Dissecting the gene regulation of Proteus vulgaris Rts1 HigB‐HigA toxin‐antitoxin system

Abstract

Bacterial toxin‐antitoxin systems are abundant genes that modulate a beneficial phenotypic switch to shift cells' focus towards survival in times of stress, including antibiotic exposure. During normal growth, toxin‐antitoxin complexes transcriptionally repress their own expression by binding at DNA operator regions that overlap with their own promoters. Toxin binding to the antitoxin‐DNA complex is important because it can result in increased repression. Upon stress, including nutrient limitation and antibiotic exposure, antitoxins are proteolyzed, releasing the toxin component. Released toxin prevents further growth by inhibiting replication, translation or cell wall synthesis. Given these important roles, it is critical to understand how toxin‐antitoxin systems regulate this phenotypic switch between a growth and dormant state that is antibiotic tolerant. The HigB‐HigA protein‐protein complex was first identified on the multi‐drug resistant Rts1 plasmid from Proteus vulgaris(Pv) associated with a post‐operative urinary tract infection. This toxin‐antitoxin complex is not responsive to antibiotics suggesting this aids in tolerance to antibiotics. HigA binding to HigB neutralizes the toxic effects of HigB and forms a heterotetramer. Most toxin‐antitoxin systems encode the antitoxin first and the toxin gene second, resulting in higher antitoxin expression to suppress toxin. However, the higB‐higA gene is unique in that its gene order is reversed with the higB toxin encoded first. Additionally, there are two putative promoters and, although it is clear that the main promoter is active (Phig), the activity of the second internal promoter (PhigA) that precedes the higA antitoxin is unknown although the antitoxin should be expressed to high levels. I hypothesize that PhigA is a strong promoter that results in high expression of the HigA antitoxin. I also hypothesize that the Phig promoter is weak, resulting in lower toxin and antitoxin expression. Here, I determine that unexpectedly, mutation of both the −35 and −10 sites, the −35 site or the −10 site causes an increase in beta‐galactosidase activity as compared to the wild‐type. These data suggest that the weak internal promotor (PhigA) is more active in regulating HigA antitoxin expression. Future work will explore how modulating the operator sites influences expression and will determine how transcriptional control may control HigB‐HigA expression.Support or Funding InformationThis research is funded by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease award 1015487 (to CMD).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.