Regulatory aspects of the Gua operon of Escherichia Coli

Pickett, Mark Anthony (1987) Regulatory aspects of the Gua operon of Escherichia Coli. University of Southampton, Doctoral Thesis.

Abstract

The mechanisms regulating expression of the guanine operon were investigated in a number of ways. (1) Gua-lac fusions were constructed in vivo using the specialised transducing phage Mud(Ap, lac). Insertion of Mud(Ap, lac) into guaA and subsequent selection for temperature resistance, yielded a recombinant, MP1004, in which the genes of the lac operon are controlled as an integral part of the gua operon, the promoter proximal guaB gene remaining intact. Upon guanine starvation of MP1004, IMP dehydrogenase (the product of the guaB gene) expression derepresses 50-fold and β-galactosidase expression simultaneously derepresses five-fold. Spontaneous and NG-induced mutations, causing defective gua control, were sought in MP1004. Mutants with constitutively derepressed levels of β-galactosidase or lactose permease were selected on minimal lactose or melibiose plates, respectively, and arose at a frequency of c.10^-5. The 24 mutants tested exhibited β-galactosidase levels raised up to 2.5-fold, but none showed IMP dehydrogenase levels raised more than 1.3-fold relative to the parent strain, MP1004. These mutants did not arise by Mud(Ap, lac) phage transposition. (2) A closer fusion of the gua and lac genes was accomplished in vitro utilising the promoter cloning plasmid pMC1403. A 2.1kb EcoRI fragment containing the gua promoter and the promoter proximal portion of guaB, was inserted into pMC1403. Correction of reading frame using exonuclease BAL31 yielded two recombinant plasmids, pMP108 and pMP112, which encoded a 140,000 mol.wt. hybrid protein consisting of an enzymatically active 116,700 mol.wt. β-galactosidase with 26,000 mol.wt. fragment (encoded by the promoter proximal portion of guaB) joined at its N-terminus. When the appropriate lac host containing either of these recombinant plasmids was starved for guanine, a six fold derepression of β-galactosidase activity was observed. a 50% repression of β-galactosidase activity was elicited by excess guanine. Thus, the genes of the lac operon are under control of the gua promoter in the plasmids pMP108 and pMP112. Attempts to isolate mutants with constitutively derepressed levels of β-galactosidase or lactose permease, were unsuccessful. (3) A putative repressor binding site overlaps the Pribnow box of the primary gua promoter. Oligonucleotide-directed mutagenesis was used to create an A to T transversion that disrupts this region of hyphenated dyad symmetry. The activities of wild-type and mutant gua promoters were compared by cloning a 310bp AluI fragment containing the promoter into the galactokinase promoter cloning plasmids, pK04 and pK06. Although the mutation caused no change in gua promoter activity, in this system even the wild-type promoter failed to respond to stimuli normally causing derepression or repression of the gua operon. (4) The DNA sequence of the gua promoter reveals several features that suggest stringent control of the operon. A relA^+/relA^- isogenic pair of strains was used to investigate such a mechanism. A `relaxed'' strain (relA^-) exhibited a 26% decrease in IMP dehydrogenase levels when starved for isoleucine. When the stringent response occurred, only a 10% decrease was observed under the same conditions. The transcriptional activity of the gua promoter was measured by hybridising labelled gua transcripts to λpguaA DNA. When the wild-type strain (W3110) was grown in medium containing excess guanine, an eight-fold repression of IMP dehydrogenase activity but only a two-fold repression of transcriptional activity, occurred. Starvation for isoleucine in a `relaxed'' strain caused a two-fold increase in transcriptional activity of the gua promoter; no such increase was observed when the stringent response occurred.

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