Two-component signal transduction is a mechanism used by nearly all bacteria to organize intracellular events and respond to changes in the environment. In a two-component signaling pathway, the upstream histidine kinase protein senses a signal and autophosphorylates on a conserved histidine residue. The phosphoryl group is then passed to a downstream response regulator protein, which generates a cellular response, such as a change in gene expression, metabolism, or motility. Although this linear paradigm is correct in most cases, some two-component proteins with important cellular roles appear to function by alternative mechanisms, or in branched networks. The project focuses on two proteins, DivK and DivL, that are essential for cell cycle progression in the aquatic bacterium Caulobacter crescentus. The aim is to determine how the response regulator DivK suppresses the activity of a histidine kinase, CckA, which functions in a separate two-component pathway. Suppression of CckA activity at the correct time is necessary for Caulobacter cells to begin chromosome replication. The other aim is to establish the function of an essential protein, DivL, which is homologous to histidine kinases, but which appears to act by a mechanism other than phosphoryl transfer. In particular, it is proposed that DivL participates in essential protein-protein interactions which are modulated by binding and hydrolysis of ATP. Experiments are proposed to measure the ATP binding and hydrolysis by DivL mutants that cause distinct cellular phenotypes, and to identify new proteins that interact with DivL to mediate its effects on the cell division cycle. This research will expand the repertoire of biochemical activities used by histidine kinases and response regulators to influence each other and cellular events.
Broader Impacts This project will provide an intensive summer lab course in microbiology for 10 students per year. This course is different from many lab classes in that the students do experiments whose results are not known beforehand. In addition to fundamental techniques in microbiology and molecular biology, they learn how to design and troubleshoot experiments so that their results are interpretable and reliable. In past years, each student has deleted a previously unstudied gene in Caulobacter crescentus and characterized the resulting mutant strain. In upcoming years, the students will do projects closely related to the scientific goals of this research, such as generating specific mutations in divL and determining their cellular effects, or performing genetic screens to identify proteins that interact with DivL or DivK. Students learn how new scientific knowledge is generated, and they gain experience that prepares them for independent research projects. Women and students from underrepresented groups are encouraged to undertake research projects in the applicant's laboratory.
Our lab studies the regulation of the cell division cycle using the aquatic bacterium Caulobacter crescentus. A key protein called CtrA must be present and activated for cell division to occur, but it must be inactivated or absent for an earlier step, chromosome replication, to occur. CtrA activity is therefore temporally regulated. We initially studied proteins in the pathway that activate CtrA by phosphorylation. One of the proteins involved in CtrA activation, DivL, was predicted to phosphorylate itself on a tyrosine residue. Since tyrosine phosphates are very stable, we reasoned that a phosphatase with the opposite activity must dephosphorylate DivL to switch it off. We therefore searched the Caulobacter genome for predicted tyrosine phosphatases. Of the four genes identified, one was essential for Caulobacter viability, suggesting that it plays an important role in the biology of this organism. Caulobacter cells depleted of the essential phosphatase, CtpA, became fused together in chains with severe outer membrane blebs before dying. CtpA is located in Caulobacter membranes and specifically migrates to the site of cell division. The phenotypes we observe seem to be entirely unrelated to DivL, the original subject of our study. Nevertheless, we identified the first tyrosine phosphatase protein that is essential for the life of a bacterium. Phosphorylation on tyrosine residues was once thought to be a switch mechanism found exclusively in plants and animals, but our work and other studies have shown that it occurs in bacteria and plays an important role in viability, cell wall integrity, and the ability of pathogenic bacterial to cause disease. The timed synthesis and degradation of proteins with specific activities is a mechanism of regulating cell behavior found in all living organisms. Just before chromosome replication in Caulobacter, the existing CtrA is dephosphorylated (to deactivate it) and completely degraded into small peptides. A key question is how CtrA degradation is regulated, so that it only occurs during a short window in the cell division cycle. We have dissected the mechanism of CtrA degradation by the protein-degrading machine, ClpXP. In Caulobacter cells, ClpXP needs three accessory proteins and a small signaling molecule, cyclic-di-GMP (cdG), to recognize and degrade CtrA. Using purified proteins, we compared reactions containing only ClpXP and CtrA with reactions containing all of the accessory factors. The accessory factors allow ClpXP to recognize and degrade CtrA at lower concentrations, making the reaction more efficient. One of the accessory proteins, PopA, binds to cdG, and in this state, it directly interacts with CtrA. Thus cdG stimulates CtrA degradation by facilitating its recognition by PopA. In Caulobacter, there is known to be a transient increase in the level of cdG coincident with CtrA proteolysis. We synthesized these findings into a model in which PopA binding to cdG stimulates the recognition of CtrA for rapid degradation. We continue to study CtrA degradation because important and puzzling questions remain about this process. For example, we don’t know why Caulobacter degrades every copy of CtrA before chromosome replication instead of merely dephosphorylating it. Dephosphorylation would be much less expensive, since CtrA would not have to be completely resynthesized from amino acids every time the cell divides. The Broader Impacts portion of this award was centered on the merging of teaching and research in an intensive summer lab course. In each course, 10 students worked in the Ryan lab on related but independent projects. Each student’s goal was to knock out a Caulobacter gene that was present in related bacteria, but whose function was completely unknown. To provide structure, the students attended lectures giving background on the techniques they were using, as well as on Caulobacter biology. They progressed at a different paces toward their goals, repeating and troubleshooting experiments. The students came together in weekly lab meetings, where they discussed their results and difficulties and practiced their presentation skills. One publication resulted from the lab course, in which we characterized a mutant lacking the BamE protein, which had previously not been identified in Caulobacter. This protein is part of a complex that helps to insert a specific class of proteins into the outer membrane. Caulobacter cells lacking BamE have shorter stalks than wild-type cells, and they have defects in outer membrane integrity, as measured by susceptibility to chemicals, detergents, and heat. We purified BamE from Caulobacter cells along with the other proteins in the BAM complex, and we identified outer membrane proteins that do not reach their proper destination when BamE is absent. This project represents research outside of our main focus that would not have been performed and published without the jump-start it received from the undergraduate lab course. The award also supported the research efforts of four graduate students, one postdoctoral researcher, and nine undergraduates, all of whom are still engaged in science in college, graduate school, medical school, nonprofit agencies, or the biotech industry.
