Monday 20 August 1 PS02 – Light and Microbial Life 421A Differential
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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 eukaryotes, 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, Argentina, 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 stromatolites and from the surrounding soil of Laguna Socompa (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. 1 PS02 – Light and Microbial Life Monday 20 August 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 cyanobacteria 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, Australia 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 2+ 3+ At circumneutral pH, under oxic conditions, Fe is rapidly oxidized to Fe 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 2 PS02 – Light and Microbial Life Monday 20 August in iron, whereas,