Response to UVB radiation and oxidative stress of marine bacteria isolated from South Pacific Ocean and Mediterranean Sea.

Marine bacterial strains isolated from South Pacific and Mediterranean Sea were studied for their resistance to UVB radiation, their repair capacity under photoreactivating light, as well as their oxidative stress response using concentrated hydrogen peroxide (H(2)O(2)), as an oxidizer. A total of 30 marine bacteria were isolated from the hyper-oligotrophic waters of the South Pacific Gyre to the eutrophic waters of the Chilean coast during the BIOSOPE cruise (2004), and 10 strains from surface Mediterranean coastal waters. One third of bacteria presented a high resistance to UVB and almost all isolates presented an efficient post-irradiation recovery. Only few strains showed cell survival to high concentration of H(2)O(2). No correlation between the sampling sites and the bacterial UVB resistance was observed. Two marine bacteria, Erythrobacter flavus and Ruegeria mobilis, were of particular interest, presenting a good response to the three parameters (UVB and H(2)O(2) resistance/efficient repair). Unexpectedly, two resistant strains were again identified as Ruegeria species underlining that this geographically widespread genus, resist to UVB regardless the environment from which the isolates originate.

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.

Effects of photochemical transformations of dissolved organic matter on bacterial metabolism and diversity in three contrasting coastal sites in the Northwestern Mediterranean Sea during summer.

The effects of phototransformation of dissolved organic matter (DOM) on bacterial growth, production, respiration, growth efficiency, and diversity were investigated during summer in two lagoons and one oligotrophic coastal water samples from the Northwestern Mediterranean Sea, differing widely in DOM and chromophoric DOM concentrations. Exposure of 0.2-microm filtered waters to full sun radiation for 1 d resulted in small changes in optical properties and concentrations of DOM, and no changes in nitrate, nitrite, and phosphate concentrations. After exposure to sunlight or dark (control) treatments, the water samples were inoculated with the original bacterial community. Phototransformation of DOM had contrasting effects on bacterial production and respiration, depending on the water's origin, resulting in an increase of bacterial growth efficiency for the oligotrophic coastal water sample (120%) and a decrease for the lagoon waters (20 to 40%) relative to that observed in dark treatments. We also observed that bacterial growth on DOM irradiated by full sun resulted in changes in community structure of total and metabolically active bacterial cells for the three locations studied when compared to the bacteria growing on un-irradiated DOM, and that changes were mainly caused by phototransformation of DOM by UV radiation for the eutrophic lagoon and the oligotrophic coastal water and by photosynthetically active radiation (PAR) for the mesoeutrophic lagoon. These initial results indicate that phototransformation of DOM significantly alters both bacterial metabolism and community structure in surface water for a variety of coastal ecosystems in the Mediterranean Sea. Further studies will be necessary to elucidate a more detailed appreciation of potential temporal and spatial variations of the effects measured.

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

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.

Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments?

The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another. Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using (3)H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production.

Use of fluorescent probes to assess physiological functions of bacteria at 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.

Marine bacterial isolates display diverse responses to UV-B radiation.

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.

A New Sensitive, Whole-Cell Hybridization Technique for Detection of Bacteria Involving a Biotinylated Oligonucleotide Probe Targeting rRNA and Tyramide Signal Amplification.

A tyramide signal amplification system with biotinylated oligonucleotide probes and streptavidin-horseradish peroxidase was used to increase the sensitivity of fluorescent in situ hybridization techniques. When applied to both gram-negative and -positive bacteria immobilized on glass slides, a 7- to 12-fold amplification of the fluorescence signal was observed relative to that of cells hybridized with fluorescently monolabeled probes. A large proportion (62 to 78%) of bacteria could be detected under starvation conditions and in natural samples from the marine environment. This amplification procedure allows new investigations in marine oligotrophic ecosystems and water quality control.

Changes in Cellular States of the Marine Bacterium Deleya aquamarina under Starvation Conditions.

In this study, we have used different fluorescent dyes and techniques to characterize the heterogeneity and changes of the physiological states encountered by the marine bacterium Deleya aquamarina during a 92-day starvation survival experiment at 20 and 5(deg)C. Changes of physiological states were investigated on a single-cell basis by flow cytometry and epifluorescence microscopy in conjunction with fluorescent dyes specific for various cellular functions and constituents. Heterogeneities within populations with regard to functions (respiration, substrate responsiveness, enzymatic activity, and cytoplasmic membrane permeability), constituent (DNA), and cell volume (light scatter) were compared to the evolution of viable plate counts (CFU). At 20(deg)C, CFU changes were divided into three stages corresponding to stability up to day 13 followed by a rapid drop between days 13 and 42 and then by stabilization at a level of 10 to 20% during the remaining survival period. Most of the cellular fractions showing a metabolic activity were close to the evolution of the culturable cells, suggesting the absence of viable but nonculturable cells. On the other hand, cells with selective cytoplasmic membrane permeability but without any metabolic activity were observed, and this stage was followed by DNA alteration occurring at different rates after the loss of membrane cytoplasmic permeability. We observed a greater maintenance of culturability, physiological functions, DNA, and cellular volume at the lower temperature. These results have different ecological implications from both methodological and conceptual viewpoints.

Ecological implications of an improved direct viable count method for aquatic bacteria.

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.

Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater.

Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.