobacteraceae and belonged to the genera Sulfitobacter and Loktanella. Molecular analyses of thirteen pufM DNA-based libraries (365 partial pufM sequences) revealed the dominance of Alphaproteobacteria and Betaproteobacteria. Alphaproteobacterial pufM sequences were mostly recovered from oceanic oligotrophic samples whereas samples influenced by the Mackenzie plume were dominated by betaproteobacterial sequences.

Since bacteriochlorophyll a synthesis is inhibited by light in AAP bacteria, we hypothesized that the infrared epifluorescence numeration method used previously could have underestimated the actual numbers of these microorganisms at the time of sampling (long periods of sunlight in summer at high latitudes). Quantitative PCR experiments confirmed the low abundance of marine AAP bacteria in the Beaufort Sea. Further analyses targeting the most abundant AAP clade retrieved in this study revealed the freshwater origin of betaproteobacterial AAPs and the active expression of their photosynthetic genes in cold offshore marine waters.

435A Combination of nanoscale zero valent iron and organochlorine respiring bacteria for the remediation of 1,1,2-trichloroethane and 1,2-dichloroethane mixtures Joanna Koenig*1, Adrian Low1, Matthew Lee1, Hardiljeet Boparai2, Denis O'Carroll2, Michael Manefield1 1University of New South Wales, Australia, 2University of Western Ontario, Canada

Due to their toxicity and recalcitrance, the chlorinated ethanes 1,2-dichloroethane (DCA) and 1,1,2- trichloroethane (TCA) cause significant soil and aquifer contamination problems around the world. One emerging approach to chlorinated ethane removal is the injection of Nanoscale Zero Valent Iron (NZVI) in the subsurface. NZVI acts as a reductant and transfers electrons to chlorinated compounds, leading to the formation of lesser chlorinated or chlorine-free innocuous hydrocarbons. While TCA is dechlorinated by NZVI at appreciable rates, DCA is invulnerable to its reductive effects, decreasing the attractiveness of NZVI remediation protocols for contaminated sites.

In anaerobic environments, several Organochlorine Respiring Bacteria (ORB) grow through the reductive dechlorination of chlorinated compounds including DCA. A microbial community was established in our laboratory, comprising a novel strain ofDesulfitobacteriumcapable of the rapid conversion of high concentrations of DCA to ethene and TCA to vinyl chloride (VC).

In this study, the effectiveness of employing either this Desulfitobacterium-containing community alone, NZVI alone, or a combination of both in treating a mixture of 0.5 mM DCA and 0.5 mM TCA was evaluated. In the presence of both compounds, the Desulfitobacterium-containing community proceeded to dechlorinate DCA to 100% ethene first, with TCA conversion to 100% VC only being initiated after all DCA was depleted. Further dechlorination of VC did not occur. With NZVI alone, TCA was converted to 70% VC and 30% ethene following pseudo first-order rate kinetics, and DCA remained. Combining the action of NZVI with ORB resulted in faster and more complete dechlorination of both compounds. In this system, the dechlorination of TCA and DCA took place in parallel and not in sequence as observed with ORB alone, and less VC was formed from TCA. Moreover, NZVI could serve as a source of energy for the ORB through the hydrogen evolved from its reaction with water. These results demonstrate that chemical and microbial approaches to pollutant remediation can work in synergy and yield better outcomes than one approach alone.

436A Phosphate availability in N:P 40:1 affects mcyD expression and microcystin synthesis in Microcystis aeruginosa PCC7806 Taís Mayumi Kuniyoshi*, Emma Sevilla, Maria Teresa Bes, María F. Fillat, Maria luisa Peleato University of São Paulo- USP/ICB2, Universidad de Zaragoza/Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Spain

Microcystins are toxins produced by some cyanobacterial strains that entail a serious ecological problem in water bodies all around the world, and a health risk for since it cause hepato- toxicosis trhough the inhibition of protein phosphatase 1 and 2A. Envirionmental and nutritional factors that trigger microcystin synthesis are still unclear, nevertheless, it has been recognized the influence of phosphate and nitrate concentration in freshwater on this cyanotoxin production. The aim of this

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study was to determine whether phosphate deficiency at a ratio of 40:1 N:P or excess of this nutrient at 1:1 N:P induces microcystin synthesis in Microcystis aeruginosa PCC7806 in batch culture. A Real time RT-PCR and semi-quantitative reverse transcription PCR analysis of mcyD transcript revealed that no significant changes were observed in the relative quantification of mcyD under excess phosphate, whereas in deficiency of this nutrient, a steady increase of mcyD during the exponential growth phase was detected, showing a maximal level on the 7th day of growth with a 6.8 fold increase over the control cells. Intracelular microcystin content was determined in each sample by HPLC and PP2A inhibition assay and displayed correlation with the trend of mcyD transcription. In conclusion, in this work we demonstrate that under phosphate limiting conditions with a ratio of 40:1 N/P, M. aeruginosa PCC 7806 showed high levels of mcyD transcript as well as intracellular microcystin content.

437A Functional proteomics of marine cyanobacterium Synechocystis sp. PCC 6906 Yeon Ju Kwak*1, Soo Jung Kim1, Jung Ha Choi1, Yeol Gyun Lee1, Jong Hyun Kim2, Jang Ryol Liu2, Seung Il Kim1, Young Ho Chung1 1Division of Life Sciences, Korea Basic Science Institute, South Korea, 2Plant Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, South Korea

Marine cyanobacteria have abilities to survive in diverse and stressful environments, making them ideal for physiological and proteomic studies of environmental adaptation. Marine cyanobacterium Synechocystis sp. PCC 6906 is arguably successful strain in hypersalinity and low light (< 5 μE/m2/s) condition. Due to its habitation in these unique conditions, the interest in this strain has greatly increased. From this point of view, it is important to understand differently expressed proteins and identify proteins, especially involved in low light adaptation and salt-tolerance. Synechocystis sp. PCC 6906 genome is fully sequenced and recently annotated. In this study, we established a 2D gel reference map for Synechocystis sp. PCC 6906, identifying 201 spots representing 160 proteins. To identify the proteins, we used MALDI-TOF/TOF mass spectrometry in combination with Mascot search program. Proteins mostly related to photosynthesis, light harvesting, cell division, carbon fixation, antioxidation and phosphate transportation were identified. We sought a proteomic outlook on the photoacclimation of marine cyanobacteria in low light conditions. This proteome map could be a useful reference for future physiology study of marine cyanobacteria. We are in progress to find the proteome difference between low and high light stress and also low and high salt stress condition.

438A Widespread occurrence of a novel class of rhodopsins Soon-Kyeong Kwon*1, Byung Kwon Kim2, Ju Yeon Song2, Min-Jung Kwak3, Choong Hoon Lee2, Dae-Hee Lee1, Jung-Hoon Yoon4, Tae Kwang Oh1, Jihyun F. Kim2 1Korea Research Institute of Bioscience and Biotechnology, South Korea, 2Yonsei University, South Korea, 3University of Science and Technology, South Korea, 4Sungkyunkwan University, South Korea

Phototrophs containing microbial rhodopsin contribute to the Earth's energy balance by converting solar light energy into chemical energy. A variety of retinal-binding rhodopsins that either function as ion pumps or have sensory roles have been found in a number of surface-ocean bacteria and archaea. We describe a novel type of microbial rhodopsin with functional residues that differ from those of other types of rhodopsins. Genes encoding these novel rhodopsins are present in recently sequenced genomes, and the corresponding organisms constitute a distinct phylogenic clade. The genome of a flavobacterium, Donghaeana dokdonensis DSW-6, which harbors genes for the novel microbial rhodopsin and for a typical proteorhodopsin, was analyzed. As a marine photoheterotroph, DSW-6 has in its genome physiological features that allow survival in marine oligotrophic environments. To infer the biological role of this novel rhodopsin in DSW-6, the gene expression levels under various environmental conditions were measured. The gene expression of the novel rhodopsin increases with increasing NaCl concentration. Sequence-based similarity searches against genomic and metagenomic datasets confirmed that these novel rhodopsins are widely distributed among organisms in various aquatic environments with high salinity.

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4390A Influence of light protectants on Persistence of Bacillus sp. BS-99 Ching Piao Liu*, Pei Haw Lin China University of Science and Technology, Dep. Biological Science and Technology, Taiwan

The development of biopesticide as environmental friendly alternatives to chemical pesticides for pest control has generated a considerable amount of interest. Therefore, the use of UV-B protectants in formulations can increase conidial survival and may enhance the efficacy of Bacillus sp. for controlling pests. Ultraviolet radiation (UV) can reduce the effectiveness of bacteria used for biological control; Exposure to simulated solar radiation for a few hours can completely inactivated the conidia of the bacteria. In the present study, we investigated the effect of exposure to an UV-B radiationon the conidial culturability and germination of selected strains of the Bacillus sp. BS-99. Formulation (0.2~0.5% w/v) in TiO2, SiO2, NaCl and sesame oil increased the germination of UV-B exposed conidia of Bacillus sp. BS-99 to levels found in the dark control without causing a delay in germination. After exposure to artificial UV-B radiation dose of 30μw cm-2, increased UV-B exposure decreased relative percent culturability for all strains. In 0.2% (v/v) nano materials and traditional reagents got better protect conidia effect and aqueous emulsions of the sesame oil and the peanut oil than by conidia formulated in water alone. Therefore, a range of compounds can provide conidia with protection from UV-B. Of these, nano materials are likely to be cost effective. From the result, the anti- UVB efficiency were TiO2 > SiO2 > Oil > NaCl > ZnO > Benphenone > Salicylate > Cinnamate. We find that using nano materials in low concentration that also have anti-UV capability. The results may help finding UV-B resistant bacteria that would prove more efficient as bioinsecticides.

440A Metagenomic analysis of aerobic and anaerobic phototrophic communities in a stratified lake Julia Maresca*1, Sean Crowe2, Edward DeLong3, Donald Canfield2 1University of Delaware, USA, 2University of Southern Denmark, Denmark, 3Massachusetts Institute of Technology, USA

Lake Matano is an ancient, stratified lake in Indonesia with active populations of photosynthetic bacteria both above and below the chemocline. The geochemistry of this lake has been intensively studied, and previous research has shown that unlike most modern stratified water bodies, Lake Matano is ferruginous rather than sulfidic, with high levels of methane accumulating below the chemocline, extreme phosphate limitation in the surface waters, and naturally high concentrations of chromate and nickel.

Although tropical lakes typically have high rates of primary production, the rate of carbon fixation in Lake Matano is very low. To better understand the limitations on primary producers in Lake Matano, we used 454 sequencing (Titanium) to sequence bulk DNA extracted from microbial communities in the surface waters and in the chemocline. In water collected from 10 m, the microbial population is dominated by the Gram-positive Actinobacteria, and cyanobacteria comprise only a small fraction of the population. However, genes from anoxygenic phototrophs and rhodopsins are also present and even abundant, indicating that in this nutrient-limited environment, phototrophy is likely a useful strategy for a variety of species. In the chemocline, more than 115 m deep, a larger proportion of the population appears to rely on sunlight: the largest taxonomic group in this population is the photolithoautotrophic green sulfur bacteria. Putative chemolithoautotrophs such as Gallionellaceae and Nitrospirae are also present. This bioinformatic analysis, in conjunction with existing geochemical data and ongoing characterization of biochemical rates, provides us with a number of testable hypotheses regarding energy conservation, electron donors and acceptors, and the constraints on primary production in this unusual ecosystem.

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441A Redox proteomics: A new approach to identifying carbonylated proteins as biomarkers of UVB radiation in the marine bacterium Photobacterium angustum S14 Sabine Matallana-Surget*1, Charles Fauconnier1, Fabien Joux1, Ruddy Wattiez2, Philippe Lebaron1 1Université Pierre et Marie Curie - CNRS - UMR7621, France, 2Department of Proteomics and Microbiology, Interdisciplinary Mass Spectrometry Center (CISMa) University of Mons Belgium

The oxidized proteome of exponentially growing cells of the marine bacterium, Photobacterium angustum S14, exposed to UVB was analyzed by the implementation of both DNPH derivatization and label-free quantitative proteomics approach. In addition to DNA damage, UVB radiation induces oxidative stress by generating reactive oxygen species (ROS) that are highly active and can damage proteins. The so-called "redox proteomics" refers to the study of the oxidative stress-induced modifications on proteins. Among them, the carbonylation, which consists of the addition of a carbonyl group (C=O), is an important irreversible modification that increases during oxidative stress. Protein carbonyl groups have many consequences on biological activities and are one of the most widely studied markers of the oxidative stress. They mainly affect four amino-acids (i.e., Proline, Lysine, Arginine, Threonine).

The purpose of our study was to present a new strategy to identify and quantify the carbonylated proteins and decipher the potential consequences/roles played by ROS induced carbonylation in the UVB resistant marine bacterium Photobacterium angustum. Detection and quantification of carbonyl groups can only be achieved using specific chemical probes, such as 2,4-dinitrophenylhyrazide (DNPH) and various techniques have been developed to quantify DNPH content in protein extracts. Here, we report the development of a new strategy that facilitates confident identification of carbonylation sites by using a combination of DNPH derivatization of the carbonyl groups and a mass spectrometry based approach (label-free quantitation).

5% of the identified proteome was found to be carbonylated. A total of 64 non-redundant proteins (452 peptides) were carbonylated. 35 proteins were quantified and showed differential abundance between UVB and Dark condition. Growth of P. angustum under UVB radiation resulted in selective protein oxidative damage. When P. angustum cells were grown under UVB radiation, alcohol dehydrogenase E, elongation factors (EF, Tu, NusA), chaperonins (GroEL, DNA K), ATPase, DNA gyrase, DNA directed RNA polymerases, outer membrane protein (OmpL) were identified as the major protein targets of UVB stress. Several metalloproteins were found to be carbonylated, which is not surprising due to the fact that the most common mechanism of protein carbonylation appears to be metal- catalyzed oxidation (MCO). After identifying proteins particularly susceptible to proteins damage, we also investigated the nature of carbonylation sites to identify hot spots. Of the carbonylated (Arg, Lys, Pro, and Thr) amino acids, Proline was found to be the most susceptible site of carbonlyation. We also presented for each carbonylated protein the impact of the carbonyl groups on its protein structure that is crucial for a proper function.

This study reports the first proteomic-based identification and characterization of carbonylated proteins in a marine bacterium exposed to UVB stress.

442A Metabolic flexibility and substrate preference by the phototrophic freshwater Fe(II)- oxidizer Rhodopseudomonas palustris TIE-1 Emily Denise Melton*1, Caroline Schmidt1, Kirsten Oswald2, Sebastian Behrens1, Andreas Kappler1 1University of Tübingen, Germany, 2Eawag, Switzerland

Iron cycling plays an important role in sedimentary processes. The distribution of sedimentary neutrophilic microbial Fe(II)-oxidizers, including photoferrotrophs, is mainly determined by local gradients of oxygen, light, nitrate and ferrous iron. However, the geochemical parameters defining their surroundings are highly variable and fluctuate according to day-night and seasonal cycles. Accordingly, many photoferrotrophs exhibit high metabolic flexibility ranging from photolithoautotrophic to photoorgano- or lithoheterotrophic modes of energy generation and carbon flow. Metabolic flexibility is a well-known phenomenon under microbes active in iron cycling and has been suggested to play an important role in maintaining the sedimentary iron cycle. In addition, competition with other Fe(II)- oxidizers for ferrous iron may force a necessity to overcome the lack of preferable electron donor by

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adapting their metabolism to use whichever electron donor is currently available. This begs the question; what transpires if more than one favourable electron donor presents itself simultaneously? And what are the ensuing consequences for the sedimentary Fe cycle? In order to answer these questions, microcosms were inoculated with the freshwater photoferrotrophic strain Rhodopseudomonas palustris TIE-1, (107 cells/ml) and various combinations of the following electron donors: ferrous iron, acetate and lactate and incubated under optimal light and temperature conditions for 21 days. Un-inoculated controls were monitored for abiotic reactions. Electron donor concentrations were followed over time by the photometric ferrozine assay (Fe(II)/Fe(III)) and HPLC (lactate/acetate) analysis. Cell growth was monitored over time through qPCR assays. By determining under which circumstances ferrous iron was the preferred electron donor, conclusions could be drawn on the role of metabolic flexibility in iron cycling and how changes in organic carbon input into the sediment affect microbial Fe(II) oxidation. This study showed that whilst the photoferrotrophic strain Rhodopseudomonas palustris TIE-1 oxidized acetate or lactate faster than ferrous iron when each was offered individually, the overall Fe(II) oxidation rate was higher when both acetate and Fe(II), or lactate and Fe(II) substrates were presented simultaneously. However, when provided in excess with a mixture of acetate and lactate in addition to ferrous iron, Fe(II) oxidation was greatly inhibited. Thus, sedimentary Fe(II) oxidation is not only controlled by the extant microbes capable of this metabolism, but also by the surrounding geochemical concentrations of available alternative electron donors in addition to, or instead of, ferrous iron.

443A Glycogen metabolism of the cyanobacterium Synechococcus sp. PCC 7002 Klaus Benedikt Möllers*1, Thomas Grochalski1, Jacob H. Jacobsen1, David Cannella2, Henning Jørgensen2, Niels Ulrik Frigaard1 1Department of Biology, University of Copenhagen, Denmark, 2Forest and Landscape, University of Copenhagen, Denmark

Cyanobacteria are photosynthetic microbes that prosper under a wide range of environmental conditions. Their sturdiness is based on the successful combination of effective metabolic pathways that hold great promise for use in sustainable production of biofuels and other bioproducts. Due to its rapid doubling time (about 4 hours), its ease of genetic manipulation, and accumulation of different storage compounds (glycogen and cyanophycin), the unicellular cyanobacterium Synechococcus sp. PCC 7002 represents a versatile research organism. It is an excellent model for cyanobacterial physiology and ecology and metabolic engineering of this organism is a promising tool for producing renewable energy resources and valuable metabolic products. The aim of this study is to study the physiological mechanisms and environmental conditions that regulate the glycogen metabolism in Synechococcus. This is done using various approaches including nutrient limitation and genetic modification. Synechococcus cells are grown under standard conditions (38°C, 250 µE m-2 s-1, 1% v/v CO2) in A+ medium containing different concentrations of sodium nitrate or potassium phosphate. A total sugar content of 56% w/w of the dry weight was obtained when Synechococcus cells were grown under nitrogen limitation (0.24 mg/ml NaNO3) for 46 hours to an optical density (OD730) of 6.5 compared to standard conditions (1 mg/ml NaNO3) that led to a total sugar content of 29% w/w at an OD730 of 11. In contrast, Synechococcus cells grown under phosphate limitation do not vary greatly in the amount of total sugar from cells grown under standard conditions. This effect might be due to the synthesis of the nitrogenous storage compound cyanophycin. The regulation mechanism still remains to be elucidated. The insights obtained in sugar metabolism have practical applications as well and we have performed fermentation of sugar-enriched cyanobacterial biomass to ethanol by an industrial production strain of the yeast Saccharomyces cerevisiae. After enzymatic treatment of Synechococcus cells the conversion yield of total glucose in the cyanobacterial biomass to ethanol was 70-90%. These results show that cyanobacterial biomass can be efficiently used as feedstock for microbial bioethanol fermentation provided the sugar content is sufficiently high.

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444A Orientation of Mayan monuments as a major determinant in spatial distribution of aeroterrestrial biofilms Benjamin Otto Ortega-Morales*1, Christine Claire Gaylarde2, Manuelita Reyes-Estebanez1, Armando Anaya-Hernández1, Manuel Jesús Chan-Bacab1, Susana De la Rosa-García1, Diana Arano-Recio1 1Universidad Autónoma de Campeche. Departamento de Microbiologia Ambiental y Biotecnologia, , 2University of Portsmouth, United Kingdom

Lithobiontic microbial communities colonize buildings belonging to the World’s cultural heritage. These communities are active geochemical agents participating both in weathering and precipitation processes in stone materials, thus affecting built heritage. Recent research evidence shows that aeroterrestrial microbial biofilms are increasing in certain urban locations of Europe (Northern Ireland), superseding pollution-related soiled stone surfaces. It is not known if aeroterrestrial microbial biofilms are increasing in Mayan monuments located in the tropics where different climatic conditions prevail and pollution levels are negligible. In this work, we studied major trends in microbial colonization patterns hypothesizing that orientation of monuments controls the composition and extent of aeroterrestrial microbial biofilms on Mayan ruins. Orange and black biofilms which are the most widespread types of biofilm on external surfaces in Mayan sites, were studied in selected sites (Becan, Chicanna and Hormiguero) located in close proximity to each other, in order to avoid the influence of climatic factors operating at the large scale. Culturing and microcopy showed that microalgae and cyanobacteria were the prevalent microbial groups; the alga Trentepohlia sp. in orange biofilms and the coccoid cyanobacterium Gloecapsa in black biofilms were dominant organisms. Orientation of buildings played a key role in the spatial distribution of these biological films. Orange biofilms, whose coloration was due to the presence of beta carotene and luteine in Trentepohlia sp. cells as evidenced by High Performance Liquid Chromatography, were predominantly associated with North and West- facing walls, or on otherwise orientated sites if under the influence of tree canopies. These Trentepohlia sp-dominated biofilms supported fungal communities, comprising the genera Myrothecium sp, Nodulisporium sp, Paecilomyces sp, and Talaromyces sp, which occurred exclusively on orange surfaces. Interestingly, Cladosporium cladosporioides recognized as a cosmopolitan fungus was an almost exclusive colonizer of these biofilms. In contrast, extensive black biofilms were mainly observed on South and West-facing walls. Different fungal genera appeared particularly associated with black biofilms including Acrodontium sp., Curvularia spp., Glyocladium spp., Stachibotrys sp., Trichoderma sp, Verticillium sp. A Geographic Information System-based assessment of total solar irradiance for the year 2010 at the site of Becan showed that in general North and East facing walls of monuments received significantly lower levels of radiation, suggesting that the major impact of orientation is to affect incident levels of damaging solar radiation, ensuring higher availability of water for Trentepohlia colonization in low-irradiance areas, while dehydration and Ultra Violet-resistant cyanobacteria can form dark biofilms in more exposed regions. Incident levels of solar radiation determine to a large extente the microclimate of Mayan monuments resulting in differential microbial colonization and probably on microbial and environmental controls of stone deterioration.

445A A xanthorhodopsin-like second bacterial rhodopsin is expressed in the flavobacterium Dokdonia donghaensis PRO95 Thomas Riedel*1, Laura Gómez-Consarnau2, Jürgen Tomasch1, Michael Jarek1, Madeleine Martin1, Thorsten Brinkhoff3, Jed Fuhrman2, Irene Wagner-Döbler1 1Helmholtz-Centre for Infection Research, Germany, 2University of Southern California, USA, 3University of Oldenburg, Germany

Proteorhodopsins are light-driven proton pumps present in a large fraction of aquatic bacteria. Photoheterotrophy based on proteorhodopsin has previously been investigated in the flavobacterial strain Dokdonia donghaensis PRO95, showing that the proteorhodopsin gene was constitutively expressed and there was no light stimulation of growth down to a carbon concentration of 9.7 mM. Here, we report the draft genome sequence of PRO95 which revealed the presence of a second bacterial rhodopsin gene encoding a xanthorhodopsin-related protein. Sequence comparisons indicated this protein to be present in a broad spectrum of bacterial phyla as well as in dinoflagellates, suggesting that it has frequently been recruited by lateral gene transfer. Direct sequencing of mRNA of PRO95 showed that expression of both rhodopsin genes as well as the pathway for the synthesis of

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the cofactor retinal was detectable. PRO95 is the first flavobacterium shown to express two rhodopsin genes.

446A Development of lux-based biosensors in Pseudomonas aeruginosa ATCC 9027 for preservative efficacy testing Niksha Shah*, David Naseby University of Hertfordshire, United Kingdom

Whole cell biosensors have been extensively used for monitoring toxicity and contamination of various compounds in environmental biology and microbial ecology. However their application in the pharmaceutical industry for preservative efficacy testing has been limited. According to several pharmacopoeias preservatives must be tested for microbial activity against five pre-defined microorganisms and the use of whole cell microbial biosensors potentially provide fast and efficient methods for this antimicrobial efficacy testing. Thus the current study aims to develop whole cell microbial biosensors in Pseudomonas aeruginosa ATCC 9027. Five constitutive promoters responsible for expressing essential genes from P. aeruginosa have been selected and successfully amplified by PCR. The lux-cassette was ligated into plasmid vector pME4510. Amplified and digested promoters were subsequently ligated upstream of the lux-cassette. The resultant plasmid clones were then transformed into E. coli cells and subsequently into P. aeruginosa. Successful transformation into P. aeruginosa led to expression studies to monitor the strength of five constitutive promoters. Preliminary data suggest that plasmids are maintained stably through the growth cycle and the lux- cassette is expressed constitutively throughout the growth cycle. Future studies will involve determining plasmid copy number by qPCR and monitoring growth throughout the growth cycle up to 28 days.

447A Metabolic and physiologic effects on Okenone and Bacteriochlorophyll a production in purple sulfur bacteria Derek Smith*1, James Scott2, Andrew Steele1, George Cody1, Shohei Ohara1, Roxane Bowden1, Marilyn Fogel1 1Carnegie Institution of Washington, USA, 2Dartmouth College, USA

Anaerobic cycling of sulfur and carbon is coupled through the metabolism of phototrophic sulfur bacteria. Okenane, the only known biomarker of purple sulfur bacteria (PSB) in the geologic record, is believed to be a reduced derivative of the carotenoid pigment okenone, which has only been documented in eleven species of Chromatiaceae. Carotenoids are responsible for light harvesting and play a substantial role in protection by removing metastable by-products of photosynthesis. Bacteriochlorophyll a (Bchl a) is a well-studied photosynthetic pigment required for photosynthesis, in those organisms that possess it. We undertook a comprehensive study examining the effects of metabolism and physiology on the production of pigments in PSB. Specific growth rates were determined in PSB grown autotrophically and photoheterotrophically. Marichromatium purpuratum DSMZ 1591, Marichromatium purpuratum DSMZ 1711, Thiocapsa marina DSMZ 5653, and FGL21, a strain isolated from Fayetteville Green Lake, New York, were the four strains tested in this study. All four strains produce the carotenoid okenone. Bchl a and okenone concentrations were quantified using Ultra Performance-Liquid Chromatogrpaphy-Mass Spectrometry (UP-LC-MS) and spectrophotometry. By quantifying the amount of okenone per cell and Bchl a per cell, a significant species and treatment effect on the production of pigment was revealed. Furthermore, a significant treatment effect was uncovered by examining the specific growth rates of metabolically differing cells and okenone per Bchl a ratios. The fact that photoheterotrophic PSB have the lowest okenone per Bchl a ratios implies a reduced requirement for okenone when provided a complex carbon source. Stable isotopic analysis, using Isotope Ratio Mass Spectrometry (IR-MS), for δ13C, δ15N, and δ34S of bulk cell biomass showed differences in substrate fractionations across different strains and treatments. The δ13C values elucidate the switching of metabolism from autotrophy to photoheterotrophy, as well as differences in carbon assimilation. The bulk cell δ15N values were relatively similar for those cells grown autotrophically, and are enriched for those cells grown photoheterotrophically, indicative of substrate limitation. The δ34S values of bulk cell biomass have an inverse relationship to atomic weight percent sulfur, reveal sulfur storage in cells during exponential phase growth, and utilization of that stored sulfur pool with further growth. Collectively, we show that there are differences in metabolism and physiology among purple sulfur bacteria, and that pigment production is significantly impacted by those differences.

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448A Photoautotrophic production and heterotrophic decomposition of climate-relevant halocarbons in Baltic and North Sea surface waters Christian Stolle*, Anna Orlikowska, Falk Pollehne, Detlef Schulz-Bull, Klaus Jürgens Leibniz-Institute for Baltic Sea Research Warnemuende, Germany

Volatile halogenated organic compounds (VHOCs) are a strong source of highly reactive halogen oxide radicals in the atmosphere, which catalyse climate relevant processes like ozone-depletion. However, emission budgets of VHOCs are still erroneous, partly due to large uncertainties about the equilibrium of biological production and decomposition processes. Several abiotic (for example photolysis) and biotic (for example diatoms, cyanobacteria) sources have been identified in aquatic systems. However, regulating factors for autotrophic VHOCs production, for example light stress and the resulting activity of haloperoxidases, have been mainly studied in laboratory experiments. Thus, knowledge of their effects in environmental habitats remains sparse. Even less information exists about microbial VHOCs degradation in the marine environment. Bromo- and chloromethane are known to serve as a carbon source for certain heterotrophic bacteria. However, the extent of bacterial VHOCs turnover and the diversity of involved species and functional enzymes remain largely unexplored.

The aim of the present study was to evaluate the effect of different light qualities (+/- UV) on autotrophic production of VHOCs. Therefore, autotrophic community composition and its physiological status were determined along with measurements of VHOCs concentration and δ13C isotope ratios. Furthermore, the bacterial degradation potential of selected VHOCs (bromoform and iodomethane) was examined using RNA-Stable Isotope Probing (RNA-SIP), with identification of the bacteria involved. Samples were taken during a cruise in the central Baltic Sea (summer 2011), and during mesocosm experiments employing different light transmission qualities, in the coastal Baltic Sea (autumn 2010, spring 2011) and in the coastal North Sea (spring 2011).

Our results show that photosynthetic activity (determined by the quantum yield) and - to a much lesser extent - bacterial heterotrophic activity (determined by 3H-thymidine uptake) was reduced at highest radiation levels, but this effect was similar in systems with and without UV-transmission. In our experiments, UV-radiation also played a negligible role in VHOCs dynamics. Nonetheless, the dynamics of several VHOCs, including iodomethane and bromoform, was related to changes in diurnal photosynthethic activity patterns, autotrophic biomass and community composition, dominated by diatoms in both the spring and autumn experiments. Iodomethane and bromoform production during the summer could be attributed to cyanobacteria, as we have first direct evidence for its production by Nodularia spumigena, a dominant cyanobacterial species of Baltic Sea surface waters. With regard to the bacterial VHOCs degradation potential, a high capacity for 14C-labelled iodomethane uptake was observed in all seasons. RNA-SIP approaches using 13C-labelled iodomethane identified methylotrophic bacteria as active degraders of this compound.

Taken together, diurnal patterns of autotrophic activity and VHOCs dynamics were observed, underlining the importance of light stress as a source of biological VHOCs production. This was, however, independent of UV radiation in our experimental setup. Furthermore, bacterial degradation of iodomethane was observed in Baltic and North Sea surface waters, with methylotrophic bacteria being actively involved. Future studies will enlighten species-specific, especially cyanobacterial, production patterns of VHOCs. Additionally, the analysis of the bacterial cmuA methyltransferase-gene distribution and diversity will provide further insights into the bacterial impact on VHOCs degradation.

449A Is it all light? Mechanisms of virus removal in waste stabilisation ponds Louise Weaver, Judith Webber, Naveena Karki, Kate Thomas, Margaret MacKenzie, Susan Lin, Wendy Williamson* Institute of Environmental Science & Research Ltd, New Zealand

Globally waste stabilisation ponds (WSP) offer a sustainable and economical method for wastewater treatment. The actual removal mechanisms occurring within WSPs are largely unknown and only a few studies have been conducted on virus removal. Most studies predict virus removal using the indicator organisms E. coli and bacteriophages. While sunlight (UV) and temperature play a major role in removal of pathogens in WSPs, these are complex systems and other mechanisms are present that contribute to removal of pathogens such as viruses. Our research aims to investigate virus removal in

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WSPs and gain knowledge of light dependent and independent mechanisms of virus removal including the effects of pH, dissolved oxygen (DO), enzymatic activity, settlement and adsorption on removal.

The removal of E. coli and F-RNA bacteriophage with enterovirus (Echovirus 7) in the presence and absence of light over two consecutive summer seasons were investigated. Previous experiments showed summer were when the highest virus removal activity occurred. The experimental system used large mesocosms to hold WSP water (21.5 L) that allowed for adjustment of the natural sunlight exposure and monitor pH, DO, temperature fluctuations regularly over 3-4 day periods. Each mesocosm was spiked with E. coli, F-RNA bacteriophage (MS2 phage) and Echovirus at the same time (at dusk). Samples were then taken at set time intervals and analysed for E. coli, MS2 phage and Echovirus. The sunlight intensity (global radiation and UV) was obtained for the experimental periods from the local NIWA CliFlo database (National Institute of Water and Air, New Zealand).

The results showed that if the pH and DO levels in the WSP remain elevated, similar levels of removal of indicator and virus are achieved in the presence and absence of direct sunlight. Removal of E. coli, MS2 phage and virus were comparable in the light and dark when pH and DO were elevated in dark mesocosm: light mesocosm removals of 6.02, 4.89 and 4.88 Log compared to dark mesocosm removals of 6.06, 5.40 and 4.13 Log for E. coli, phage and Echovirus respectively. In dark mesocosms with no pH and DO elevation a comparable removal was seen in 2011 but not in 2012: removal was between 4 - 6 Log in 2011 compared with 1 -5 Log in 2012. Higher temperatures seen in 2011 could explain the higher dark removal in 2011. Also, weather data for the two periods is being processed to investigate if higher UV intensities occurred during summer 2011. The level of continuous sunshine could activate a higher level of algae species and or algal biomass, which may also increase the removal rates by maintaining high pH and DO. Algal species and biomass is being analysed to identify any differences in the two years.

Although sunlight is an important factor in virus removal in WSPs, other mechanisms are influencing virus removal. It appears that high pH and high DO, which may be generating photoxidative by- products, are also a mechanism of removal in the dark sections of WSPs.

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