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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM036296-17S1
Application #
6746522
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Anderson, James J
Project Start
1987-08-01
Project End
2004-02-29
Budget Start
2002-04-01
Budget End
2004-02-29
Support Year
17
Fiscal Year
2003
Total Cost
$64,618
Indirect Cost
Name
University of Washington
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Bosch, Gundula; Skovran, Elizabeth; Xia, Qiangwei et al. (2008) Comprehensive proteomics of Methylobacterium extorquens AM1 metabolism under single carbon and nonmethylotrophic conditions. Proteomics 8:3494-505
Chistoserdova, Ludmila; Crowther, Gregory J; Vorholt, Julia A et al. (2007) Identification of a fourth formate dehydrogenase in Methylobacterium extorquens AM1 and confirmation of the essential role of formate oxidation in methylotrophy. J Bacteriol 189:9076-81
Marx, Christopher J; Van Dien, Stephen J; Lidstrom, Mary E (2005) Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism. PLoS Biol 3:e16
Kalyuzhnaya, Marina G; Korotkova, Natalia; Crowther, Gregory et al. (2005) Analysis of gene islands involved in methanopterin-linked C1 transfer reactions reveals new functions and provides evolutionary insights. J Bacteriol 187:4607-14
Chistoserdova, Ludmila; Laukel, Markus; Portais, Jean-Charles et al. (2004) Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol. J Bacteriol 186:22-8
Marx, Christopher J; Lidstrom, Mary E (2004) Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch. Microbiology 150:9-19
Marx, Christopher J; Laukel, Markus; Vorholt, Julia A et al. (2003) Purification of the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 and demonstration of its requirement for methylotrophic growth. J Bacteriol 185:7169-75
Marx, Christopher J; Chistoserdova, Ludmila; Lidstrom, Mary E (2003) Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1. J Bacteriol 185:7160-8
Marx, C J; Lidstrom, M E (2001) Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147:2065-75
Chistoserdova, L; Gomelsky, L; Vorholt, J A et al. (2000) Analysis of two formaldehyde oxidation pathways in Methylobacillus flagellatus KT, a ribulose monophosphate cycle methylotroph. Microbiology 146 ( Pt 1):233-8

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