Dataset Information

Expanding the CarD interaction network: CrsL is a novel transcription factor in Mycobacterium smegmatis

ABSTRACT: Bacterial transcription is a tightly regulated process critical for gene expression, adaptation and cell survival. CarD is an essential transcription factor in mycobacteria involved in regulation of gene expression. Here, we searched for CarD interaction partners in the model organism Mycobacterium smegmatis and identified two candidate proteins: ApeB (MSMEG_5828) and an uncharacterized protein, which we named CrsL (MSMEG_5890). While ApeB interacted with CarD only when CarD was overexpressed, CrsL associated with CarD at its physiological levels. We therefore focused on CrsL. It is a small protein of 5.7 kDa that we show by NMR to be intrinsically disordered. Genes encoding CrsL homologs are present in Actinobacteria including pathogenic species such as Mycobacterium tuberculosis. We demonstrate that CrsL directly interacts with CarD and both proteins bind to RNAP. Moreover, we show by ChIP-seq that CrsL associates with promoter regions of actively transcribed genes and ~75 % of these regions are associated also with CarD. By RNA-seq experiments with crsL and carD knockdown strains, we reveal ~50 % and ~66 % overlap between differentially expressed genes in the two strains in exponential and stationary phase, respectively. CrsL represses the expression of DesA desaturase and RNA helicase MSMEG_1930 gene, which are important for adaptation to cold stress. Furthermore, CrsL promotes the growth of M. smegmatis at elevated temperature. In summary, this study identifies CrsL as a novel actinobacterial transcription factor and provides a basis for its further investigation.

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ORGANISM(S): Mycobacterium Smegmatis

SUBMITTER: Martin Hubalek

LAB HEAD: Martin Hubálek

PROVIDER: PXD058166 | Pride | 2025-12-22

REPOSITORIES: Pride

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Expanding the CarD interaction network: CrsL is a novel transcription regulator in actinobacteria.

Shoman Mahmoud M Černý Martin M Jirát Matějčková Jitka J Schwarz Marek M Borah Nabajyoti N Vaňková Hausnerová Viola V Neva Silvia S Šiková Michaela M Šanderová Hana H Halada Petr P Hubálek Martin M Dvořáková Věra V Převorovský Martin M Holubová Jana J Staněk Ondřej O Krásný Libor L Žídek Lukáš L Hnilicová Jarmila J

Nucleic acids research 20251101 22

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 tuberculos ...[more]

PMID: 41404806

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

			Action	DRS
	211005_ApeB_Ex.msf	Msf
	211005_ApeB_Ex.raw	Raw
	211005_ApeB_St.msf	Msf
	211005_ApeB_St.raw	Raw
	211005_CarD_Ex.msf	Msf