Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalase-peroxidase hydroperoxidase I in Escherichia coli.

A locus unlinked to either katE or katF that affected catalase levels in Escherichia coli was identified and localized between metB and ppc at 89.2 min on the genome. The locus was named katG. Mutations in katG which prevented the formation of both isoenzyme forms of the bifunctional catalase-peroxidase HPI were created both by nitrosoguanidine and by transposon Tn10 insertions. All katG+ recombinants and transductants contained both HPI isoenzymes. Despite the common feature of little or no catalase activity in four of the catalase-deficient strains, subtle differences in the phenotypes of each strain resulted from the different katG mutations. All three mutants caused by nitrosoguanidine produced a protein with little or no catalase activity but with the same subunit molecular weight and with similar antigenic properties to HPI, implying the presence of missense mutations rather than nonsense mutations in each strain. Indeed one mutant produced an HPI-like protein that retained peroxidase activity, whereas the HPI-like protein in a second mutant exhibited no catalase or peroxidase activity. The third mutant responded to ascorbate induction with the synthesis of near normal catalase levels, suggesting a regulatory defect. The Tn10 insertion mutant produced no catalase and no protein that was antigenically similar to HPI.     

Intracellular location of catalase-peroxidase hydroperoxidase I of Escherichia coli

The catalase-peroxidase hydroperoxidase I of Escherichia coli has been confirmed to be located in the cytoplasm using two independent methods. Catalase activity was found predominantly (> 95%) in the cytoplasmic fraction following spheroplast formation. The cytoplasmic enzyme glucose-6-phosphate dehydrogenase and the periplasmic enzyme alkaline phosphatase were used as controls. The second method of immunogold staining for the enzyme in situ revealed an even distribution of the enzyme across the cell.     

Purification and characterization of hydroperoxidase II of Escherichia coli B.

The second heme-containing hydroperoxidase isozyme (HP-II) has been isolated from aerobic cultures of Escherichia coli B. The protein exists as a stable tetramer of subunits of equal size, with a combined molecular weight of 312,000. The heme spectrum of HP-II is unusual, in that it exhibits two absorbance maxima at 407 and 591 nm; the alkaline pyridine hemochromogen spectrum shows maxima at 425, 559, and 609 nm. HP-II differs in several respects from the HP-I isozyme previously reported (Claiborne, A., and Fridovich, I. (1979) J. Biol. Chem. 254, 4245-4252). Thus HP-II is virtually devoid of peroxidatic activity toward o-dianisidine but has a 6-fold higher catalatic activity than HP-I. Antisera to HP-II do not cross-react with HP-I, and analyses of chymotryptic and cyanogen bromide digests suggest differences in primary structure between these two isozymes.     

Physical characterization of katG, encoding catalase HPI of Escherichia coli

The gene encoding the bifunctional catalase-peroxidase HPI from Escherichia coli was located on a 3.8-kb HindIII fragment of the Clarke and Carbon plasmid pLC36-19 using transposon Tn5 insertions. This fragment was subcloned into the HindIII site of pAT153 to create pBT22. The size of the insert was reduced by BAL 31 digestion of one end to an apparent minimum size for catalase expression of approx. 2.5 kb as determined by complementation and expression in maxicell strains. Further reduction in size or digestion from the opposite end inactivated the gene. The location and orientation of the promoter at the 0 kb end of the insert in pBT22 was confirmed by cloning a 320-bp BglII fragment into the promoter-cloning vector pKK232-8. Differences in the Southern blots of genomic DNA from a wild-type strain and a katG17::Tn10 mutant digested with HincII and probed with pBT22 confirmed that the transposon previously mapped in katG was located in the 2.5-kb coding region for HPI.     

Role of glutathione in regulation of hydroperoxidase I in growing Escherichia coli.

To examine role of glutathione in regulation of catalases in growing Escherichia coli, katG::lacZ and katE::lacZ fusions were transformed into a glutathione-deficient Escherichia coli strain and wild-type parent. In the absence of H2O2 and in the presence of the low H2O2 concentrations (0.1-3 mM), the gshA mutation stimulated katG::lacZ expression and the total catalase activity in exponential phase. In the absence of H2O2, the mutation in gshA also stimulated katE::lacZ expression. At higher H2O2 concentrations, the gshA mutation suppressed katG::lacZ expression and catalase activity. In stationary and mid-exponential phases, the intracellular concentrations of H2O2 in the gshA mutant were markedly increased compared to those in the wild type. These results suggest that glutathione may be involved in regulation of catalases.     

Interactions between mutations affecting ribosome synthesis in Escherichia coli

RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA. The ribosome mutation did not by itself lead to relaxedness. The relaxed mutation could be expressed in organisms that contained the ribosome mutation.     

Regulatory gene mutations affecting arginine biosynthesis in Escherichia coli

Summary Mutants of Escherichia coli with altered regulation of arginine biosynthesis were isolated. The alterations in all of the mutants with increased levels of the biosynthetic enzymes were found to map in the argR locus. The mutants were grouped into three classes based on their effect on the regulatory behavior. Complementation studies with stable merodiploid strains demonstrated that the derepressed synthesis in the mutants was recessive to wild-type regulation.     

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

Summary A streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.     

Spectinomycin resistance mutations affecting the stability of sex-factors in Escherichia coli

Some mutations to Spectinomycin resistance prevent the maintenance of sex-factors in Escherichia coli cells growing at high temperature (41 °C).     

Isolation and characterization of regulatory mutations affecting the expression of the guaBA operon of Escherichia coli K-12

Summary We isolated strains of Escherichia coli K 12 in which the lac structural genes were fused to the structural genes of the guaBA operon. These strains were used to isolate regulatory mutations that increased the expression of the guaBA operon under normal repressing conditions as compared to the wild type parental fusion strain. Three classes of guaBA specific regulatory mutations were identified. Class I regulatory mutations were trans-acting and unlinked to the guaBA operon as shown by bacteriophage P1 transduction. Class II regulatory mutations were tightly linked to the guaBA operon, cis-dominant to the wild type allele in a cis-trans analysis and were regarded as control region mutations. Class III regulatory mutations were tightly linked to the guaBA operon and trans-recessive to the wild type allele in a cis-trans analysis. We have designated the locus responsible for the class III regulatory mutations as guaR. The guaR locus is tightly linked and was mapped to the counterclockwise side of the guaBA operon. The guaR locus is proposed to specify a trans acting regulatory element involved in the regulation of the guaBA operon.