We describe a technique that allows the insertion of any DNA fragment into the EcoRI-site-containing malPpa, the promoter of malPQ, one of the three maltose operons of Escherichia coli. DNA fragments were cloned into the unique EcoRI site of the pBR322-derived plasmid pOM40, which carries malPpa. In the next step these fragments were transposed into the chromosome by homologous recombination events occurring on both sides of malPp. Cells in which such insertion of the entire recombinant plasmid have occurred can be conveniently selected. Excision and curing of the vector plasmid could then occur spontaneously at a high frequency, leaving behind the inserted fragment that can be manipulated as any chromosomal marker. When the inserted fragment contains a properly positioned promoter, its promoting activity can be estimated by assaying amylomaltase, the product of malQ. When required, the inserted fragment can be easily transferred back onto pOM40. As examples of application we have transferred two different fragments into the chromosome of E. coli: one contained the ceaC-ceiC operon, which encodes colicin E3 and its immunity protein, and the other contained the lac promoter of E. coli.

Base Sequence↗

PubMedDate not supplied

Genetic studies on the promoter of malT, the gene that encodes the activator of the Escherichia coli maltose regulon.

We report the construction of a chromosomal malT-lacZ gene fusion that is expressed under the control of the malT promoter in Escherichia coli K12. The resulting hybrid protein is soluble and stable in crude cellular extracts, which allowed us to measure very low levels of malTp activity. In this note, we confirm and extend previous observations on the regulation of malTp. We show that the promoter is 40-times less active in the absence of cAMP receptor protein (CRP) than in its presence, that CRP works by binding to the site centred at position -70.5, and that all of the elements necessary and sufficient for the regulation by CRP are located downstream from position -122.

Bacterial Proteins↗

PubMedDate not supplied

[Preliminary characterization of a new system allowing maltose assimilation by Escherichia coli].

We showed that Klebsiella pneumoniae and Erwinia herbicola possessed a pathway for maltose metabolism which could function in parallel with that encoded by the classical maltose regulon. Indeed, specific DNA fragments isolated from these two bacteria allowed growth on maltose of any of the mal mutants of Escherichia coli when in a multicopy number. The preliminary characterization of the E. herbicola DNA fragment indicated that a 4-Kb region was sufficient to complement the mal mutations of E. coli, and that this region encoded at least one 50-Kd protein. This protein is probably bound to the cytoplasmic membrane and its synthesis is induced by maltose independently of malT, the positive regulator gene of the maltose regulon.

DNA, Bacterial↗