Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors

Abstract

A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed.

Cloning and expression of the distal portion of the histidine operon of Escherichia coli K-12

“…The results are reported in Table 2. The product of the hisC gene, not present in the cloned fragment (14), was used to establish the activity of the chromosomal genes expressed under the primary hisGp promoter. One of the activities of the hisB gene, histidinol phosphatase, was used to estimate the levels of expression of the cloned genes.…”

“…Expression of this biosynthetic operon in both species is regulated at the transcriptional level by an overall mechanism termed attenuation (9,48). Recent studies from our and other laboratories have been concerned with the genetic analysis of the regulatory region (28), DNA sequencing of the regulatory region (4,20,44) and of regulatory mutants (29), and cloning and expression (transcription and translation) of the proximal part of the operon (5,14,23). There are several other aspects of the his operon organization which are of potential interest: internal promoters (1,21), intercistronic regions (40), and multifunctional gene products (42).…”

“…Bacterial strains used are listed in Table 1. Strain FB251 was constructed by selecting a spontaneous thy mutant with thymidine-trimethoprim selection (36) in strain FB186 and mating this derivative with an Hfr strain thy' recA56 (N1200), selecting for thy' recombinants, and scoring for UV sensitivity (14). Phage lysates and transduction tests were performed as previously de-scribed (2).…”

“…The restriction map of the plasmid DNA was determined by using restriction sites uniquely present in the vector or cloned DNA, as described below. Fine mapping of the HindIII 4,500-bp fragment was performed by the technique of Smith and Birnstiel (41), as previously described (14).…”

“…Plasmids pVG2 and pVG3 were obtained by mixing the his DNA and the pBR313 DNA, both restricted with HindIII. The DNA mixtures were ligated and used to transform strain FB251, as previously described (14). Plasmids pVG4 and pVG5 were constructed as described below.…”