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

病毒影响组成微生物群落的有机体的数量和类型、生物地球化学循环以及海洋表层水域中碳和营养物质的通量和化学性质。尽管对病毒活动对微生物过程的影响进行定量测量越来越可行，但我们对引起生态规模特性的大量宿主-病毒相互作用之间的相互作用的理解仍处于初级阶段。对于溶原性(温带)病毒尤其如此，即能够整合到宿主基因组中并保持“休眠”的病毒，直到受到外部或内部线索的提示，启动裂解周期，导致细胞破裂和病毒释放。最近的证据表明，溶原性病毒是海洋病毒的主要类型。因此，这表明在海洋微生物群落中，“裂解或不裂解”的决定是一个普遍但知之甚少的过程。尽管病毒对海洋系统产生了公认的深远影响，但在确定与环境相关的宿主-病毒相互作用方面所做的工作相对较少。这个项目将研究感染异养海洋细菌中具有重要生态意义的玫瑰杆菌分支成员的溶原性病毒。在沿海环境中，玫瑰杆菌的丰度最高，浮游植物大量繁殖，并与有机颗粒结合在一起，这些微生物经常与多种其他物种一起生长在被称为生物膜的凝胶状基质中。事实上，生物膜是微生物和病毒活动的热点，并为更好地确定病毒对宿主生理和功能的影响提供了机会。这项研究将解决基因组整合病毒如何影响宿主的新陈代谢和行为，以及这些宿主与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四名毕业生的跨学科培训，其中一人将参与国际科学家的培训。田纳西大学目前资助的微生物学和化学REU项目中描述的培训机会将被利用并扩展到参与本文所述研究的本科生。所有私人投资机构将继续以演示文稿、论文和其他形式及时传播其研究成果，并将利用公共储存库共享数据。该研究计划分为三个目标：(I)表征生长条件对溶源-裂解开关的作用，(Ii)确定决定溶源-裂解开关的遗传决定因素，(Iii)量化宿主-病毒相互作用对单一和混合物种生物膜中微生物生物膜发育和功能的影响。为了实现这些目标，研究人员将使用基于质谱学的高通量代谢组学和脂质组学方法来检验生长底物将不同地影响宿主代谢，特别是细胞内cAMP浓度的假设，并导致不同的裂解和裂解速率。该项目将使用一种经典但高效的技术来分离噬菌体(透明斑块突变体)中的随机突变，这些突变使它们无法整合到宿主基因组中(即它们将固定在裂解状态)。噬菌体突变体将通过生理研究得到确认，并进行序列分析。为了更好地了解宿主-病毒相互作用对与海洋碳循环和生物泵相关的微生物活动的影响，研究人员将在浮游植物Emily huxleyi的裂解物上用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化学成分的差异。这项研究的结果将为更广泛地了解病毒对海洋细菌群落的生态相关组成部分的影响提供必要的基础步骤。由此，就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.