A haloarchaeal VapBC toxin-antitoxin system: regulatory mechanism and role in establishing population heterogeneity to facilitate survival of subpopulations under stress conditions

Abstract

ABSTRACT Toxin-antitoxin (TA) systems are ubiquitously distributed in bacteria and archaea and have been implicated in various functions such as plasmid maintenance, phage defense, biofilm formation, stress response, and persistence, but their regulatory mechanisms and roles in haloarchaea are largely unknown. In this study, we found that four of the eight putative VapBC TA systems of Natrinema gari J7-2 are functional in strain J7-2 and Haloferax volcanii. Among them, the NgVapBC1 system is composed of the antitoxin NgVapB1 and the toxin NgVapC1, and the two proteins could form a NgVapBC1 complex. The NgVapBC1 system could be transcriptionally autoregulated, wherein a pseudo-palindromic sequence in the vapBC1 operon promoter acts as a negative cis-regulatory element, and the NgVapBC1 complex is a stronger repressor than NgVapB1. The qRT-PCR and in vivo toxicity analyses showed that strain J7-2 maintains a high vapB1/vapC1 mRNA ratio through truncation of the vapBC1 operon transcript within the toxin-coding region for preventing the synthesis of excess toxin NgVapC1 to release toxicity abnormally. Mutational analyses showed that differential start codon usage by vapB1 and vapC1 and a −4 nt overlap of their stop and start codons contribute to maintaining a higher vapB1/vapC1 mRNA ratio, and the −4 nt overlap-mediated translational coupling of vapB1 and vapC1 would enable both post-transcriptional and translational regulation of the NgVapBC1 system. The phenotypic comparison of strain J7-2 and its ΔvapBC1 mutant revealed that the NgVapBC1 system contributes to population heterogeneity of decline-phase rather than exponential-phase strain J7-2 and facilitates the survival of subpopulations under stress conditions. IMPORTANCE Although TA systems are regarded as versatile modulators of prokaryotic cell fate, their bona fide physiological roles remain elusive. Moreover, so far, there is no report on the regulatory mechanism and function of haloarchaeal VapBCs, which constitute the major group of TA systems in haloarchaea. Our results demonstrate that a VapBC system of the haloarchaeon Natrinema gari J7-2 is regulated at multiple levels and mediates the establishment of population heterogeneity, thereby facilitating the survival of the strain J7-2 population as a whole in diverse and changing environments. This study provides the first experimental evidence of the regulatory mechanism of the biological role of the VapBC system in conferring fitness advantages to haloarchaea. Although TA systems are regarded as versatile modulators of prokaryotic cell fate, their bona fide physiological roles remain elusive. Moreover, so far, there is no report on the regulatory mechanism and function of haloarchaeal VapBCs, which constitute the major group of TA systems in haloarchaea. Our results demonstrate that a VapBC system of the haloarchaeon Natrinema gari J7-2 is regulated at multiple levels and mediates the establishment of population heterogeneity, thereby facilitating the survival of the strain J7-2 population as a whole in diverse and changing environments. This study provides the first experimental evidence of the regulatory mechanism of the biological role of the VapBC system in conferring fitness advantages to haloarchaea.