Abstract
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.
| Original language | English |
|---|---|
| Article number | e1007131 |
| Pages (from-to) | e1007131 |
| Journal | PLoS Genetics |
| Volume | 13 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - Dec 2017 |
Funding
This work has received funding from the Innovative Medicines Initiative Joint Undertaking, resources of which are composed of financial contributions from the European Union's Seventh Frame-work Programme (FP7/2007–2013) and EFPIA (European Federation of Pharmaceutical Industries and Associations) companies (imi (Innovative Medicines Initiative)), under Grant 115337 (https://www.imi.europa.eu). Funding for development of TF mutants and RNAseq pipeline was provided by the National Institute of Health (NIH) National Institute of Allergy and Infectious Disease (NIAID) 1U19AI106761-01, "Omics for TB"(https://www.niaid.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Christina M. J. E. Vandenbroucke-Grauls, Astrid van der Sar, Susanna Commandeur, for their helpful ideas and fruitful discussions. We thank Jessica Winkler and Kinki Jim for technical assistance. The authors would also like to sincerely thank Bob Morrison and Kyle Minch for their invaluable help with the ChIP-sequencing experiment. We would also like to thank Bill Jacobs, Michelle Larsen, and Torin Weisbrod for sharing with us the phage, methods, and advice used in generating the iniR knockout. Lastly, we thank Stewart Cole for the providing PBTZ169.
| Funders | Funder number |
|---|---|
| National Institute of Allergy and Infectious Diseases | U19AI106761 |
Keywords
- Journal Article
- Research Support, Non-U.S. Gov't
