Six mutants of Escherichia coli K12 AB1157 were isolated, the catalase activity of which was about from 10% to 60% of the activity of the wild type. Four mutants showed nomal composition and contents of the cytochromes . Two mutants showed nomal cytochrome composition, although having slightly less amounts of cytochromes than those of wild type. Measurement of the pyridine hemochrome spectra with the mutant cells having the 10west catalase activity, 10% of the parent, showed that protoheme content of the mutant was about 70% of the parent/s level. These results suggest that the defects in catalase activity of mutants are not due to heme deficiency, but due to some defect in synthesis of catalase apoprotein, caused by mutation of a cat gene as described previouly. All of the catalase-less mutants isolated in this work were most probably caused by one point mutation, so that the two mutants with about 10% of the catalase activity of parent mayby useful for the analysis of the catalase gene in Escherichia coli

Hashimoto Tateo

Nagasaki University's Academic Output SITE: NAOSITE

Sci. Bull. Eac. Educ, Nagasaki Univ. No. 30, pp. 45-50 (1979)Regulation of Catalase Synthesis in Escherichia coli K12I. Further Isolation of Catalase-less Mutantscaused by Deficiency Apo-proteinsTateo HASHIMOTOScience Education, Faculty of EducationIntroductionCatalase (EC 1. 11. 1. 6) is constituted of protoheme and apoprotein, therefore itssynthesis is regulated by the synthesis of two components. Several studies have been re-ported as for the regulation of catalase synthesis in microbes.u.2,3,4.5,6,7) g云S左rman et al.(1968) determined the loci of the gene that controls the catalase synthesis in Escherichiacolt, but the catalase-less mutants he used were the hemin less-mutants (8). All of theother catalase-less mutants already reported in some bacteria were heminless-mutants.We have isolated a catalase-less mutant, which is defective in apoprotein moiety, froma Hfr strain of Escherichia coliiw'. In this study, using E. coli K12 AB1157 which is aF -strain and is applicable for genetic analysis, it was attempted to isolate the catalase-less mutants that are deficient in catalase apoprotein.Materials and MethodsOrganism ;Escherichia colt K12 AB1157, whose genetic markers are F~, gal~, his",arg , str+, xyl~, mt「, thi , thr , ara~ leu , pro , lac , wasused in this study.Isolation of Mutants ; The procedure for isolation of mutants was followed as descri-bed before<10)Assay of Catalase Activity ; (i) Titration with permanganate : The activity wasassayed with intact cells as described beforecio). (ii) Spectrophotometry with Extracts :cells were disrupted with a Bronson Sonifier (150w) treating tree times each for 30seconds. Extracts were obtained after unbroken cells and cell debris were removed bycentrifugation at 6,500 x g for 15min. Catalase activities were measured with HitachSpectro Photometer Model 356 in quarty cuvettes of lOmm light path. The assay procedurefor measurement of catalase was followed by refering the method of Beers and Sizercn)Reference cuvette contained 2ml of cell extracts, 0.8ml of 0.02M phosphate buffer (pH7.0) and 0.4ml of water. Sample cuvette contained 2ml of cell extracts, 0.8ml of the0.02M phosphate buffer, 0.2ml of water and 0.2ml of 0.2M hydrogen peroxide. Reactionswere started by the addition of hydrogen peroxide and followed at 240nm for 2min at room46 Tateo　HAsHIMoTotemperature．Specific　activities　were　calculated　as　the　decrease　of　the　optical　density　ofhydrogen　peroxide　per　min　per　mg　nitrogen　of　the　extracts．　　　Determination　of　Nitrogen；The　nitrogen　contents　of　the　intact　cells　were　determinedindirectry　using　a　standard　curve　drawn　between　the　optical　dencity　value　at660nm　andmicro　Kjeldahl　method．The　nitrogen　contents　of　the　ce11－free　extracts　were　determinedindirectry　using　a　standard　curve　drawn　between　the　amount　of　protein　and　nitrogen　perml　of　extracts，assayed　by　the　buiret　and　micro－Kjeldahl　method，respectively．　　　Measurement　of　Spectra　l　The　reduced　cytchrome　spectra　and　pyridine　hemochromespectra　were　measured　by　the　method　described　befor（10）．　Oxidized　minus　reductedcytchrome　spectra　were　measured　by　recording　the　optical　density　of　the　reduced　sampleagainst　oxidized　sample．The　cuvett　of　oxidized　sample　contained　cells（6。75mg　nitrogenper　cuvett）and　O．02M　phosphate　buffter　in　total　volume　of3ml　and　cytochromes　wereoxidized　by　addition　of　two　drops　of　H202．The　cuvette　of　the　reduced　sample　containedcells（6．75mg　nitrogen　per　cuvett），0．02M　phosphate　buffer　and　a　few　grains　of　Na2S204in　total　volume　of3m1．Spectra　were　measured　with　a　Hitachi　two　wavelength　spectrophotometer　Model356．ResultsandDiscussion　　　After　nitrosoguanidine　treatment　was　repeated　three　times，25colonies　that　didn／tform　bubbles　from　hydrogen　peroxide　were　isolated　from　about5000colonies　on　nutrient－agar　plats。These　colonies　were　transfered　to　agar　slants，cells　grown　under　the　definedcondition　and　subjected　to　duplicate　quantitative　assay　of　catalase　activity　with　titrationmethod．The　result　of　this　measurement　showed　that　cells　from19colonies　had　almostnomal　Ievel　of　catalase　activity，but　cells　from　six　colonies，designated　by　the　symbols，cat2，cat7，cat8，cat11，cat14，cat17，showed　distinctIy　low　activity．　　　The　measurement　of　catalase　activity　by　cells　from6colonies　were　repeated　fivetimes　and　the　activity　of　parent　cells　was　usually　measured　together　with　the　mutants．The　relative　activities　of　the　cells　from6colonies　were　shown　in　Fig1．The　reIativeactivities　were　calculated　as　the　mean　value　of　catalase　activities　of　mutants　obtained　infive　independent　measurements　divided　by　the　mean　value　of　parent／s　catalase　activity．The　mutant　having　the　lowest　activity　was　designated　as　cat　ll　and　the　activity　was　about10percent　relative　to　the　activity　of　parent。The　mutant　having　the　highest　activity　wasdesignated　as　cat17，whose　activity　was　about70percent　reIative　to　the　activity　of　parent．To　confirm　these　results，photo　electric　method　were　used　to　measure　the　enzyme　activitywith　ce11－free　extracts．The　results　were　shown　in　Fig2．　　　The　pattem　of　the　relative　activities　shown　in　Fig2were　as　same　as　that　shown　inFig1．As　evident　from　Fig　l　and　Fig2the　genes　controling　catalase　in　Eschε万ch∫α60露K12AB1157were　damaged　to　the　different　extent　by　the　nitrosoguanidine　treatment．Itmight　be　that　cat　ll　and　cat1410st　the　most　important　site　of　genes　controling　catalase，while　cat2and　cat1710st　the　most　minor　site　of　the　genes　or　lost　more　easily　reversiblesite　of　the　genes．No　colony　that　completely　lost　catalase　activity　was　isolated。In　thisRegulat圭on　of　Catalase　Synthesis　in　E5‘hεパ6hズαcoあ　K12 47parentCat2cat7cat8cat11．cat14cat　I7Rel飢ive　Activity50　　　　　　　　　100parentcat2・cat7cat8cat11cat14’cat17Relatfve　Actlv重ty　50 100Fig1．　Relative　activity　of　catalase　of　intact　　　　　　　cells　measured　with　titration　by　　　　　　　KMnO4　　The　assay　mixture　and　the　assay　procedurewas　discribed　beforeく10）．　The　measurementof　catalase　activity　was　done　five　times．Thevalue　in　this　figure　are　the　mean　of　fiverelative　activities．The　relative　activities　areculculated　by　the　catalase　activities　in　mutantsdevided　by　that　of　the　parent．Fig2．　　Relative　activity　of　catalase　in　extracts　　　　　　　measured　with　spectrophotometric　　　　　　　method　The　proce（lure　to　obtain　ce11－free　extracts　andassay　of　the　activity　are　discribed　iガ電Materialsand　Method”。The　relative　activities　in　thisfigure　are　the　means　of　four　determinations．respect，the　genes　controling　catalase　might　be　difficult　to　be　damaged　by　nitrosoguani－dine　treatment．From　Fig　l　and　Fig2，it　was　shown　that　the　result　of　the　measurementsof　catalase　activity　with　intact　cell　were　almost　the　same　as　that　of　the　measurement～vithceII－free　extracts．　　　The　curves　in　Fig3are　reduced　cytochrome　spectra　of　parent　and　catalase－less　mu－tants．The　peaks　and　shoulders　of　the　spectra　are　due　to　the　presence　of　cytochrome　b1（559，530，428nm），cytochrome　d（635nm），and　cytochrome　a1（595nm）．The　peak　at500nm　of　the　spectra　of　cells　from　parent，cat2，　cat　11，　cat　14，　cat　17　colonies　areprobably　due　to亡he　presence　of　porphyrin　because　the　peak　disapPeared　in　the　oxidized－minus　reduced　cytochrome　spectra（Fig4）．　　The　known　catalase－1ess　mutants　except　one　mutant（10）so　far　reported　lacked　cyto－chrome　components　but　the　mutants　reported　in　this　study　have　the　nomal　quantity　ofcytochrome　as　shown　in　Fig3and　Fig4，although　the　quantity　of　cytochrome　in　cat7was　about60％of　that　of　the　parellt。The　mutant　cat　ll　has　almost　the　same　quantity　ofcytochromes　as　that　of　the　parent　but　has　only10％of　catalase　activity　of　the　parent．Toexamine　the　ability　of　the　present　catalase－less　mutants　to　synthesize　heme，the　pyridine48 Tateo　HAS田MOTOゆ1卜．cat17cat14catユrcat8cat2pa（r畦ntcat7諺　　ocat17cat7cat14cat　llcat8cat2parehtり　§cat17．cat14cat7cat1工cat8cat2parent400　450　500　550　600　650　700WAVELENGTH（NMン400　450　P500　550　600　650　700WAVELENdTH（NM）Fig3．　Reduced　cytochrome　spectra　with　　　　　　intact　ce11S　　Each　cuvette　conteined6．7mg　nitrogen　ofintact　cells．Cytochromes　were　reduced　withNa2S204and　spectm　were　measured　asdiscribed　before（10）．Fig4．Oxidized　minus　reduced　cytochrome　　　　　spectra　with　intact　cells　　Each　cuvette　contained6。75mg　nirogen　ofintact　celL　Cytochrone　were　reduced　withNa2S204and　oxidized　with2drops　of3％H202．Procedures　for　measurement　are　described　in璽Materials　and　Methods”81卜parenセcat11hemochrome　spectra　of　the　mutant，cat11，was　measured（Fig5）．The　protohemecontens　of　parent　and　cat　ll　were　culcu－1ated　from　difference　in　absorbancy　bet－ween　at557nm（peak）and　at570nm，divided　by　pmtein－nitrogen　per　ml　of　thece11－free　extracts　in　optical　cuvettes．Theresult　of　such　calculation　showed　that400　450　500　550　600　650　700　　　　　　　　　　　WAVELENGTH（暦M）一Fig5．　Pyridine　hemochrome　spectra　　Each　curette　conteined3．1mg　nitrogen　ofcell－free　extracts．Procedures　for　measurementwere　discribed　before（10）．protoheme　content　of　cat　ll　was　about70％　o壬that　of　the　parent．　　　Although　the　ability　of　the　presentcata1＆se－1ess　mutants　to　synthesize　pmto－heme　fell　down　a　little　from　that　of　theparent， the　contents　of　cytochrom　mRegulation of Catalase Synthesis in Eschericnia coli K12 49 the mutants was almost the same with that of the parent. These facts suggest that the decrease of catalase activities in the present mutants was caused by the deficiency in catalase-apoprotein but not by the deficiency in heme-prosthetic group. The mutant having no catalase activity was not isolated through this and previous work and the mutant with the most diminished catalase activity still had 10~ of the catalase activity of the parent. It is possible that a catalase isozyme occuping nomaly about 10% of the total activity is present in Escherichia coli. Although the mutant isolated in the previous study required casamino acids for growth, all of the mutants isolated in this work required only those amino acids that are required by the parent. Probably, all of the present mutants were caused by one point mutation . By the present method for isolating the catalase-less mutants , no mutant that completely lost the ability to synthesize heme has not been isolated. Su m mary Six mutants of Escherichia coli K12 AB1157 were isolated, the catalase activity of which was about from 10~ to 60% of the activity of the wild type. Four mutants showed nomal composition and contents of the cytochromes . Two mutants showed nomal cytochro-me composition, although having slightly less amounts of cytochromes than those of wild type. Measurement of the pyridine hemochrome spectra with the mutant cells having the 10west catalase activity, 10% of the parent, showed that protoheme content of the mutant was about 70% of the parent/s level. These results suggest that the defects in catalase activity of mutants are not due to heme deficiency, but due to some defect in synthesis of catalase apoprotein, caused by mutation of a cat gene as described previouly. All of the catalase-1ess mutants isolated in this work were most probably caused by one point mutation, so that the two mutants with about 10% of the catalase activity of parent may by useful for the analysis of the catalase gene in Escherichia coli. 1) 2) 3) 4) 5) Literature Cited Chantrence , H. & Courtois, C. 1974 Formation de catalase induite par 1/0xygene chez la levure. Biochim. Biophys. Acta 14 : 379-400 Claytone , R. K . 1960 a. Physiology of induced catalase synthesis in Rhodopseudomonas spheroides. J. Cell. Comp. Physiol. 55 : 1-7 Claytone , R . K . 1960 b Protein synthesis in the induced formation of catalase in Rhodopseudo-monas spheroides. J. Biol. Chem. 235 : 405-407 Cox, R . & Charles, H . P. 1973 Porphyrin-accumlating mutants of Escherichia coli. J . Bacteriol. 113 : 122-132 Finn, G.J. & Condon, S. 1975 Regulation of catalase Salmonella typhimurium J. Bacteriol. 123 : 570-579 50 6) 7) 8) 9) 1 O) 11) Tateo HASHIMOTO Ishida , A . 1976 Effect of glucose and adenosine cyclic 31, 5f-mono phosphate on the induced formation of catalase in Escherichia coli. J. Gen. Appl. Microbiol. 22 : 35-41 Jacobs, N .J. , Maclosky. E.R. & Jacobs, J.M. 1967 Role of oxygen and heme in heme synthe-sis and the development of hemo protein activity in an anaerobically grown Staphylococcus. Biochim. Biophys. Acta 148 : 645-654 S~s~rman, A. , Surdeanu, M. , Szgli, G. , Horodniceanu, T. . Greceanu, V. & Dumitrescu, A. 1968. Hemin-deficient mutants of Escherichia coli K12. J . Bacteriol. 96 : 570-572 S~s~rman, A. , Sanderson, K.E. , Surdeanu, M. & Sonea, S. 1970 Hemin-deficient mutants of Salmonella Typhimurium. J . Bacteriol. 102 : 531-536 Hashimoto, T. , Nanda, G. , and Hino, S. 1979 Isolation of a Mutant of Escherichia coli defective in catalase apoprotein. J . Science of the Hiroshima Univ. vol. 17 Beers, R. F. JR. and I. W. Sizer 1952 A spectrophotometric method for the breakdown of hydrogen peroxide by catalase . J . Biol. Chem. vol. 195 

Regulation of Catalase Synthesis in Escherichia coli K12 : 1. Further Isolation of Catalase-less Mutants caused by Deficiency Apo-proteins

Six mutants of Escherichia coli K12 AB1157 were isolated, the catalase activity of which was about from 10% to 60% of the activity of the wild type. Four mutants showed nomal composition and contents of the cytochromes . Two mutants showed nomal cytochrome composition, although having slightly less amounts of cytochromes than those of wild type. Measurement of the pyridine hemochrome spectra with the mutant cells having the 10west catalase activity, 10% of the parent, showed that protoheme content of the mutant was about 70% of the parent/s level. These results suggest that the defects in catalase activity of mutants are not due to heme deficiency, but due to some defect in synthesis of catalase apoprotein, caused by mutation of a cat gene as described previouly. All of the catalase-less mutants isolated in this work were most probably caused by one point mutation, so that the two mutants with about 10% of the catalase activity of parent may
by useful for the analysis of the catalase gene in Escherichia coli

Hashimoto, Tateo

Nagasaki university's Academic Output SITE

http://naosite.lb.nagasaki-u.ac.jp/dspace/bitstream/10069/32694/1/kyoikuS30_045.pdf

Regulation of Catalase Synthesis in Escherichia coli K12 : 1. Further Isolation of Catalase-less Mutants caused by Deficiency Apo-proteins

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