Fabien Joux

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems

ABSTRACT 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.

Use of fluorescent probes to assess physiological functions of bacteriaat single-cell level

A wide diversity of fluorescent probes is currently available to assess the physiological state of microorganisms. The recent development of techniques such as solid-phase cytometry, the increasing sensitivity of fluorescence tools and multiparametric approaches combining taxonomic and physiological probes have improved the effectiveness of direct methods in environmental and industrial microbiology.

Resistance of Marine Bacterioneuston to Solar Radiation

ABSTRACT 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 γ-proteobacteria and 14% and 8% for α-proteobacteria and the Cytophaga/Flavobacterium/Bacteroides (CFB) group, respectively. Within the γ-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.

Responses of enteric bacteria to environmental stresses in seawater

Abstract The effects of different environmental factors (nutrient deprivation, hyperosmotic shock, exposure to light) on enteric bacteria which have been transferred into the marine environment, have been studied experimentally (microcosms) by considering demographic, physiological and genetic responses in Escherichia coli or Salmonella typhimurium populations. Short-term experiments (≤ 48 h) showed that nutrient deprivation induced limited changes in measured bacteriological variables, but when combined with hyperosmotic shock, it results in an energy charge decrease and inactivation of membrane transport. Light exposure mainly affects the colony-forming capacity of bacterial populations. Combining different stress factors confirmed the rapid appearance of a viable, but nonculturable state (VBNC) in populations of E. coli and S. typhimurium. It has been shown that cellular forms other than those previously described in the literature can be generated following incubation in seawater. It was also established that pre-adaptation phenomena may occur, leading to better survival (e.g. pre-incubation in seawater in darkness enhanced survival under light exposure). An explanation concerning these phenomena can be found by looking at the rpoS gene which controls the expression of numerous genes and can trigger a general anti-stress response under different adverse conditions. Although the results provide better comprehension of the fate of enteric bacteria in the marine environment, they also raise numerous questions related to fundamental and applied problems, given in the conclusion of this paper.

The response of the marine bacterium Sphingopyxis alaskensis to solar radiation assessed by quantitative proteomics

The adaptive response of the marine bacterium Sphingopyxis alaskensis RB2256 to solar radiation (both visible and ultraviolet) was assessed by a quantitative proteomic approach using iTRAQ (isobaric tags for relative and absolute quantification). Both growth phase (mid-log and stationary phase) and duration (80 min or 8 h) of different light treatments (combinations of visible light, UV-A and UV-B) were assessed relative to cultures maintained in the dark. Rates of total protein synthesis and viability were also assessed. Integrating knowledge from the physiological experiments with quantitative proteomics of the 12 conditions tested provided unique insight into the adaptation biology of UV and visible light responses of S. alaskensis. High confidence identifications were obtained for 811 proteins (27% of the genome), 119 of which displayed significant quantitative differences. Mid-log-phase cultures produced twice as many proteomic changes as stationary-phase cultures, while extending the duration of irradiation exposure of stationary-phase cultures did not increase the total number of quantitative changes. Proteins with significant quantitative differences were identified that were characteristic of growth phase and light treatment, and cellular processes, pathways and interaction networks were determined. Key factors of the solar radiation adaptive response included DNA-binding proteins implicated in reducing DNA damage, detoxification of toxic compounds such as glyoxal and reactive oxygen species, iron sequestration to minimize oxidative stress, chaperones to control protein re/folding, alterations to nitrogen metabolism, and specific changes to transcriptional and translational processes.

Sunlight-induced DNA Damage in Marine Micro-organisms Collected Along a Latitudinal Gradient from 70°N to 68°S

We examined ultraviolet radiation (UVR)‐induced DNA damage in marine micro‐organisms collected from surface seawater along a latitudinal transect in the Central Pacific Ocean from 70°N to 68°S. Samples were collected predawn and incubated under ambient UVR in transparent incubators at in situ temperatures until late afternoon at which time they were filtered into primarily bacterioplankton and eukaryotic fractions. Cyclobutane pyrimidine dimers (CPDs) and (6‐4) photoproducts [(6‐4)PDs] were quantified in DNA extracts using radioimmunoassays. UVB was lowest in the polar regions and highest near the equator and correlations between UVB and DNA damage were observed. The eukaryotic fraction showed significant CPDs across the entire transect; (6‐4)PDs were detected only in the tropics. The bacterial fraction showed no accumulation of (6‐4)PDs at any latitude, although residual (6‐4)PDs were observed. Bacterial cell volumes were greatest in the sub‐Arctic and northern temperate latitudes and lower in the tropics and southern hemisphere, a unique observation that parallels Bergmann’s rule. A strong negative correlation was observed between cell volume and CPDs. The environmental impact of solar UVR on marine micro‐organisms in the open ocean is complex and our results suggest that several factors such as DNA repair, cell size, temperature, salinity, nutrients and species composition are important in determining relative sensitivity.

Ultraviolet Radiation in the Rhône River Lenses of Low Salinity and in Marine Waters of the Northwestern Mediterranean Sea: Attenuation and Effects on Bacterial Activities and Net Community Production

The high content in nutrients of freshwater outflows induces highly productive and buoyant plumes spreading over marine waters (MW). As a consequence, the growth of organisms developing in these low‐salinity waters (LSW) might be potentially affected by UV‐R (280–400 nm). This study investigated the penetration of UV‐R and its impact on net community production (NCP) and bacterial protein (BPROTS) and DNA (BDNAS) synthesis in mesotrophic‐LSW formed from the Rhône River and in oligotrophic MW of the Northwestern Mediterranean Sea (Gulf of Lions) in May 2006. High concentrations of chlorophyll a (up to 8 μg L−1) measured in the LSW (<37.8 psu, 0–10 m) were the main factor influencing the diffuse attenuation coefficients (Kd) of both UV‐R and photosynthetically active radiation (PAR). The mean ratio of the Kd measured between the LSW and the MW increased with wavelength from 2.4 at 305 nm to 2.9 at 380 nm and 3.1 for PAR indicating more similarity in the UV region. NCP was severely inhibited by UV‐R at the surface of the LSW, whereas no effect was measured in the surrounding MW. In contrast, BPROTS and BDNAS were affected deeper by UV‐R in the MW (up to 8 m depth) compared to the LSW where inhibition was only observed at the surface. Differences in response of bacteria in LSW and MW are largely explained by differences in UV‐R transparency; however, transplant experiments indicate that bacterial assemblages from the MW were also more sensitive to UV‐R than those present in the LSW. We also observed that higher activity of bacteria after nutrient additions increased their sensitivity to UV‐R during the day, but favored their recovery during the night incubation period for both LSW and MW. Results suggest that riverine and nutrient inputs may alter the effects of UV‐R on microbial activity by attenuating the UV‐R penetration and by modifying the physiology of bacteria.

Evidence of heterotrophic prokaryotic activity limitation by nitrogen in the Western Arctic Ocean during summer

Global warming in the Arctic Ocean may result in changes to the stock and dynamics of nutrients that drive the activity of heterotrophic prokaryotes, a key component of the microbial food web. We performed 14 enrichment experiments during summer in the Beaufort and Chukchi Seas (Western Arctic Ocean), with C (acetate and/or glucose), N (nitrate and/or ammonium) and P (phosphate) amendments. In 8 out of 11 experiments performed with surface waters, prokaryotic heterotrophic production was limited by N, either alone (5 experiments) or in co-limitation with C (3 experiments). This contrasted with the experiments performed using waters from the chlorophyll maxima, where N was never limiting. Diversity analyses (DNA- and RNA-based fingerprinting) suggest that limitation was not restricted to specific operational taxonomic units but common to the different groups. This is the first report of N limitation of prokaryotic heterotrophic production in Arctic surface waters. This control by N may gain importance in future scenarios of higher productivity in the area.

Proteome Analysis of the UVB-Resistant Marine Bacterium Photobacterium angustum S14

The proteome of the marine bacterium Photobacterium angustum S14 was exposed to UVB and analyzed by the implementation of both the post-digest ICPL labeling method and 2D-DIGE technique using exponentially growing cells. A total of 40 and 23 proteins were quantified in all replicates using either the ICPL or 2D-DIGE methods, respectively. By combining both datasets from 8 biological replicates (4 biological replicates for each proteomics technique), 55 proteins were found to respond significantly to UVB radiation in P. angustum. A total of 8 UVB biomarkers of P. angustum were quantified in all replicates using both methods. Among them, the protein found to present the highest increase in abundance (almost a 3-fold change) was RecA, which is known to play a crucial role in the so-called recombinational repair process. We also observed a high number of antioxidants, transport proteins, metabolism-related proteins, transcription/translation regulators, chaperonins and proteases. We also discuss and compare the UVB response and global protein expression profiles obtained for two different marine bacteria with trophic lifestyles: the copiotroph P. angustum and oligotroph Sphingopyxis alaskensis.

Physiological Alteration of the Marine Bacterium Vibrio angustum S14 Exposed to Simulated Sunlight During Growth

Growth experiments on the marine bacterium Vibrio angustum S14 were conducted under four light conditions using a solar simulator: visible light (V), V + ultraviolet A (UV-A), V + UV-A + UV-B radiation, and dark. Growth was inhibited mainly by UV-B and slightly by UV-A. UV-B radiation induced filaments containing multiple genome copies with low cyclobutane pyrimidine dimers. These cells did not show modifications in cellular fatty acid composition in comparison with dark control cultures and decreased in size by division after subsequent incubation in the dark. A large portion of the bacterial population grown under visible light showed an alteration in cellular DNA fluorescence as measured by flow cytometry after SYBR-Green I staining. This alteration was not aggravated by UV-A and was certainly due to a change in DNA topology rather than DNA deterioration because all the cells remained viable and their growth was not impaired. Ecological consequences of these observations are discussed.