title: Mycobacterial cells have dual nickel-cobalt sensors: sequence relationships and metal sites of metal-responsive repressors are not congruent.
abstract: A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterized from mycobacteria. Mutants of Mycobacterium tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays and fluorescence anisotropy show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. Incubation of KmtR with cobalt inhibits DNA complex assembly and metal-protein binding was confirmed. KmtR is the second, to NmtR, characterized ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, whereas NmtR retains a response to nickel and cobalt under these conditions. KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB-related proteins are listed in databases. KmtR has none of the previously defined metal-sensing sites. Substitution of His88, Glu101, His102, His110, or His111 with Gln generated KmtR variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory motifs revealing that only the latter should be used to predict metal sensing.
authors.affiliation: Life Sciences, University of Manchester, 1.800 Stopford Building, Manchester M13 9PT, United Kingdom.
authors.forename: Duncan R
authors.initials: DR
authors.lastname: Campbell
authors.forename: Kaye E
authors.initials: KE
authors.lastname: Chapman
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Waldron
authors.forename: Stephen
authors.initials: S
authors.lastname: Tottey
authors.forename: Sharon
authors.initials: S
authors.lastname: Kendall
authors.forename: Gabriele
authors.initials: G
authors.lastname: Cavallaro
authors.forename: Claudia
authors.initials: C
authors.lastname: Andreini
authors.forename: Jason
authors.initials: J
authors.lastname: Hinds
authors.forename: Neil G
authors.initials: NG
authors.lastname: Stoker
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Robinson
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Cavet
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003035:Cobalt / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis; D009532:Nickel / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D003035:Cobalt; D009532:Nickel; D015032:Zinc