The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis

Abstract

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria. The bacterial cell envelope is home to an array of important functions including, energy conservation, motility, influx/efflux of nutrients and toxins, modulation of cell morphology and division, cell-cell interaction, and biofilm formation. Consequently, it is a major target of antibiotics and antimicrobial agents that inhibit these essential processes. Key to the recognition of environmental stressors or stimuli are bacterial TCSs, however systems that monitor or directly regulate cell envelope assembly and homeostasis are not widely conserved amongst bacteria. Here, we use Rhodobacter sphaeroides as a model to investigate the function of the CenKR TCS in this and other α-proteobacteria. We show that this essential TCS plays a key role in maintenance of the cell envelope through the regulation of outer membrane integrity and division, cell wall remodeling and homeostasis, and an alternate sigma factor that controls global cellular stress response. We provide evidence that this TCS and its function is widely conserved in α-proteobacteria and identify genes of unknown function as candidates for the study of cell envelope assembly in this and related bacteria.