Our laboratory has three primary research interests, one concerned with transcriptional and metabolic regulation in bacteria, a second with transport protein evolution, and a third with the recently identified process of transposon-mediated directed mutation. We also maintain the IUBMB-approved Transporter Classification Database, TCDB, which classifies transport systems found in all living organisms on Earth into five categories: class, subclass, family, subfamily and transport system.
Our laboratory takes a multidisciplinary approach to science, using biochemical, molecular genetic, physiological, and computational approaches. Directed mutation is a proposed process that allows mutations to occur at higher frequencies when they are beneficial than when detrimental. Until recently, the existence of such a process has been controversial. However, we have described a novel mechanism of directed mutation mediated by the transposon, IS5 in Escherichia coli. crp deletion mutants mutate specifically to glycerol utilization (Glp+) at rates that are enhanced by glycerol or the loss of the glycerol repressor (GlpR), depressed by glucose or glpR overexpression, and RecA-independent. Of the four tandem GlpR binding sites (O1–O4) upstream of the glpFK operon, O4 specifically controls glpFK expression while O1 primarily controls mutation rate in a process mediated by IS5 hopping to a specific site on the E. coli chromosome upstream of the glpFK promoter. IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish an example of transposon-mediated directed mutation, identify the protein responsible and define the mechanism involved. Most recently, we have identified two additional operons in E. coli that appear to be subject to transposon-mediated directed mutation: The flhDC flagellar master switch operon controlling motility and the fucAO operon controlling L-fucose and propanediol utilization.
Our 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 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. It thereby mediates catabolite repression of catabolic operons by an indirect mechanism. Most recently, we have demonstrated that Cra regulates growth by activating expression of the crp 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). 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.
Milton Saier received his Ph.D. from UC Berkeley and was a postdoctoral fellow at Johns Hopkins University.