Regulation of Newly Evolved Enzymes. III Evolution of the ebg Repressor during Selection for Enhanced Lactase Activity

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.     

Regulation of Newly Evolved Enzymes II. the EBG Repressor

The ebg (evolved β-galactosidase) regulatory locus has been mapped. The map order is argG–ebgA–ebgR–tolC–metC , and there is 1.6% recombination between ebgR and ebgA . Studies with ebgR^-/ebgR⁺ merdiploids have shown that ebgR^- is recessive, and it is concluded that the synthesis of ebg enzyme is under negative control.     

Regulation of Newly Evolved Enzymes. IV. Directed Evolution of the EBG Repressor

In Escherichia coli, the wild-type repressor of ebg (evolved beta-galactosidase) enzyme synthesis, specified by the ebgR+ gene, responds very weakly to lactulose (fructose-beta-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 X 10(-10) . EBGR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.     

Evolution of Transposons: Natural Selection for Tn5 in ESCHERICHIA COLI K12

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.     

Negative Dominant Mutations of the uidR Gene in ESCHERICHIA COLI: Genetic Proof for a Cooperative Regulation of uidA Expression

The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI I. Isolation and Specificity of Host and Plasmid Mutations

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.     

Phenethyl Alcohol Resistance in ESCHERICHIA COLI. III. a Temperature-Sensitive Mutation (dnaP) Affecting DNA Replication

A temperature-sensitive DNA replication mutant of E. coli K-12 was isolated among the mutants selected for phenethyl alcohol resistance at low temperatures. This mutation, designated as dnaP18, affects sensitivity of the cell to phenethyl alcohol, sodium deoxycholate and rifampicin, presumably due to an alteration in the membrane structure. At high temperatures (e.g., 42 degrees ), synthesis of DNA, but not RNA or protein, is arrested, leading to the formation of "filaments" in which no septum formation is apparent. Nucleoids observed under electron microscope seem to become dispersed and DNA fibrils less condensed, which may explain the loss of viability under these conditions. Genetic analyses, including reversion studies, indicate that a recessive dnaP mutation located between cya and metE on the chromosome is responsible for both alterations of the membrane properties and temperature sensitivity. The dnaP18 mutation does not affect growth of phage T4 or lambda under conditions where host DNA replication is completely inhibited. Kinetic studies of DNA replication and cell division in this mutant after the temperature shift from 30 to 42 degrees , and during the subsequent recovery at 30 degrees , accumulated evidence suggesting that DNA replication comes to a halt at 42 degrees upon completion of a cycle already initiated before the temperature shift. Since the recovery of DNA synthesis after exposure to 42 degrees does not depend on protein or RNA synthesis or other energy-requiring processes, the product of the mutant dnaP gene appears to be reversibly inactivated at 42 degrees . Taken together with the recessive nature of the present mutation, it was suggested that one of the membrane proteins involved in initiation of DNA replication is affected in this mutant.