JOBNAME: No Job Name PAGE: 1 SESS: 9 OUTPUT: Tue Apr 24 15:49:11 2012 SUM: BCD8E856 /v2451/blackwell/journals/emi4_v0_i0/emi4_350 Environmental actinorhodopsin expression revealed by a new in situ filtration and fixation sampler Christian Wurzbacher, 1 Ivette Salka 1 and Hans-Peter Grossart 1,2 * 1 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhütte 2, 16775 Stechlin, Germany. 2 Potsdam University, Institute for Biochemistry and Biology, Am Neuen Palais 10, 14469 Potsdam, Germany. Summary Freshwater Actinobacteria are an important and dominant group of bacterioplankton in most temper- ate freshwater systems. Recently, metagenomic studies discovered rhodopsin-like protein-coding sequences present in Actinobacteria which could be a decisive hint for their success in freshwater ecosys- tems. We analysed the diversity of actinorhodopsin (ActR) in Lake Stechlin (northern Germany) and assessed the actR expression profile during a diurnal cycle. We obtained 85 positive actR clones which could be subsequently grouped to 17 operational taxonomic units assuming a 90% sequence similarity. The phylogenetic analysis points to a close relation- ship of all obtained sequences to the acI lineage of Actinobacteria, forming six independent clusters. For the first time, we followed in situ transcription of actR in Lake Stechlin revealing a rather constitutive circa- dian gene expression. For analysing in situ expres- sion patterns of functional genes in aquatic ecosystems, such as actR, we invented a new in situ filtration and fixation sampler (IFFS). The IFFS enables the representative investigation of microbial transcriptomes in any aquatic ecosystem at all water depths. The IFFS sampler is simple and inexpensive, and we provide all engineering plans for an easy rebuild. Consequently, our IFFS is suitable to reliably study expression of any known functional gene of any aquatic microorganism. Introduction In aquatic ecosystems, culture-independent methods are increasingly applied to study bacterioplankton communi- ties and are successful to discover globally distributed abundant bacterial groups, e.g. the marine SAR11 group or the freshwater acI Actinobacteria, which lack represen- tative numbers of isolates. Actinobacteria provide a significant fraction of bacterioplankton in freshwater eco- systems worldwide. By using molecular approaches, War- necke and colleagues (2004) identified four different actinobacterial lineages (acI–acIV) of which the acI sub- cluster represents one of the most important bacterial groups in temperate aquatic systems. In Lake Stechlin, acI lineages represent the dominant group within the Acti- nobacteria contributing up to 47% of the total bacterial community (Allgaier and Grossart, 2006). Despite all efforts no pure cultures of this specific lineage could be obtained and solely a mixed culture has been isolated recently (Jezbera et al., 2009). Therefore, functional genes of these uncultivated environmental lineages are frequently assessed by metagenomic approaches (Béjà et al., 2000; Sabehi et al., 2005; Legault et al., 2006; Martín-Cuadrado et al., 2007; Philosof et al., 2009) or genome analysis of sorted single cells (Stepanauskas and Sieracki, 2007; Martinez-Garcia et al., 2011). Recently, these approaches resulted in the discovery of the actinorhodopsin gene (actR) from the Global Ocean Sampling campaign (Sharma et al., 2008; 2009). Actinorhodopsin is assumed to be a light-driven proton pump (Sharma et al., 2008; 2009). Whereas bacterial pro- teorhodopsins enhance environmental fitness under light conditions (Gómez-Consarnau et al., 2007), the ecologi- cal role of ActRs is still unknown. Consequently, up to now its functionality remains theoretical and largely specula- tive, primarily based on related genes found in sequence databases (Sharma et al., 2008; 2009; Martinez-Garcia et al., 2011). Although a light-dependent expression could be expected based on its possible function, nothing is known about in situ ActR gene expression patterns. Non- directed sequencing of metatranscriptomes as well as quantitative PCR provide promising approaches to study gene regulation since they enable studies on even unknown or related genes which are transcribed during certain environmental conditions by natural microbial Received 31 December, 2011; revised 5 April, 2012; accepted 10 April, 2012. *For correspondence. E-mail hgrossart@igb-berlin.de; Tel. (+49) 33082 69991; Fax (+49) 33082 69917. 1 1 emi4_350 Environmental Microbiology Reports (2012) doi:10.1111/j.1758-2229.2012.00350.x © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92