Ecophysiology of prokaryotes WS 2010/11 AG Cypionka / Paleomicrobiology 28. 02. 2011 – 25.03. 2011 Seminar plan Mo 28.02. Tu 01.03. We 02.03. Th 03.03. Fr 04.03. Group Assignment Open questions Introduction A + B Introduction C + D Introduction E + F Mo 07.03. Tu 08.03. We 09.03. Th 10.03. Fr 11.03. Preliminary reports A - C Preliminary reports D - F Mo 14.03. Tu 15.03. We 16.03. Th 17.03. Fr 18.03. Literature seminar 1 + 2 Literature seminar 3 + 4 Microbiol. Colloquium Literature seminar 5 + 6 Literature seminar 7 + 8 Mo 21.03. Tu 22.03. We 23.03. Th 24.03. Fr 25.03. Literature seminar 9 + 10 Literature seminar 11 + 12 Final reports D - F Final reports A - C Projekt A (Judith Lucas) Diversity, abundance and isolation of Roseobacter -affiliated bacteria from marine sediments Projekt B (Monika Sahlberg): Abundance of Rhizobium radiobacter and Roseobacter sp. within sediments of the Benguela upwelling system Projekt C (Maya Soora): Influence of light on the survival of Dinoroseobacter shibae during starvation Projekt D (Tim Engelhardt): Characterization of bacteriophages in sulfate-reducing bacteria (SRB) from deep-subsurface environments Projekt E (Bert Engelen): Microbial abundance and diversity within a subterranean gas-storage facility Projekt F (Heribert Cypionka): Cultivation of sulfate-reducing bacteria from a subterranean gas-storage facility Seminar list Project A 1. Wietz M, Gram L, Jorgensen B, Schramm A (2010) Latitudinal patterns in the abundance of major marine bacterioplankton groups. Aquat Microb Ecol (61) 179-189 2. Giebel HA, Kalhoefer D, Lemke A, Thole S, Gahl-Janssen R, Simon M, Brinkhoff T (2010) Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate. The ISME Journal 1-12 Project B 3. Schäfer H, Ferdelman TG, Fossing H, Muyzer G (2007) Microbial diversity in deep sediments of the Benguela Upwelling System. Aquatic Microbial Ecology 50: 1-9. 4. Mollenhauer G, Schneider RR, Müller PJ, Spieß V, Wefer G (2002) Glacial/Interglacial variability in the Benguela upwelling system: Spatial distribution and budgets of organic carbon accumulation. Global Biogeochemical Cycles 16: 81-1 - 81-15. Project C 5. Holert, J., Hahnke, S. and Cypionka, H. (2011) Influence of light and anoxia on chemiosmotic energy conservation in Dinoroseobacter shibae. Environmental Microbiology Reports, 3: 136–141. doi: 10.1111/j.1758-2229.2010.00199.x 6. Gómez-Consarnau L, Akram N, Lindell K, Pedersen A, Neutze R, et al. (2010) Proteorhodopsin Phototrophy Promotes Survival of Marine Bacteria during Starvation. PLoS Biol 8(4): e1000358. doi:10.1371/journal.pbio.1000358 Project D 7. Williamson S J, Cary S C, Williamson K E, Helton R R, Bench S R, Winget D, Wommack K E (2008) Lysogenic virus–host interactions predominate at deep-sea diffuse-flow hydrothermal vents. ISME J 2:1112-1121. 2 8. Eydal H SC, Jägevall S,Hermansson M, Pederson K (2009) Bacteriophage lytic to Desulvovibrio aespoeensis isolated from deep groundwater. ISME J 3:1139-1147. Project E 9. Orphan, V.J., et al. (2000) Culture-dependent and culture-independent characterization of microbial assemblages associated with high-temperature petroleum reservoirs. Applied and Environmental Microbiology 66(2): p. 700-711. 10. Mochimaru H, Yoshioka H, Tamaki H, Nakamura K, Kaneko N, Sakata S, Imachi H, Sekiguchi Y, Uchiyama H, Kamagata Y (2007) Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan. Extremophiles 11:453–461 Project F 11. Sass H, Cypionka H (2004) Isolation of sulfate-reducing bacteria from the terrestrial deep subsurface and description of Desulfovibrio cavernae sp. nov. System Appl Microbiol 27:541-548 12. Rozanova, E.P., et al. (2001) Desulfacinum subterraneum sp nov., a new thermophilic sulfate- reducing bacterium isolated from a high-temperature oil field. Microbiology, 70(4): p. 466-471. 3 Project A (Judith Lucas) Diversity, abundance and isolation of Roseobacter-affiliated bacteria from marine sediments Background The Roseobacter clade is a major lineage of the family Rhodobacteraceae of theAlphaproteobacteria. Its members form one of the most abundant and successful groups in the marine environment. No other phylogenetically coherent clade occurs in such a variety of marine habitats and ecosystems of different geographic regions and has such a diverse physiology. Members of the Roseobacter clade have been detected in the water column, in biofilms, oxic and anoxic sediments and in association with other organisms (Buchan et al 2005, Wagner-Döbler & Biebl 2006). Although this phylogenetic lineage exhibits a broad metabolic versatility, members of the Roseobacter clade are generally considered to be obligate aerobes. Nevertheless, there are some exceptions with a facultatively anaerobe metabolism (e.g. Roseobacter denitrificans, Roseovarius crassostreae, Dinoroseobacter shibae) and others are even capable of performing aerobic anoxgenic photosynthesis (AAP). Sediments have been identified as the third-most important habitat for the Roseobacter-clade (Buchan et al., 2005). Molecular surveys showed that this group contributes with an average of 3% to the microbial communities thriving in marine surface sediments (e.g. Li et al. 1999, Madrid et al. 2001, Mills et al. 2003), but they were also detected in deep anoxic layers. However, the abundance, distribution and metabolic potential of sediment-dwelling Roseobacter have not been studied systematically so far. To elucidate their role in marine sediments in comparison to other surfaces, algal and sediment samples were taken from Helgoland island and analyzed on different phylogenetic levels. DGGE was performed using primers specific for Bacteria, Rhodobacteraceae and Phaeobacter sp.. All phylogenetic groups were analyzed by quantitative PCR. To confirm the results, numbers of Bacteria and Rhodobacteraceae were determined by CARD-FISH. During another extended cultivation study (Köpke et al., 2005), five facultatively anaerob Roseobacter-affiliated strains were isolated from tidal-flat sediments. One strain NB43 seems to form a new genus. Some physiological analyses like determination of the optimal growth temperature and ph still have to be done. Aim of the project: While investigated numbers of Roseobacter-affiliated bacteria within the Helgoland samples are unlikely high (CARD-FISH: ~ 20% qPCR: 40-60%), Phaeobacter sp. could hardly be found by applying specific PCR. Questions and tasks which will be addressed are: • Does the DNA-concentration influence the quantification via qPCR? • And if so, is there a correlation between DNA-concentration and cell-quantification? • Verification of qPCR results via CARD-FISH • Detection of Phaeobacter sp. with new specific primers. • Determination of temperature- and pH-Optimum of the tidal-flat isolate NB43. 4 Methods/ working program DNA extraction and quantification DNA will be extracted from Helgoland sediments by using the MolBio-Kit for soil and quantified afterwards via PicoGreen on a microtiter plate reader. General primers for the bacterial 16S rRNA gene will be used to make sure that the extracted DNA is amplifiable. PCR A PCR using primers PHA-16S-129f and PHA-ITS-111r specific for Phaeobacter will be applied to detect Phaeobacter sp. in the Helgoland samples. Real-time PCR The extracted and quantified DNA serves as a template to determine the abundance of Rhodobacteraceae. and Phaeobacter sp. by qPCR. The template DNA will be diluted (1:100, 1:200, 1:300, 1:400) to analyze the influence of different template concentrations on the cell quantification. CARD-FISH To verify and prove the qPCRs results, CARD-FISH will be performed using a Rhodonacteraceae specific probe (HRP-Ros536). Determination of growth parameters In order to determine optimal growth temperature and pH for NB43 growth curves should be recorded. Growth of NB43 will be monitored through cell counting after staining with SybrGreen I as direct detection and quantification method. In addition the turbidity will be measured photometrically (OD436). Time schedule: First week: Monday: General introduction Tuesday: DNA-extraction/Quantification of DNA-amount Wednesday: PCR/Preparation of qPCR-standard Thursday: Preparation dilution series and qPCR Roseobacter Friday: evaluation qPCR/2nd qPCR Roseobacter Second week: CARD-FISH, SybrGreenI-cell counts, Phaeobacter PCR Third week: Preparation of medium/ Growth curves temperature-optimum Fourth week: Preparation of buffers and media/Growth curves pH-optimum Literature: Buchan A, González JM, Moran MA (2005) Overview of the marine Roseobacter lineage. Appl Environ Microbiol 71: 5665-5677 Köpke B, Wilms R, Engelen B, Cypionka H, Sass H (2005) Microbial diversity in coastal subsurface sediments - a cultivation approach using various electron acceptors and substrate gradients. Appl Environ Microbiol 71:7819-7830 Li L, Guenzennec J, Nichols P, Henry P, Yanagibayashi M, and Kato C (1999) Microbial diversity in Nankai Trough sediments at a depth of 3,843 m. J. Oceanogr. 55:635-642. Madrid VM, Aller JY, Aller RC, and Chistoserdov AY (2001) High prokaryote diversity and analysis of community structure in mobile mud deposits off French Guiana: identification of two new bacterial candidate divisions. FEMS Microbiol. Ecol. 37:197-209 Mills HJ, Hodges C, Wilson K, MacDonald IR, and Sobecky PA (2003) Microbial diversity in sediments associated with surface-breaching gas hydrate mounds in the Gulf of Mexico. FEMS Microbiol. Ecol. 46:39- 52. Wagner-Döbler I, Biebl H (2006) Environmental