MnoSR removal in triggers broad transcriptional response to 1,3-propanediol and glucose as sole carbon sources.
Renata P��oci��ska, Katarzyna Stru��, Ma��gorzata Korycka-Macha��a, Przemys��aw P��oci��ski, Magdalena Kuzio��a, Anna ��aczek, Marcin S��omka, Jaros��aw Dziadek
Author Information
Renata P��oci��ska: Institute of Medical Biology of the Polish Academy of Sciences, ����d��, Poland.
Katarzyna Stru��: Institute of Medical Biology of the Polish Academy of Sciences, ����d��, Poland.
Ma��gorzata Korycka-Macha��a: Institute of Medical Biology of the Polish Academy of Sciences, ����d��, Poland.
Przemys��aw P��oci��ski: Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of ����dz, ����d��, Poland.
Magdalena Kuzio��a: Institute of Medical Biology of the Polish Academy of Sciences, ����d��, Poland.
Anna ��aczek: Department of Microbiology, College of Medical Sciences, University of Rzesz��w, Rzesz��w, Poland.
Marcin S��omka: Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of ����d��, ����d��, Poland.
Jaros��aw Dziadek: Institute of Medical Biology of the Polish Academy of Sciences, ����d��, Poland.
Introduction: The two-component signal transduction systems play an essential role in the adaptation of bacteria to changing environmental conditions. One of them is the MnoSR system involved in the regulation of methylotrophic metabolism in M. smegmatis. Methods: Mycobacterium smegmatis mutant strains ��mnoS, ��mnoR and ��mnoS/R lacking functional mnoS, mnoR and both genes were generated using a homologous recombination approach. MnoR recombinant protein was purified by affinity column chromatography. The present study employs molecular biology techniques: cloning strategies, global RNA sequencing, qRT-PCR, EMSA, Microscale thermophoresis, and bioinformatics analysis. Results and discussion: The ���mnoS, ���mnoR, and ���mnoS/R mutant strains were generated and cultured in the presence of defined carbon sources. Growth curve analysis confirmed that inactivation of the MnoSR impairs the ability of M. smegmatis cells to use alcohols such as 1,3-propanediol and ethanol but improves the bacterial growth on ethylene glycol, xylitol, and glycerol. The total RNA sequencing method was employed to understand the importance of MnoSR in the global responses of mycobacteria to limited carbon access and in carbon-rich conditions. The loss of MnoSR significantly affected carbon utilization in the case of mycobacteria cultured on glucose or 1,3-propanediol as sole carbon sources as it influenced the expression of multiple metabolic pathways. The numerous transcriptional changes could not be linked to the presence of evident MnoR DNA-binding sites within the promotor regions for the genes outside of the mno operon. This was confirmed by EMSA and microscale thermophoresis with mutated MnoR binding consensus region. Our comprehensive analysis highlights the system's vital role in metabolic adaptability, providing insights into its potential impact on the environmental survival of mycobacteria.
