Bacillus anthracis is the causative agent of anthrax. This Gram-positive bacterium poses a substantial risk to human health due to high mortality rates and the potential for malicious use as a bioterror weapon. To survive within the vertebrate host, B. anthracis relies on two-component system (TCS) signaling to sense host-induced stresses and respond to alterations in the environment through changes in target gene expression. HitRS and HssRS are cross-regulating TCSs in B. anthracis that respond to cell envelope disruptions and high heme levels, respectively. In this study, an unbiased and targeted genetic selection was designed to identify gene products that are involved in HitRS and HssRS signaling. This selection led to the identification of inactivating mutations within dnaJ and clpX that disrupt HitRS- and HssRS-dependent gene expression. DnaJ and ClpX are the substrate-binding subunits of the DnaJK protein chaperone and ClpXP protease, respectively. DnaJ regulates the levels of HitR and HitS to facilitate signal transduction, while ClpX specifically regulates HitS levels. Together these results reveal that the protein homeostasis regulators, DnaJ and ClpX, function to maintain B. anthracis signal transduction activities through TCS regulation. One sentence summary: Use of a genetic selection strategy to identify modulators of two-component system signaling in Bacillus anthracis .

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.

Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δcas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

AbstractBacterial two component systems (TCSs) have been studied for decades; however, most work has focused on individual domains or proteins. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that has been implicated in detecting cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. This study establishes the use of unbiased genetic selections to study TCS signaling, provides a comprehensive mechanistic understanding of an entire TCS, and lays the foundation for development of novel antimicrobial therapeutics against this important infectious threat.