Unraveling the complex interactions of host-virus interactions on marine microbial physiology

病毒影响海洋表层沃茨微生物群落的数量和类型、海洋地球化学循环以及碳和营养盐的通量和化学性质。虽然它是越来越可行的，使病毒活动对微生物过程的影响的定量措施，我们的理解之间的相互作用，引起生态规模的属性主机病毒的相互作用的众多仍然处于起步阶段。这对于溶原性（温和性）病毒尤其如此，即，病毒具有整合到其宿主基因组中并保持“休眠”的能力，直到受到外部或内部提示而启动导致细胞破裂和病毒释放的裂解周期。最近的证据表明溶原性病毒是海洋病毒的主要类型。因此，这表明“溶解或不溶解”的决定是海洋微生物群落中一个普遍但知之甚少的过程。尽管病毒对海洋系统的影响是公认的和深远的，但相对较少的努力被放在表征宿主-病毒相互作用的环境相关性上。本项目将研究感染具有重要生态意义的海洋异养细菌Rosebrum分支成员的溶原性病毒。在沿海环境、浮游植物大量繁殖和与有机颗粒相关的地方，玫瑰藻的丰度最大，这些微生物经常与其他多种物种一起生长在凝胶状基质中，称为生物膜。事实上，生物膜是微生物和病毒活动的热点，并提供了一个机会，以更好地确定病毒对宿主生理和功能的影响。这项研究将解决基因组整合的病毒如何影响宿主的代谢和行为，反过来，这些宿主病毒的相互作用如何影响海洋微生物生物膜的结构和功能。所述项目将直接支持对四名毕业生进行跨学科培训，其中一名毕业生将接受国际科学家的培训。根据目前资助的REU项目在微生物学和化学在田纳西大学描述的培训机会将被利用，并扩展到本科生参与这里提出的研究。所有专业研究机构将继续及时以演讲、论文和其他形式传播其研究成果，并将利用公共知识库共享数据。该研究计划分为三个目标：（i）表征生长条件对溶原性裂解开关的作用，（ii）确定决定溶原性裂解开关的遗传决定因素，（iii）量化宿主-病毒相互作用对微生物生物膜发育和单一和混合物种生物膜功能的影响。为了实现这些目标，研究人员将使用基于质谱的高通量代谢组学和脂质组学方法来测试生长底物将差异地影响宿主代谢，特别是细胞内cAMP浓度，并导致不同的溶解速率与溶原性的假设。 该项目将使用一种经典但高效的技术来分离噬菌体中的随机突变（透明噬斑突变体），使它们无法整合到宿主基因组中（即，它们将被固定在裂解状态）。噬菌体突变体将通过生理学研究进行确认，并通过序列分析进行表征。为了更好地了解宿主-病毒相互作用对与海洋碳循环相关的微生物活动的影响，研究人员将在浮游植物Emiliana huxleyi的裂解物上生长的单一和混合物种生物膜中对我们的宿主溶原菌进行一系列实验。研究人员将量化生物膜生物量，形态结构，微生物活性和细胞外基质的化学成分的差异。这项研究的结果将提供必要的基础步骤，以更广泛地了解病毒对海洋细菌群落生态相关组成部分的影响。由此，将有可能开始推断和量化这种相互作用在区域尺度碳循环中的作用。

Viruses influence the number and types of organism that compose microbial communities, biogeochemical cycles and the flux and chemical character of carbon and nutrients in marine surface waters. Though it is increasingly feasible to make quantitative measures of the impact of virus activity on microbial processes, our understanding of the interplay between the multitude of host-virus interactions that give rise to ecological scale properties remains in its infancy. This is especially true for lysogenic (temperate) viruses, i.e., viruses with the ability to integrate into their host's genome and remain "dormant" until prompted, by external or internal cues, to initiate a lytic cycle leading to cell rupture and virus release. Recent evidence suggests lysogenic viruses are the dominate type of marine viruses. Thus, indicating that the decision to "lyse or not to lyse" is a prevalent, but poorly understood, process in marine microbial communities. Despite the recognized and profound influence of viruses on marine systems, relatively little effort has been placed on characterizing host-virus interactions of environmental relevance. This project will study the lysogenic viruses that infect members of the ecologically important Roseobacter clade of heterotrophic marine bacteria. Roseobacter abundance is greatest in coastal environments, phytoplankton blooms and in association with organic particles, where these microbes frequently grow in a gel-like matrix, called biofilms, with multiple other species. Indeed, biofilms are hot spots for both microbial and virus activity, and provide an opportunity to better define the impact that viruses have on host physiology and function. This research will address how genome-integrated viruses influence host metabolism and behavior and, in turn, how these host-viruses interactions influence marine microbial biofilm structure and function. The described project will directly support the interdisciplinary training of four graduates, one of which will be involved in training by international scientists. Training opportunities described under currently funded REU programs in both Microbiology and Chemistry at the University of Tennessee will be leveraged and extended to undergraduates participating in the research presented here. All PIs will continue to disseminate their research results in presentations, papers and other forms on a timely basis and data will be shared using public repositories. The research plan is organized into three objectives: (i) Characterize the role of growth conditions on the lysogenic-lytic switch, (ii) identify genetic determinants that dictate the lysogenic-lytic switch, (iii) quantify the effect of host-virus interactions on microbial biofilm development and function in single and mixed species biofilms. To achieve these objectives, the investigators will use mass spectrometry based high-throughput metabolomics and lipidomics approaches to test the hypothesis that growth substrate will differentially influence host metabolism, particularly intracellular cAMP concentrations, and result in different rates of lysis vs. lysogeny. The project will use a classic, yet highly effective, technique to isolate random mutations in phage (clear plaque mutants) that render them incapable of integration into the host genome (i.e., they will be fixed in the lytic state). Phage mutants will be confirmed by physiological studies and characterized by sequence analysis. To better understand the influence of host-virus interactions on microbial activities that are relevant to ocean carbon cycling vis-a-vis the Biological Pump, the investigators will perform a series of experiments with our host lysogens in single and mixed species biofilms grown on lysate from the phytoplankton species Emiliana huxleyi. The investigators will quantify differences in biofilm biomass, morphological structure, microbial activity and chemical composition of the extracellular matrix. The results from this research will provide the foundational steps necessary to develop a broader understanding of virus effects on an ecologically relevant component of oceanic bacterial communities. From this, it will be possible to begin to extrapolate and quantify the role of this interaction in regional scale carbon cycling.