title: LytB1 and LytB2 of Mycobacterium tuberculosis Are Not Genetically Redundant.
abstract: Mycobacterium tuberculosis synthesises isoprenoid precursors via the MEP/DOXP pathway and at least five enzymes in the pathway (Dxs1, Dxr/IspC, IspD, IspF, and GcpE/IspG) are required for growth in vitro. We investigated the role of LytB (IspH) in M. tuberculosis; M. tuberculosis is unusual in that it has two homologs-LytB1 and LytB2. We were unable to delete the lytB2 gene unless we provided an additional copy elsewhere, demonstrating that this is the essential homolog. We expressed lytB1 from the lytB2 promoter and confirmed that this could not complement for loss of function of lytB2, despite LytB1 possessing all the previously described conserved critical residues. Interestingly the sole LytB homolog of Mycobacterium smegmatis was able to compensate for loss of LytB2 in M. tuberculosis. We tested translational fusions of LytB1 and LytB2 for functionality in M. tuberculosis, but only a fusion with 90% N-terminal LytB2 and 10% C-terminal LytB1 was functional. In order to identify the key difference between the two proteins, site directed mutagenesis was used to change LytB2 residues into their counterparts in LytB1. None of these amino acid substitutions was essential for function and all lytB2 mutant alleles were functional. In contrast, mutation of the key residues for [Fe4S4] cluster formation, as well as a catalytic residue in LytB1 did not result in functional complementation. Thus, although LytB1 and LytB2 are not genetically redundant, this is not dependent on small amino acid changes, but is likely to be a result of major overall structural differences.
authors: Queen Mary University of London, London, E1 2AD, United Kingdom; Barts & the London School of Medicine and Dentistry, London, E1 2AD, United Kingdom.
authors: Amanda Claire
authors: AC
authors: Brown
authors: TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, 98102, United States of America.
authors: Rachel
authors: R
authors: Kokoczka
authors: Queen Mary University of London, London, E1 2AD, United Kingdom; Barts & the London School of Medicine and Dentistry, London, E1 2AD, United Kingdom; TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, 98102, United States of America.
authors: Tanya
authors: T
authors: Parish
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D020043:Genes, Essential; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D020033:Protein Isoforms; D010088:Oxidoreductases