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

这项研究的重点是新的分子技术的发展和应用，以识别活跃生长的浮游细菌物种，并估计单个浮游细菌类群的新细胞产量。这项工作建立在最近发表的技术基础上。这项研究的动机是希望提供一种强大的、多功能的模拟现有方法来测量细菌产量。~3H-胸腺嘧啶核苷(~3H-TdR)掺入是推断自然种群细菌生物量生产力最常用的方法。它的使用是基于这样的假设，即受试者种群中的所有细菌都可以将外源TDR结合到新生的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，还不可能检验这些假设，从而验证~3H-TdR掺入在海洋浮游细菌产量测量中的应用。这是因为目前几乎所有的优势海洋细菌都是不可培养的。我们开发了一套应用溴脱氧尿苷(BrdU)的技术，它是一种免疫原性TDR类似物，用于分析自然种群中的微生物生长。与~3H-TdR掺入相比，BrdU技术的主要优点是免疫化学纯化含BrdU的DNA，结合已建立的分析16S rRNA基因的方法，可用于鉴定天然样品中含有BrdU的细菌。这提供了一种方法来确定哪些社区成员对使用BrdU进行的细菌生产力测量做出了贡献，从而使该技术得以验证。此外，随着本建议中概述的实验的完成，有可能使用BrdU来估计单个浮游细菌类群的生物量产量。拟议的研究包括利用培养的细菌和沿海浮游细菌进行实验，以优化BrdU的技术，校准BrdU和~H-TdR掺入到不可培养的类群的生长，鉴定浮游细菌中含有BrdU的类群，并估计它们的类群特定生长。为了完整，其他实验将比较浮游细菌中BrdU阳性与~3H-葡萄糖和~H-亮氨酸掺入细胞的数量，评估浮游细菌中明显无效的类群的BrdU掺入潜力，并确定BrdU和H-TdR是否被相同的浮游细菌类群掺入。最后，将使用BrdU方法来确定不同的浮游细菌类群是否在一天的不同周期中合成DNA。在这项建议中的实验包括分析在俄勒冈州海岸采集的一些浮游细菌样本，并解决不同的类群是否在一天中的不同时间合成DNA的问题。然而，实验设计并不旨在分析活跃的浮游细菌类群的全球模式，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来加强和校准BrdU方法，以便未来的实验可以在良好的理论和技术基础上进行。这项研究应该会为海洋学家的分子生物学工具箱提供一个强有力的新补充。

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.