Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in the regulation of gene expression. We searched for CarD interaction partners in Mycobacterium smegmatis and identified a novel uncharacterized protein, named CrsL (MSMEG_5890). CrsL is a 5.7 kDa protein shown by NMR to be intrinsically disordered. CrsL homologs are present in actinobacteria, including pathogenic species such as Mycobacterium tuberculosis. CrsL interacts directly with CarD, adopting an ordered structure in the complex, and also binds RNAP, controlling CarD-RNAP association. ChIP-seq showed that CrsL associates with the promoters of actively transcribed genes and ∼75% of these regions are also associated with CarD. RNA-seq revealed ∼50% and ∼66% overlap in differentially expressed genes between CrsL and CarD knockdowns during the exponential and stationary phases, respectively. Among CrsL-regulated genes are DesA desaturase (MSMEG_5773) and DEAD/DEAH-box RNA helicase MSMEG_1930, which contribute to cold stress adaptation. CrsL supports the growth of M. smegmatis at elevated temperature but limits growth in cold environments. In summary, these findings identify CrsL as a novel, conserved CarD-interacting protein playing a key role in mycobacterial stress responses by modulating CarD function.

Central European Institute of Technology Masaryk University Brno 62500 Czech Republic

Department of Cell Biology Faculty of Science Charles University Prague 12800 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 16000 Czech Republic

Laboratory of Bioinformatics Institute of Microbiology of the Czech Academy of Sciences Prague 14220 Czech Republic

Laboratory of Microbial Genetics and Gene Expression Institute of Microbiology of the Czech Academy of Sciences Prague 14220 Czech Republic

Laboratory of Molecular Biology of Bacterial Pathogen Institute of Microbiology of the Czech Academy of Sciences Prague 14220 Czech Republic

Laboratory of Regulatory RNAs Faculty of Science Charles University Prague 12844 Czech Republic

Laboratory of Structural Biology and Cell Signaling BIOCEV Institute of Microbiology of the Czech Academy of Sciences Vestec 25250 Czech Republic

Military Health Institute Military Medical Agency Prague 16200 Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 62500 Czech Republic

National Institute of Public Health Prague 10000 Czech Republic

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