Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase.

Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase (EC 2.3.2.2) were isolated after mutagenesis of cells with ethyl methanesulfonate. They lost the enzyme activity to different extents. The mutations of two mutants that had lost the enzyme activity completely were mapped at 76 min of the E. coli K-12 linkage map. These mutations made the cells neither nutrient requiring nor cold sensitive. The mutants leaked much more glutathione into the medium than the wild type. We propose the symbol ggt for these mutations.     

Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions.

The major cotransduction gap of the Escherichia coli chromosome extends from mini 31 to 34. We have inserted transposons through this gap which, by sequential transduction, link sbcA (min 29.8) with manA (min 35.7) and thus eliminate the gap. These results indicate that the length of DNA in the region, as measured by transduction, is not significantly different from the length obtained by conjugational time of entry. Since this segment of the E. coli chromosome has few known genes, these transposon insertions will be useful for genetic manipulations in the region of the gap. We describe the usefulness of these markers for rapidly mapping mutations which may be isolated in the region from min 27 to 37.     

Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli

The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.     

Isolation and characterization of Escherichia coli dnaA amber mutants.

Specialized transducing phage lambda (formula, see text) dnaA-2 was mutagenized, and two derivatives designated lambda (formula) dnaA17(Am) and lambda (formula) dnaA452(Am) were obtained. They did not transduce such mutations as dnaA46, dnaA167, and dnaA5 when an amber suppressor was absent, but they did so in the presence of an amber suppressor. By contrast, they transduced the dna-806 and tna-2 mutations in the absence of an active amber suppressor. The dna-806 and tna-2 mutations are known to be located very close to the dnaA gene, but in separate cistrons. When ultraviolet light-irradiated uvrB cells were infected with the derivative phages and proteins specified by them were analyzed by gel electrophoresis, a 50,000-dalton protein was found to be specifically missing if an amber suppressor was absent. This protein was synthesized when an amber suppressor was present. The dnaA17(Am) mutation on the transducing phage genome was then transferred by genetic recombination onto the chromosome of an Escherichia coli strain carrying a temperature-sensitive amber suppressor supF6(Ts), yielding a strain which was temperature sensitive for growth and deoxyribonucleic acid replication. The temperature-sensitive trait was suppressed by supD, supE, or supF. We conclude that, most likely, the derivative phages acquired amber mutations in the dnaA gene whose product is a 50,000-dalton protein as identified by gel electrophoretic analysis.     

Isolation and characterization of isoprene mutants of Escherichia coli.

Isoprenoid compounds are found in all organisms. In Escherichia coli the isoprene pathway has three distinct branches: the modification of tRNA; the respiratory quinones ubiquinone and menaquinone; and the dolichols, which are long-chain alcohols involved in cell wall biosynthesis. Very little is known about procaryotic isoprene biosynthesis compared with what is known about eucaryote isoprene biosynthesis. This study approached some of the questions about isoprenoid biosynthesis and regulation in procaryotes by isolating and characterizing mutants in E. coli. Mutants were selected by determining their resistance to low levels of aminoglycoside antibiotics, which require an electron transport chain for uptake into bacterial cells. The mutants were characterized with regard to their phenotypes, map positions, enzymatic activities, and total ubiquinone content. In particular, the enzymes studied were isopentenyldiphosphate delta-isomerase (EC 5.3.3.2), farnesyldiphosphate synthetase (EC 2.5.1.1), and higher prenyl transferases.     

Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli.

A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolic repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce beta-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3',5-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.     

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.     

Isolation and characterization of T-even ghost-tolerant mutants of Escherichia coli.

Mutants of Escherichia coli tolerant to the ghosts of T-even phages (T2, T4, and T6) have been isolated from a strain supersensitive to T6 phage. First, T6 supersensitive mutants were isolated from mutagenized E. coli W2252 by replica plating to T6 phage-overlaid agar. One of them, strain NM101, was mutagenized again, grown, and then plated with a high multiplicity of T4 and T6 ghosts. Surviving cells were checked for tolerance to ghosts and adsorption of phages. One such ghost-tolerant mutant, strain GT29, was tolerant to ghosts of both T4 and T6 phages and sensitive to T2 ghosts. This mutant was also sensitive to ethylenediaminetetraacetic acid and penicillin G and intermediately sensitive to acriflavine, sodium dodecyl sulfate, sodium deoxycholate, actinomycin D, and lysozyme. Another mutant, strain GT62, was tolerant not only to T4 and T6 ghosts but also to T2 ghosts. It was sensitive to sodium dodecyl sulfate, sodium deoxycholate, penicillin G, acridine orange, actinomycin D, phenethyl alcohol, and novobiocin and intermediately sensitive to acriflavine and lysozyme. Spontaneous revertants of strain GT62 were isolated with a frequency of 2.7 X 10(-9). It is suggested that ghosts attack host bacteria indirectly through the cell surface by a mechanism similar to the transmission hypothesis that was originally adopted by Nomura (1967) to explain the mechanism of the action of colicins, and that our ghost-tolerant mutants presumably have defects in the cell surface.     

Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.     

Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation

Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways.     

Properties of Escherichia coli mutants lacking membrane-derived oligosaccharides

Membrane-derived oligosaccharides (MDO) consist of branched substituted beta-glucan chains and are present in the periplasmic space of Escherichia coli and other gram-negative bacteria. A procedure for the isolation of mutants defective in MDO synthesis is described. Their phenotype was compared with a mdoA mutant previously identified, and they are mapped in the mdoA region. Mutants lacking MDO showed imparied chemotaxis on tryptone swarm plates, a reduced number of flagella, and an enhanced expression of the OmpC porin. Revertants able to form swarm rings again had regained the ability to synthesize MDO and showed the wild-type porin pattern. A second group of chemotactic revertants were mutated in the ompB gene region involved in osmoregulation, and they were still devoid of MDO. These findings provide evidence for a link between MDO biosynthesis and other functions of E. coli related to its adaptation to the environment.     

Isolation and characterization of cAMP suppressor mutants of Escherichia coli K12

Summary We have isolated spontaneous and chemically induced revertants of cya mutant strains of Escherichia coli. Three different classes of revertants were obtained. One class consisted of primary site revertants; a second class was pseudorevertants that had phenotypically reverted to wild type but retaining the original cya mutation and the third class of revertants, designated csm, were pseudorevertants hypersensitive to exogenous cAMP. Transductional analysis of the csm mutation indicated the mechanism of suppression in these strains was intergenic. The csm mutation and hypersensitivity to cAMP map in or near the crp gene. Growth of the csm strains of PTS (phosphoenolpyruvate phosphotransferase system) and non-PTS substrates was inhibited by 5 mM cAMP. The csm strains were found to accumulate toxic levels of methylglyocal when grown on non-PTS substrates in the presence of exogenous cAMP. All csm strains were sensitive to catabolite repression mediated by -methylglucoside. Revertants selected as resistant to cAMP fell into four major classes that could be distinguished by their fermentation patterns in the presence and absence of cAMP as well as by their growth response to streptomycin in the presence of cAMP.Paper No. 6623 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650, USA     

Isolation and characterization of respiratory-deficient mutants of Escherichia coli K-12.

Several mutants of Escherichia coli K-12 defective in aerobic metabolism were isolated. One such mutant was found to be deficient in cytochromes, heme, and catalase. Aerobically grown cells did not consume oxygen and could grow only on fermentable carbon sources. Supplementation of the growth medium with delta-aminolevulonic acid, protoporphyrin IX, or hemin did not restore aerobic metabolism. The lack of heme and catalase in mutant cells grown on glucose was not due to catabolite repression, since the addition of exogenous cyclic AMP did not restore the normal phenotype. When grown aerobically on complex medium containing glucose, the mutant produced lactic acid as the principal fermentation product. This pleotropic mutation was attributed to an inability of the cells to synthesize heme, and preliminary data mapped the mutation to between 8 and 13 min on the E. coli genome.     

Isolation and characterization of cysK mutants of Escherichia coli K12.

cysK mutants, deficient in O-acetylserine sulphydrylase A [O-acetyl-L-serine acetate-lyase (adding hydrogen-sulphide); EC 4.2.99.8], were isolated as strains resistant to selenite or giving a black colour reaction on bismuth citrate indicator medium. All were resistant to the inhibitor I,2,4-triazole. Four independent mutants were found which possessed lowered levels of O-acetylserine sulphydrylase activity and also partially constitutive levels of NADPH-sulphite reductase [hydrogen-sulphide: NADP+ oxidoreductase; EC I.8.I.2]. Strains containing both a cysE mutation and a cysK mutation lacked the constitutive levels of NADPH-sulphite reductase showing that these levels were due to the in vivo concentration of the inducer, O-acetylserine. The cysK locus was found to be 81% cotransducible with the ptsI gene.     

Isolation and characterization of visible light-sensitive mutants of Escherichia coli K12

Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated. Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA. The sixth mutant, which was isolated from bacteria that carried the visA+/visA+ diploid allele, is defective in a gene that maps near 63 minutes on the linkage map, which has been designated visB. These mutant strains of bacteria are killed by illumination with visible light. The effective wavelength of the light is around 460 nm. The nucleotide sequence of the visA gene was determined. As a result of a search for homologous products, we found that visA may be identical to hemH, the structural gene for ferrochelatase which catalyzes a final step in the biosynthesis of heme. A possible mechanism for the killing of the visA mutant bacteria is discussed.