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

本研究的重点是新的分子技术的开发和应用，以确定积极增长的浮游细菌物种和估计新的细胞生产的个别浮游细菌类群。这项工作建立在最近发表的技术。这项研究的动机是希望提供一个强大的，多功能的类似物，以建立测量细菌生产的方法。3 H-TdR掺入法是推断自然界细菌生物量的最常用方法。它的使用是基于这样的假设，即受试者群体中的所有细菌都可以将外源TdR掺入新生DNA中，并且H-TdR掺入和生产力之间的关系在物种之间几乎相等。然而，它一直不可能测试这些假设，从而验证应用3 H-TdR掺入海洋浮游细菌生产的测量。这是因为几乎所有占优势的海洋细菌目前都是不可培养的。我们已经开发了一套技术，应用溴脱氧尿苷（BrdU），免疫原性TdR类似物，在自然人群中的微生物生长的分析。BrdU技术相对于3 H-TdR掺入的主要优点是，含BrdU的DNA的免疫化学纯化，结合已建立的16 S rRNA基因分析方法，可用于鉴定天然样品中掺入BrdU的细菌。这提供了一种确定哪些群落成员有助于使用BrdU测量细菌生产力的方法，从而能够验证该技术。此外，随着本提案中概述的实验的完成，有可能使用BrdU来估计单个浮游细菌分类群的生物量生产。拟议的研究包括使用培养细菌和沿海浮游细菌进行实验，以优化BrdU技术，校准BrdU和3 H-TdR掺入，以适应不可培养分类群的可培养亲属的生长，鉴定浮游细菌中结合BrdU分类群并估计其分类群特异性生长。为了完整起见，额外的实验将比较浮游细菌中BrdU阳性细胞与3 H-葡萄糖和3 H-亮氨酸结合细胞的数量，评估浮游细菌中明显不活跃的类群的BrdU结合潜力，并确定BrdU和H-TdR是否被相同的浮游细菌类群结合。最后，BrdU的方法将被用来确定是否不同的浮游细菌类群合成DNA在不同的部分的昼夜cycle.The实验在这个建议包括一些浮游细菌样本的分析收集了俄勒冈州海岸和地址的问题，是否不同的类群合成DNA在不同的时间在开放的海洋和沿海环境。然而，实验设计并不旨在分析全球活跃的浮游细菌类群的模式，它们在食物网中的作用，或特定类群的生物量生产。这些类型的大规模实验是未来的目标。在这里，我们提出了一系列有序的实验，以加强和校准BrdU方法，使未来的实验可以进行良好的理论和技术基础。这项研究应该为海洋学家的分子生物学工具箱提供一个强大的新补充。

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.