Two systems for the dissimilation of glycerol have been found in enteric bacteria: the glp regulon specifying a respiratory network and the dha genes specifying a fermentative network. Operations of the glp system requires an exogenous electron acceptor. In contrast, operation of the dha system requires no exogenous electron acceptor, because a portion of glycerol can be used to generate an endogenous one. In the glp system, sn-glycerol 3-phosphate (C3P) is the central intermediate that is oxidized to dihydroxyacetone phosphate by either of two dehydrogenases of the flavoprotein kind: an aerobic dehydrogenase glpD product) thought to pass the electrons from G3P via ubiquinone-8 to either O2 or nitrate, and an anaerobic dehydrogenase (glpACB product) thought to pass electrons from G3P via menaquinone to either nitrate or fumarate. Function of both electron transport chains results in the generation of proton motive force. In Klebsiella pneumoniae, which possesses both systems, the glp system is preferentially induced aerobically and the dha system is preferentially induced anaerobically. Moreover, a switch from anaerobic to aerobic metabolism leads to the inactivation of two NAD+-oxidoreductases of the dha system. This project encompasses three major aims: (1) The degree of ubiquinone specificity of the two G3P dehydrogenases will be probed in appropriately constructed Escherichia coli strains, and attempts will be made to select mutants with altered specificity. If successful, Dr. Lin will use this class of mutants to map the protein domains involved by comparing DNA sequences. He will also search for mutants with altered control in quinone synthesis. (2) The dha regulon of K. pneumoniae will be analyzed to determine the location of its five known genes, the number of operons involved, and their direction of transcription. Transcriptional regulation of this system will be studied with dha-lacZ protein fusion, and a search will be made for unlinked mutations that relieve aerobic repression of the regulon. (3) The nature and source of the oxidizing agent that inactivates the two NAD+-linked enzymes in the dha system after the switch from an anaerobic to an aerobic environment will be explored, as well as the target site of enzyme inactivation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM011983-28
Application #
3268342
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-05-01
Project End
1995-04-30
Budget Start
1991-05-01
Budget End
1992-04-30
Support Year
28
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Iuchi, S; Aristarkhov, A; Dong, J M et al. (1994) Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase. J Bacteriol 176:1695-701
Iuchi, S (1993) Phosphorylation/dephosphorylation of the receiver module at the conserved aspartate residue controls transphosphorylation activity of histidine kinase in sensor protein ArcB of Escherichia coli. J Biol Chem 268:23972-80
Dong, J M; Taylor, J S; Latour, D J et al. (1993) Three overlapping lct genes involved in L-lactate utilization by Escherichia coli. J Bacteriol 175:6671-8
Iuchi, S; Lin, E C (1993) Adaptation of Escherichia coli to redox environments by gene expression. Mol Microbiol 9:9-15
Fu, H A; Iuchi, S; Lin, E C (1991) The requirement of ArcA and Fnr for peak expression of the cyd operon in Escherichia coli under microaerobic conditions. Mol Gen Genet 226:209-13
Iuchi, S; Cole, S T; Lin, E C (1990) Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control. J Bacteriol 172:179-84
Sweet, G; Gandor, C; Voegele, R et al. (1990) Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product. J Bacteriol 172:424-30
Iuchi, S; Matsuda, Z; Fujiwara, T et al. (1990) The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon. Mol Microbiol 4:715-27
Eiglmeier, K; Honore, N; Iuchi, S et al. (1989) Molecular genetic analysis of FNR-dependent promoters. Mol Microbiol 3:869-78
Iuchi, S; Furlong, D; Lin, E C (1989) Differentiation of arcA, arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilus formation and enzyme regulation. J Bacteriol 171:2889-93

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