Cross-regulation in a three-component cell envelope stress signaling system of Brucella.

IMPORTANCE: As intracellular pathogens, Brucella must contend with a variety of host-derived stressors when infecting a host cell. The inner membrane, cell wall, and outer membrane, i.e. the cell envelope, of Brucella provide a critical barrier to host assault. A conserved regulatory mechanism known as two-component signaling (TCS) commonly controls transcription of genes that determine the structure and biochemical composition of the cell envelope during stress. We report the identification of previously uncharacterized TCS genes that determine Brucella ovis fitness in the presence of cell envelope disruptors and within infected mammalian host cells. Our study reveals a new molecular mechanism of TCS-dependent gene regulation, and thereby advances fundamental understanding of transcriptional regulatory processes in bacteria.

Cross regulation in a three-component cell envelope stress signaling system of Brucella.

A multi-layered structure known as the cell envelope separates the controlled interior of bacterial cells from a fluctuating physical and chemical environment. The transcription of genes that determine cell envelope structure and function is commonly regulated by two-component signaling systems (TCS), comprising a sensor histidine kinase and a cognate response regulator. To identify TCS genes that contribute to cell envelope function in the intracellular mammalian pathogen, Brucella ovis, we subjected a collection of non-essential TCS deletion mutants to compounds that disrupt cell membranes and the peptidoglycan cell wall. Our screen led to the discovery of three TCS proteins that coordinately function to confer resistance to cell envelope stressors and to support B. ovis replication in the intracellular niche. This tripartite regulatory system includes the known cell envelope regulator, CenR, and a previously uncharacterized TCS, EssR-EssS, which is widely conserved in Alphaproteobacteria. The CenR and EssR response regulators bind a shared set of sites on the B. ovis chromosomes to control transcription of an overlapping set of genes with cell envelope functions. CenR directly interacts with EssR and functions to stimulate phosphoryl transfer from the EssS kinase to EssR, while CenR and EssR control the cellular levels of each other via a post-transcriptional mechanism. Our data provide evidence for a new mode of TCS cross-regulation in which a non-cognate response regulator affects both the activity and protein levels of a cognate TCS protein pair.

The Brucella Cell Envelope.

The cell envelope is a multilayered structure that insulates the interior of bacterial cells from an often chaotic outside world. Common features define the envelope across the bacterial kingdom, but the molecular mechanisms by which cells build and regulate this critical barrier are diverse and reflect the evolutionary histories of bacterial lineages. Intracellular pathogens of the genus Brucella exhibit marked differences in cell envelope structure, regulation, and biogenesis when compared to more commonly studied gram-negative bacteria and therefore provide an excellent comparative model for study of the gram-negative envelope. We review distinct features of the Brucella envelope, highlighting a conserved regulatory system that links cell cycle progression to envelope biogenesis and cell division. We further discuss recently discovered structural features of the Brucella envelope that ensure envelope integrity and that facilitate cell survival in the face of host immune stressors.

The ChvG-ChvI Regulatory Network: A Conserved Global Regulatory Circuit Among the Alphaproteobacteria with Pervasive Impacts on Host Interactions and Diverse Cellular Processes.

The ChvG-ChvI two-component system is conserved among multiple Alphaproteobacteria. ChvG is a canonical two-component system sensor kinase with a single large periplasmic loop. Active ChvG directs phosphotransfer to its cognate response regulator ChvI, which controls transcription of target genes. In many alphaproteobacteria, ChvG is regulated by a third component, a periplasmic protein called ExoR, that maintains ChvG in an inactive state through direct interaction. Acidic pH stimulates proteolysis of ExoR, unfettering ChvG-ChvI to control its regulatory targets. Activated ChvI among different alphaproteobacteria controls a broad range of cellular processes, including symbiosis and virulence, exopolysaccharide production, biofilm formation, motility, type VI secretion, cellular metabolism, envelope composition, and growth. Low pH is a virulence signal in Agrobacterium tumefaciens, but in other systems, conditions that cause envelope stress may also generally activate ChvG-ChvI. There is mounting evidence that these regulators influence diverse aspects of bacterial physiology, including but not limited to host interactions.

An Inducible T7 Polymerase System for High-Level Protein Expression in Diverse Gram-Negative Bacteria.

A broad host range (BHR)-inducible T7 RNA polymerase system was developed, enabling induction with isopropyl-ß-d-thiogalactopyranoside (IPTG), similar to the Escherichia coli strain BL21(DE3) protocol, but it is now applicable in a wide range of bacteria. This system allows for high protein yields and purification from diverse Gram-negative bacteria, including the native host.

Motility control through an anti-activation mechanism in Agrobacterium tumefaciens.

Many bacteria can migrate from a free-living, planktonic state to an attached, biofilm existence. One factor regulating this transition in the facultative plant pathogen Agrobacterium tumefaciens is the ExoR-ChvG-ChvI system. Periplasmic ExoR regulates the activity of the ChvG-ChvI two-component system in response to environmental stress, most notably low pH. ChvI impacts hundreds of genes, including those required for type VI secretion, virulence, biofilm formation, and flagellar motility. Previous studies revealed that activated ChvG-ChvI represses expression of most of class II and class III flagellar biogenesis genes, but not the master motility regulator genes visN, visR, and rem. In this study, we characterized the integration of the ExoR-ChvG-ChvI and VisNR-Rem pathways. We isolated motile suppressors of the non-motile ΔexoR mutant and thereby identified the previously unannotated mirA gene encoding a 76 amino acid protein. We report that the MirA protein interacts directly with the Rem DNA-binding domain, sequestering Rem and preventing motility gene activation. The ChvG-ChvI pathway activates mirA expression and elevated mirA is sufficient to block motility. This study reveals how the ExoR-ChvG-ChvI pathway prevents flagellar motility in A. tumefaciens. MirA is also conserved among other members of the Rhizobiales suggesting similar mechanisms of motility regulation.

Short, Rich, and Powerful: a New Family of Arginine-Rich Small Proteins Have Outsized Impact in Agrobacterium tumefaciens.

Due to minute size and limited sequence complexity, small proteins can be challenging to identify but are emerging as important regulators of diverse processes in bacteria. In this issue of the Journal of Bacteriology, Kraus and coworkers (A. Kraus, M. Weskamp, J. Zierles, M. Balzer, et al., J Bacteriol 202:e00309-20, 2020, https://doi.org/10.1128/JB.00309-20) report a comprehensive analysis of a fascinating subfamily of arginine-rich small proteins in Agrobacterium tumefaciens, conserved among Alphaproteobacteria Their findings reveal that these small proteins are under complex regulation and have a disproportionately large impact on metabolism and behavior.