Our laboratory is investigating the structure and mechanism of the enzyme/permease responsible for the coupled transport and phosphorylation of the hexitol D-mannitol in Escherichia coli. This protein is an integral membrane component of the bacterial phosphotransferase system (PTS) and also serves as a chemotactic receptor for D-mannitol in E, coli. Over the past decade, its structure and catalytic mechanism have been extensively studied by biochemical, biophysical and genetic techniques. The mannitol permease remains the most attractive transport protein of the PTS to study because it has been purified, its gene has been cloned and sequenced, and its overall disposition with respect to the membrane is known. The broad objective of these studies is to determine in as much molecular detail as possible the mechanism by which the mannitol permease carries out its receptor and transport functions. This is not completely known for any membrane transport protein, and the results of these studies should therefore add fundamental knowledge to the understanding of transport protein structure and function. Since all cells rely on proper functioning of transport proteins for interaction with their environment, and some diseased cells may lack one or more of these functions, the study of these fundamental processes is essential in understanding the basic principles underlying growth and metabolism in both normal and diseased cells. Moreover, since many pathogenic bacteria have a PTS, but this system is absent in eucaryotes, it is a potential target for antibacterial agents. In the proposed project continuation, the aims are to: 1) continue to isolate and characterize mutants in the mannitol permease; 2) use complementation studies and the isolation of pseudorevertants to characterize important interactions in the permease; 3) study the structure of the permease including attempts to crystallize it; and 4) study a structurally related protein, ORF162, which may be a regulatory link between the PTS and nitrogen assimilation in bacteria. A combination of biochemical, biophysical, molecular biological and genetic techniques will be used to accomplish these aims. Collaborations and consultations with other workers in this area will ensure a complementary, rather than a competitive expenditure of time and effort in this project. Successful completion of this work will lead to significant progress toward our overall goal of understanding the molecular basis of transport and chemoreception in this system.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Boston University
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Saraceni-Richards, C A; Jacobson, G R (1997) A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease. J Bacteriol 179:1135-42
Saraceni-Richards, C A; Jacobson, G R (1997) Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins. J Bacteriol 179:5171-7
Begley, G S; Warner, K A; Arents, J C et al. (1996) Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease. J Bacteriol 178:940-2
Begley, G S; Jacobson, G R (1994) Overexpression, phosphorylation, and growth effects of ORF162, a Klebsiella pneumoniae protein that is encoded by a gene linked to rpoN, the gene encoding sigma 54. FEMS Microbiol Lett 119:389-94
Weng, Q P; Jacobson, G R (1993) Role of a conserved histidine residue, His-195, in the activities of the Escherichia coli mannitol permease. Biochemistry 32:11211-6
Postma, P W; Lengeler, J W; Jacobson, G R (1993) Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57:543-94
Jacobson, G R; Saraceni-Richards, C (1993) The Escherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system. J Bioenerg Biomembr 25:621-6
Briggs, C E; Khandekar, S S; Jacobson, G R (1992) Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization. Res Microbiol 143:139-49
Weng, Q P; Elder, J; Jacobson, G R (1992) Site-specific mutagenesis of residues in the Escherichia coli mannitol permease that have been suggested to be important for its phosphorylation and chemoreception functions. J Biol Chem 267:19529-35
Sugiyama, J E; Mahmoodian, S; Jacobson, G R (1991) Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions. Proc Natl Acad Sci U S A 88:9603-7

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