Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in several bacterial species, but the mechanisms are often unclear. Here, we report that c-di-GMP promotes biofilm formation in mycobacteria in a manner dependent on the nucleoid-associated protein Lsr2. We show that c-di-GMP specifically binds to Lsr2 at a ratio of 1:1. Lsr2 upregulates the expression of HadD, a (3R)-hydroxyacyl-ACP dehydratase, thus promoting the synthesis of keto-mycolic acid and biofilm formation. Thus, Lsr2 acts as a c-di-GMP receptor that links the second messenger’s function to lipid synthesis and biofilm formation in mycobacteria.

D-xylose is the most abundant fermentable pentose, which usually represents an architectural component of the bacterial cell wall. However, its regulatory function and the involved signaling pathway in bacteria remain largely unclear. Here, we show that D-xylose can act as a signaling molecule to regulate the lipid metabolism and affect multiple physiological characteristics in mycobacteria. D-xylose directly interacts with XylR and inhibits its DNA-binding ability, thus blocking XylR-mediated repression. The xylose inhibitor, XylR, plays a global regulatory role and affects the expression of 166 mycobacterial genes that are involved in lipid synthesis and metabolism. Furthermore, we show that the xylose-dependent gene regulation of XylR affects the multiple physiological characteristics of Mycobacterium smegmatis, including bacterial size, colony phenotype, biofilm formation, cell aggregation, and antibiotic resistance. Finally, we found that XylR inhibited the survival of Mycobacterium bovis BCG in the host. Our findings provide novel insights into the molecular mechanism of lipid metabolism regulation and its correlation with bacterial physiological phenotypes.

Mycobacterium tuberculosis is a formidable pathogen capable of establishing persistent infections within macrophages. To survive and thrive within the host environment, it has evolved intricate regulatory networks, including a diverse array of transcription factors that enable adaptation to various stresses encountered within the host. However, the mechanisms by which transcription factors regulate biofilm formation in M. tuberculosis remain incompletely understood. This study aimed to investigate the role of serC, encoding phosphoserine aminotransferase, and its regulation by NapR, a transcription factor, in mycobacterial physiology. NapR regulates serC through directly binding to its promoter. Notably, the regulatory effect and corresponding phenotypes vary due to distinct binding affinities of NapR for the serC promoter in different mycobacterial species. In Mycobacterium smegmatis, NapR_Msm positively regulates biofilm formation, growth on solid media, and the transition from microcolonies to microcolonies by activating serC_Msm. In the BCG vaccine, on the contrary, NapR_BCG represses serC_BCG, thus negatively regulating colony size and alleviating the growth inhibition caused by high concentrations of serine. Furthermore, proteomic analysis suggested NapR serves as a global transcriptional regulator in BCG vaccine strains by simultaneously modulating four metabolic pathways. These findings underscore the complex and strain-specific regulatory mechanisms governing serine metabolism in mycobacteria and provide valuable insights into the interplay between metabolism, gene regulation, and bacterial physiology.

Isoniazid (INH) targets cell wall biosynthesis and is a potent antimycobacterial agent. Elucidating the regulatory networks that govern drug susceptibility in mycobacterial models is fundamental to understanding intrinsic resistance in pathogenic species. The iniBAC operon plays a crucial role in INH tolerance and envelope permeability, yet the transcriptional regulatory mechanisms controlling its expression in response to INH-induced stress remain incompletely understood. The second messenger cyclic di-GMP (c-di-GMP) regulates drug susceptibility in several bacteria, but its downstream receptors and regulatory pathways in mycobacteria have not been explored. Here, we demonstrate that c-di-GMP reduces INH susceptibility via the receptor HpoR. Mechanistically, c-di-GMP alleviates HpoR-mediated repression of the iniBAC operon in a concentration-dependent manner, which decreases envelope permeability and consequently modulates multidrug susceptibility in both M. bovis BCG and M. smegmatis. This regulatory paradigm is likely conserved in pathogenic mycobacteria.