The non-pathogenic bacterium Mycobacterium smegmatis mc2155 has been widely used as a model organism in mycobacterial research, yet a detailed study about its transcription landscape remains to be established. Here we report the transcriptome, expression profiles and transcriptional structures through growth-phase-dependent RNA sequencing (RNA-seq) as well as other related experiments. We found: (1) 2,139 transcriptional start sites (TSSs) in the genome-wide scale, of which eight samples were randomly selected and further verified by 5′-RACE; (2) 2,233 independent monocistronic or polycistronic mRNAs in the transcriptome within the operon/sub-operon structures which are classified into five groups; (3) 47.50% (1016/2139) genes were transcribed into leaderless mRNAs, with the TSSs of 41.3% (883/2139) mRNAs overlapping with the first base of the annotated start codon. Initial amino acids of MSMEG_4921 and MSMEG_6422 proteins were identified by Edman degradation, indicating the presence of distinctive widespread leaderless features in M. smegmatis mc2155. (4) 150 genes with potentially wrong structural annotation, of which 124 proposed genes have been corrected; (5) eight highly active promoters, with their activities further determined by β-galactosidase assays. These data integrated the transcriptional landscape to genome information of model organism mc2155 and lay a solid foundation for further works in Mycobacterium.
Immunocytes dynamically reprogram their gene expression profiles during differentiation and immunoresponse. However, the underlying mechanism remains elusive. Here, we develop a single-cell Hi-C method and systematically delineate the 3D genome and dynamic epigenetic atlas of macrophages during these processes. We propose “degree of disorder” to measure genome organizational patterns inside topologically-associated domains, which is correlated with the chromatin epigenetic states, gene expression, and chromatin structure variability in individual cells. Furthermore, we identify that NF-κB initiates systematic chromatin conformation reorganization upon Mycobacterium tuberculosis infection. The integrated Hi-C, eQTL, and GWAS analysis depicts the atlas of the long-range target genes of mycobacterial disease susceptible loci. Among these, the SNP rs1873613 is located in the anchor of a dynamic chromatin loop with LRRK2, whose inhibitor AdoCbl could be an anti-tuberculosis drug candidate. Our study provides comprehensive resources for the 3D genome structure of immunocytes and sheds insights into the order of genome organization and the coordinated gene transcription during immunoresponse.
Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.
As an intracellular pathogen,
Mycobacterium tuberculosis
could avoid host cell immune clearance using multiple strategies for its long-term survival. Understanding these processes could facilitate the development of new approaches to restrict intracellular
M. tuberculosis
survival.
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