Book Description

Prokaryotes depend on a number of two-component systems (TCSs) to rapidly sense and respond to environmental stimuli. These TCSs, comprised of a signaling histidine kinase and cognate response regulator, are critical for bacterial signal integration and cellular response to external or internal cues. Commonly, these systems function to regulate the expression of target genes, or initiate larger regulatory cascades, to coordinate this response. Bacteria rely on several well-studied envelope stress response TCSs to monitor extracytoplasmic and cell envelope homeostasis as well as respond to antibiotics, toxins, host immune factors, osmotic and desiccation stress to promote survival. Other TCSs are important regulators of essential processes including cell cycle dynamics and cell wall biosynthesis. However, these systems tend to be poorly conserved and/or studied in Gram-negative bacteria. As a result, little is known about this regulation in Îł-proteobacteria. Therefore, one goal of my thesis project was to provide insight into these processes using the Gram-negative Îł-proteobacterium Rhodobacter sphaeroides as a model. This purple non-sulfur bacterium remodels the cell envelope under low oxygen conditions by increasing lipid content and creating intracytoplasmic membrane invaginations (ICM) that protrude from the cytoplasmic membrane and house the cell's photosynthetic machinery. Thus, we hypothesized that Rb. sphaeroides would be amenable to perturbation to identify the genetic basis and regulation of cell envelope remodeling and biosynthesis. First, I characterized a previously unidentified histidine kinase (CenK) with no known cognate response regulator that when disrupted, produced cells with altered cell morphology and hypersensitivity to compounds inhibiting peptidoglycan synthesis. This work defined the TCS that this kinase belongs in and identified an essential response regulator (CenR) as its cognate pair. Utilizing gain of function and loss of function alleles, I assayed the predicted function of CenR in transcriptional regulation and determined that this TCS regulates the expression of cell division, outer membrane biogenesis, and peptidoglycan cell wall biosynthesis and assembly. Consistent with its activity in Rb. sphaeroides, I found that many Îł-proteobacteria contain a homologous TCS and provide evidence for its conserved function in these microbes. Expanding on this work, I utilized the cell morphological changes produced by increasing the activity of the CenKR TCS to probe poorly understood spatial and temporal cell division processes in Rb. sphaeroides. I showed that the localization of the outer membrane lipoprotein Pal is key to proper localization of peptidoglycan biosynthesis and the coordination of bacterial cytoskeletal proteins MreB and FtsZ. By monitoring the localization of these essential proteins, I developed a model for how these systems function in wild type cells as well as how their disruption leads to TCS mutant phenotypes. Therefore, I propose CenKR is an unusual and essential regulator of outer membrane homeostasis and division. I further propose that genetic manipulation of this TCS has and can continue to provide new insights into poorly understood process of cell elongation, cell division, and cell envelope biogenesis in Rb. sphaeroides and other Îł-proteobacteria.