PubMed HealthSearch

PubMed · 6329713

Sequence elements determining ampC promoter strength in E. coli.

Abstract

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

B Jaurin, T Grundström, S Normark. 1982. Sequence elements determining ampC promoter strength in E. coli.. https://doi.org/10.1002/j.1460-2075.1982.tb01263.x

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Adjacent upstream superhelical writhe influences an Escherichia coli promoter as measured by in vivo strength and in vitro open complex formation.

This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the -35 consensus sequence of Escherichia coli promoters. A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately upstream of the beta-lactamase promoter in plasmid pUC19. All the mutants showed reduced promoter activities in E. coli cells. However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment. Leftward writhe greatly repressed transcription. A plane curve showed an intermediate effect. This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter. The positive effect of rightward writhe was also observed in vivo for the promoter of the tetracycline resistance gene of pBR322. The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity.

Ampicillin

Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway.

An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.

Ampicillin

SopB protein-mediated silencing of genes linked to the sopC locus of Escherichia coli F plasmid.

Expression of a high level of F-plasmid-encoded SopB protein in Escherichia coli is found to repress genes linked to sopC, a sequence element of F consisting of 12 tandemly joined imperfect repeats of a 43-bp motif. Repression of a gene can occur over a distance of at least 10 kb from the sopC element and is not affected by the relative orientation of sopC. In the repressed state, accessibility of intracellular DNA to cellular proteins is greatly reduced in the region containing sopC, as monitored by the trapping of the covalent intermediate between DNA and DNA gyrase and by Dam methylase-catalyzed DNA methylation. These results signify the formation of a nucleoprotein structure emanating from sopC and are discussed in terms of position-dependent silencing of genes in general and the IncG type of plasmid incompatibility in particular.

Ampicillin