Altered transcription activation specificity of a mutant form of Bacillus subtilis GltR, a LysR family member

Abstract

A mutation (gltR24) that allows Bacillus subtilis glutamate synthase (gltAB) gene expression in the absence of its positive regulator, GltC, was identified. Cloning and sequencing of the gltR gene revealed that the putative gltR product belongs to the LysR family of transcriptional regulators and is thus related to GltC. A null mutation in gltR had no effect on gltAB expression under any environmental condition tested, suggesting that gltR24 is a gain-of-function mutation. GltR24-dependent transcription of gltAB, initiated at the same base pair as GltC-dependent transcription, was responsive to the nitrogen source in the medium and required the integrity of sequences upstream of the gltAB promoter that are also necessary for GltC-dependent expression. Expression of the gltC gene, transcribed divergently from gltA from an overlapping promoter, was not affected by GltR. Both wild-type GltR and GltR24 negatively regulated their own expression. The gltR gene was mapped to 233؇ on the B. subtilis chromosome, very close to the azlB locus.Glutamate synthase, encoded by the gltAB genes, synthesizes glutamate from 2-ketoglutarate and glutamine and is an essential component of the major pathway for ammonia assimilation in Bacillus subtilis (39). Glutamate synthase gene expression is positively regulated in a nitrogen source-dependent manner by the product of the gltC gene. In the absence of GltC protein, gltAB transcription is drastically reduced under normally activating growth conditions, e.g., when ammonium ions are available as nitrogen source (7,8). GltC may not be the only factor controlling gltAB expression, however, since the low, residual expression of gltA in a gltC mutant decreases further under nonactivating growth conditions, e.g., when glutamate or proline serves as sole nitrogen source (5). We sought to identify a second factor involved in gltAB regulation by looking for mutants with elevated expression of gltA despite the absence of GltC.GltC belongs to the LysR family of bacterial transcriptional regulators (20, 38), a family which now comprises about 100 members. The members of this family share some common properties (38): (i) they have near their N termini a conserved helix-turn-helix region that is thought to be responsible for DNA binding; (ii) their target genes are frequently linked to and transcribed divergently from the genes for the LysR-type regulators; (iii) binding sites for many LysR-type proteins have a consensus sequence T-N 11 -A, with elements of dyad symmetry around the two conserved nucleotides, often separated by an (AϩT)-rich core; centers of these sequences are located near position Ϫ65 with respect to the transcription start sites of the target genes; (iv) for some LysR proteins the binding site extends beyond the consensus site or they have an additional binding site between the consensus sequence and the transcription start site of the target gene; and (v) the genes encoding LysR-type proteins are frequently negatively autoregulated. All of these properties apply to GltC (7,8).Th...

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

“…1B). This indicates that CztR negatively regulates its own expression, in agreement with the findings reported for regulators of this family (3,19). The introduction of the cztR gene in a multicopy plasmid under the control of a xylose-inducible promoter in SP2012(pUJcztR) restores the ␤-galactosidase activity to levels comparable to those of the wild-type strain, even in the absence of xylose in the medium (Fig.…”

“…Transcriptional regulators of the LysR family usually regulate the divergent gene (3,19). The gene divergent to cztR, cztA, encodes a putative permease of the SbtA family (31).…”

“…The prototype of this family is Synechocystis SbtA, which plays the main role in HCO 3 Ϫ transport and is energized by the primary Na ϩ pump NtpJ. It has been suggested that NtpJ establishes an electrochemical gradient of Na ϩ across the plasma membrane that could drive the transport of HCO 3 Ϫ by SbtA, given that this transport is Na ϩ dependent (31). Interestingly, sbtA is regulated by the LTTR CcmR in response to inorganic carbon (C i ) limitation (38).…”

CbbR, the Master Regulator for Microbial Carbon Dioxide Fixation

“…Amino acid substitutions that confer constitutive activity are thought to change the conformation of the LTTR tetramer to mimic the conformation seen when it is bound with the effector or to change the conformation of the LTTR/promoter complex to produce a favorable interaction with RNA polymerase to activate transcription. Studies of LTTR* proteins from several LTTR family members, including NodD, AmpR, OccR, CysB, OxyR, NahR, GtlR, XapR, and Cbl, have been previously published (70)(71)(72)(88)(89)(90)(91)(92)(93)(94)(95)(96).…”

“…Many of these amino acid substitutions are centered at the effector pocket, but substitutions in other areas of the LTTR, such as at residues within the linker helix or hinge region or throughout RD-I and RD-II, can generate constitutive activity (70,72,76,86,(88)(89)(90)(91)(92)(93)(94). Typically, single-amino-acid substitutions encompass the vast majority of the reported changes identified for constitutive proteins, and most constitutive proteins bind their effectors but may behave differently from the wild-type LTTR in gel mobility shift assays, DNase I footprinting assays, or in vitro transcription assays (76,88,89,(92)(93)(94). Amino acid substitutions that confer constitutive activity are thought to change the conformation of the LTTR tetramer to mimic the conformation seen when it is bound with the effector or to change the conformation of the LTTR/promoter complex to produce a favorable interaction with RNA polymerase to activate transcription.…”