title: Deletion of SenX3-RegX3, a key two-component regulatory system of Mycobacterium smegmatis, results in growth defects under phosphate-limiting conditions.
abstract: Two component regulatory systems are key elements in the control of bacterial gene expression in response to environmental perturbations. The SenX3-RegX3 system is implicated in the control of phosphate uptake in Mycobacterium smegmatis and Mycobacterium tuberculosis. regX3 is reported to be essential in M. smegmatis, but not in M. tuberculosis. We attempted to construct complete senX3-regX3 operon deletion strains of M. smegmatis; initially we found that the operon could only be deleted when another functional copy was provided. Using a strain in which the only functional copy of the operon was present on an integrating plasmid, we attempted to replace the functional copy with an empty vector. Surprisingly, we obtained strains in which the functional copy had been deleted from the chromosome at a low frequency. We deleted the senX3 gene in a similar fashion, but it was not possible to delete regX3 alone. To identify possible compensatory mutations we sequenced the whole genome of two deletion strains and the wild-type. A synonymous single nucleotide polymorphism (SNP) in a lipoprotein was found in all deletion strains, but not the parental strains, and a frameshift mutation in nhaA was identified in three of the four deletion strains. Operon deletion strains were more sensitive to phosphate limitation, showing a reduced ability to grow at lower phosphate concentrations. The M. tuberculosis operon was able to functionally complement the growth phenotype in M. smegmatis under phosphate-replete conditions, but not under low phosphate conditions, reinforcing the difference between the two species. Our data show that, in contrast with previous reports, it is possible to delete the operon in M. smegmatis, possibly due to the accumulation of compensatory mutations, and that the deletion does affect growth in phosphate.
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Jade N
authors.initials: JN
authors.lastname: James
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Zeeshaan-Ul
authors.initials: ZU
authors.lastname: Hasan
authors.affiliation: Department of Computer Science and Engineering, Texas A&M University, College Station, TX, USA.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Ioerger
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Brown
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Yoann
authors.initials: Y
authors.lastname: Personne
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Carroll
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Melanie
authors.initials: M
authors.lastname: Ikeh
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Natasha L
authors.initials: NL
authors.lastname: Tilston-Lunel
authors.affiliation: Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Palavecino
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
authors.affiliation: TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA.|Queen Mary University of London, Barts and the London, London, UK.
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010710:Phosphates; C110608:RegX3 protein, Mycobacterium; D010770:Phosphotransferases; C110607:SenX3 protein, Mycobacterium