Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase.

A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA.     

The melibiose permease system of Escherichia coli K12

The melibiose permease system of E. coli K12 has been explored using a strain deficient in lactose permease: 300 P. The accumulation of 1-S-methyl-beta-D-thiogalactopyranoside (TMG) was observed. The uptake system was inducible by melibiose and a number of analogs at 30 degrees C. At higher temperatures the differential rate of synthesis decreases until becoming negligible at 42 degrees C. The uptake tends toward a steady state which corresponds to an accumulation several hundredfold over the sugar concentration in the medium. At a given temperature the steady state level was proportional to the initial rate of uptake whatever the degree of induction and the substrate concentration. Lowering the temperature decreased the initial rate of uptake but increased the steady state level. This uptake system was pH dependent with better efficiency at pH 8. It was also dependent on the presence of sodium or lithium ions active at 5 mM whereas potassium at 170 mM enable only about half maximal uptake. The uptake in a medium with choline chloride was less than one fifth of optimal activity. Addition of Li+ brought about half maximal activation at approximately 0.5 mM. The activation consists mainly in a decrease of apparent Km. The emphasis of this study was put on the similarities and differences with lactose permease which is able to transport the same sugar to approximately the same extent. Inducer specificities and substrate specificities were compared and a method of measuring the two activities in the same cells was devised.     

Aggregation deficient Escherichia coli K-12 female mutants with altered lipopolysaccharide.

Two F- mutants deficient in conjugation with F-donors have been characterized. They map at about 83 minut position, show resistance to T3 and T7 bacteriophages, and form mating aggregates in the liquid medium with lowered efficiency. Mutants have no detectable alterations in their outer membrane protein composition.     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Expression of the human alpha-galactosidase A in Escherichia coli K-12

We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.     

Actinomycin sensitive mutants of Escherichia coli K-12

Summary Actinomycin sensitive mutants of E. coli K12 have been isolated and shown to have pleiotropic defects in the fermentation of sugars. The locus of a gene controlling actinomycin resistance is very close to that of the lactose gene.     

Active-site-directed photolabeling of the melibiose permease of Escherichia coli

Covalent photolabeling of the melibiose permease (MelB) of Escherichia coli has been undertaken with the sugar analogue [(3)H]-p-azidophenyl alpha-D-galactopyranoside ([(3)H]-alpha-PAPG) with the purpose of identifying the domains forming the MelB sugar-binding site. We show that alpha-PAPG is a high-affinity substrate of MelB (K(d) = 1 x 10(-)(6) M). Its binding to or transport by MelB is Na-dependent and is competitively prevented by melibiose or by the high-affinity ligand p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG). Membrane vesicles containing overexpressed histidine-tagged recombinant MelB were photolabeled in the presence of [(3)H]-alpha-PAPG by irradiation with UV light (lambda = 250 nm). Eighty-five percent of the radioactivity covalently associated with the vesicles was incorporated in a polypeptide corresponding to MelB monomer. MelB labeling was completely prevented by an excess of melibiose or alpha-NPG during the assay. Radioactivity analysis of CNBr cleavage or limited proteolysis products of the purified [(3)H]-alpha-PAPG-labeled transporter suggests that several domains of MelB are targets for labeling. One of the labeled CNBr cleavage products is a peptide with an apparent molecular mass of 5.5 kDa. It is shown that (i) its amino acid sequence is that of the Asp124-Met181 domain of MelB (7.5 kDa), which includes the cytoplasmic loop 4-5 connecting helices IV and V, the hydrophobic helix V, and the outer loop connecting helices V-VI, and (ii) that Arg141 in loop 4-5 is the only labeled amino acid of this peptide. Labeling of loop 4-5 provides independent evidence that this specific domain plays a significant role in MelB transport. Comparison with the well-characterized equivalent domain of LacY suggests that sugar transporters with similar structure and substrate specificity may have conserved domains involved in sugar recognition.     

Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress

In Escherichia coli, the coordinate action of two antioxidant enzymes, superoxide dismutase and hydroperoxidase (catalase), protect the cell from the deleterious effects of oxyradicals generated during normal aerobic respiration. To evaluate the relative importance of these two classes of enzymes, strains of E. coli deficient in superoxide dismutase and (or) hydroperoxidase were constructed by generalized transduction and their physiological responses to oxygen and oxidant stress examined. Superoxide dismutase was found to be more important than hydroperoxidase in preventing oxygen-dependent growth inhibition and mutagenesis, and in reducing sensitivity to redox-active compounds known to generate the superoxide anion. However, both types of enzymes were required for an effective defense against chemical oxidants that generate superoxide radicals and hydrogen peroxide.     

New types of Escherichia coli K-12 facultative recombination deficient mutants resistant to ultraviolet.

Numerous facultative temperature sensitive recombination deficient mutants of Escherichia coli K-12 strain 108 were isolated after mutagenization with nitrosoguanidine. The majority of the mutants were resistant to UV irradiation. Three mutants, KBP72, KBP169 and KBP610, with marked recombination deificiency (300 to 15,000 times) at 42 degrees C, were UV resistant; their sensitivity to mitomycin C was altered only slightly or not at all. Mutation KBP72 was co-transduced with ilv (83 unit on E. coli genetic map). The mutant is not able to form a functional recombinat structure. Two other mutations are located between 0 and 19 unit of the genetic map.     

Assembly-defective OmpC mutants of Escherichia coli K-12.

Novel ompC(Dex) alleles were utilized to isolate mutants defective in OmpC biogenesis. These ompC(Dex) alleles also conferred sensitivity to sodium dodecyl sulfate (SDS), which permitted the isolation of SDS-resistant and OmpC-specific phage-resistant mutants that remained Dex+. Many mutants acquired resistance against these lethal agents by lowering the OmpC level present in the outer membrane. In the majority of these mutants, a defect in the assembly (metastable to stable trimer formation) was responsible for lowering OmpC levels. The assembly defects in various mutant OmpC proteins were caused by single-amino-acid substitutions involving the G-39, G-42, G-223, G-224, Q-240, G-251, and G-282 residues of the mature protein. This assembly defect was correctable by an assembly suppressor allele, asmA3. In addition, we investigated one novel OmpC mutant in which an assembly defect was caused by a disulfide bond formation between two nonnative cysteine residues. The assembly defect was fully corrected in a genetic background in which the cell's ability to form disulfide bonds was compromised. The assembly defect of the two-cysteine OmpC protein was also mended by asmA3, whose suppressive effect was not achieved by preventing disulfide bond formation in the mutant OmpC protein.     

Identification and characterization of a cyanate permease in Escherichia coli K-12.

Escherichia coli contains an inducible enzyme, cyanase, that catalyzes the decomposition of cyanate into ammonia and bicarbonate. The gene encoding cyanase, cynS, was cloned and found to be on a DNA fragment that contained the lac operon. Characterization of a plasmid encoding cyanase indicated that a 26-kilodalton (kDa) protein of unknown function was also induced by cyanate (Y-C. Sung, D. Parsell, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 169:2639-2642, 1987). The gene encoding the 26-kDa protein was located between cynS and its promoter, indicating the existence of a cyn operon. The 26-kDa protein was identified as a cyanate permease that transports exogenous cyanate by active transport. E. coli was shown to contain a cyanate transport system that is energy dependent and saturable by cyanate.     

Threonine analogue resistant mutants of Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 resistant to a threonine analogue (-amino--hydroxy valeric acid) were predominantly resistant to ethionine and overproduced both threonine and methionine (2 mg/ml each). Novelty of the mutants is discussed.