title: Revisiting the essentiality of glutamate racemase in Mycobacterium tuberculosis.
abstract: Glutamate racemase (MurI) converts l-glutamate into d-glutamate which is an essential component of peptidoglycan in bacteria. The gene encoding glutamate racemase, murI has been shown to be essential for the growth of a number of bacterial species including Escherichia coli. However, in some Gram-positive species d-amino acid transaminase (Dat) can also convert l-glutamate into d-glutamate thus rendering MurI non-essential for growth. In a recent study the murI gene of Mycobacterium tuberculosis was shown to be non-essential. As d-glutamate is an essential component of peptidoglycan of M. tuberculosis, either Dat or MurI has to be essential for its survival. Since, a Dat encoding gene has not been reported in M. tuberculosis genome sequence, the reported non-essentiality of murI was unexplainable. In order to resolve this dilemma we tried to knockout murI in the presence of single and two copies of murI, in wild type and merodiploid strains respectively. It was found that murI could not be inactivated in the wild type background indicating that it could be an essential gene. Also, inactivation of murI could not be achieved in the presence of externally supplied d-glutamate in 7H9 medium suggesting that M. tuberculosis is unable to take up d-glutamate under the conditions tested. However we could generate murI knockout strains at high frequency when two copies of the gene were present indicating that at least one murI gene is required for cellular viability. The essential nature of MurI in M. tuberculosis H37Rv suggests that it could be a potential drug target.
authors.affiliation: Present address: Biocon Bristol Myer squib R&D Centre, Biocon Park, Jigani Link Road, Bommasandra, Bangalore, India.
authors.forename: Sapna
authors.initials: S
authors.lastname: Morayya
authors.affiliation: Present address: Strand Center for Genomics & Personalized Medicine, Strand Life Sciences Pvt. Ltd., Veterinary College Campus, Bellary Road, Bangalore, India.
authors.forename: Disha
authors.initials: D
authors.lastname: Awasthy
authors.affiliation: AstraZeneca India Pvt. Ltd., "Avishkar", Bellary Road, Hebbal, Bangalore, India.
authors.forename: Reena
authors.initials: R
authors.lastname: Yadav
authors.affiliation: Present address: Biocon Bristol Myer squib R&D Centre, Biocon Park, Jigani Link Road, Bommasandra, Bangalore, India.
authors.forename: Anisha
authors.initials: A
authors.lastname: Ambady
authors.affiliation: Present address: GangaGen Biotechnologies Pvt. Ltd, No 12, 5th Cross, Raghavendra Layout, Tumkur Road, Yeshwantpur, Bangalore, India. Electronic address: umender1912@gmail.com.
authors.forename: Umender
authors.initials: U
authors.lastname: Sharma
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D051046:Amino-Acid N-Acetyltransferase / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D019295:Computational Biology; D018274:Electroporation; D017353:Gene Deletion; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010457:Peptidoglycan / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D010457:Peptidoglycan; D051046:Amino-Acid N-Acetyltransferase; D000591:Amino Acid Isomerases; C062292:glutamate racemase
keywords: Gene knockout; Glutamate racemase; M. tuberculosis; MurI essential; Peptidoglycan