Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)- producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of ActBm, a σ 54 regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium

Beatriz S. Méndez

Gustavo J. Vázquez Gustavo J. Vázquez

M. Julia Pettinari

Gustavo J. Vázquez, Gustavo J. Vázquez

Pettinari, M. Julia

Méndez, Beatriz S.

Hemeroteca Cientifica Catalana

RESEARCH ARTICLEEvidence of an association between poly(3-hydroxybutyrate)accumulation and phosphotransbutyrylase expressionin Bacillus megateriumReceived: 11 March 2003 / Accepted: 8 April 2003 / Published online: 24 June 2003 Springer-Verlag and SEM 2003Abstract Molecular analysis of a genomic region ofBacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of agene coding for the enzyme phosphotransbutyrylase(Ptb). Enzyme activity was measured throughout thediﬀerent growth phases of B. megaterium and wasfound to correlate with PHB accumulation during thelate-exponential growth phase. Ptb expression wasrepressed by glucose and activated by the branchedamino acids isoleucine and valine. Overexpression ofActBm, a r54 regulator from B. megaterium whosegene is located upstream from ptb, caused an increasein Ptb activity and PHB accumulation in B. megate-rium.Keywords Bacillus megaterium Æ PolyhydroxybutyratePhosphotransbutyrylaseIntroductionThe presence and/or expression of genes involved in thesynthesis of ATP at the substrate level, including thosecoding for the enzymes phosphotransacetylase (Pta),acetate kinase (Ack), phosphotransbutyrylase (Ptb),and butyrate kinase (Buk), have been reported for themodel organism of the Bacillus genus, Bacillus subtilis[6].In previous experiments, we cloned and molecularlyanalyzed a 2.53-kb region of the B. megaterium chro-mosome, in which three complete open reading frames(ORFs) were identiﬁed: actBm, ORF2, and ptb. Thesequence of ORF2 was found to be homologous to thatof sensory transduction histidine kinases [16]. actBmencodes an activator homologous to r54 regulators thatis able to activate in trans the Escherichia coli genes forthe degradation of short-chain fatty acids [12]. Thisregulator is highly homologous in structure and functionto the BkdR regulator of B. subtilis, involved in bran-ched amino acids degradation [3]. The third ORF [16]revealed extensive similarity with ptb genes [17]. InClostridium acetobutylicum and other Clostridium spe-cies, the product of ptb is involved in butyric acid syn-thesis from butyryl-CoA by means of a pathway thatresults in the production of butyryl phosphate followedby the generation of ATP and butyrate [5]. B. megate-rium, unlike B. subtilis, is a polyhydroxybutyrate (PHB)producer. PHB is a member of the family of the poly-hydroxyalkanoates, biodegradable plastics and elas-tomers, that accumulate in several bacterial speciesunder growth conditions characterized by an excess ofcarbon source and the lack of one or more essentialnutrients. PHB acts as a carbon storage compound andas a sink for reducing equivalents [11]. Previous researchhas shown that low extracellular pH correlates with thestart of PHB accumulation in this species [7]. The en-zyme phosphotransbutyrylase is involved in butyrateexcretion in Clostridium, and as a consequence in theacidiﬁcation of the medium. In addition, butyrylCoA, apossible intermediate in PHB synthesis, is also a sub-strate of the Ptb enzyme. Thus, we studied expression ofthis enzyme in order to analyze its possible relation toPHB production.This work describes ptb expression in B. megateriumthroughout the diﬀerent phases of growth and in asso-ciation with PHB accumulation as well as acidiﬁcationof the culture medium.Int Microbiol (2003) 6: 127–129DOI 10.1007/s10123-003-0120-5Gustavo J. Vazquez Æ M. Julia PettinariBeatriz S. Me´ndezG. J. Vazquez Æ M. J. Pettinari Æ B. S. Me´ndez (&)Departamento de Quı´mica Biolo´gica,Universidad de Buenos Aires,Facultad de Ciencias Exactas y Naturales,Buenos Aires, ArgentinaE-mail: bea@qb.fcen.uba.arTel.: +54-1145763334Fax: +54-1145763342Present address: G. J. VazquezMicrobiology Laboratory,Brookhaven National Laboratory,Environmental Sciences Department,Upton, New York, USAMaterials and methodsStrains and growth conditionsThe bacterial strains and plasmids used in this study were: plasmidpS14 [16] containing the actBm gene in pAT18 [14], B. megateriumPV447 [15], and its pS14 transconjugant. B. megaterium was growneither in supplemented nutrient broth (SNB)[15], or in minimalmedium (MM) [4] supplemented with glucose or gluconate asindicated. SNB contains 5 g glucose/l, unless otherwise indicated.The concentrations used were: glucose and gluconate 5 g/l, valine1.5 g/l, and isoleucine 3.5 g/l. The medium contained suﬃcientammonium so that amino acids were not utilized as a nitrogensource. Ptb speciﬁc activity and PHB were measured under eachgrowth condition. Samples were taken at the early stationarygrowth phase. Cultures of B. megaterium PV447 and of the samestrain bearing pS14, a recombinant multicopy plasmid containingact Bm, were grown in SNB supplemented with 20 g glucose /l untilthe early stationary phase of growth.Analytical proceduresPHB and short-chain fatty acids were determined with a Gow MacSeries 580 gas chromatograph equipped with a ﬂame ionizationdetector. PHB was esteriﬁed as previously described [1], and theresulting methyl esters were run in a 1.80-m glass column packedwith Carbowax 20M-TPA on Chromosorb WAW (80/100mesh).Short-chain fatty acids were measured by acidifying the culturesupernatants with H2SO4 (167 g/l ﬁnal concentration) followed byextraction with diethyl ether. The corresponding ethyl esters wereseparated in a 1.80-m glass column packed with 10% Sp 1000-1%H3PO4 on Chromosorb WAW (100/120mesh). Determinationswere repeated twice in two parallel cultures.Five ml of bacterial cultures, grown as indicated in eachexperiment, were used for crude extract preparations followingprocedures previously described [12]. Ptb activity was determinedas described [2], following the increase in A412 due to formation ofthe complex DTNB-CoA (=1.36·104/M·cm) after liberation ofCoA-SH from butyryl-CoA. Proteins were measured according tothe method described in [10], with bovine serum albumin as thestandard. Measurements were repeated twice in two parallel cul-tures.Results and discussionKinetics of Ptb activity in B. megateriumAs mentioned above, low pH values of the medium areassociated with the start of PHB accumulation in SNB[7]. Thus, in accordance with the functions predictedfrom the sequence of the B. megaterium region analyzed,a decrease in the pH of the medium was expected tocorrelate with an increase in Ptb activity. These twoparameters were measured throughout the exponentialand stationary phase of growth. The results are shown inFig. 1. Enzyme activity and PHB accumulation startedwhen the pH of the medium reached its lowest value,and were highest during stationary phase. Acetic acidwas the only short-chain fatty acid detected extracellu-larly. These results suggest either that short-chain fattyacids other than acetate were not synthesized in detect-able amounts or that they were immediately metabo-lized.The addition of 50 mM butyrate to late-exponentialphase cultures of B. megaterium in SNB caused a 68%reduction of Ptb activity (data not shown). Therefore,the possible metabolism of butyrate might proceed by apathway not involving this enzyme.Eﬀect of branched amino acids and carbohydrates onPHB accumulationBranched amino acids were found to induce theexpression of ptb, as determined by using lac geneticfusions in B. subtilis [3]. In order to study the inductionof Ptb synthesis and its possible inﬂuence on PHBcontent, B. megaterium was grown in MM supplementedas indicated in Materials and methods. This mediumcontained suﬃcient ammonium so that amino acids werenot used as a nitrogen source. Ptb speciﬁc activity andPHB were measured under each growth condition (Ta-ble 1).The results showed that the increase in enzymeactivity caused by the addition of branched amino acidscorrelated with an increase in PHB accumulation.Branched short-chain fatty acids were not detectedextracellularly, as in previous experiments.Eﬀect of ActBm on PHB accumulationThe results indicated a correlation between PHB accu-mulation and Ptb activity. In order to exclude the pos-sibility that the increase in PHB content was solely dueto the use of the amino acids as a carbon source, Ptbactivity was induced without the addition of its naturalinducers. It was predicted that overexpression of ActBm,an activator of Ptb synthesis, would as a consequenceFig. 1 Ptb expression, PHB accumulation and pH of the mediumthroughout the growth of Bacillus megaterium in supplementednutrient broth128increase the intracellular concentration of PHB.B. megaterium strain PV447 and a transconjugant of thisstrain containing plasmid pS14 were grown in SNBsupplemented with 20 g glucose/l. The results shown inTable 2 indicate that the strain carrying actBm in amulticopy plasmid had higher Ptb activity and a higherPHB content than the wild-type strain.These experiments suggest an association betweenPtb activity and PHB accumulation in B. megaterium forthe following reasons: (1) ptb expression and polymeraccumulation occur simultaneously during growth; (2)branched amino acids, which are inducers of enzymesynthesis, enhance PHB accumulation; (3) multiplecopies of actBm activate ptb expression and, at the sametime, enhance PHB production, in accordance with theresults obtained after induction of the enzyme withbranched amino acids. ptb from C. acetobutylicum hasbeen used for PHB production in E. coli recombinants[8].Enzyme activity and PHB accumulation increasedfrom the mid-exponential to the stationary phase ofgrowth. These ﬁndings are in line with what is knownabout the transition from the exponential to the sta-tionary phase in the sporulating model organism B.subtilis. This transient state, previous to sporulation, ischaracterized by the expression of a variety of genes thatare necessary for the scavenging of nutrients [13]. Wehave previously shown that B. megaterium PHB) mu-tants commit to sporulation earlier than the wild-typestrain [9]. Thus, PHB, as a carbon and energy source,allows this species to remain in the vegetative statelonger. Therefore, it is possible that both Ptb expressionand PHB accumulation are included in the global reg-ulation system characteristic of the transient state ofBacillus.Acknowledgements This work was supported by grants from theUniversidad de Buenos Aires (UBA) and Consejo Nacional deInvestigaciones Cientı´ﬁcas y Te´cnicas (CONICET). GJV had agraduate student fellowship from UBA. BSM is a career investi-gator from CONICET.References1. Braunegg G, Sonnleitner R, Laﬀerty R (1978) A rapid gasChomatographic method for the determination of poly-b-hy-droxybutiric acid in microbial biomass. Eur J Appl MicrobiolBiotechnol 6:29–372. Cary JW, Petersen DJ, Papoutsakis ET, Bennett GN (1988)Cloning and expression of Clostridium acetobutylicum phos-photransbutyrylase and butyrate kinase genes in Escherichiacoli. J Bacteriol 170:4613–46183. Debarbouille M, Gardan R, Arnaud M, Rapoport G (1999)Role of BkdR, a transcriptional activator of the SigL-depen-dent isoleucine and valine degradation pathway in Bacillussubtilis. J Bacteriol 181:2059–20664. English J, Vary P (1986) Isolation of recombination-defectiveand UV-sensitive mutants of Bacillus megaterium. J Bacteriol165:155–1605. Jones D, Woods D (1986) Acetone-butanol fermentationrevisited. Microbiol Rev 50:484–5246. Kunst F et al. (1997) The complete genome sequence of theGram-positive bacterium Bacillus subtilis. Nature 390:249–2567. Lach DA, Sharma VK, Vary PS (1990) Isolation and charac-terization of a unique division mutant of Bacillus megaterium.J Gen Microbiol 3:545–5538. Liu S-J, Steinbu¨chel A (2000), A novel genetically engineeredpathway for synthesis of poly (hydroxyalkanoic acids) in Esc-herichia coli. Appl Environ Microbiol 66:739–7439. Lo´pez N, Floccari M, Garcı´a A, Steinbu¨chel A, Me´ndez B(1995) Eﬀect of poly (3-hydroxybutyrate) content on the star-vation survival of bacteria in natural waters. FEMS MicrobEcol 16:95–10210. Lowry O, Rosebrough N, Farr L, Randall R (1951) Proteinmeasurement with the Folin phenol reagent. J Biol Chem193:265–27511. Madison LL, Huisman GW (1999) Metabolic engineering ofpoly(3-hydroxyalkanoates): From DNA to plastic. MicrobiolMol Biol Rev 63:21–5312. Pettinari MJ, Vazquez GJ, Kruger N, Vary P, Steinbu¨chel A,Me´ndez BS (1998) Trans activation of the Escherichia coli atostructural genes by a regulatory protein from Bacillus megate-rium: potential use in polyhydroxyalkanoate production. ApplMicrobiol Biotechnol 49:737–74213. Strauch M, Hoch J, (1993) Transition-state regulators: sentinelsof Bacillus subtilis post-exponential gene expression. MolecMicrobiol 7:337–34214. Trieu-Cuot P, Carlier C, Poyart-Salmeron C, Courvalin P(1991) Shuttle vectors containing a multiple cloning site and alacZ alpha gene for conjugal transfer of DNA from Escherichiacoli to gram-positive bacteria. Gene 102:99–10415. Vary P, Tao YP (1988) Development of genetic methods inBacillus megaterium. In: Ganesan AT, Hoch JA (eds) Geneticsand Biotechnology of Bacilli, vol 2. Academic , New York, pp403–40716. Vazquez GJ, Pettinari MJ, Me´ndez BS (2001) Phosphotrans-butyrylase expression in Bacillus megaterium. Curr Microbiol42:345–34917. Walter KA, Nair RV, Cary JN, Bennett GN, Papoutsakis ET(1993) Sequence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824.Gene 134:107–111Table 1 Eﬀect of branched amino acids on phosphotransbutyry-lase expression and polyhydroxybutyrate (PHB) accumulation.Bacillus megaterium was grown in minimal medium (MM) sup-plemented with carbon sources and amino acidsGrowth medium Ptb speciﬁcactivity (U/mg protein)PHB (% celldry weight)MM+glucose 791 8.83MM+glucose,valine, and isoleucine6,616 19.05MM+gluconate 5,183 11.58MM+gluconate,valine, and isoleucine43,555 31.05Table 2 Eﬀect of ActBm on PHB accumulation in B. megateriumgrown in SNB supplemented as describedPlasmid Ptb speciﬁcactivity (U/mgprotein)PHB (% celldry weight)None 17,190 12.4pS14 23,520 22.3129

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in Bacillus megaterium

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Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in Bacillus megaterium

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