Control of the gua operon of Escherichia coli K-12

Spibey, Norman (1983) Control of the gua operon of Escherichia coli K-12. University of Southampton, Doctoral Thesis.

Abstract

114P dehydrogenase-less (guaB) mutants of Escherichia coli were isolated following chemical mutagenesis. Two ua mutants (I:S1021 and PL1138) were found to be temperate-sensitive. A cell-free extract of NS1021 gave only weak precipitin lines on Ouchterlony double diffusion gels whereas a cell-free extract of PL1138 gave precipitin lines as intense as those from a g„RT strain. Complementation in vitro between paB mutants revealed two complementing groups. One group has only two memmers NS1021 and PL1138, the other group is comprised of all other CR14 mutants. Complementation is only observed between the two groups and not within either group. Complementation using NS1021 always resulted in lower enzyme activities compared with PL1138. Alkylation of mutant extracts using Cl-31D prior to complementation revealed that both NS1021 and PL1138 provide the catalytic subunits in the hybrid enzyme. The regulation of the gua operon of E.coli was investigated using lac fusion strains isolated by the general method of Casadaban 1976, J.Nol.Biol. 104, 51+1). The lao genes were shown to be regulated as part of the gua,opercn by cnse to the addition of purines and purine analogues to the growth medium. Expression of the lac genes was derepressed during growth in guanine-limited medium. Both guaA-lac and guaB-lac fusions exhibited the same degree of derepxession of j3-galactesidase synthesis upon guanine starvation (approximately seven-fold) compared with fifty-fold for IMP dehydrogenase (in a guaA mutant) and twenty-fold for GMP synthetase (in a guaB mutant). The reason for the similar low derepression ratios may be due to the presence of a rho-dependent transcription termination sequence between Lua and lac. Regulatory mutants were sought from the ua--lac fusions. Mutants with increased levels of lac expression were isolated using a variety of procedures, however none of these mutants had altered expression of R. The apparent regulatory mutants were probably due to mutations in the -L--D DNA beeween gua and lac. The introduction of a placid carrying the R promoter and promoter proTimal portion of guaB into a strain with a aaa-lac fusion caused an alteration in expression of the fused lac genes which was dependent upon the growth medium. In minimal glucose medium the introduction of of the gua plasmid caused an increase in chromosomal gua expression whereas in L-broth the presence of the plasmid had the opposite effect. This suggests that gua operon expression may be under metabolic control. Analysis of transcription in vitro revealed only one promoter on the 2.1 kb DNA fragment used to construct the gua plasmid mentioned above. The transcript produced from this isolated DNA fragment was approximately 630 bases. Experiments to demonstrate the specific nucleoside or nucleotide involved in control of the qua, operon proved inconclusive.

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