The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.
Bacterial populations inhabiting the sea surface microlayer from two contrasted Mediterranean coastal stations (polluted vs. oligotrophic) were examined by culturing and genetic fingerprinting methods and were compared with those of underlying waters (50 cm depth), for a period of two years. More than 30 samples were examined and 487 strains were isolated and screened. Proteobacteria were consistently more abundant in the collection from the pristine environment whereas Gram-positive bacteria (i.e., Actinobacteria and Firmicutes) were more abundant in the polluted site. Cythophaga-Flavobacter-Bacteroides (CFB) ranged from 8% to 16% of total strains. Overall, 22.5% of the strains showed a 16S rRNA gene sequence similarity only at the genus level with previously reported bacterial species and around 10.5% of the strains showed similarities in 16S rRNA sequence below 93% with reported species. The CFB group contained the highest proportion of unknown species, but these also included Alpha- and Gammaproteobacteria. Such low similarity values showed that we were able to culture new marine genera and possibly new families, indicating that the sea-surface layer is a poorly understood microbial environment and may represent a natural source of new microorganisms. Genetic fingerprinting showed, however, no consistent differences between the predominant bacterial assemblages from surface microlayer and underlying waters, suggesting that the presence of a stable and abundant neustonic bacterial community is not a common trait of coastal marine environments.
A total of 90 bacterial strains were isolated from the sea surface microlayer (i.e., bacterioneuston) and underlying waters (i.e., bacterioplankton) from two sites of the northwestern Mediterranean Sea. The strains were identified by sequence analysis, and growth recovery was investigated after exposure to simulated solar radiation. Bacterioneuston and bacterioplankton isolates were subjected to six different exposure times, ranging from 0.5 to 7 h of simulated noontime solar radiation. Following exposure, the growth of each isolate was monitored, and different classes of resistance were determined according to the growth pattern. Large interspecific differences among the 90 marine isolates were observed. Medium and highly resistant strains accounted for 41% and 22% of the isolates, respectively, and only 16% were sensitive strains. Resistance to solar radiation was equally distributed within the bacterioneuston and bacterioplankton. Relative contributions to the highly resistant class were 43% for ␥-proteobacteria and 14% and 8% for ␣-proteobacteria and the Cytophaga/Flavobacterium/Bacteroides (CFB) group, respectively. Within the ␥-proteobacteria, the Pseudoalteromonas and Alteromonas genera appeared to be highly resistant to solar radiation. The majority of the CFB group (76%) had medium resistance. Our study further provides evidence that pigmented bacteria are not more resistant to solar radiation than nonpigmented bacteria.
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