A mutation defining ultrainduction of the Escherichia coli gal operon

Abstract

Tn1O insertion in the galS (ultrainduction factor) gene of Escherichia coli allows the gal operon to be constitutively expressed at a very high level, equal to that seen in a AgalR strain in the presence of an inducer. The insertion has been mapped by criss-cross Hfr matings and by marker rescue into Kohara phages at 46 min on the E. coli chromosome.The gal operon of Escherichia coli is regulated both positively and negatively at the level of transcription initiation. There are two gal promoters, P1 and P2; P1 is activated by the cyclic AMP-cyclic AMP receptor protein complex bound in the -40 region, whereas P2 is inhibited by this binding. To isolate a mutation in the gene encoding the second repressor, we started with a strain that was deleted for the galR gene and had a galE-lacZ protein fusion (JT247). (The strains used in this study are shown in Table 1.) In such a strain, ultrainduction was indicated by a threefold increase in ,-galactosidase activity when the inducer was added (10). Colonies of such a strain appeared red on MacConkey lactose plates, even in the absence of an inducer, because of the high level of expression (approximately 500 Miller units; see Table 2). When the P-galactosidase competitive inhibitor thio-ethyl-phenyl-,-D-galactoside (TEPG) (5) of these MC4100 Tcr colonies were pooled, and phage P1 was grown on the bacterial pool. The resulting P1 lysate was used to infect JT247, selecting for tetracycline resistance on Luria broth plates containing 15 ,ug of tetracycline per ml. Tetracycline-resistant colonies were replicaplated to MacConkey lactose plates containing 15 ,ug of tetracycline per ml and 1 mM TEPG. Red colonies were found at a frequency of approximately 1.0%.To ensure that the red phenotype on MacConkey lactosetetracycline-TEPG plates was caused by the ATnJO insertion, phage P1 was used to transfer the ATnJO from six of the mutants into a fresh JT247 strain. In one of the six, there was no linkage (O of 226 tetracycline-resistant colonies) between tetracycline resistance and the MacConkey lactose plate phenotype. In each of the other five, 100% of at least 2,000 tetracycline-resistant colonies were red on the MacConkey lactose-tetracycline-TEPG plates. Only these five were studied further.,-Galactosidase in the mutants was measured and compared with that in the parent strain. Results are shown in Table 2. Two classes of mutants were found: class I, four mutants that in the absence of an inducer had a high level of gal-lacZ expression which increased further upon addition of an inducer (represented by strain AG708); and class II, one mutant that in absence of an inducer had a high level of expression of gal-lacZ which did not increase upon addition of an inducer (strain AG701). The phenotype of the second class was what we expected from a mutation in a gene encoding the proposed second gal repressor. Expression was at a high level regardless of whether an inducer was present. We call the gene into which the ATnJO was inserted galS. The mutation in AG708 is probably not allelic...

Functional characterization of roles of GalR and GalS as regulators of the gal regulon

“…They indicate that the sets of genes controlled by these repressors overlap. In a galR deletion strain the gal operon can be further induced by galactose, and the ultimate level of expression is higher than that which can be obtained by adding galactose to wild-type cells (9). On the basis of this phenomenon, called ultrainduction, the existence of the galS gene was predicted and subsequently verified (9,15).…”

“…GalR, the first of these proteins to be identified and which has been purified, functions as the major repressor of the gal operon and is sufficient for repression of its genes in the absence of galactose (1). GalS was identified because in strains in which galR was deleted, the gal operon could be further induced by galactose, a phenomenon called ultrainduction (9). Subsequent genetic studies indicated that GalS functions to control its own expression and that of the ␤-methylgalactoside transport system, the galactose transporter in E. coli (16).…”

Synergism between the Trp repressor and Tyr repressor in repression of the aroL promoter of Escherichia coli K-12

“…There are few examples of E. coli promoters that are repressed by two different repressor proteins (6). However, the gal promoter appears to be another example of a promoter that is subject to dual repression (12).…”

“…When The other a n t/c G T A C T a/c G T t oligonucleotide (TTAAGCGGATCCGCAAGGG) was de- 12 Fig. 2.)…”

Locations and orientations on the Escherichia coli physical map of the mgl operon and galS, a new locus for galactose ultrainduction

“…This phenomenon has been termed ultrainduction (8). A ATnJO insertion abolished ultrainduction of the gal operon in an E. coli AgalR strain, and the locus, called galS, was mapped to a location between 42 and 50 min on the chromosome (2).…”