Research

We are principally interested in two topics:

1. The features and mechanisms of bacterial stress responses, using Bacillus subtilis as a model species, and
2. The metabolism and behavior of the opportunistic human pathogen Pseudomonas aeruginosa, with a special focus on its ability to produce pyocins, phage tail-like intraspecific killing complexes.

Our research uses many different techniques, from bacterial molecular genetics and traditional culture techniques to modern "omics" approaches and advanced microscopy. We benefit particularly from microfluidic technology, which permits us to observe cells under a light microscope as they grow and has several advantages over traditional analytical methods. For example, because the cells are constantly bathed in fresh growth medium, we can follow cells for tens or hundreds of generations in constant and uniform growth conditions. We can manipulate the composition of the growth medium, adding or removing stressors or other compounds, so that we can observe the immediate and long-term responses of cells to the compounds that we add. Because the growth conditions are uniform, we can examine the true responses of cells apart from self-induced changes to the local environment caused by nutrient depletion or waste produce buildup. Finally, because we observe cells under the microscope, we achieve single-cell resolution, allowing us to assess cell-to-cell variability across a population of cells. We typically use fluorescent reporters to observe the inner workings of cells as they grow and respond to changes in their environment.

 

Bacterial Stress Responses

Because of their small size, bacteria experience the presence of stressors in their local environments virtually instantaneously. This means that bacteria must have systems in place that sense stress and are poised to respond very rapidly but also with an appropriate magnitude and duration. As in eukaryotic systems such as the NF-κB stress-response system, a rapid response is ensured by making a stress-response protein ahead of time and then holding it in an inactive state until a stress signal is received. In the model bacterium B. subtilis, the stress-response protein is a sigma factor known as σB. Sigma factors are proteins that tell RNA polymerase where to bind to DNA and transcribe (express) genes. When σB is active, it directs the transcription of a group of some 150 stress-response genes that confer a so-called "general stress response" that helps cells survive the sudden onset of many different types of stress. However, most of the specific functions of the general-stress-response proteins remain unknown.

 

When cells are not under stress, σB is kept in an inactive state by an anti-sigma factor called RsbV (or just "V" for simplicity). V binds to σB and prevents it from bringing RNA polymerase to the general stress-response genes. When cells sense stress, a signaling cascade is initiated (figure) that leads to the dephosphorylation (i.e., removal of a phosphate group) of V, which makes it let go of σB and instead bind an anti-anti-sigma factor, RsbW ("W"). Now σB is free to activate the general stress response.

 

 

The upstream signaling cascades are illustrated in the figure. Energy or nutritional stress, sensed as the depletion of ATP (the cellular energy currency), is sensed through an unknown mechanism by RsbQ and RsbP—P is a phosphatase that can dephosphorylate V. On the other hand, environmental stress—ethanol, heat, acid, salt, antibiotics, etc.—is sensed by large 80-protein complexes called stressosomes. Stressosomes are composed of RsbR and RsbS proteins, and they also bind to a protein called RsbT. Environmental stressors cause stressosomes to release T in a process that involves T phosphorylating R and S, and the free T then activates RsbU—U is another phosphatase that can dephosphorylate V.

 

Further complicating things is that the stressosome contains a mixture of four "flavors" (paralogs) of R proteins that are similar but distinct. Why does B. subtilis make these similar but different proteins? Our work thus far has taken advantage of microfluidic technology to uncover differences among these R proteins in their stress-response profiles when responding to ethanol, a classic stressor. We now seek to discover the molecular bases of these differences and to understand how different stress-response profiles can confer survival advantages to cells.

 

Here are some of the fundamental questions we are asking:

• What is actually sensed by the R proteins in the stressosome?
• Do the different flavors of R proteins respond differently to different stresses?
• What parts of the R proteins govern their distinct response profiles?
• Which amino-acid residues play key roles in stress sensing by R proteins?
• How do different combinations of R proteins affect overall response profiles?
• How do different response profiles confer survival advantages or disadvantages in the presence of different stresses?

As you can see, we have many promising avenues of research. Understanding how different stress-response profiles are generated and how they influence cell survival will give greater insights into the strategies that cells have evolved to maximize their fitness while being prepared for ever-changing conditions.

 

Interested in working on these projects? Contact us—we are always looking for motivated students who love to learn.

 

Metabolism and Behavior

P. aeruginosa is an important opportunistic human pathogen, meaning that it typically does not infect healthy persons but often infects those already sick or immunocompromised, including those in the hospital.

Our research on this bacterium can divided into three broad areas: biofilm formation, metabolism, and pyocin production.

 

Biofilm Formation

Biofilms are communities of bacterial cells that are encased in a self-produced extracellular matrix—sort of like a bunch of cells stuck in a gluey substance. Biofilms are important, both in medicine and in industry. Nuisance biofilms can form in pipes and in industrial equipment, while infectious biofilms are difficult for doctors to treat. Because they are encased in a matrix, biofilm cells are relatively shielded from antibiotics, the immune system, and other therapeutics; moreover, because some cells in the interior of a biofilm are not growing, they are especially resistant to antibiotics, making biofilm infections notoriously difficult to eradicate. Therefore, scientists are actively looking for other ways to combat biofilm infections. One of the ways we can do this is to gain a better understanding of how biofilms form—the signals and mechanisms that tell cells to settle down and secrete an extracellular matrix.

 

 

One of the initial research thrusts in the Cabeen lab was to study biofilm formation by P. aeruginosa, which can infect many body sites but is particularly well-known for lung infections, particularly in individuals with cystic fibrosis (a genetic disorder). Because of its medical importance, much is known about how biofilms are signaled and constructed in P. aeruginosa. Upstream signals induce the formation of a bacterial second-messenger compound known as cyclic-di-GMP (cdG), a cyclic dinucleotide molecule that can be thought of as a sort of "internal office memo" in the bacterial cell. High levels of cdG in the cell generally encourage biofilm formation, yet we still have much to learn about all the proteins that control cdG levels or respond to cdG to govern biofilm formation.

 

We used a screening approach that differs from previous screens in P. aeruginosa. We use colony morphology as a way to assess biofilm formation: wrinkled colonies indicate biofilm formation, whereas smooth and flat colonies indicate that no biofilm has formed (Figure 2). In this way, we can easily assess by visual inspection whether a mutant strain has increased (more wrinkled) or decreased (less wrinkled) biofilm relative to a parental (non-mutant) strain. This approach has uncovered several genes with previously unappreciated roles in biofilm formation, and we continued running the screen to uncover even more genes with roles in biofilm formation while following up on the genes we have already discovered.

 

This approach has been fruitful, and we currently have manuscripts in preparation or revision in which we characterize genes with previously unappreciated roles in biofilm formation. 

 

Metabolism

Postdoctoral fellow (2020-2022) Simon Underhill has been leading a research direction in the lab exploring signaling and metabolic pathways in P. aeruginosa. He first examined the utilization of citrate and cis-aconitate by P. aeruginosa, finding a number of previously unknown citrate-utilization proteins that are most likely membrane-bound transporters and uncovering extensive redundancy in citrate transport. Teaming up with PhD student Somalisa Pan, he also examined phosphorylation in the nitrogen-related phosphotransfer system (Nitro-PTS) of P. aeruginosa, for the first time showing native phosphostates of the PtsN protein and uncovering new putative regulatory targets of PtsN (Underhill, Pan et al., in revision). Finally, before leaving the lab he conducted experiments linking growth on glycerol with biofilm formation and worked out some of the glycerol metabolism pathway (Underhill et al., in preparation).

 

Pyocin Production

Pyocins are interbacterial but intraspecific killing molecules. The best-studied among them are the R-type pyocins, which are like phage tails without heads that literally spear competing cells with a microscopic, iron atom-tipped spike, depolarizing the membrane and killing them. While studying a biofilm-related gene, we unexpectedly discovered that P. aeruginosa strains deficient in or deleted for a tyrosine recombinase called XerC overproduce pyocins, which are typically only produced in response to DNA damage via the RecA protein. This second, non-canonical, RecA-independent pathway for pyocin expression is now a major research focus in our lab, as we seek to learn the molecular mechanism of non-canonical pyocin expression and to leverage it for therapeutic advantage.