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.     

Catalase HPI influences membrane permeability in Escherichia coli following near-UV stress

The katG gene in Escherichia coli encodes catalase HPI, which is involved in membrane transport and protects the cell during oxidative stress. Hydrogen peroxide (H2O2) induces synthesis of HPI. We examined the role of HPI in membrane permeability (proline uptake) following exposure to near-ultraviolet radiation (NUV). We found that NUV resulted in the same type of induction as H2O2. KatG::Tn10 cells experienced a large drop in uptake after NUV exposure, and levels remained low following incubation. A strain carrying a katG+ plasmid, however, showed considerably less decrease in uptake after NUV, and uptake quickly resumed upon incubation. Further, in an srd mutant which lacks 4-thiouracil, NUV resulted in only a small drop in proline uptake, which was immediately resumed.     

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, less than 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to beta-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.     

Growth of Escherichia coli in iron-enriched medium increases HPI catalase activity

Escherichia coli has two catalases, HPI and HPII. HPI is induced during logarithmic growth in response to low concentrations of hydrogen peroxide. This induction is OxyR-dependent. On the other hand, HPII is not peroxide-inducible but is induced in entry to the stationary phase. We demonstrate here that E. coli displayed higher HPI catalase activity when compared to the cultures that were grown in a normal medium, if grown in a medium supplemented with iron-citrate. Iron supplementation had no effect on HPII catalase. This increase of HPI activity was OxyR-independent and not observed in a Deltafur mutant. The physiological significance of the increase of HPI activity is unclear, but it appears that the katG gene that codes for HPI catalase is among the genes that are regulated by Fur.     

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.     

Catalases HPI and HPII in Escherichia coli are induced independently

Three strains of Escherichia coli differing only in the catalase locus mutated by transposon Tn10 were constructed. These strains produced only catalase HPI (katE::Tn10 and katF::Tn10 strains) or catalase HPII (katG::Tn10). HPI levels increased gradually about twofold during logarithmic growth but did not increase during growth into stationary phase in rich medium. HPII levels, which were initially threefold lower than HPI levels, did not change during logarithmic growth but did increase tenfold during growth into stationary phase. HPI levels increased in response to ascorbate or H2O2 being added to the medium but HPII levels did not. In minimal medium, any carbon source derived from the tricarboxylic acid cycle caused five- to tenfold higher HPII levels during logarithmic growth but had very little effect on HPI levels. Active electron transport did not affect either HPI or HPII levels.     

Oxidative stress effects on conjugational recombination and mutation in catalase-deficient Escherichia coli

The objective of the present investigation was to determine the effects on genetic recombination and mutation in Escherichia coli of either endogenous increases in oxygen radicals resulting from catalase deficiencies, or exogenous increases resulting from H2O2 treatment. Using the classical paradigm of Escherichia coli bacterial conjugation, strains deficient in the production of hydroperoxidase I (HPI) and/or hydroperoxidase II (HPII) were used as recipients in Hfr x F- matings. 'Background' recombination rates, measured by the rate of appearance of threonine prototrophs, was similar to wild-type levels in the HPI-deficient (katG) strain, but were significantly decreased in HPII- (katE) mutants. The addition of relatively nontoxic H2O2 concentrations (0.25 mmoles dm-3) to the mating mixtures stimulated recombination rates in wild-type and katE strains, but decreased rates in katG and katEkatG strains. A 0.5 mmoles dm-3 concentration of H2O2 inhibited recombination rates in all strains. In order to gauge the level of recA-dependent 'SOS' processes occurring under the experimental conditions, 'background' mutation rates were determined in both fluctuation and forward mutation (thyA) assays. Mutation rates in aerobically-grown cultures were increased up to 2.2-fold in katG and katEkatG strains. Treatment with relatively nontoxic H2O2 concentrations elevated the thyA mutagenesis up to 8-fold in catalase-deficient cultures. Furthermore, these studies along with data presented elsewhere show that the SOS phenotype of katEkatG is more resistant than that of katG strains. These studies clearly show that cellular oxidative stress occurring from catalase deficiency interferes with normal DNA metabolism.     

Homology among bacterial catalase genes

Catalase activities in crude extracts of exponential and stationary phase cultures of various bacteria were visualized following gel electrophoresis for comparison with the enzymes from Escherichia coli. Citrobacter freundii, Edwardsiella tarda, Enterobacter aerogenes, Klebsiella pneumoniae, and Salmonella typhimurium exhibited patterns of catalase activity similar to E. coli, including bifunctional HPI-like bands and a monofunctional HPII-like band. Proteus mirabilis, Erwinia carotovora, and Serratia marcescens contained a single band of monofunctional catalase with a mobility intermediate between the HPI-like and HPII-like bands. The cloned genes for catalases HPI (katG) and HPII (katE) from E. coli were used as probes in Southern hybridization analyses for homologous sequences in genomic DNA of the same bacteria. katG was found to hybridize with fragments from C. freudii, Ent. aerogenes, Sal. typhimurium, and K. pneumoniae but not at all with Ed. tarda, P. mirabilis, S. marcesens, or Er. carotovora. katE hybridized with C. freundii and K. pneumoniae DNAs and not with the other bacterial DNAs.     

The role of antioxidant systems in response of bacteria Escherichia coli to heat shock]

Shifting the temperature from 30 to 45 degrees C in an aerobic Escherichia coli culture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor. The expression was evaluated by measuring beta-galactosidase activity in E. coli strains that contained fusions of the antioxidant gene promoters with the lacZ operon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type and sodA sodB mutant cells to heat shock was almost the same. In the E. coli culture adapted to growth at 42 degrees C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30 degrees C. The temperature-adapted cells grown aerobically at 42 degrees C showed an increased ability to express the fused katG-lacZ genes.     

Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide, but not to isonicotinic acid: differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (Mr, 137) to isonicotinamide (Mr, 122), not to isonicotinic acid (Mr, 123), in the presence or absence of H2O2. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide,but not to isonicotinic acid: Differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (M_r, 137) to isonicotinamide (M_r, 122), not to isonicotinic acid (M_r, 123), in the presence or absence of H₂O₂. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.     

Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.     

结核分枝杆菌katG基因突变的研究

探讨编码过氧化氢-过氧化物酶的katG基因突变与结核分枝杆菌异烟肼(INH)耐药性的相关关系。根据结核分枝杆菌GenBank中的katG序列,自行设计特异性寡聚核苷酸引物,采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析和直接测序法(DS)分析结核分枝杆菌中katG基因突变情况。以HR37Rv标准株为对照。所有23株敏感菌均未有SSCP结果异常;35株耐药菌中,有2株(5．7％)katG基因扩增阴性,且发生在高度耐药菌中。进一步分析发现,SSCP法突变检出23株(65．7％),测序法突变检出24株(68．6％),符合率为95．8％(23／24)。参照测序法对耐药菌突变序列的分析结果,PCR—SSCP敏感、特异,可快速检测结核分枝杆菌katG耐药基因突变,有利于耐药结核分枝杆菌耐药性的快速检测。     

Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin. Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats

The biological activities of several derivatives of human proinsulin (HPI) containing peptide bond cleavages or peptide deletions in the connecting peptide region were examined in vivo in rats and in several in vitro systems. The two derivatives which were tested in vivo, split (32-33)HPI and des-(64,65)HPI, both demonstrated greater potency in lowering blood glucose than did intact HPI. The receptor binding affinities of split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, des-(33-56)HPI, des-(31,32)HPI, split (32-33)HPI, and split (56-57)HPI were examined in cultured IM-9 lymphocytes, freshly isolated rat adipocytes, and purified rat liver membranes and were compared to the binding of intact HPI and insulin. In these systems, HPI averaged approximately 1% of the activity of insulin. Modification of proinsulin in the connecting peptide region near the A-chain of insulin to form split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, or des-(33-56)HPI resulted in an increase in affinity for receptor binding ranging from 11 to 27-fold over that of intact HPI. In contrast, modifications near the B-chain of insulin to form either des-(31,32)HPI or split (32-33)HPI resulted in roughly a 5-fold increase in affinity, whereas a cleavage within the connecting peptide to form split (56-57)HPI showed only a 2-fold increase in affinity as compared to intact HPI. The biological potencies of these materials were examined in isolated rat adipocytes. At high concentrations (10(-7) M), each derivative produced the same maximal response. At lower concentrations, differences in the relative potencies paralleled the differences in receptor binding affinity previously noted.