Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.
Author summary The mycobacterial cell wall is a complex and unique structure that protects extremely well against harmful compounds. Understanding the biogenesis and functioning of this cell envelope is essential to be able to effectively target mycobacteria. One way to uncover cell envelope functionality is to study stress mechanisms that are induced when the cell envelope is damaged. Here, we describe the identification of a major cell envelope stress regulator and the inducing signal. As stress inducers we have used antimycobacterial drugs that target the biogenesis of the mycobacterial cell envelope, as these have previously been shown to specifically induce the major cell wall stress operon iniBAC. We have identified a multi-domain regulator that is essential for the induction of this operon to transduce cell envelope stress and named this IniR. Importantly, we were also able to show that cell envelope stress signaling was induced by free trehalose. Trehalose is a central unit in many mycobacterial lipids and mycobacteria have a dedicated trehalose salvage pathway that is used when lipids are degraded and recycled. We hypothesize that lipid turnover and concomitant release of free trehalose in the cell envelope is a signal for cell envelope stress in mycobacteria.