Levering, P. R.

English

Many microorganisms are able to utilize C1 compounds, i.e. compounds which do not contain carbon-carbon bonds, as carbon- and energy sources for growth. In order to synthesize cell constituents from these C1 compounds special metabolic pathways are employed by such organisms. Although a great deal of knowledge currently exists on the physiology and biochemistry of microorganisms able to grow on Cl compounds (autotrophs and methylotrophs), relatively little is known about the regulation of the synthesis of enzymes specifically involved in the various C1 assimilation pathways. About ten years ago such regulatory studies were initiated in our laboratory. As a first approach the regulation of the synthesis of Calvin (RuBP) cycle enzimes in autotrophic bacteria was investigated and a considerable body of information on this subject is currently available. In many bacteria able to grow aerobicalty on Cl compounds, however, carbon assimilation is initiated by fixation of formaldehyde via the ribulose monophosphate (RuMP) cycle. The regulation of the synthesis of RuMP cycle enzymes constitutes the main topic of this thesis. ... Zie: Summar

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   University of GroningenMetabolic regulation in the facultative methylotroph arthrobacter P1Levering, P. R.IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1985Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Levering, P. R. (1985). Metabolic regulation in the facultative methylotroph arthrobacter P1. University ofGroningen.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022S U M M A R Yl lany nicroorganisms are able to ut i l ize C1 conpounds,  i .e.  conpoundsrhich do not contain carbon-carbon bonds, as carbon- and energy sources forgrowth.  In order to synthesize cel l  const i tuents f rdn these C1 col t t t roundsspecial rnetabolic pathnays are employed by such organisrns. Although a greatdeal  of  knowledge current ly  exists on the physio logy and biochemistry ofnicroorganisms able to gron on Cl conpounals (autotrophs and methylotrophs),relatively lit,tle is known about the regulation of Èhe synthesis of enzynesspeci f ical ly  involved in the var ious C1 assimi lat ion pathways.  About tenyears ago such regulatory studies were in i t iated in our laboratory.  As af i rst  approach the regulat ion of  the synthesis of  Calv in (RUBP) cyclêenzlmes in autotrophic bacteria was investigated and a considerable body ofinformat ion on th is subject  is  current ly  avai lable.  In many bacter ia ableto grow aerobical ty on Cl  compounds,  however,  carbon assirn i lat ion is  in i -tiated by fixation of formaldehycle via the ribulose nonophosphate (RUMP)cycle.  The regulat ion of  thê synthesis of  RUMP cycle enzymes const i tutesthe rnain topic of  th is thesis.A survey of  the l i terature avai lable on methylotrophic microorganisms,which assinilate carbon in the RuÈíP cycle, revealed that no suitable nodelorganisns for  thêse studies had yet  been descr ibed (chapter 1) .  Therefore 'at tempts were made to isolate such bacter ia f rorn enr ichnent cul tures con-taining the C1 compounds nethylamine or trimethylamine as sole source ofcarbon and energy.  Af ter  screening several  of  the isolates obtained in th isway for fast growth on c1 compounds, their abiliÈy to grow on various sub-strates contain ing carbon-carbon bonds,  such as sugars and organic acids,and for the presence of RuttlP cycle enzlzmes, an Àrthrobactêr species waschosen for  fur ther physio logical  and biochemical  studies (chapter 21.Dur ing growth of  th is bacter ium (Arthrobactêr  Pl  )  on methylanine i tappeareil that the enzyÍne amine oxidase performs a key role in the netab-ol ism of  th is compound. This enzyme catalyzes the oxidat ion of  nethylaninewith mol-ecular oxygen to produce formalclehyde, hydrogen peroxide and ammo-nia. Cytochemical staining experiments showed that anine oxidase activityis  rnain ly located on invaginat ions of  the cytoplasmic menbrane. Assimi la-t ion of  the formaldehyde, produced f rqn the oxidat ion 'of  nethylanine,  isaccomplished by the RUMP cyclê via the enzymes hexulose phosphate synthase(HPS) and hexulose phosphate isomerase (HPI) .  Since enzymes for  the l inearoxidation of fornaldehyde (via formate to carbon dioxide) were undetectablein methylamine-grown cel ls  of  th is organism, i t  was speculated that  dur inggrowth of Arthrobacter P1 on C1 conpounds energy generation proceeds viathe so-cal led diss in i latory RUMP cycle.  The operat ion of  th is cycle,  invol-ving the RUMP cycle enzynes mentioned above and enzyrnes of the oxidativepentosê phosphate pathnay, is rather corntnon among RUMP cycle rnethylotrophs.In thesê studies i t  was also observed that  the enzynes speci f ical lyr z  Iinvolved in C1 metabol isn in Arthrobactêr  p l  are normal ly  not  found incel ls  grown on "heterotrophic"  substrates,  such as glucose or  acetate,indicat ing that  the synthesis of  these enzlmes in th is facul tat ive methylo-t roph is  regulated in an ef f ic ient  nanner.Further studies of  methyl-amine metabol isn in Arthrobacter  P1 revealedthat  conversion of  forrnaldehyde into C3 conpounds,  the precursors for  thebiosynthesis of  cel l  mater ia l ,  proceeds v ia the energet ical ly  nost  favour-able var iant  of  the RUMP cycle.  The resul ts (chapter  3)  suggest  the opera-t ion of  the EBP aldolase (Enbden-Meyerhof)  route for  sugar phosphate c lea-vage and the t ransketolase/ t ransaldolase sequence of  regenerat ion of  theformaldehyde-acceptor molecule r ibulose rnonophosphate.General ly ,  in methylotrophic rn icroorganisms only one part icular  C1 assi-n i lat ion pathway operates as the najor  route of  C1 carbon leading to thefornat ion of  ceI1 const i tuents.  In chapter 4 enzynat ic evidence ispresented which indicates that  in Arthrobacter  P1 the except ional  s i tuat ionexists that  both the Rul{P cycle as wel l  as (part  of)  the ser ine pathway areinvolved in the netabol ism of  chol ine.  In th is organism chol ine is  brokendown in a ser ies of  react ions y ie ld ing glyc ine and formaldehyde in a rat ioof  one to thrée.  I t  is  postulated that  g lyc ine and one of  the forrnaldehydemolecules combine to y ie ld ser ine,  which is  subsequent ly convert ,ed to phos-phoglyceratê through typical  ser ine path$ray enzymes, whereas the remainingfornaldehyde nolecules are f ixed in the RUMP cycle.In chapters 5 and 6 resul ts of  studies on the regulat ion of  the syn-thesis of  key enzymes in the speci f ic  methylotrophic and heterotrophiccarbon assimi lat ion pathways in Arthrobacter  P1 are presented,  obtained insubstrate t ransi t ion exper inents,  f rorn methylanine to ethylarnine,  and v iceversa,  and dur ing growth on nixtures of  methylamine and acetate,  in batchas wel l  as cont inuous cul tures.  In th is organism the C2 conpound ethylanineand the C1 compound nethylamine are Ínetabol ized in i t ia l ly  in an ident icalfashion,  v ia the respêct ive aldehydes.  Transfer  of  ethylamine-grown cel lsinto a nediun vr i th methylanine resul ted in i rnmediate excret ion of  lowIevels of  formal-dehyde (max. 0.5 nM).  In the reverse exper iment excret ionof  much higher acetaldehyde levels were observed.  In fur ther exper iments,some of  which involved mutants of  Arthrobacter  P1 blocked in a ldehyCleut i l izat ion,  i t  became clear that  in t ransient  state s i tuat ions accumula-t ion of  toxic leveIs of  formaldehyde f ron nethylamine oxidat ion is  pre-venteal  because of  a rapid synthesis of  HPS up to h igh levels and by feêd-back inhib i t ion of  formaldehyde on the act iv i t ies of  the nethylam!-ne uptakesystêm and amine oxidase.When Arthrobacter  P1 $ras grov,n on nixtures of  acetate and methylanine inbatch cul ture in the presence of  ammonia,  acetate turnecl  out  to be the pre-ferred growth substrate i r respect ive of  the way cel1s were pregrown. Underthese condi t ions acetate did not  ( fuI Iy)  repress the synthesis of  enzymesspeci f ic  for  methylamine netabol ism. The observed sequent ia l  ut i l izat ion ofthese t$ro substrates was subsequently shown to be due to a strong (non-con-I 2 8pe t i t i ve )  i nh i b i t i on  ex .methylamine. Iilhen ammon:plus nethylanine the orgto amnonia to fu l f i l  i tsg rown  ce l l s  i n  t h i s  med :substrates,  but  the growthe very poor rate of  amrnixtures of  acetate pluscont inuous cul tures thewas undetectably low. Thboth substrates at  d i lu jst rates.  In these experdependent on the rat io onetabol isrn of  the Cl  subsis of  the enzlmes invoacetate,  indicat ing thatenergy source.  At  D = 0only occurred when aceta 'the feed of  a methylaninrSince fornaldehyde anamine and i ts  subsequer( chap te r s  5  and  6 )  ,  i t  w icon t ro l l i ng  t he  syn thes i Íin exper iments involv ingg row th  subs t ra te .  I n  v i (naking i t  v i r tual ly  impcba t ch  cu l t u re ,  A r t h robac jcu l t u res  ( chap te r  71 .enzynes invoLved in conformaldehyde f ixat ion isamine t ransport  systêm a:ence of  methylamine,  wheby fornaldehyde, presentcel lu lar  product ion f romn i sm o f  ca tabo l i t e  r ep re Íenzlmes vras subsequent lys t r a te  ( g l ucose  o r  ace t ,r esu l t ed  i n  s imu l t aneousacetate and a coÍnplete s lHPS .  Th i s  i nd i ca ted  t haenzyme  i s  i ndeed  sens i t io l i sm  o f  t hese  nhe te ro t r (suspens ions  o f  A r t h robachyde is indeed the indtenzyme HPS (chapter 8)  .  ' lpetitive) inhibition exerted by acetate on the uptake anil oxidation ofrnethylanÍne. When ammonia is omitted frqn the medirm containing acetateplus nethylanine the organism is dependent on the oxidation of nethylanineto amnonia to fulfil its need for nitrogen. Incubation of nethylamine-pre-grown ce1ls in th is medium did resul t  in s inul taneous ut i l izat ion of  bothsubstrates,  but  the growth rate nas very low and most l ikely deternined -bythê very poor rate of arnmonia generation. vihen Arthrobacter P1 lras grovrn onmixtures of  acetate plus nethylanine in carbon- and energy source- l imi tedcont inuous cul tures the residual  concentrat ion of  acetate in the cul turenas undetectably Iow. This a l lowed s imul taneous and conplete ut i l izat ion ofboth substrates at  d i lut ion rates belon the Umax on ei ther of  these sub-strates.  In these exper imênts,  however,  the observat ion was nade that ,dependent on the ratio of acetate and nethylamine in the nediun supply, thenetabolism of the Cl substrate nethylanine nas able to rePress the synthê-sis of the enzymes involved in carbon assinilation from the C2 substrateacetate, indicat.ing that under these conditions acêtate was only used as anenergy source.  At  D = 0.10 h- l  synthesis of  the glyoxylate cycle enzyrnesonly occurred when acetate was added at concentrations above 7.5 - 10 tnM tothe feed of a nethylanine-limited (Sg = 25 mM) chernostat culture.Since formaldehyde and acetaÈe strongly influence the uptake of methyl-anine and its subsequent oxidation to fornaldehyde in Arthrobacter Pl(chapters 5 and 6)  ,  i t  was real ized that  a c lear p icture of  the mechanisns,controlling the synthesis of the RUMP cycle enzyrnes, might only be obtainedin experinents involving fornaldehyde itself rather than rnêthylanine as thegronth substrate.  In v iew of  thê extrenely toxic nature of  formal i lehyde,naking it virtually impossible to grovr microorganisms on this compound inbatch cul ture,  Arthrobacter  P1 was grown in fornal i lehyde- l in i ted cont inuouscul tures (chapter 11.  Enzlme analyses revealed that  the synthesis ofenzlmes involved in conversion of nethylanine into fornaldehyde and inforrnaldehyde fixation is induced sequentially in this organisrn. The nêthyl-amine transport systen and arnine oxidase are only synthesized in the pres-ence of methylanine, whereas the RuMP cycle enzymes are apparently inducedby fornaldehyde, present in the nediun reservoir or following its intra-cellular production frorn nethylamine. The possible involvement of a mecha-nism of  catabol i te repression in regulat ing the synthesis of  the RUMP cycleenzl'mes was subsequently studied by adding orcess of a "heterotrophic[ sub-strate (g lucose or  acetate)  i l i rect ly  to these cont inuous cul turês.  Thisresulteal in sinultaneous utiLization of both fornaldehyde and glucose oracetate ancl a complete switch-off of the synthesis of thê RuMP cycle enzlrneHPS. This indicated that ,  under these condi t ions,  the synthesis of  th isenzymê is indeed sensitive to catabolite repression exerted by the netab-ol isn of  these nheterotrophic"  substrates.  In exper iments wi th rest ing cel lsuspensions of Arthrobacter Pl clear evidence was obtained that fornalde-hyde is indeed the inducing signal for the synthesis of the RUMP cycleenzyne IIPS (chapter 8). The rêIative importance of induction ard repressionL Z amechanisms in regulat ing the synthesis of  HPs vtas subsequent ly invest igatedin experinents where fornalclehyde was added at a low' constant rate tobatch cul tures of  Arthrobacter  P' l  growing exponênt ia l ly  on glucose ora c e t a t e ( c h a p t e r S ) . I n ê a c h c a s e t h i s r e s u l t e d i n t h e i n m e d i a t e a n d v e r yrapid synthesis of  ï tPS, whi le no s igni f icant  accurnulat ion of  formaldehydeoccurred.  I t  is  therefore concluded that  induct ion by formaldehyde is theoverr id ing contro l  mechanism for  the synthesis of  I IPS. Repression may actas an auxi l iary rnechanisn and i ts  main funct ion may be found uni ler  condi-t ions,  character ized by hiqh (excess) levels of  HPS, requir ing rapid a ' laPt-at ion of  the cel ls  to a changing environment 'The present stualy establ ished the presence of  t ransal-c lo lase isoenzlrynesin Arthrobacter  Pl  (chapter  9)  '  This enzyme is invorved in thê pentosephosphate pathway as weII as in the RuMP cycle' One of these isoenzl'mesa p p e a r e d t o b e s y n t h e s i z e d c o n s t i t u t i v e l y , w h e r e a s t h e s e c o n i l i s s p e c i f i c -aI Iy induced dur ing growth on c l  conpounds.  A mutant  lacking the "const i -tut ive, ,  t ransaldolase was unable to grow on gluconate or  xy lose,  s ince th ise n z } r m e i s o b l i g a t o r y i n t h e m e t a b o l i s n o f t h e s e c o n p o u n d s ( c h a p t e r 3 ) .studies wi th th is mutant  revealed that  normal  growth on gluconate was,  hoet-ever,  possib le providecl  methylamine was suppl ied as a ni t rogen source'  Thisresul ted in the induct ion of  the "methylotrophic"  t ransaldolase to h ighIevels.  This indicates that ,  funct ional ly ,  there are no di f ferences betweenboth isoenzynes.  To study th is in nore detai l ,  the t ransaldolases werepart ia l ly  pur i f ied and i t  was subsequent ly shown that  they did not  d i f ferwi th respect  to k inet ic  propert ies.  Howêver,  the molecular  weights appearedto be s l ight ly  d i f ferent  and,  moreover,  the "nethylotrophic"  t ransaldolase$ras very heat  sensi t ive.  I t  is  postulated that  dur ing growth on c1 com-pounds the "nethylotrophic"  t ransaldolase serves to ensure a suf f ic ient lyhigh rate of  regenerat ion of  the formaldehyde-acceptor molecule r ibulosenonophosphate.1 J UVeIe nicroorganismen :d i ngen  d i e  geen  koo l s t o :bron van koolstof  en encelbestanddelen te synth(a l e  me tabo l i s che  rou tes .over de fys io logie en bickunnen groeien (autotrof ,ove r  de  regu le r i ng  van  iass im i l a t i e rou tes  be t rok lI abo ra to r i un  een  beg Ín  g 'benader ing werd de reguenzymen in autotrofe bacinformat ie over d i t  onbacter iën die aeroob opgeass im i l ee rd  v i a  f i xa t(RUMP) cyclus.  De regulelhet  voornaamste onderwer lBestuder ing van de b€nisnen, d ie hun koolstoI icht  dat  nog geen geschlte voeren waren beschrever i ën  t e  i so l e ren  vanu i tamine of  t r imethylaminescheic lene,  op deze manie:op  C1  ve rb i nd ingen ,  hunstof-koolstof  b indingen Ide aanwezigheid van Rugekozen voor êen nader(Hoo fds tuk  2 ) .  He t  b l ee lbac te r  P1 ,  op  me thy lam in€het metabol isme van dezemethylanine net  molecul .oxyde en ammoniak gevorÍItechnieken werd aanget(gelocaJ- iseerd is  op rnv,van  he t  u i t  de  oxyda t i ein de Rulr lP cyclus v ia d<lose  f os faa t  i somerase  ir ou te  van  f o rma ldehYde  ( ri n  ce l l en  gekweek t  op  mer e r i n g  t i j d e n s  d e  g r o e i ,

Metabolic regulation in the facultative methylotroph arthrobacter P1

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Metabolic regulation in the facultative methylotroph arthrobacter P1

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