Molecular Genetics of Biofilm Formation

描述(由申请人提供)：附着在表面的微生物种群通常被称为生物膜。在大多数自然环境中，细菌主要存在于生物膜中，但多年来对细菌生理学的研究主要集中在细菌细胞的浮游状态。人们普遍认识到，生物膜影响着无数的环境，这导致了各方共同努力，以更好地了解这些社区发展的分子机制。 最近的研究结果表明，生物膜的形成是一个复杂的发育过程，它发生在对环境提示的反应中。浮游细菌如何从环境感知到成熟生物膜形成的工作模型现在正在指导许多研究人员的研究。然而，最受关注的是在塑料和玻璃等非生物固体表面形成的生物膜。生物膜在生物表面的形成也很普遍，并在环境和临床环境中产生重要影响。在我们的第一个实验方法中，我们建议扩展我们对模式细菌铜绿假单胞菌进行的研究，以调查它如何在活的真菌细丝上形成生物膜。为此，我们将：1)通过生理、生化和显微分析确定细菌-真菌相互作用的特征；2)选择特定的基因产生突变并测试它们对细菌-真菌相互作用的影响；3)进行突变筛选并使用微阵列进行转录谱分析，以确定参与细菌-真菌相互作用的其他基因。 人们普遍认为，生物膜内存在细胞分化。然而，人们对生物膜分化过程背后的分子机制知之甚少。在第二个实验方法中，遵循与我们第一个方法类似的方法，我们将通过研究枯草芽孢杆菌中具有良好特征的孢子形成过程来解决生物膜中细胞分化的问题。我们将：1)利用报告融合技术以及光、电子和激光共聚焦扫描显微镜技术分析孢子特异基因转录的空间和时间模式；2)为选定的基因产生额外的报告基因融合并分析其表达的空间和时间模式，以开发更完整的生物膜功能解剖；3)测试参与细胞-细胞信号和环境感知的突变对生物膜内细胞分化的影响。

DESCRIPTION (provided by applicant): Populations of surface-attached microorganisms are commonly referred to as biofilms. In most natural settings bacteria are found predominantly in biofilms, yet for many years studies of bacterial physiology focused on the planktonic state of bacterial cells. The widespread recognition that biofilms impact a myriad of environments has led to concerted efforts to gain a better understanding of the molecular mechanisms that underlie the development of these communities. Recent results have revealed that biofilm formation is a complex developmental process that occurs in response to environmental cues. Working models for how planktonic bacteria proceed from environmental sensing to the formation of mature biofilms are now guiding many investigators in their research. However, most of the attention has been placed on biofilms that form on abiotic solid surfaces such as plastics and glass. The formation of biofilms on living surfaces is also widespread and has important impacts in environmental and clinical settings. In our first experimental approach we propose to extend the studies we have carried out with a model bacterium, Pseudomonas aeruginosa, to investigate how it forms a biofilm on living fungal filaments. To this end we will: 1) Characterize the bacterial-fungal interactions through physiological, biochemical and microscopic analyses, 2) Select specific genes in which to generate mutations and test their effects on bacterial-fungal interactions and 3) Carry out a mutant screen and perform transcriptional profiling using microarrays to identify additional genes involved in the bacterial-fungal interactions. It is generally accepted that there is cellular differentiation within biofilms. Yet, relatively little is known about the molecular mechanisms that underlie differentiation processes in bioflims. In a second experimental approach that follows a path analogous to our first approach, we will address the question of cellular differentiation in biofilms through the study of a well-characterized sporulation process in Bacillus subtilis. We will: 1) Analyze the spatial and temporal patterns of transcription of a spore-specific gene using reporter fusions and light, electron and confocal scanning laser microscopic techniques, 2) Generate additional reporter gene fusions to selected genes and analyze their spatial and temporal patterns of expression in order to develop a more complete functional anatomy of the biofilm and 3) Test the effects of mutations involved in cell-cell signaling and environmental sensing on cellular differentiation within the biofilm.