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.

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We measured prokaryotic production and respiration in the major water masses of the North Atlantic down to a depth of ,4,000 m by following the progression of the two branches of North Atlantic Deep Water (NADW) in the oceanic conveyor belt. Prokaryotic abundance decreased exponentially with depth from 3 to 0.4 3 10 5 cells mL 21 in the eastern basin and from 3.6 to 0.3 3 10 5 cells mL 21 in the western basin. Prokaryotic production measured via 3 H-leucine incorporation showed a similar pattern to that of prokaryotic abundance and decreased with depth from 9.2 to 1.1 mmol C m 23 d 21 in the eastern and from 20.6 to 1.2 mmol C m 23 d 21 in the western basin. Prokaryotic respiration, measured via oxygen consumption, ranged from about 300 to 60 mmol C m 23 d 21 from ,100 m depth to the NADW. Prokaryotic growth efficiencies of ,2% in the deep waters (depth range 1,200-4,000 m) indicate that the prokaryotic carbon demand exceeds dissolved organic matter input and surface primary production by 2 orders of magnitude. Cell-specific prokaryotic production was rather constant throughout the water column, ranging from 15 to 32 3 10 23 fmol C cell 21 d 21 in the eastern and from 35 to 58 3 10 23 fmol C cell 21 d 21 in the western basin. Along with increasing cell-specific respiration towards the deep water masses and the relatively short turnover time of the prokaryotic community in the dark ocean (34-54 d), prokaryotic activity in the meso-and bathypelagic North Atlantic might be higher than previously assumed.

Diel and seasonal variations in abundance, activity, and structure of particle-attached vs free-living bacterial communities were investigated in offshore NW Mediterranean Sea (0-1000 m). Attached bacteria were always less abundant and less diverse but generally more active than free-living bacteria. The most important finding of this study was that the activity of attached bacteria showed pronounced diel variations in the upper mixed water column with higher activities at night. Under mesotrophic conditions, the contribution of attached bacteria to total bacterial activity increased from less than 10% at day time to 83% at night time. At high chlorophyll a concentration, the highest cell-specific activities and contribution to total bacterial activity were due to free-living bacteria at day and to attached bacteria at night. Under summer oligotrophic conditions, free-living bacteria dominated and contributed to the most important part of the bacterial activity at both day and night, whereas attached bacteria were much less abundant but presented the highest cell-specific activities. These diel and seasonal variations in activities were concomitant to changes in bacterial community structure, mainly in the upper layer. The number of attached ribotypes was fairly constant suggesting that particles are colonized by a relatively limited number of ubiquitous ribotypes. Most of these ribotypes were also free-living ribotypes suggesting that attached bacteria probably originate from colonization of newly formed particles by free-living bacteria in the upper layer. These results reinforce the biogeochemical role of attached bacteria in the cycling of particulate organic carbon in the NW Mediterranean Sea and the importance of diel variability in these processes.

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Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186-194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.

The surface film of the hydrosphere covers more than 70% of the world's surface. The sea surface microlayer (SML) or "skin" of the ocean is a sink for natural and anthropogenic material originating from the atmosphere and the water column. Organisms living in this SML are called "neuston." Our knowledge of the biology of the SML is still in its infancy. Research of the sea surface microlayer requires the use of appropriate sampling techniques and strategies, and the question of what is the most suitable device has not yet been answered. In the present study, we have compared the efficiency of the Harvey glass plate (GP) and the Garrett metal screen (MS) to analyze a wide range of microbiological parameters in SML samples collected at two coastal stations in the NW Mediterranean Sea. Two types of membranes (Teflon and polycarbonate) were also used to collect bacterioneuston. The MS was the most appropriate technique for most biological parameters providing higher enrichment factors as compared to the GP and, therefore, the highest enrichment factors compared with underlying waters (UW). Control experiments with UW demonstrated that the enrichment reported for the MS was not biased by any selectivity of the sampler itself. Therefore, we recommend the use of the MS when the aim is to compare different biological parameters. In contrast, there is clear evidence that hydrophobic and hydrophilic membranes have an important drawback and should not be used for quantification purposes. AcknowledgmentsThis work was supported by the European Commission (Research Directorate General-Environment Program-Marine Ecosystems) through the AIRWIN project "Structure and role of biological communities involved in the transport and transformation of persistent pollutants at the marine AIR-Water Interface" (contract EVK3-CT2000-00030). The AIRWIN project is part of the EC IMPACTS cluster. We thank Frédérique François-Carcaillet for helpful comments on statistical analysis and Danielle Boissé for language improvements. We are also grateful to the two anonymous reviewers for valuable comments on a previous version of the manuscript.

In most aquatic environments, at least 2 subpopulations of bacterial cells can be discriminated by flow cytometry based on their nucleic acid content. Recent investigations have shown that the cells with a high nucleic acid (HNA) content have a higher cell-specific activity (CSA) cell than those with a low nucleic acid (LNA) content. In this study, the CSA and biomass-specific activities (BSA) of HNA and LNA cells from different aquatic ecosystems, including marine, brackish and freshwater, were investigated using radioactive leucine incorporation and cell sorting by flow cytometry. The genetic diversity of natural assemblages, HNA and LNA cells was investigated using the SSCP (PCR single-strand conformation polymorphism) method. Data showed that both CSA and BSA of HNA cells were always significantly higher than CSA and BSA of LNA cells. In addition, HNA cells had a dominant contribution to the production of the total community (77 to 98%). For the different samples, the SSCP fingerprints from the natural assemblage and from the 2 sorted fractions were not significantly different. This clearly suggests that HNA and LNA subpopulations were composed by the same dominant species and, thus, confirms an important heterogeneity of physiological states within most natural populations.