The community structure of attached and free-living bacteria in the Aegean Sea (eastern Mediterranean Sea) was analyzed with use of terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting. Since the Aegean Sea is characterized by rather small temperature fluctuations between surface and deep-water layers, it represents an ideal study site to determine the variations in the community structure of bacteria with depth, since environmental factors other than temperature are likely to determine depth zonation of bacteria. The analysis of 132 T-RFLP electropherograms indicated pronounced differences among the attached and free-living bacterial communities defined as operational taxonomic units (OTUs). Distinct vertical differences of attached and free-living OTUs were found between mesopelagic waters (Ͼ200 m depth) and the upper mixed water column (ϳ10-200 m). Attached and free-living OTUs differed considerably throughout the water column, with only ϳ35% for the South Aegean and ϳ24% for the North Aegean of all OTUs in both free-living and attached OTUs. Approximately 50% of attached and free-living OTUs were present throughout the water column. Fingerprinting analysis using 16S rRNA indicated that only ϳ14% of the attached and ϳ33% of the free-living OTUs were identical to the 16S rDNA fingerprints. The distribution of free-living versus attached bacteria as obtained in this study suggests that even in the absence of temperature as a major selective factor, a distinct deep-water bacterial community exists (particularly in the free-living mode). The deep-water free-living bacterial community appears to be as compositionally complex as the surface water free-living bacterial community.

Clones from the same marine bacterioplankton community were sequenced, 100 clones based on DNA (16S rRNA genes) and 100 clones based on RNA (16S rRNA). This bacterioplankton community was dominated by alpha-Proteobacteria in terms of repetitive DNA clones (52%), but gamma-Proteobacteria dominated in terms of repetitive RNA clones (44%). The combined analysis led to a characterization of phylotypes otherwise uncharacterized if only the DNA or RNA libraries would have been analyzed alone. Of the DNA clones, 25.5% were found only in this library and no close relatives were detected in the RNA library. For clones from the RNA library, 21.5% of RNA clones did not indicate close relatives in the DNA library. Based on the comparisons between DNA and RNA libraries, our data indicate that the characterization of the bacterial community based on RNA has the potential to characterize distinct phylotypes from the marine environment, which remain undetected on the DNA level.

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.

We examined the phylogenetic diversity of microbial communities associated with marine basalts, using over 300 publicly available 16S rDNA sequences and new sequence data from basalt enrichment cultures. Phylogenetic analysis provided support for 11 monophyletic clades originating from ocean crust (sediment, basalt and gabbro). Seven of the ocean crust clades (OCC) are bacterial, while the remaining four OCC are in the Marine Group I (MGI) Crenarchaeota. Most of the OCC were found at diverse geographic sites, suggesting that these microorganisms have cosmopolitan distributions. One OCC in the Crenarchaeota consisted of sequences derived entirely from basalts. The remaining OCC were found in both basalts and sediments. The MGI Crenarchaeota were observed in all studies where archaeal diversity was evaluated. These results demonstrate that basalts are occupied by cosmopolitan clades of microorganisms that are also found in marine sediments but are distinct from microorganisms found in other marine habitats, and that one OCC in the ubiquitous MGI Crenarchaeota clade may be an ecotype specifically adapted to basalt.

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