Lac messenger RNA in lac Z gene mutants of Escherichia coli caused by insertion of bacteriophage Mu

The messenger RNA made under conditions of induction of the lac operon has been studied in various lac mutants in which the mutation was caused by integration of bacteriophage Mu into the Z gene. The percentage of RNA hybridizing specifically to lac DNA is proportional to the distance from the beginning of the gene at which a given mutation is located. It thus appears that only lac RNA proximal to the site of insertion is transcribed in these mutants. This may account for the complete polarity of Mu-induced lacZ mutants.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

Binding of p-nitrophenyl alpha-D-galactopyranoside to lac permease of Escherichia coli

Binding of the substrate analogue p-nitrophenyl alpha-D-galactopyranoside (NPG) to lac permease of Escherichia coli in different membrane preparations was investigated. Binding was assayed with an improved version of the centrifugation technique introduced by Kennedy et al. [Kennedy, E.P., Rumley, M.V., Armstrong, J.B. (1974) J. Biol. Chem. 249, 33-37]. Two binding sites for NPG were found with dissociation constants of about 16 microM and 1.6 mM at pH 7.5 and room temperature. With purified lac permease reconstituted into proteoliposomes, it could be shown that one permease molecule binds two substrate molecules. Oxidation of lac permease with the lipophilic quinone plumbagin or alkylation with the sulfhydryl reagent N-ethylmaleimide caused a 12-fold increase in the first dissociation constant. The second dissociation constant seemed to be increased as well, but its value could not reliably be estimated. Ethoxyformylation of lac permease with diethyl pyrocarbonate totally abolished NPG binding. The implications of these results for the catalytic performance of the enzyme are discussed.     

cys154 Is important for lac permease activity in Escherichia coli

The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport.     

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.     

Targeting the Escherichia coli lac repressor to the mammalian cell nucleus

We have previously shown that about 90% of total Escherichia coli lac repressor synthesized in mammalian cells is located in the cytoplasm [Hu and Davidson, Cell 48 (1987) 555-566]. To target a functional lac repressor to the nucleus, we mutated 10 nucleotides at the 3' end of the coding sequence, thus adding the nuclear localization signal of the simian virus 40 large-T antigen to the C terminus of the repressor. The mutant lacI gene and the wild-type (wt) gene, both in standard animal cell expression vectors, driven by the promoter of the Rous sarcoma virus long terminal repeat, were stably transfected into three rodent cell lines. In confirmation of our previous results, only about 10% of the wt repressor, but all of the mutant protein, was localized in the nucleus. DNase I footprint analyses showed that the mutant repressor retained the same operator DNA-binding specificity as wt repressor. Furthermore, both repressor-operator complexes could be dissociated by addition of isopropyl-beta-D-thiogalactopyranoside in vitro. However, the ratio of number of repressor molecules per nucleus that, by in vitro assay, could bind to the operator sequence to the number of monomer repressor polypeptides per nucleus, as determined by Western blotting, was about 1:4 for the wt repressor and about 1:30 for the mutant repressor. This suggests that: (a) the mutant repressor assembles into tetramers inefficiently; and/or (b) it has reduced binding affinity to the operator sequence; and/or (c) it has higher binding affinity to nonspecific DNA.     

Corepressor system for catabolite repression of the lac operon in Escherichia coli

Acetylated amino sugars, normally used in the biosynthesis of cell walls and cell membranes, were found to play a role as corepressors for catabolite repression of the lac operon in Escherichia coli. This conclusion was derived from studies conducted on mutants of E. coli that were able to assimilate an exogenous source of N-acetylglucosamine (AcGN) but were unable to dissimilate or grow on this compound. At concentrations less than 10(-4)m, AcGN caused severe catabolite repression of beta-galactosidase synthesis in cultures grown under either nonrepressed or partially repressed conditions. This repression occurred in the absence of any effect of AcGN on either the carbon and energy metabolism or the growth of the organism. In addition, this repression by AcGN occurred in a mutant strain that is constitutive for beta-galactosidase production, demonstrating that the AcGN effect does not involve the uptake of inducer. This model for the corepressor system of catabolite repression is discussed in relation to the existing theories on repression of the lac operon.     

Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.     

Structure of the lac carrier protein of Escherichia coli

Circular dichroic measurements on the lac carrier protein purified from the cytoplasmic membrane of Escherichia coli indicate that 85 +/- 5% of the amino acid residues comprising this integral membrane protein are arranged in helical secondary structures. Analysis of the sequential hydropathic character of this protein by the method of Kyte and Doolittle (J. Mol. Biol. (1982) 157, 105-132) indicates that the protein is composed of at least 12 hydrophobic segments with a mean length of 24 +/- 4 residues/segment. Approximately 70% of the 417 amino acids in the lac carrier are found in these domains. The hydropathic profile, together with the circular dichroic measurements, suggest that the 12 hydrophobic segments are largely in a helical conformation. If the segments are assumed to be alpha-helical, the mean length of each domain approximates the thickness of the most hydrophobic portion of the lipid bilayer. Based on these considerations, it is proposed that the lac carrier protein consists of at least 12 alpha-helical segments that traverse the membrane in a perpendicular sense, i.e. in a fashion similar to bacteriorhodopsin.