title: Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis.
abstract: The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (TopoI) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. TopoI is essential for Mtb survival. However, the necessity of TopoI or other relaxases in Msm has not been investigated. To recognize the importance of TopoI for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of TopoI in Msm. The TopoI-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in TopoI level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the TopoI-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of TopoI in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in TopoI level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Wareed
authors.initials: W
authors.lastname: Ahmed
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Shruti
authors.initials: S
authors.lastname: Menon
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Pullela V
authors.initials: PV
authors.lastname: Karthik
authors.affiliation: Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.|Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Valakunja
authors.initials: V
authors.lastname: Nagaraja
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020102:Mycobacterium smegmatis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D010641:Phenotype
chemical_list: D001426:Bacterial Proteins; D004264:DNA Topoisomerases, Type I