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Journal of Plant Physiology 164 (2007) 312—317

0176-1617/$ - sdoi:10.1016/j.

Abbreviationzone; DCCD, dphenyl)-1, 1-diweight; MSX, L

xybutyrate�Correspond

fax: +91 3222 2E-mail addr

www.elsevier.de/jplph

Poly-b-hydroxybutyrate accumulation inNostoc muscorum: Effects of metabolic inhibitors

Nirupama Mallick�, Laxuman Sharma, Akhilesh Kumar Singh

Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur-721302, India

Received 14 December 2005; accepted 17 January 2006

KEYWORDSMetabolic inhibitors;Nostoc muscorum;Poly-b-hydroxybuty-rate

ee front matter & 2006jplph.2006.01.012

s: CCCP, carbonylcyaniicyclohexylcarbodiimidemethylurea; DNP, 2, 4-d-methionine-DL-sulfoxim

ing author. Tel.: +91 32282244.ess: nm@agfe.iitkgp.er

SummaryPoly-b-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied inpresence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophe-nyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation inphosphate-limited N. muscorum under photoautotrophic growth condition. PHBaccumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dryweight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) anddicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4-dinitrophenol (DNP)supplementation depicted insignificant effect on PHB pool of the test cyanobacter-ium. Supplementation of L-methionine-DL-sulfoximine (MSX) and azaserine was alsofound to increase PHB accumulation in N2-fixing and NH4

+-grown N. muscorum, butnot in NO3

�-grown cells. The stimulatory action of monofluoroacetate on PHBaccumulation was suppressed in presence of a-ketoglutarate and DCMU. Interest-ingly, 2,3-butanedione supplementation was not only found inhibitory foraccumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grownN. muscorum but suppression of PHB synthesis was also evident in control cultures inpresence of 2,3-butanedione. The possible mechanisms are discussed.& 2006 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights rese

de m-chlorophenylhydra-; DCMU, 3-(3, 4-dichloro-initrophenol; dw, dryine; PHB, poly-b-hydro-

2 283166;

net.in (N. Mallick).

Introduction

The occurrence of poly-b-hydroxybutyrate (PHB)in a group of microorganisms that lacks a completetricarboxylic acid cycle is very intriguing since thebreakdown of this reserve polymer can play only aminor role in cell metabolism, because the acetyl-CoA which arises from its depolymerization can beutilized neither for significant energy production

rved.

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Inhibition of PHB accumulation in Nostoc 313

nor for the synthesis of more than a few cellconstituents (Smith, 1982). Though the role of PHBin cyanobacterial metabolism is still unclear, it iswell known that nutrient deficient conditions,particularly deficiency of nitrogen and phosphorus,stimulate synthesis of the polymer in photoauto-trophically growing cultures. In Synechococcus sp.MA19 accumulation up to 27% and 55% of dry weight(dw) were reported, respectively, under nitrogen-and phosphorus-limited conditions (Miyake et al.,1996; Nishioka et al., 2001). In photoautotrophi-cally grown Nostoc muscorum, PHB contentreached the level of 23% and 25% of dw, respec-tively, under phosphorus and nitrogen deficiencies(Sharma and Mallick, 2005a). Imbalance in theratios of C:N and NADPH:ATP, respectively in N- andP-deficient cells is being suggested to trigger PHBbiosynthesis (De Philippis et al., 1992a, b; Miyakeet al., 1997). In this report, studies have beenconducted to confirm the above two phenomena bydisrupting the balance of C/N and NADPH/ATP withthe help of some specific metabolic inhibitors.

Materials and methods

N. muscorum Agardh was grown axenically inNO3-free BG11 medium (Rippka et al., 1979) at2571 1C, pH 8.0, under 14 h light (75 mmolphotonm�2 s�1 PAR): 10 h dark cycles. N-deficiencywas achieved by transferring the stationary phasecultures of N. muscorum to NO3-free BG11 mediumdevoid of Na2MoO4. Further, ferrous ammoniumcitrate and Co(NO3)2 � 6H2O were also substitutedby equimolar concentrations of ferric citrate andCoCl2 � 6H2O. Phosphate deficiency was obtained byreplacing K2HPO4 with equimolar concentration ofKCl. For chemoheterotrophic growth condition,cultures supplemented with 0.4% acetate wereincubated under complete darkness (Sharma andMallick, 2005b).

The inhibitors selected for this study were:3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU):competes with QB for binding site in PSII; carbo-nylcyanide m-chlorophenylhydrazone (CCCP) and2,4-dinitrophenol (DNP): uncouplers; dicyclohexyl-carbodiimide (DDCD): blocks proton flow through Foand CFo; L-methionine-DL-sulfoximine (MSX): inhi-bitor of glutamine synthetase; azaserine: inhibitorof glutamate synthetase (GOGAT); monofluoroace-tate: inhibitor of aconitase of TCA cycle and 2,3-butanedione: inhibitor of phosphotransacetylase.

Biomass containing PHB was centrifuged andsuspended in methanol (4 1C, overnight) for re-moval of pigments. The pellet obtained aftercentrifugation was dried at 60 1C and PHB was

extracted in hot chloroform followed by precipita-tion with cold diethyl ether. The precipitate wascentrifuged (11,000g, 20min), washed with acet-one and was dissolved again in hot chloroform. PHBconcentration was determined following the pro-panolysis method of Riis and Mai (1988) using a GC(Clarus 500, Perkin Elmer) in split mode (1:50, v/v),equipped with Elite-1 dimethylpolysiloxane capil-lary column (30m� 0.25mm� 0.25 mm) and flameionization detector. Benzoic acid was used as theinternal standard.

Results and discussion

DCMU supplementation under phosphatedeficiency

De Philippis et al. (1992a, b) postulated that PHBsynthesis is stimulated under phosphate starvation,when reducing power may be in excess, becauseATP synthesis is known to decrease markedly withthe onset of phosphate limitation, while reductionof NADP through non-cyclic photosynthetic electronflow is not inhibited (Bottomley and Stewart, 1976;Konopka and Schnur, 1981). To check this hypoth-esis, experiment was designed to suppress NADPHsynthesis in phosphate-starved cells by DCMUtreatment. DCMU inhibits photosystem II throughbinding to the QB binding site vis-a-vis the non-cyclic electron flow and NADPH production (Rippka,1972). More recently, blue fluorescence, an indica-tor of NADPH concentration was also found todecrease in Chlorella vulgaris and Thalassiosirarotula under DCMU treatment (Steigenbergeret al., 2004). In this study (Fig. 1), PHB accumulationdepicted an increasing trend only after 24h ontransferring N. muscorum cells to P-deficient med-ium. The accumulation, however, was amounted upto 23% from an initial PHB content of 8.5% of dw onday 5 of phosphate deficiency. Contrary to this, adeclining trend in PHB pool was evident followingDCMU treatment to P-deficient cultures even fromday 1 of treatment. Thus the inhibition of PHBaccumulation in phosphate-limited DCMU-treated N.muscorum cells supports the view of De Philippiset al. (1992a, b) that in photoautotrophically growncultures NADPH synthesis through non-cyclic elec-tron transfer plays a major role in PHB accumulationunder phosphate deficiency.

Impact of uncouplers

In order to confirm whether disrupting thebalance between phosphorylation and NAD(P)

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Figure 2. Effects of CCCP, DCCD and DNP on PHB pool ofphotoautotrophically grown N. muscorum: control (B),CCCP 10 mM (n), CCCP 20 mM (m), DCCD 10 mM (&), DCCD20 mM (’), DNP 1mM (J) and DNP 2mM (K).

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Figure 1. Effect of DCMU supplementation on PHB poolof phosphate-deficient N. muscorum. All the culturesexcept control were starved for phosphate: control (B),phosphate-deficient (m), DCMU 10 mM (&) and DCMU20 mM (J).

N. Mallick et al.314

reduction really stimulate PHB biosynthesis, theuncoupler, CCCP was added to the standard mineralmedium. Under photoautotrophic condition, theintracellular PHB accumulation was stimulated,reached a maxima of 21% of dw in 20 mM CCCP-treated cultures (Fig. 2). Similar rise was alsoobserved following DCCD treatment, where the PHBpool reached up to 17% of dw in 20 mM DCCD-treated cells. The DNP supplementation, however,did not register any significant change in PHBcontent.

Uncouplers abolish phosphorylation without in-hibiting electron transport. The electron transport,freed of the restraints imposed by the couplingmechanism, is often greatly accelerated (Izawa andGood, 1972). Under such a situation, a rise in theconcentration of reducing power is expectedthrough the accelerated non-cyclic electron trans-fer of photosynthetic light reaction. CCCP is a well-known uncoupler of both oxidative- and photopho-sphorylation (Izawa and Good, 1972). A significantdecrease in ATP pool of Synechocystis PCC 6803under CCCP treatment was reported by Ryu et al.(2004). Similar decline in ATP content of Natrono-bacterium pharaonis under light-driven ATP synthe-sizing condition was recorded under DCCDtreatment (Avetisyan et al., 1998). DCCD inhibitsATP synthesis by blocking proton flow through Foand CFo. Under such situations, an imbalance in theratio between NAD(P)H and ATP was created, whichmight be the cause for the increased PHB accumu-lation (Fig. 2). However, no such rise in PHB poolwas observed in DNP-treated cultures (Fig. 2). Thephenols are classical uncouplers of oxidativephosphorylation and DNP even at 10�3 M has only

mild effects on chloroplast (Neumann and Jagen-dorf, 1964). In photoautotrophically growing cellsas photosynthetic light reaction is the main sourceof energy (Smith, 1982), uncoupling oxidativephosphorylation by DNP might not be resulted intosignificant imbalance in NAD(P)H and ATP ratio,thus not depicting any impact on PHB pool.Experiment conducted in photosynthetically grow-ing N. muscorum did not exhibit any significantimpact of 2mM DNP on the ATP pool size (data notshown), thus reconfirming the above view thatimbalance in ATP and NAD(P)H ratio is a majorcause for PHB accumulation.

Impact of MSX and azaserine

Nitrogen starvation, induced by transferring N.muscorum filaments into the medium devoid of themicronutrient Mo, was found stimulating for PHBaccumulation (25% of dw, Sharma and Mallick,2005a). When nitrogen starvation was imposed byaddition of MSX and azaserine, specific inhibitors ofglutamine synthetase and glutamate synthetase(GOGAT), respectively, to N2-fixing and NH4

+-grownN. muscorum, thus inhibiting the nitrogen incor-poration, a rise in PHB pool was evident (Table 1).This could be attributable to the disrupted C/Nratio, as proposed by Miyake et al. (1997). Never-theless, no such rise in PHB content was evidentwhen MSX and azaserine were added to NO3

�-grownN. muscorum (Table 1). This agrees with the findingof De Philippis et al. (1992a) where the polymersynthesis was not detected in Spirulina maxima,when nitrogen incorporation was disrupted by

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Table 1. Effect of MSX and azaserine on PHB accumu-lation potential of N2-fixing, NH4

+- and NO3�-grown

N. muscorum

Culture condition PHB (% dw)

N2-fixingControl 8.370.54a

MSX (50 mM) 15.271.23b

Azaserine (20 mM) 17.171.13b

NH4+-grown

Control 2.670.15a

MSX (50 mM) 12.270.65b

Azaserine (20 mM) 10.271.95b

NO3�-grown

Control 2.570.34a

MSX (50 mM) 2.370.46a

Azaserine (20 mM) 2.370.30a

All values are mean7SE, n ¼ 3. Values in the column super-scripted by different letters are significantly (Po0:05) differentfrom each other (Duncan’s new multiple range test). Separateanalysis was done for each group.

Inhibition of PHB accumulation in Nostoc 315

azaserine treatment, without interfering withnitrate reduction. These workers suggested thatthe polymer synthesis would not be stimulated innitrogen-starved cells if nitrate reduction wasallowed to compete for reducing powers. However,the results of this study ruled out this possibility, asstimulation in PHB accumulation was observedunder MSX/ azaserine treatment in N2-fixing cells(Table 1), where nitrogenase was competing for thereducing power. One possible reason for this couldbe that the dependence of nitrogen fixation on thereductant pool of the fixed carbon compoundsrather than the photosynthetic NADPH generation(Stewart and Rowell, 1975). Various authors notedthat C2H2 reduction of Anabaena cylindrica pro-ceeded unimpaired in presence of DCMU, wherePSII activity was totally blocked (Cox, 1966; Botheand Loos, 1972). The acetylene reduction even didnot show an Emerson-enhancement effect, con-trary to photosynthetic CO2 fixation. C2H2 reduc-tion is severely affected in carbon-limited culturesof Anabaena cylindrica (Fay, 1976). All theseobservations indicate that reductants for N2 fixa-tion come from the pool of fixed carbon com-pounds. Contrary to this, nitrate reduction inphotosynthesizing cells is directly dependent onphotosynthetic reducing power (Flores et al.,2005).

As mentioned earlier, in photoautotrophicallygrowing cyanobacteria photosynthesis is the mainsource of energy (Smith, 1982), and nitrate reduc-tion is directly dependent on the photosynthetic

reducing power. Therefore, the cells with activenitrate reductase activity might not be able toaccumulate the photosynthetically generatedNADPH substantially, thus depicting no noticeablechange in the PHB pool. The dependence of N2

fixation on photosynthesis is indirect (Bothe et al.,1982), therefore may not affect the photosyntheticNADPH pool significantly. Under such situation PHBsynthesis might have been stimulated without theconstraint of NADPH.

Effect of monofluoroacetate

Figure 3 presents the effect of monofluoroace-tate, an inhibitor of the enzyme aconitase of TCAcycle, on PHB pool of N. muscorum grown photo-autotrophically in usual N2-fixing medium. A sig-nificant rise in PHB content (19% of dw) was evidentin 50mg L�1 monofluoroacetate supplemented ves-sels. This stimulation was, however, suppressedwhen a-ketoglutarate was supplemented in combi-nation with monofluoroacetate (Fig. 3a). Similarsuppression was also noticed in monofluoroaceta-te+DCMU-supplemented vessels (Fig. 3b).

Monofluoroacetate blocks the conversion ofcitrate to isocitrate by inhibiting the enzymeaconitase of TCA cycle (Quastel, 1963), thusresulting into reduced/inhibited synthesis ofa-ketoglutarate, the prerequisite for amino acidbiosynthetic pathway. Under such a condition, PHBbiosynthesis might have been stimulated as nitro-gen incorporation was blocked due to unavailabilityof a-ketoglutarate. Nonetheless, no rise in PHB poolwas evident in cultures where a-ketoglutarate andmonofluoroacetate were supplemented simulta-neously (Fig. 3a), thus again providing testimonyto the hypothesis that disruption in nitrogenincorporation or imbalance in C/N ratio stimulatesPHB accumulation. However, the suppression ofPHB accumulation in monofluoroacetate+DCMU-supplemented vessels (Fig. 3b) points towards therole of NADPH in PHB accumulation even in thecultures with disrupted C/N ratio.

Effect of 2,3-butanedione

As hypothesized by Asada et al. (1999), incyanobacteria the enzyme PHB synthase is regu-lated post-translationally, and that the enzyme isactivated by acetyl phosphate, synthesized byanother enzyme, phosphotransacetylase. Phospho-transacetylase catalyzes the reversible acetylgroup transfer between CoA and orthophosphate:

CoASHþ CH3CO2PO32�!CH3COSCoAþ HPO4

2�:

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Figure 3. (A) Impact of monofluoroacetate and a-keoglu-tarate on PHB pool of N. muscorum: control (B),monofluoroacetate 25mgL�1 (n), monofluoroacetate50mgL�1 (m), monofluoroacetate 25mgL�1+a-ketogluta-rate 25mgL�1 (&) and monofluoroacetate 50mgL�1+a- ketoglutarate 25mgL�1 (’). (B) Impact of monofluor-oacetate and DCMU on PHB pool of N. muscorum: control(B), monofluoroacetate 25mgL�1 (n), monofluoroacetate50mgL�1 (m), monofluoroacetate 25mgL�1+DCMU 10mM(&) and monofluoroacetate 50mgL�1+DCMU 20mM (’).

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Figure 4. Effect of 2,3-butanedione (10mM) on PHBaccumulation potential of (A) P-deficient cells of N.muscorum: control (K), P-deficient (m), control+2,3-butanedione (J) and P-deficient+2,3-butanedione (n).(B) N-deficient cells of N. muscorum: control (K), N-deficient (m), control+2,3-butanedione (J) and N-defi-cient+2,3-butanedione (D). (C) Chemoheterophically(0.4% acetate)-grown N. muscorum: control (K), che-moheterotrophy (m), control+2,3-butanedione (J) che-moheterotrophy+2,3-butanedione (n).

N. Mallick et al.316

2,3-butanedione is known to be a specificinhibitor of the enzyme, phosphotransacetylase(Iyer and Ferry, 2001). PHB accumulation reachedup to 23%, 25% and 43% of dw, respectively, inphosphate-deficient, nitrogen-deficient and che-moheterotrophically grown N. muscorum cells(Sharma and Mallick, 2005a, b). As presented inFig. 4, supplementation of 10mM 2,3-butanedioneto N. muscorum cells resulted in complete suppres-sion of PHB accumulation not only in thosestimulated cells, but also in control cultures. Thissupports the hypothesis that PHB synthase isactivated post-translationally by acetyl phosphate.

In conclusion, accumulation of NADPH or a highratio of reducing power to ATP stimulates PHB

accumulation. The reducing power pool also seemsto play a major role in PHB accumulation even inthe cultures with disrupted carbon-to-nitrogen

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Inhibition of PHB accumulation in Nostoc 317

ratio. Results of DCMU and DNP supplementation(Figs. 1 and 2) and studies on PHB accumulation inNO3�-grown and N2-fixing N. muscorum under MSX

and azaserine treatment (Table 1) suggest that PHBaccumulation in cyanobacteria might be linked tophotophosphorylation rather than oxidative phos-phorylation, which needs further elucidation.Synthesis of acetyl phosphate is, however, foundessential for PHB biosynthesis in the test cyanobac-trium, N. muscorum.

Acknowledgement

Financial support from Department of Biotech-nology, Ministry of Science and Technology, India, isthankfully acknowledged.

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