Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette

Abstract

We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. These Kmr insertions were then crossed directly into the bacterial chromosome. We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants. We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes. The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT. We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-l and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively. Of these genes, only the functions of usg-2 (pdrB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-l or dsg-l for function. usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37°C. In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter. The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT. The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons.

The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis

“…Howard et al achieved 100% of the activity of a n f control To determine what role, if any, the newly identified nif genes nifw and nzj2 have in nitrogen fixation, chromosomal mutations in nzfw and nzfl were constructed by inserting kanamycin cassettes into the coding regions of these genes, thereby disrupting their function. It was anticipated that the insertion of the cassette would result in a reduction of expression of genes downstream in the operon, as reported by Arps and Winkler in their study of the E. coli hisT operon [15]. However analysis of the phenotypes of the nifmutants suggested that the cassettes were not polar if the direction of transcription of the cassette was in the same direction as that in the nifoperon ('B orientation), and only partially polar if in the opposite 'A' orientation.…”

“…In orientation 'A' the direction of transcription of the kanamycin gene (kan) is in the opposite direction to transcription in the nzfCiSV operon; in orientation 'B' transcription is in the same direction. Since the kanamycin cassette has been found to reduce the expression of downstream genes [15], any phenotype observed from the mutant strains may be due polarity onto n$M, in the case of the nij2::kan and nifA W Z : : kan strains, and due to polarity onto nifZ and nifM in the nzfW::kan strains. To compensate for any polarity effects, the mutant strains were transformed with pWP13 (a NifM-producing plasmid) or pWP14 (a NifZ,M-producing plasmid).…”

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations

“…Characterization of these suppressors is described below. Because insertion 2 Chromosomal insertions 7 and 8 did not impair colony growth on LBC medium, which suggested that they were not polar onpdx-dpj expression (Table 4). However, insertions 7 and 8 resulted in strong UV sensitivity, indicative of a recombination-or repair-deficient mutation.…”

Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations

“…2). Insertions imparting antibiotic resistances were crossed from linearized plasmids into the chromosome of recBC sbc mutant JC7623 as detailed before (1,63) pdxH::MudI-8 chromosomal mutants and plasmids pNU216 and pNU217 were isolated previously in this laboratory (33). Plasmid pTX281 (Fig.…”