Noninducible Tet repressor mutations map from the operator binding motif to the C terminus

Hecht, B; Müller, Gerhard; Hillen, Wolfgang

doi:10.1128/jb.175.4.1206-1210.1993

Cited by 61 publications

(62 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results of a large number of biochemical, structural, and mutagenesis studies on TetR(B) and TetR(D) are in good agreement, which provides an exceptionally detailed picture of the induction process (55,60,135,193,194,205). In addition to identifying amino acids involved in the induction process, random mutagenesis has even produced a TetR mutant that exhibits increased, not decreased, affinity for the tet operator upon inducer binding, which, in combination with the relative nontoxicity of tetracycline to eukaryotic cells, has facilitated its use to tightly regulate gene expression in eukaryotes (16,44,62).…”

Section: Tetr-ligand Interactionssupporting

confidence: 51%

Regulation of Bacterial Drug Export Systems

Grkovic

Brown

Skurray

2002

Microbiol Mol Biol Rev

361

339

View full text Add to dashboard Cite

The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed

show abstract

Section: Tetr-ligand Interactionssupporting

confidence: 51%

Regulation of Bacterial Drug Export Systems

Grkovic

Brown

Skurray

2002

Microbiol Mol Biol Rev

361

339

View full text Add to dashboard Cite

show abstract

“…A possible explanation is that multiple mutations may be required in PmtaC1 to result in a deregulated but still active promoter. On the other hand, a single point mutation can render a regulatory protein unable to bind its target site (16). It should be noted that proteins other than a transcriptional regulator, regulatory RNA, or metabolites, which might interact with DNA sequences and/or components of the transcription machinery, could be affected by the mutation observed.…”

Section: Discussionmentioning

confidence: 99%

Methanol-Dependent Gene Expression Demonstrates that Methyl-Coenzyme M Reductase Is Essential in Methanosarcina acetivorans C2A and Allows Isolation of Mutants with Defects in Regulation of the Methanol Utilization Pathway

et al. 2005

View full text Add to dashboard Cite

Methanosarcina acetivorans C2A is able to convert several substrates to methane via at least four distinct methanogenic pathways. A common step in each of these pathways is the reduction of methyl-coenzyme M (CoM) to methane catalyzed by methyl-CoM reductase (MCR). Because this enzyme is used in each of the known pathways, the mcrBDCGA operon, which encodes MCR, is expected to be essential. To validate this prediction, a system for conditional gene inactivation was developed. A heterologous copy of the mcrBDCGA operon was placed under the control of the highly regulated mtaC1 promoter, which directs the expression of genes involved in methanol utilization, and recombined onto the M. acetivorans chromosome. This allowed for disruption of the endogenous mcr operon in the presence of methanol. Because the PmtaC1 promoter is transcribed only during growth on methanol, mcrBDCGA was rendered methanol dependent and the strain was unable to grow in trimethylamine media, strongly suggesting that mcrBDCGA is essential. Upon prolonged incubation, suppressed mutants which expressed mcrBDCGA constitutively could be selected. Expression analysis of PmtaC1::uidA gene fusions in several isolated suppressed mutants suggests that they carry transactive mutations leading to deregulation of all genes under control of this promoter. Subsequently, proteome analysis of one such suppressed mutant revealed that all known proteins derived from mtaC1 promoterdependent expression were constitutively expressed in this mutant. This genetic system can therefore be employed for the testing of essential genes and for the identification of genes under a common regulatory mechanism by making regulatory mutations phenotypically selectable.

show abstract

“…1). Deletion mutagenesis analyses of the TetR protein have shown that the residues needed for the activity of the repressor are distributed all along the protein sequence (6,7,18,37,41). The N-terminal region contains the DNA-binding domain; and the C-terminal region contains the dimerization domain, the region required for the binding of tetracycline, and regions involved in the folding and stability of the protein.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of SmeT, a Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

Sánchez

Alonso

Martínez

2002

Antimicrob Agents Chemother

View full text Add to dashboard Cite

We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5 end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PsmeT. The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.

show abstract

Noninducible Tet repressor mutations map from the operator binding motif to the C terminus

Cited by 61 publications

References 20 publications

Regulation of Bacterial Drug Export Systems

Regulation of Bacterial Drug Export Systems

Methanol-Dependent Gene Expression Demonstrates that Methyl-Coenzyme M Reductase Is Essential in Methanosarcina acetivorans C2A and Allows Isolation of Mutants with Defects in Regulation of the Methanol Utilization Pathway

Cloning and Characterization of SmeT, a Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

Contact Info

Product

Resources

About