RESUMEN
The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ~44 bp of the DNA sequence preceding and/or overlapping the predicted -35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR "recognition motifs" at different responsive promoters appears to be limited.
Asunto(s)
Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas/genética , Azufre/metabolismo , Factores de Transcripción/metabolismo , Fusión Artificial Génica , Secuencia de Bases , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Operón , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Two genes encoding transcriptional regulators involved in sulfur assimilation pathways in Burkholderia cenocepacia strain 715j have been identified and characterized functionally. Knockout mutations in each of the B. cenocepacia genes were constructed and introduced into the genome of 715j by allelic replacement. Studies on the utilization of various sulfur sources by 715j and the obtained mutants demonstrated that one of the B. cenocepacia regulators, designated CysB, is preferentially involved in the control of sulfate transport and reduction, while the other, designated SsuR, is required for aliphatic sulfonate utilization. Using transcriptional promoter-lacZ fusions and DNA-binding experiments, we identified several target promoters for positive control by CysB and/or SsuR--sbpp (preceding the sbp cysT cysW cysA ssuR cluster), cysIp (preceding the cysI cysD1 cysN cysH cysG cluster), cysD2p (preceding a separate cluster, cysD2 cysNC), and ssuDp (located upstream of the ssuDCB operon)--and we demonstrated overlapping functions of CysB and SsuR at particular promoters. We also demonstrated that the cysB gene is negatively controlled by both CysB and SsuR but the ssuR gene itself is not significantly regulated as a separate transcription unit. The function of B. cenocepacia CysB (in vivo and in vitro) appeared to be independent of the presence of acetylserine, the indispensable coinducer of the CysB regulators of Escherichia coli and Salmonella. The phylogenetic relationships among members of the "CysB family" in the gamma and beta subphyla are presented.
Asunto(s)
Proteínas Bacterianas/fisiología , Burkholderia/metabolismo , Regulación Bacteriana de la Expresión Génica , Azufre/metabolismo , Factores de Transcripción/fisiología , Alcanosulfonatos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Burkholderia/genética , Clonación Molecular , ADN/metabolismo , Genoma Bacteriano , Datos de Secuencia Molecular , Fenotipo , Filogenia , Regiones Promotoras Genéticas , Serina/análogos & derivados , Serina/farmacología , Sulfatos/metabolismoRESUMEN
Cbl is a member of the large family of LysR-type transcriptional regulators (LTTRs) common in bacteria and found also in Archaea and algal chloroplasts. The function of Cbl is required in Escherichia coli for expression of sulphate starvation-inducible (ssi) genes, associated with the biosynthesis of cysteine from organic sulphur sources (sulphonates). Here, we report the crystal structure of the cofactor-binding domain of Cbl (c-Cbl) from E. coli. The overall fold of c-Cbl is very similar to the regulatory domain (RD) of another LysR family member, CysB. The RD is composed of two subdomains enclosing a cavity, which is expected to bind effector molecules. We have constructed and analysed several full-length Cbl variants bearing single residue substitutions in the RD that affect cofactor responses. Using in vivo and in vitro transcription assays, we demonstrate that pssuE, a Cbl responsive promoter, is down-regulated not only by the cofactor, adenosine phosphosulphate (APS), but also by thiosulphate, and, that the same RD determinants are important for the response to both cofactors. We also demonstrate the effects of selected site-directed mutations on Cbl oligomerization and discuss these in the context of the structure. Based on the crystal structure and molecular modelling, we propose a model for the interaction of Cbl with adenosine phosphosulphate.
Asunto(s)
Adenosina Fosfosulfato/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , Tiosulfatos/química , Factores de Transcripción/química , Sitios de Unión , Cristalografía por Rayos X , Regulación hacia Abajo , Proteínas de Escherichia coli/genética , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Estructura Secundaria de Proteína , Factores de Transcripción/genéticaRESUMEN
Cbl (CysB-like protein) is a member of the family of LysR-type transcriptional regulators (LTTRs) and controls genes engaged in sulfur assimilation in Escherichia coli. It has been postulated that adenosine 5-phosphosulfate (APS) is responsible for abolishing Cbl-activated transcription from the ssu promoter (Bykowski et al., 2002). To elucidate the structural basis of Cbl function and to confirm the role of APS as an anti-inducer, the cofactor-binding domain of Cbl (c-Cbl, MW = 26 kDa) was cloned, purified and crystallized in the presence of APS. The crystals belong to space group C222(1), but show substantial variation of the unit-cell parameters and diffraction anisotropy. Despite this, X-ray data extending to 3.0 A resolution have been collected and solution of the structure by molecular replacement is in progress.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Factores de Transcripción/química , Adenosina Fosfosulfato/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/aislamiento & purificación , Unión Proteica/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sincrotrones , Factores de Transcripción/biosíntesis , Factores de Transcripción/aislamiento & purificaciónRESUMEN
CysB is a LysR-type transcriptional regulator (LTTR) controlling the expression of numerous genes involved in bacterial sulphur assimilation via cysteine biosynthesis. Our previous mutational analysis of CysB identified several residues within the N-terminal domain crucial for DNA-binding function. Here, we focus on the functional significance of CysB residues localized in the turn between the alpha2 and alpha3 helices forming an N-terminal helix-turn-helix motif. On the basis of the characteristics of alanine-substituted mutants, we propose that CysB residues Y27, T28 and S29, lying in this turn region, comprise an 'activating region' (AR) that is crucial for positive control of the cysP promoter, but not for DNA binding and inducer response activities of CysB. Using a library of alanine substitutions in the C-terminal domain of the RNAP alpha subunit (alpha-CTD), we identify several residues in alpha-CTD that are important for CysB-dependent transcription from the cysP promoter. After probing potential protein-protein contacts in vivo with a LexA-based two-hybrid system, we propose that the '273 determinant' on alpha-CTD, including residues K271 and E273, represents a target for interaction with CysB at the cysP promoter.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Unión Periplasmáticas/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Desoxirribonucleasa I , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Secuencias Hélice-Giro-Hélice , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Unión Periplasmáticas/genética , Plásmidos/genética , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The utilization of organosulphur compounds as sources of sulphur by Escherichia coli is strongly repressed by sulphate. To search for the signal enabling E. coli to alternate gene expression according to the sulphur source, we investigated the transcriptional control of the ssuEADCB operon, required for the transport and desulphonation of aliphatic sulphonates. We demonstrate that, of the two LysR-type regulators involved in expression from the ssu promoter, Cbl acts as a direct and sufficient activator of transcription in vivo and in vitro, whereas CysB downregulates the promoter efficiency. Most importantly, the Cbl-mediated transcription initiation at the ssu promoter in vitro is abolished in the presence of an early metabolite of the sulphate assimilatory pathway, adenosine 5'-phosphosulphate (APS). This role for APS was confirmed in vivo by measuring the expression of beta-galactosidase from a transcriptional ssu-lacZ fusion in strains containing different mutations blocking the synthesis and consumption of APS. Our data comprise the first evidence that APS may act as the negative cofactor of the transcriptional regulator Cbl, and that APS, and not sulphate itself, serves as the signalling molecule for sulphate excess.