Marine microbial diversity studied via 16S rRNA sequences: Cloning results from coastal waters and counting of native archaea with fluorescent single cell probes

New molecular approaches relying on 16S rRNA sequences allow qualitative and quantitative analysis of marine microbial diversity. Here we report on (1) continued development of 'lists' of taxa present in marine environments, in temperate coastal waters, and (2) new fluorescent in situ hybridization (FISH) approaches to quantify taxonomic compositions, with an initial focus on archaea. Our cloning results come from Long Island Sound on the Atlantic coast (February and August), and Malibu (April) and offshore Monterey Bay (September), California. The clones were dominated (39 of 45 total clones) by proteobacteria, with the α subdivision (33 clones), and the SAR11 cluster (17) in particular, being quite abundant. There were also clones from the β (2) and γ (4) subdivisions, the cyanobacteria (4, from Monterey Bay only) and the Cytophaga group (2). Some clones were very similar to those previously reported from open ocean or deep sea environments, but others were not close relatives of any of those previously reported. The FISH results used doubly-labeled probes that were 'universal', bacterial, and archaeal (single and multiple), in combination with chloramphenicol treatment and probe detection by intensified video microscopy. Universal probes detected ca. 75-95% of total DAPI counts. Of 2 depth profiles from mesotrophic-oligotrophic California waters, a September one, to 300 m, indicated a low but detectable presence of archaea (about 10% above control values) as measured with single probes. A second profile in May with 4 archaeal probes showed <5% at 100 m depth, but the percentage relative to total DAPI counts increased to about 40% at 600 m depth. Samples from the French Mediterranean coast showed few detectable archaea (analyzed with single probes) in surface waters of Villefranche-sur-Mer Bay, but about 60% archaea at 200 m depth outside the bay. These results point in general to the suitability of this single cell FISH method to quantify taxonomic composition of marine samples, and the specific results indicate the high abundance of archaea in at least some midwater locations.