A Molecular Genetic Analysis of Polyhydroxyalkanoate Metabolism and Inclusion-Body Structure in Bacillus Megaterium

巨大芽孢杆菌聚羟基脂肪酸酯代谢和包涵体结构的分子遗传学分析

• 批准号: 9604450
• 负责人: Maura Cannon
• 金额: $ 6万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-15 至 1999-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604450&HistoricalAwards=false
• 关键词: Molecular; Genetic; Analysis; Polyhydroxyalkanoate; Metabolism

Cannon 9604450 Polyhydroxyalkanoates (PHA) are polyesters that accumulate within inclusion-bodies in many bacteria and archaea as carbon and energy reserves. Despite the fact that the gene for PHA synthase has been cloned and sequenced from more than twenty strains of bacteria, the genetic regulation and the links between PHA accumulation and other metabolic pathways are poorly understood. Most of our present knowledge in this area comes from studies of Pseudomonas and Alcaligenes. Apart from some inclusion-body structural studies, very little of our present knowledge comes from studies of the Bacillus genus. The decision to diversify and pursue this study of PHA accumulation in B. megaterium is likely to reveal important new insights into the regulation of PHA accumulation and its links with other cellular processes. An understanding of the regulation of PHA inclusion-body synthesis and its coordinated regulation with other aspects of cellular metabolism is the subject of this research. The following strategy is being undertaken: (1) It has been shown that PHA accumulates in a growth phase dependent manner, and the sequence and characterization of the phaB/phaC region of B. megaterium has been determined. Three novel genes and two divergent promoters associated with PHA accumulation have been identified. This research seeks to demonstrate the functions of the three novel gene products and to determine what regulates the two divergent promoters (pl and p2). The effects of over-expression (gene dosage) and deletion (knock-out by marker substitution), of each of these three genes, individually and in combinationq with each other, will be tested. Effects of these mutations will be measured in relation to transcription from the pl and p2 promoters, translation of the messages, PHA accumulation, and size and shape of inclusion-bodies throughout growth of the culture. (2) It is hypothesized that spore formation is not occurring during PHA accumulation, based on promoter studies of the genes involved. This suggests that PHA accumulation has a regulatory role in spore formation. This hypothesis will be tested by examining the relationship of transcription from the p2 promoter to spore formation. For example, replacement of p2 with a constitutive promoter would be expected to block sporulation, while a deletion mutation in the p2 promoter, may result in very high sporulation rates. (3) A PHA over-producing transposon mutant of B. megaterium has been isolated and the tagged DNA has been identified as a cluster of gas vesicle genes (gvp). The relevance of gas vesicles to PHA inclusion-bodies in B. megaterium is being examined. This is being done by measuring the effects of deletion or over-expression of gvp genes on PHA accumulation (as in 1, above). The accumulation in bacteria of the biodegradable polymer PHA is the result of the combined effects of pha gene products and prevailing environmental conditions, which are reflected in many cellular processes. The results of these experiments should increase our understanding of the integrated genetic regulation of these processes with PHA accumulation.

Cannon 9604450聚羟基链烷酸酯（PHA）是一种聚酯，在许多细菌和古细菌的包涵体中积累，作为碳和能量储备。尽管事实上，PHA合成酶的基因已被克隆和测序超过20株细菌，遗传调控和PHA积累和其他代谢途径之间的联系知之甚少。我们目前在这一领域的知识大多来自假单胞菌和产碱菌的研究。除了一些包涵体结构的研究，我们目前的知识很少来自芽孢杆菌属的研究。在B中进行PHA蓄积研究的多样化决定。megaterium可能揭示重要的新见解PHA积累的调节及其与其他细胞过程的联系。 了解PHA包涵体合成的调节及其与细胞代谢其他方面的协调调节是本研究的主题。正在采取以下战略： (1)已经表明PHA以生长期依赖性方式积累，并且B.已经确定了。三个新的基因和两个不同的启动子与PHA积累已被确定。本研究试图证明这三种新基因产物的功能，并确定是什么调控两种不同的启动子（p1和p2）。将测试这三个基因中的每一个单独和彼此组合的过表达（基因剂量）和缺失（通过标记物置换敲除）的影响。这些突变的影响将相对于来自pl和p2启动子的转录、信息的翻译、PHA积累以及整个培养物生长过程中包涵体的大小和形状来测量。 (2)根据对相关基因的启动子研究，推测在PHA积累期间不发生孢子形成。这表明PHA积累在孢子形成中具有调节作用。这一假设将通过检查p2启动子转录与孢子形成的关系来检验。例如，用组成型启动子替换p2预期会阻断孢子形成，而p2启动子中的缺失突变可能导致非常高的孢子形成率。 (3)一种PHA高产转座子突变体B。已分离出巨大芽孢杆菌，标记的DNA已被鉴定为一簇气泡基因（gvp）。B中气泡与PHA包涵体的相关性。正在检查中。这是通过测量gvp基因的缺失或过表达对PHA积累的影响来完成的（如上文1中所述）。 生物可降解聚合物PHA在细菌中的积累是PHA基因产物和主要环境条件的组合作用的结果，这反映在许多细胞过程中。这些实验的结果应该增加我们的理解，这些过程与PHA积累的综合遗传调控。