Our laboratory has two primary research interests, one concerned with transcriptional and metabolic regulation in bacteria, the other with transport protein evolution. As transport proteins play central regulatory roles, these two interests are related. We take a multidisciplinary approach to science, using biochemical, molecular genetic, physiological, and computational approaches, and we collaborate with x-ray crystallographers and multidimensional NMR spectroscopists in order to solve the 3-dimensional structures of central regulatory proteins we have overproduced and purified. As many complex transcriptional and metabolic regulatory phenomena in bacteria are still poorly understood, we additionally study the coordination of carbon metabolism with nitrogen, phosphorous, sulfur and iron metabolism as well as with oxygen availability. In this capacity we study the interactions of various global regulatory systems with each other. Our interests also extend to the differences in regulatory mechanisms exhibited by evolutionarily divergent bacterial groups
Our recent efforts have revealed three basic mechanisms of transcriptional control concerned with catabolite repression/activation in bacteria. Two of these occur in E. coli, and one occurs in B. subtilis. In E. coli, two DNA binding proteins, the cyclic AMP receptor protein (Crp) and the catabolite repressor/activator (Cra) protein, mediate transcriptional regulation of hundreds of genes encoding key enzymes of carbon and energy metabolism. Virtually every pathway of carbon metabolism is subject to these regulatory constraints. Crp generally controls the initiation of exogenous carbon source metabolism and senses cytoplasmic cyclic AMP levels. These levels are controlled by complex, poorly understood mechanisms, involving phosphorylated proteins of the sugar-transporting phosphotransferase system (PTS). Cra generally controls the flux of carbon through metabolic pathways and senses cytoplasmic metabolite concentrations. Cra usually controls gene expression independently of Crp, but it sometimes acts cooperatively with or antagonistically to Crp, depending on the target gene.
In B. subtilis and other Gram-positive bacteria, a metabolite-activated protein kinase phosphorylates a serine residue in a protein of the PTS called HPr. Phosphorylated HPr allosterically controls the activities of many target proteins (transport proteins, enzymes and transcription factors (see figure). It thereby controls the cytoplasmic concentrations of inducers as well as the activities of transcription factors that mediate catabolite repression. We are coming to realize that the mechanisms of catabolite control are very different for phylogenetically divergent bacteria.
Phylogenetic analyses of integral membrane transport protein sequences have yielded a plethora of information about the times of appearance, the routes of evolution, and the relative rates of divergence of the proteins and protein domains which comprise various families of transport systems. These studies have shown that families of transport proteins of similar topology have evolved independently of each other, at different times in evolutionary history, using different routes. They have also revealed extensive domain shuffling in some such families but not in others. The probable means by which energy coupling became superimposed on transport during the evolutionary process has also come to light.
Choi, S.-K. and Saier, M.H., Jr. (2005). Regulation of sigL expression by the catabolite control protein CcpA involves a roadblock mechanism in Bacillus subtilis: potential connection between carbon and nitrogen metabolism. J. Bacteriol. 187: 6856-6861.
Barabote, R.D. and Saier, M.H., Jr. (2005). Comparative genomic analyses of the bacterial phosphotransferase system. Microbiol. Mol. Biol. Rev. 69: 608-634.
Saier, M.H., Jr., Tran, C.V. and Barabote, R.D. (2006). TCDB: The transporter classification database for membrane transport protein analyses and information. Nucleic Acids Res. 34: D181–D186 (Database issue).
Kim, S.H., Chao, Y., and Saier, M.H., Jr. (2006). Protein-translocating trimeric autotransporters of Gram-negative bacteria. J. Bacteriol. 188: 5655-5667.
Soberón, X. and Saier, M.H., Jr. (2006). Engineering transport protein function: theoretical and technical considerations using the sugar-transporting phosphotransferase system of E. coli as a model system. J. Mol. Microbiol. Biotechnol. 11: 302-307.
Milton Saier received his Ph.D. from UC Berkeley and was a postdoctoral fellow at Johns Hopkins University.