AQUATIC MICROBIAL ECOLOGY Vol. 46: 209–224, 2007 Published March 13 Aquat Microb Ecol Depth-specific distribution of Bacteroidetes in the oligotrophic Eastern Mediterranean Sea Martina Blümel, Jörg Süling, Johannes F. Imhoff* Leibniz-Institut für Meereswissenschaften an der Universität Kiel, Düsternbrooker Weg 20, 24105 Kiel, Germany ABSTRACT: Representatives of the phylum Bacteroidetes are repeatedly reported to be abundant members of oceanic bacterioplankton, probably because of their ability to degrade complex organic matter. 16S rDNA analysis was performed in order to address the importance of this phylum in a highly oligotrophic region such as the Eastern Mediterranean Sea and to investigate its distribution patterns and community composition. A new antisense primer was designed for PCR and used in combination with the general eubacterial sense primer 27F to specifically target Bacteroidetes repre- sentatives. Data were correlated with water depth and water mass properties. Denaturant gradient gel electrophoresis (DGGE) analysis and sequencing of environmental clone libraries revealed spe- cific distribution patterns. A major fraction of the sequences was assigned to the AGG58 cluster, a branch of yet uncultured members of the Bacteroidetes lineage. Our results demonstrate a consider- able diversity within the Bacteroidetes lineage in the oligotrophic Eastern Mediterranean Sea. Differing water mass properties are considered to be of major relevance for the spatial distribution with depth; several environmental clone sequence clusters could be specifically assigned to a de- fined water mass or to the deep waters of investigated locations. Depth-specific distribution of Bacteroidetes is demonstrated for the first time by the results of this study. KEY WORDS: Bacteroidetes · Depth specific distribution · Eastern Mediterranean Sea · Bacterial diversity Resale or republication not permitted without written consent of the publisher INTRODUCTION isolated from marine and marine-derived habitats in Antarctica (Bowman et al. 2003). The phylum Bacteroidetes is a widely distributed Representatives of Bacteroidetes were found to group of chemoheterotrophic bacteria in marine habi- comprise 14% of bacterial isolates from a marine tats and is abundant within marine bacterioplankton habitat (Uphoff et al. 2001), but the majority of Bac- (Bowman et al. 1997, Glöckner et al. 1999, Cottrell & teroidetes sequences detected in environmental sam- Kirchman 2000, Fry 2000, Kirchman 2002). It is a major ples by molecular methods remains as yet uncultured eubacterial group and can be divided into different (DeLong et al. 1993, Suzuki et al. 1997, Eilers et al. lineages based on 16S ribosomal DNA sequences. 2000). Major branches are the Cytophaga–Flavobacterium Members of the Bacteroidetes represented 10 to lineage, the Bacteroides lineage as well as the families 40% of total bacterial numbers (DAPI counts) in ocean Sphingobacteriaceae, Flexibacteraceae and Creno- waters (Glöckner et al. 1999, Abell & Bowman 2005a) trichaceae (Kirchman 2002). A new family within the as observed by fluorescence in situ hybridisation phylum, the Cryomorphaceae, has recently been pro- (FISH). In lakes, a growth rate 2-fold faster than that posed (Bowman et al. 2003), phylogenetically branch- of other bacterial groups was observed for the ing between the families Flavobacteriaceae and Bac- Cytophaga–Flavobacterium lineage (Jürgens et al. teroidaceae. The type genus of this new family is the 1999), providing a possible explanation for the high genus Cryomorpha, type species C. ignava, originally abundances of this group in aquatic systems. *Corresponding author. Email: [email protected] © Inter-Research 2007 · www.int-res.com 210 Aquat Microb Ecol 46: 209–224, 2007 In contrast to the data obtained by FISH, only few cal conditions (O’Sullivan et al. 2004). O’Sullivan et al. Bacteroidetes sequences have been found in clone (2004) found comparably low similarity values of Bac- libraries from open-ocean environments using general teroidetes clones to GenBank sequences and con- eubacterial primers (5 to 20%; Kirchman 2002, Kirch- cluded that the phylogenetic diversity of this group is man et al. 2003). Bacteroidetes sequences are often not as well investigated as that for the proteobacteria. underrepresented in clone libraries and consequently A specific prokaryotic community is considered to in DNA sequence databases (O’Sullivan et al. 2004), inhabit the deep Mediterranean Sea (Zaballos et al. because general eubacterial primers are likely to dis- 2006). Zaballos et al. (2006) found substantial differ- criminate against this phylum (Suzuki et al. 2001). ences in the bacterial community composition com- Although data from FISH suggest that this phylum pared to other ocean environments. In general, the represents an important part of bacterial communities ubiquitous alpha-bacterial SAR-11 cluster contributed in ocean waters, little is known of its ecological func- a major fraction of the total bacterial community of tion and specific distribution (Reichenbach 1991, open ocean environments, whereas in the Ionian Basin Hagström 2000, Hagström et al. 2000, Kirchman 2002). (Eastern Mediterranean Sea), Gammaproteobacteria Representatives of the Bacteroidetes and especially dominated the bacterioplankton assemblage (Zaballos the Flavobacterium lineage are known to be degraders et al. 2006). Bacteroidetes were the most heterogenous of complex organic matter. In recent years, the distrib- group retrieved in clone libraries of seawater samples ution of marine representatives of Bacteroidetes has from the Greenland Sea and the Ionian Sea, indicating attracted increasing attention, mainly due to their sup- a high diversity within this group in these waters. posed essential role in vertical energy and carbon flow To date, depth-specific distribution has been reported (Kirchman 2002, Bowman et al. 2003). Abell & Bow- for several bacterial phyla, e.g. the green non-sulfur man (2005a) hypothesized an opportunistic response of bacteria (Gordon & Giovannoni 1996), the ubiquitous Flavobacteria to the organic carbon present. As major SAR11 cluster (Field et al. 1997) and Deltaproteobacte- decomposers, uptake and degradation (especially of ria (Wright et al. 1997). Lee & Fuhrman (1991) reported high molecular weight, HMW) of organic matter is a varying bacterial community composition with assumed for this bacterial group (Cottrell & Kirchman depth, but the phylum Bacteroidetes has never been 2000, Kirchman 2002). Pinhassi et al. (2004) further the focus of studies dealing with depth-specific distrib- emphasized the particular importance of Bacteroidetes ution. However, with changing availability of HMW in the processing of organic matter during algal blooms. organic matter with depth, a depth-specific distribu- Crump et al. (1999) detected a high proportion of tion particularly of Bacteroidetes representatives as Bacteroidetes in the particle-attached fraction of an major degraders seems likely. Seritti et al. (2003) estuarine system, underlining the importance of this reported a water-mass-specific distribution of dissolved bacterial phylum especially in the degradation of organic carbon (DOC) in the Eastern Mediterranean marine snow aggregates. DeLong et al. (1993) found Sea. Bacteroidetes as major mineralizers of particulate that particle-attached bacterial assemblages shared no organic carbon (POC) may contribute a major fraction identical rRNA types with free-living assemblages and of DOC, further indicating a possible depth- or even suggested major compositional differences between water-mass-specific distribution of this phylum. the 2 assemblages. DeLong et al. (1993) also retrieved The Mediterranean Sea can be primarily character- the first sequence of the so-called ‘AGG58’ cluster of ized by unusually high deep water temperatures the Bacteroidetes (O’Sullivan et al. 2004) from marine (>13°C, CIESM 2003) and extremely low nutrient con- snow in the Santa Barbara Channel, USA. The AGG58 centrations (Krom et al. 1991). The Eastern Mediter- cluster is a coherent branch with closest similarity to ranean is divided into 2 main sub-basins, the Levan- the Flavobacteria lineage, now incorporated into the tine Basin and the Ionian Basin (Theocharis et al. new family Cryomorphaceae (O’Sullivan et al. 2004, 1993). Characteristic surface water masses of the Abell & Bowman 2005b). The AGG58 cluster com- Eastern Mediterranean Sea are the Levantine Surface prises sequences from such various environments as Water (LSW, origin: Levantine Basin) and the Modified the Santa Barbara Channel (DeLong et al. 1993), the Atlantic Water (MAW, origin: Atlantic Ocean). Inter- Delaware Estuary (Kirchman et al. 2003), the Arctic mediate water masses are the Levantine Intermediate Ocean (Bano & Hollibaugh 2002) and coastal seawater Water (LIW, main origin: Rhodes cyclonic gyre; Theo- off North Carolina (Rappé et al. 1997). It is therefore charis et al. 1999) and the newly formed Cretan Inter- considered to be cosmopolitan (O’Sullivan et al. 2004). mediate Water (CIW, origin: Aegean Sea; Schlitzer et The different branches of the cluster showed changes al. 1991, Manca et al. 2003). Deep water circulation has in dominance during the course of a phytoplankton changed over the last decade: the Adriatic Sea as pri- bloom, leading to the assumption that different mem- mary source of Eastern Mediterranean Deep Water bers of the cluster are specialized to different ecologi- (EMDWAdr; Kress et al. 2003) has been replaced