Isolation and mapping of Escherichia coli K12 mutants defective in Tn9 transposition

Summary Five mutants (called tnm) of Escherichia coli with impaired ability for transposition of Tn9 were isolated after treatment with ethyl methanesulfonate (EMS) or N-methyl-N-nitro-N-nitrosoguanidine (NG).The map locations of the tnm mutations were deterimined by a combination of Hfr matings, F episome complementation and P1 transductional mapping. The data obtained show that the five tnm mutations are located near 91 min on the Escherichia coli linkage map and are cotransducible with the metA marker with a frequency of 3%–4%. Introduction of F plasmids containing this region complements the Tnm^- phenotype for the two mutants tested i.e. tnm-1 and tnm-2 are recessive in tnm ⁺/tnm^-merodiploids.     

Isolation of haemin-requiring mutants of Escherichia coli K12.

Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen III cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC.     

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.     

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 a new type of Escherichia coli K12 phoB mutants

A new type of Escherichia coli K12 phoB mutant was isolated as 5-fluorouracil-plus-adenosine-resistant derivatives of a upp phoS,T strain. Such phoB mutants (called type III) differ from previously described phoB mutants (types I and II) in the synthesis pattern of phosphate-regulated periplasmic proteins P4a and 30.5 K, sensitivity to toxic compounds, and several other phenotypic characters. The analysis of isogenic strains carrying phoB mutations (type I, II or III) showed that; the phoB gene product exerted (i) a positive control over the synthesis of periplasmic proteins 30.5 K, 11.5 K, and 9 K, inner membrane proteins 32 K and 17.5 K, and outer membrane protein Tsx, (ii) and a direct or indirect negative control over the synthesis of a 20 K phosphate-inducible periplasmic protein.     

Genetic recombination in Escherichia coli. IV. Isolation and characterization of recombination-deficiency mutants of Escherichia coli K12

19 independent recombination-deficient mutants were isolated. 7 carried mutations that mapped near or in the recB and recC genes between thyA and argA. 10 mutants carried mutations cotransducible with pheA and exhibited no complementation with recA in temporary zygotic diploids.     

Positive control of sulphate reduction in Escherichia coli. Isolation, characterization and mapping of cysteineless mutants of E. coli K 12

To determine to what extent the biosynthesis of cysteine in Escherichia coli resembles that in Salmonella typhimurium, the following experiments were performed. (1) Mutants of E. coli K 12 deficient in the biosynthesis of cysteine were isolated. (2) These mutants were classified by nutritional and biochemical criteria; some mutants lacked a single enzyme of sulphate reduction, other mutants appeared to lack two or more enzymes. (3) The genetic map predicted from the biochemical data alone is shown to be incorrect, and an alternative map, consistent with the genetic data, is proposed for the cys mutants of E. coli.     

Isolation of Escherichia coli K12 mutants with deficient in precise excision of transposons

Plasmid pNM1, the derivative of R100.1, has been constructed by insertion of transposon Tn5 into structural tet genet (Tn10) of the parental plasmid. The frequency of precise excision of Tn5 from plasmidic genome is 10(-5). The high frequency of precise excision obtained in this system permits one, to use it for isolation of mutants having low frequencies of precise excision. Two mutants were isolated in which the frequencies of precise excision of Tn5 were decreased for two orders. The pex1 and pex2 mutations responsible for the effect decrease the precise excision of Tn5 from R100.1 as well as from RP4 genomes.     

Microcin-E492-insensitive mutants of Escherichia coli K12

Mutations in three Escherichia coli K12 genes, tonB, exbB and the newly discovered semA, reduce sensitivity to the low Mr polypeptide antibiotic microcin E492. The products of the tonB and exbB genes were previously shown to be involved in the uptake of siderophore-complexed iron and in the action of a number of colicins. Strains mutated at or close to semA (collectively referred to as sem mutations) remained fully sensitive to these colicins, and grew as well as wild-type strains under conditions of iron starvation. Expression of a number of sem-lacZ operon fusions was not affected by iron limitation, and sem mutations did not affect the production of iron-regulated outer membrane proteins which are known or thought to be involved in iron uptake. Hfr conjugation and P1 phage transduction experiments indicated that semA is located close to pabB at 40 min on the E. coli K12 chromosome. This places semA close to the mng locus, wherein mutations result in decreased manganese sensitivity. However, strains carrying the semA mutation exhibited increased manganese sensitivity.     

Isolation of vitamin B 12 transport mutants of Escherichia coli

Escherichia coli KBT001, a methionine-vitamin B(12) auxotroph, was found to require a minimum of 20 molecules of vitamin B(12) (CN-B(12)) per cell for aerobic growth in the absence of methionine. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and penicillin selection, two kinds of B(12) transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B(12) to the cell and were unable to grow in the absence of methionine even with CN-B(12) concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B(12) uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B(12) taken up into other cobalamins. In the absence of methionine, some of these strains (e.g., KBT103) were able to grow on media containing 1 ng of CN-B(12)/ml, whereas others (e.g., KBT041) were unable to grow with any of the CN-B(12) concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B(12) but gave a substantial decrease in the secondary rate. Trace amounts of B(12)-binding macromolecules were released from the cells by the osmotic shock, but only from strains such as KBT001 and KBT041 which possessed an active initial phase of CN-B(12) uptake. These results are interpreted as being consistent with the view that the initial CN-B(12) binding site which functions in this transport system is probably bound to the cell membrane.     

Isolation and identification of mutants constitutive for aspartokinase III synthesis in Escherichia coli K 12.

We devised a procedure in order to isolate, in Escherichia coli, constitutive mutants for aspartokinase III synthesis, the first enzyme of the lysine regulon. It consists of the introduction of a limiting step in lysine biosynthesis, by the use of the partial suppression of a nonsense mutation. For the first time we could isolate many constitutive mutants. Their characteristics (cotransduction with the lysC structural gene; no effect on the synthesis of other enzymes of the regulon; cis-dominance) lead to classify these mutations as operator-type. The fact that no repressor mutations could be isolated is discussed.     

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.