Development and Application of Bromodeoxyuridine Immunochemical Techniques for the Molecular Analysis of Actively Growing Bacterioplankton

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The focus of this study is the development and application of new, molecular techniques to identify actively growing bacterioplankton species and estimate new cell production by individual bacterioplankton taxa. The work builds upon recently published techniques. The motivation for the research is the desire to provide a powerful, multifunctional analog to established methods for measuring bacterial production. 3H-thymidine (3 H-TdR) incorporation is the most commonly used method for inferring bacterial biomass productivity in natural populations. Its use is based upon the assumption that all bacteria in subject populations can incorporate exogenous TdR into nascent DNA, and that the relationship between H-TdR incorporation and productivity is nearly equal among species. However, it has not been possible to test these assumptions, and thereby validate application of 3 H-TdR incorporation to measurements of marine bacterioplankton production. This is because nearly all dominant marine bacteria are currently unculturable. We have developed a suite of techniques applying bromodeoxyuridine (BrdU), an immunogenic TdR analog, to the analysis of microbial growth in natural populations. The main advantage of BrdU techniques over 3 H-TdR incorporation is that immunochemical purification of BrdU-containing DNA, combined with established methods for the analysis of 16S rRNA genes, can be used to identify bacteria incorporating BrdU` in natural samples. This provides a means of determining which community members are contributing to measurements of bacterial productivity made using BrdU, enabling validation of the technique. In addition, with the completion of experiments outlined in this proposal, it may be possible to use BrdU to estimate biomass production by individual bacterioplankton taxa.The proposed research includes experiments using cultured bacteria and coastal bacterioplankton to optimize BrdU` techniques, calibrate BrdU and 3 H-TdR incorporation to growth for culturable relatives of unculturable taxa, identify BrdU-incorporating taxa in bacterioplankton and estimate their taxon-specific growth. For completeness, additional experiments will compare numbers of BrdU-positive to 3 H-glucose and 3 H-leucine-incorporating cells in bacterioplankton, assess the BrdU-incorporation potential of apparently inactive taxa in bacterioplankton, and also determine whether BrdU and H-TdR are incorporated by the same bacterioplankton taxa. Finally, BrdU methods will be used to determine whether different bacterioplankton taxa synthesize DNA during different portions of the diurnal cycle.The experiments in this proposal include analyses of some bacterioplankton samples collected off the Oregon coast and address the question of whether different taxa synthesize DNA at different times of day in open ocean and coastal environments. However, the experimental design does not purport to analyze global patterns of active bacterioplankton taxa, their roles in food webs, or biomass production by specific taxa. These types of large-scale experiments are a goal for the future. Here, we propose an ordered series of experiments to strengthen and calibrate BrdU methods so that future experiments can be conducted on sound theoretical and technical bases. This research should provide a powerful new addition to the oceanographer's molecular biology toolbox.