Regulation of the Escherichia coli K-12 uvrB operon

The UV light inducibility of the uvrB operon of Escherichia coli K-12 was previously demonstrated by exploiting a strain in which the gene for the enzyme beta-galactosidase was inserted into the uvrB operon. This insert is now shown to be located within the structural gene for the uvrB enzyme, leaving the regulatory sequences of the operon intact. Analyses to quantitate the induction of this system show that derepression of the operon is first detectable 5 min after UV exposure, with the rate of synthesis increasing to four to six times the uninduced rate during the subsequent 30 min. Induction is unaffected by mutations in other components of nucleotide excision repair. The control of uvrB was found to result from direct repression by the lexA gene product, with the recA gene product playing an indirect role. Nucleotide excision repair thus seems to be part of the SOS response.     

The cloning of the Escherichia coli K-12 deoxyribonucleoside operon

A 6.1-kb EcoRI DNA fragment containing the four structural genes (deoC, deoA, deoB, deoD) of the deoxyribonucleoside operon has been cloned into the plasmid pMFS53. By use of a unique, asymmetrically positioned HindIII site on the 6.1 kb insert, plasmids containing the deoC,deoA genes (pMFS50) or the deoB,deoD genes (pMFS55) have been constructed. Enzyme assays performed on extracts prepared from clones harboring pMFS53, pMFS50 or pMFS55 revealed that each clone possessed amplified deo enzyme levels and that the spectrum of enzyme amplification corresponded to the genetic composition of the plasmids carried by each clone. A plasmid (pMFS50l) having functional deoA, deoB and deoD genes but devoid of the deo regulatory region and a portion of the deoC structural gene has been isolated following treatment of BamHI cleaved pMFS53 and BAL31 nuclease. Comparison of the deo enzyme levels for clones harboring pMFS53 and pMFS501 suggest that plasmid pMFS53 possesses a functional deo regulatory region in addition to the four structural genes of the operon.     

Secondary promoter of the guanine operon of Escherichia coli K-12.

Evidence of a secondary promoter for the guaA gene within the guaB gene was obtained by using lambdapguaA transducing phage. The technique is generally applicable to distinguish a promoter present within a bacterial deoxyribonucleic acid segment, which has replaced the lambda b2 region of transducing phage, from the phage pI promoter.     

Regulatory mutants of the aroF-tyrA operon of Escherichia coli K-12.

The regulatory region of the aroF-tyrA operon was fused to the chloramphenicol acetyltransferase (cat) gene on a plasmid vector. Expression of the cat gene was subject to repression by tyrR+. This fusion was used to isolate regulatory mutants with increased expression of the cat gene in which repression by tyrR+ was affected. Nucleotide sequencing of these mutants has led to the identification of three sites involved in the repression of aroF by tyrR+. The existence of a functional promoter divergently transcribing from the aroF regulatory region was also demonstrated by using the cat fusion vector. The expression of this promoter is also regulated by tyrR+.     

Operator-constitutive mutants in the threonine operon of Escherichia coli K-12.

Three Escherichia coli K-12 mutant strains resistant to DL-alpha-amino-beta-hydroxyvaleric acid were isolated in which the expression of the thr operon is constitutive. The localization and dominance properties of the mutations involved, called thrO, are those of operator mutations. The gene sequence is OABC as suggested by earlier studies.     

Effect of triethyltin on Escherichia coli K-12.

Triethyltin (TET) stimulated the basal respiration of Escherichia coli K-12 membrane vesicles in chloride (Cl-) medium but it had little effect on respiration in sulphate (SO4(2-)) medium. Since this uncoupling activity was Cl- dependent it was attributed to the Cl-/hydroxide (OH-) exchange reaction known to be mediated by TET [1,2]. TET inhibited the oxidation of succinate by intact E. coli in both Cl- and SO4(2-) medium, but at the same concentration of TET, inhibition was always more extensive in Cl- than SO4(2-) medium. In Cl- medium uncoupling in membrane vesicles and inhibition of succinate oxidation in intact bacteria occurred over the same concentration range and it appeared that the same mechanism, i.e. Cl-/OH- exchange, was responsible for both effects. Inhibition of succinate oxidation in SO4(2-) medium was not substantial until the concentration of TET was greater than 10(-5) M. Although the nature of this inhibition could not be determined by experiments with membrane vesicles indirect evidence from growth experiments indicated that it was due to impairment of oxidative phosphorylation. The relationship between these biochemical findings and the bacteriocidal action of TET was examined by using various concentrations of anion and substrate in the growth medium. Growth was inhibited in media containing either Cl- or SO4(2-) as the main anion but at a particular concentration of TET, inhibition was greater in Cl- medium. Growth was also inhibited to a greater extent in succinate than glucose medium. Furthermore in either Cl- or SO4(2-) glucose medium, lactic acid production increased as the concentration of TET was increased. These findings imply that the bacteriocidal action of TET is related to its effect(s) on oxidative phosphorylation.     

Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.