REGULATION OF C1 METABOLISM IN METHYLOTROPHS

甲烷和甲醇氧化细菌（甲基营养菌）能够 以甲烷或甲醇作为唯一的碳源和能源。 它们独特的代谢途径和酶使它们成为研究的对象。 从生物技术和环境的角度都有很大的兴趣。 从细胞生物学的角度来看，它们将所有细胞 碳通过有毒中间体（甲醛）使他们感兴趣 就他们如何平衡甲醛的生产和消费而言， 基质流动条件的宽范围。 很明显， 所使用的机制之一是划分关键的甲醛产生系统， 进入周质。 这导致了发展的详细 用于合成、组装和成熟复杂周质的系统 这已成为一个富有成果的研究领域。 但不少 关于这一问题的时间和空间方面， 过程，以及各方面如何协调。 该实验室对基因组织特别感兴趣， 在甲基营养型中表达，该项目的长期目标是 了解甲基营养生理学以及复杂的调节子如何参与 在甲基化营养中，它们是根据细胞的需要而协调的。 在先前NIH的支持下， 已被用来研究C1代谢的兼性甲醇利用者 扭脱甲基杆菌AM1. 因此，我们获得了重大的新 深入了解甲基营养代谢的四个部分，甲醇氧化， 甲胺利用，通过丝氨酸循环和氧化同化 甲醛。 现在建议继续这一努力， 方法。 首先，我们对丝氨酸循环遗传学的理解 将使用测序和后生方法进行扩展。 第二、 丝氨酸循环基因的调节，甲醛氧化 基因，和Mau（甲胺利用）系统将被调查 使用转录融合，并且在可能的情况下， 将与有关转录本、转录起始 位点、编码基因产物的蛋白质水平和编码基因产物的活性。 内切酶 基因融合也将用于分离调控基因， Mau系统，然后将其特征化。 类似的工作有 已经为混合氧化物燃料系统进行了，但我们建议， 专门寻找编码可能的负调节因子的基因， Mox系统，以及可能在不同的细胞之间协调的基因。 途径。

Methane- and methanol-oxidizing bacteria (methylotrophs) are capable of growth on methane or methanol as their sole source of carbon and energy. Their unique metabolic pathways and enzymes have made them a subject of great interest from both biotechnological and environmental perspectives. From a cell biology perspective, their ability to funnel all of their cell carbon through a toxic intermediate (formaldehyde) make them of interest in terms of how they balance formaldehyde production and consumption under a wide regime of substrate flow conditions. It has become clear that one of the mechanisms used is to partition key formaldehyde producing systems into the periplasm. This has resulted in the development of elaborate systems for synthesis, assembly and maturation of complex periplasmic dehydrogenases, which has become a fruitful area of study. However, many questions still remain concerning temporal and spatial aspects of this process, and how all facets are coordinated. This laboratory is particularly interested in gene organization and expression in methylotrophs, and the long-term goal of this project is to understand methylotrophic physiology and how the complex regulons involved in methylotrophy are coordinated in response to the needs of the cell. Under previous NIH support a combined genetic and physiological approach has been used to study C1 metabolism in the facultative methanol-utilizer Methylobacterium extorquens AM1. As a result, we have gained major new insights into four parts of methylotrophic metabolism, methanol oxidation, methylamine utilization, assimilation via the serine cycle and oxidation of formaldehyde. It is now proposed to continue this effort by two approaches. First, our understanding of the genetics of the serine cycle will be extending using a sequencing and metagenesis approach. Second, the regulation of the serine cycle genes, the formaldehyde oxidation genes, and the Mau (methylamine utilization) system will be investigated using transcriptional fusions and where possible, data from these fusions will be compared to data concerning transcripts, transcriptional start sites, protein levels of encoded gene products, and activities of encoded enzymes. Gene fusions will also be used to isolate regulatory genes for the Mau system, which will then be characterized. Similar work has already been carried out for the Mox system, but we propose in addition to search specifically for genes encoding possible negative regulators of the Mox system, and for genes that might coordinate between the different pathways.