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

Abstract

The operator-distal genes hisBHAFI(E) of the Escherichia coli K-12 histidine operon were mapped on a DNA fragment 4,500 base pairs long. This fragment, originally present in a X transducing phage, was cloned in the vector plasmid pBR313. A restriction map was determined, allowing identification of the orientation of the genes in the fragment. The cloned genes were expressed in appropriate hosts, independent of the orientation of the DNA fragment, as shown by transformation tests and by enzyme assays of one of the gene products, hisB, histidinol phosphatase. An internal transcription initiation site was identified by isolation of the cellular RNA, hybridization to specific DNA probes, and mapping by S1 nuclease.With the development of recombinant DNA technology, the histidine operon of the enterobacteria Escherichia coli and Salmonella typhimurium has been the subject of extensive studies (9). Most of the work has been concerned with the elucidation of the mechanisms of operon regulation. 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). As a prerequisite to studying some of these features, we report here the cloning of the distal portion of the E. coli K-12 his operon, a restriction map of this region, the orientation and expression of its genes, and the mapping of an internal transcription initiation site.MATERIALS AND METHODS Bacterial strains, phage, and plasmids. 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). Details of the construction of histidine recombinant plasmids are given below.Media, growth conditions, and enzyme assays. Liquid media were LB broth (36) and minimal medium (45) supplemented with 0.5% glucose. Solid media contained 1.2% agar (Difco) and were nutrient broth (36) and minimal medium (45) supplemented with 0.5% glucose. Amino acids were added at 0.5 mM; Lhistidine was added at 0.1 mM, and histidinol was added at 1.0 mM. Tetracycline and ampicillin were added to both liquid and solid media at 25 and 50 ,ug/ ml, respectively. Strains for enzyme assays or for RNA preparation were grown in minimal medium to an absorbancy at 650 nm of 0.8. Assay procedures for th...

Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector

“…Nucleic acids were extracted with neutral phenol-chloroform (1:1) and finally with chloroform and were precipitated with 2.5 volumes of ethanol at -20°C. RNAs were also isolated by lysing the protoplasts in 8 M guanidine hydrochloride as previously described (9). This method gave identical results with the proteinase K-SDS procedure for bacteria grown in minimal medium, whereas our attempts to demonstrate, by the same methods, intact RNAs from bacteria grown in L-broth failed.…”

Intracistronic transcription termination in polysialyltransferase gene (siaD ) affects phase variation in Neisseria meningitidis

“…Total bacterial RNA was extracted from logarithmically growing cells by the guanidine hydrochloride procedure previously described (Grisolia et al ., 1982).…”

Structure and function of the internal promoter (hisBp) of the Escherichia coli K-12 histidine operon

“…The bacterial strains used are listed in Table 1. Construction of plasmids pCB3 (7) and pVG2, pVG4, and pVG5 (18) has already been described. The multicopy plasmids pKO0, pKO4, and pKG1800 (26) were obtained from M. Rosenberg.…”

“…Several features of the proximal region of this operon and the mechanism of coupled translation-transcription termination (attenuation) by which the operon expression is regulated have been analyzed and are summarized in reference 4. In an attempt to better define the still poorly known features of the distal portion of this operon, we previously reported the cloning of this region of the E. coli K-12 chromosome comprising the hisBHAFI(E) genes and the mapping of an internal transcription initiation site (18). We have now extended these studies and report here the cloning, in a vector plasmid, and the restriction map of the entire operon, the location of several genes by subcloning and complementation tests, the sequence of a DNA fragment comprising the end of the hisC gene and the beginning of the hisB gene, and the structure and function of the internal hisBp promoter.…”