Light Requirement forInduction andContinuous Accumulation of anAmmonium-Inducible NADP-Specific Glutamate
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Theammonium-inducible NADP-specific glutamate dehydrogenase of Chlorella sorokiniana wasshown torequire light forbothits induction by ammonia inuninduced cels, anditscontinuous accumulation infully induced cells. Addition ofammonia touninduced cells inthelight resulted ina35-minute induction lagfollowed bylinear andcoincident increases in enzyme activity andantigen. Enzyme activity wasnotinduced inthedark; however, transfer ofthese cells tothelight resulted inanimmediate increase inenzyme activity andantigen. Theabsence ofaninduction lag suggested that mRNAsequences and/or anenzyme precursor with differentantigenic properties thantheactive holoenzyme accumulated incells inthedarkinammonium medium. Whenfully induced cells weretransferred tothedark, theactivity oftheenzyme quickly ceased toaccumulate. Incontrast totheNADP-speclfic isozyme, thecells also contain aconstitutive NAD-specific isozyme which wasshown toaccumulate incells inthe darkineither ammonium ornitrate medium. synchronous cells growing inthecontinuous presence ofinducer foranentire cell cycle (10, 11). Thislatter experimental approach revealed theoperation ofaregulatory system whichalters the timing between genereplication andtheexpression ofnewly. replicated genes incells growing atdifferent rates inammonium medium. Underthese different cell cycle conditions, theactivity oftheNAD-GDHincreased inatypical steppattern during the last 0.5hofthecell cycle. Instudies onthecell cycle inducibility oftheNADP-GDH,it wasobserved that theinitial rate ofenzymeinduction (i.e., enzyme potential) wasproportional totherateofaccumulation oftotal cellular protein (21, 22). Since theaccumulation rateofcellular protein hasbeenobserved tobeproportional totheeffective light intensity percell (17, 19), itseemed possible that theinduction or accumulation oftheNADP-GDHmight belight-dependent. The experimental evidence described inthepresent paperisconsistent withalight requirement forboththeinduction andcontinuous accumulation oftheNADP-GDHinChlorella cells inammonium medium.Keywords:
Chlorella sorokiniana
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Fusion plasmids withlacZunderthecontrol ofthekatE(encoding catalase orhydroperoxidase HPII)and katF(encoding a sigmafactor-like protein required forkatEexpression) promoters wereconstructed. Expression frombothkatEandkatFpromoters waslowinrich mediumbutelevated inpoormediumduring log-phase growth. Furthermore, theslowdown ingrowth ascells entered thestationary phase inrich medium, aresult ofcarbon source depletion, wasassociated withanincrease inkatEandkatFexpression. A simple reduction inthecarbon source level asthecells entered thestationary phase wasnotresponsible fortheincrease inexpression, because transferring theculture toamediumwithnoglucose didnotinduce expression from either promoter. Spent rich mediumfromstationary-phase cells wascapable ofinducing expression, aswere simple aromatic acids suchasbenzoate, o-hydroxybenzoate, andp-aminobenzoate addedtonewmedium. Anaerobiosis didnotcause anincrease inexpression, nordiditsignificantly change thepattern ofexpression. Regardless ofthemedium, katFexpression wasalways turned onbefore orcoincidently withkatEexpression; inthepresence ofbenzoate katFwasfully induced, whereas katEwasonlypartially induced, suggesting that afactor inaddition toKatFprotein wasinvolved inkatEexpression. During prolonged aerobic incubation, cells lacking katFdiedoffmorerapidly thandidcells lacking either katEorkaiG. Escherichia coli produces twocatalases orhydroperoxidases, thebifunctional catalase-peroxidase HPIandthe monofunctional catalase HPII,encoded bythegeneskatG (10)andkatE(6), respectively, Purification andphysical characterization haveshownthatthetwoenzymes differ significantly fromeachother andfromatypical catalase. HPIisactive asatetramer of81,000-Da subunits andtwo protoheme IXgroups(3),whereasHPIIisactive asa hexamer of93,000-Da subunits andsixhemed-like groups (1, 7). Thelevels ofthetwocatalases respond todifferent stimuli; HPIsynthesis isinduced byH202addedtothemedium, andHPIIsynthesis isinduced during growth into thestationary phaseorduring growth ontricarboxylic acidcycle intermediates (8). Themechanisms controlling these induction patterns haverecently becometheobject ofstudy ina numberoflaboratories. ThekatGgenehasbeenshowntobe partoftheOxyRregulon, whichresponds tooxidative stress, including thepresence ofhydrogen peroxide (2,25)as part ofabroader response illicited byredox-cycling reagents (4). Themechanism controlling theexpression ofkatEis quite different andrequires afunctional katFgeneasa positive effector (9,15,20). Sequence analysis ofkatFhas shownthat KatFprotein closely resembles knownoffactors, suggesting that itmaycontrol theexpression ofanumberof genes, including katE(14). Thegeneforexonuclease III, xthA, hasbeenshowntobeunder thecontrol ofkatF(18), andlesions inkatFhavebeenshowntoresult insensitivity tobroad-spectrum UV radiation (19), confirming this suggestion. Various metabolic changes havebeenstudied asto their influence onkatEexpression, confirming thatkatE expression isturned onascells enter thestationary phase, is unaffected byhydrogen peroxide, isunaffected byanaerobiosis, could beinduced byacomponent ofstationary-phase culture supernatant, andmight beaffected byintracellular pH (20). Ithasalsobeenshownthatashift toanaerobic conditions induces HPIIsynthesis andthathigh concentra
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Themutation ofanuclear geneinpeanut(Arachis hypogaea L.)plants results inareducedlight-dependent development of chloroplast finestructure, soluble protein, ribulose-1, 5-diP carboxylase, NADP-glyceraldehyde-3-P dehydrogenase, fructose1,6-diP aldolase, glycerate-3-P kinase, phosphoenolpyruvate carboxylase, malatedehydrogenase, anddarkrespiration during the72-hour lagperiod ofchlorophyll synthesis indark-grown leaves exposed tocontinuous light. Themutation haspleiotropic affects. Kinetic analysis showsthereisalso a72-hour lagperiod inthelight-dependent development ofNADP-glyceraldehyde3-Pdehydrogenase andfructose-1,6-diP aldolase inthemutant leaves, whereas there isnolaginthedevelopment ofNAD-malatedehydrogenase anddarkrespiration. Thereisminimaldevelopmentofthechloroplast during the72-hourmutationally inducedlagperiod, butthereispronounced cytoplasmic and mitochondrial activity during thisphase. Thereisa 24-hour lag period inthelight-dependent enlargement ofthemutantleaves. Atthecompletion ofleafenlargement, chloroplast differentiationisinitiated. Themutation doesnotresult inanychloroplast deletions, itonlyaffects thetiming ofthesynthesis ofthese components. Elimination ofthelagperiod inleafenlargement andchloroplast development (potentiation) requires apreliminary 72-to 96-hour darkperiod beforeexposing thedark-grown leaves to continuous light. Thereisextensive development oftheetioplasts during thisdarkperiod. Theseresults establish thatthe nuclear genemutation affects theearly stages ofplastid developmentandnotthelight-dependent synthesis ofplastid components. Thenuclear genemaycodefortheregulation ofthesynthesis ofacomponent (nutrient) inthedark(orduring thelag phaseinthelight) whichisessential forthedevelopment of mesophyll cells andplastids. Although, thechloroplast isasemiautonomousorganelie, nuclear genecontrol ofchloroplast differentiation maynotbeindependent ofcellular growth.
Phosphoenolpyruvate carboxylase
Glyceraldehyde
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Starvation ofEscherichia coli forpotassium, phosphate, ormagnesium ions leads toareversible increase intherate ofprotein degradation andaninhibition ofribonucleic acid (RNA)synthesis. Incells deprived ofpotassium, thebreakdown ofthemorestable cell proteins increased two- tothreefold, whereas thehydrolysis ofshort-lived proteins, bothnormal onesandanalog-containing polypeptides, did notchange. Themechanisms initiating theenhancement ofproteolysis during starvation forthese ionswereexamined. Uponstarvation foraminoacids or aminoacyl-transfer RNA(tRNA), protein breakdown increases inreA+(but not reA)cells asaresult oftherapid synthesis ofguanosine-5'-diphosphate-3'diphosphate (ppGpp). However, alack ofaminoacyl-tRNA doesnotappear to beresponsible fortheincreased protein breakdown incells starved forinorganic ions, since protein breakdown increased intheabsence ofthese ions inbothrelA + andreAcultures, andsince alarge excess ofaminoacids didnotaffect this response. Inbacteria inwhich energy production isrestricted, ppGpplevels also rise, andprotein breakdown increases. Theion-deprived cultures didshowa40to 75%reduction inadenosine-5'-tri phosphate levels, similar tothatseenupon glucose starvation. However, this decrease inATPcontent doesnotappear to cause theincrease inprotein breakdown orlead toanaccumulation ofppGpp. No consistent change inintracellular ppGpplevels wasfound inreA+orreAcells starved forthese ions. Inaddition, inreiX mutants, removal ofthese ions ledto accelerated protein degradation eventhough relXcells areunable toincrease ppGpplevels orproteolysis whendeprived of' acarbon source. Inthepotassium-, phosphate-, andmagnesium-deprived cultures, theaddition ofchoram
Degradation
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