JOURNAL OF BACTERIOLOGY, May 2004, p. 3015–3021 Vol. 186, No. 100021-9193/04/$08.00�0 DOI: 10.1128/JB.186.10.3015–3021.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

PhaQ, a New Class of Poly-�-Hydroxybutyrate (PHB)-ResponsiveRepressor, Regulates phaQ and phaP (Phasin) Expression in

Bacillus megaterium through Interaction with PHBTian-Ren Lee, Jer-Sheng Lin, Shih-Shin Wang, and Gwo-Chyuan Shaw*

Institute of Biochemistry, School of Life Science, National Yang-Ming University,Taipei, Taiwan, Republic of China

Received 31 October 2003/Accepted 2 February 2004

Bacillus megaterium can produce poly-�-hydroxybutyrate (PHB) as carbon and energy storage materials. Wenow report that the phaQ gene, which is located upstream of the phasin-encoding phaP gene, codes for a newclass of transcriptional regulator that negatively controls expression of both phaQ and phaP. A PhaQ bindingsite that plays a role in this control has been identified by gel mobility shift assays and DNase I footprintinganalysis. We have also provided evidence that PhaQ could sense the presence of PHB in vivo and that artificialPHB granules could inhibit the formation of PhaQ-DNA complex in vitro by binding to PhaQ directly. Thesesuggest that PhaQ is a PHB-responsive repressor.

Poly-�-hydroxybutyrate (PHB) and other polyhydroxyal-kanoates (PHAs) are biodegradable polyesters that are pro-duced by a wide variety of bacteria as intracellular carbon andenergy storage materials (1, 13, 31). PHB synthases (PhaC)and phasins (PhaP) are proteins that play important roles inPHB production and granule formation. Phasin, an abundantgranule-associated protein, forms a boundary layer on thePHB surface to sequester hydrophobic PHB from the cyto-plasm. Thus, phasin can inhibit individual granules from coa-lescing and promotes PHB synthesis by regulating the ratio ofsurface area to volume of PHB granules (27–29). PhaP mayalso have a protective function to reduce the passive attach-ment of cytoplasmic proteins to PHB surface (16, 27). It isgenerally thought that the synthesis of phasin proteins is highlyregulated (8). In Ralstonia eutropha, expression of the phaPgene is negatively controlled by the autoregulated repressorPhaR (21, 30). The accumulation of PhaP in the R. eutrophacells is strictly dependent on PHB production (28). After theonset of PHB biosynthesis, PhaR can sense the presence ofPHB and bind to nascent PHB granules, leading to derepres-sion of phaP. In Paracoccus denitrificans the phaR gene, whichis located immediately downstream of phaP, encodes a repres-sor that regulates phaP expression. PhaR can sense the pres-ence of PHA and interact with PHA granules (14, 15).

Although Bacillus megaterium is already known to be able tosynthesize PHB and accumulate PHB granules, genes that areinvolved in PHB synthesis and encode PHB granule-associatedproteins were not cloned until recently (17). Among a clusterof five pha genes of B. megaterium, phaP and phaQ are tran-scribed in one direction, whereas phaR, phaB, and phaC aredivergently transcribed as a tricistronic operon. phaP codes fora phasin protein. phaB and phaC encode NADPH-dependentacetoacetyl coenzyme A reductase and a novel PHB synthase,

respectively (17). This novel PHB synthase requires both PhaCand PhaR for activity (18). It should be noted that the desig-nation of the B. megaterium phaR gene does not follow theconventional rule. Rather than encoding a transcriptional reg-ulator like the PhaR protein of R. eutropha or P. denitrificans,the B. megaterium phaR gene probably encodes a subunit of aheterodimeric PHB synthase (18, 25). Nevertheless, the pro-tein that can sense the presence of PHB has not been identi-fied, and how the phasin-encoding phaP gene is regulatedremains unknown. Here we report that phaQ encodes a newclass of transcriptional regulator that can interact directly withPHB and regulate phaP expression.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterial strains and plasmidsused in this study are listed in Table 1. Escherichia coli and B. megaterium cellswere grown in Luria-Bertani (LB) medium (24). Antibiotics were used at thefollowing concentrations (in micrograms per milliliter): ampicillin, 100; chlor-amphenicol, 2 (for selection of B. megaterium integrants of pSG1151 derivatives)or 7 (for selection of B. megaterium transformants of pLC4); tetracycline, 10.

Construction of plasmids. Green fluorescent protein (GFP) tagging vectorpSG1151 (11) was used for the construction of various integrative plasmids. Toconstruct the integrative plasmid pGS1042, a 288-bp DNA fragment carryingcodons 104 through 199 of phaR and flanked by HindIII and EcoRI sites wasamplified by PCR and cloned between the HindIII and EcoRI sites of plasmidpSG1151, resulting in the phaR104–199::gfp in-frame fusion. To construct theintegrative plasmid pGS1071, a 336-bp DNA fragment carrying the Shine-Dal-gano sequence plus codons 1 through 100 of phaP and flanked by HindIII andEcoRI sites was generated by PCR and cloned between the HindIII and EcoRIsites of plasmid pSG1151, resulting in the phaP1–100::gfp in-frame fusion. Toconstruct the integrative plasmid pGS1111, a 297-bp DNA fragment carryingcodons 59 through 157 of phaC and flanked by HindIII and EcoRI sites wasamplified by PCR and was cloned between the HindIII and EcoRI sites ofplasmid pSG1151, resulting in the phaC59–157::gfp in-frame fusion.

Various DNA fragments in plasmids pGS1060, pGS1108, pGS1120, pGS1132,and pGS1142 as shown in Fig. 1A were amplified by PCR and cloned individuallybetween the EcoRI and HindIII sites of pLC4 (22). The DNA fragment inplasmid pGS1143, which contains a single-base substitution in the phaQ gene (Tto C at nucleotide position �161 relative to the phaQ translational start site), andthe DNA fragment in plasmid pGS1151, which carries a two-base substitutionwithin the 19-bp inverted repeat (AC to GT at positions �6 and �5), weregenerated by a two-step PCR method (6) followed by cloning the resulting DNA

* Corresponding author. Mailing address: Institute of Biochemistry,School of Life Science, National Yang-Ming University, Taipei 112,Taiwan. Phone: 886-2-2826-7127. Fax: 886-2-2826-4843. E-mail: gcshaw@ym.edu.tw.

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fragments individually between the EcoRI and HindIII sites of pLC4. The mu-tated sequences were verified by DNA sequencing.

To construct a plasmid that overproduces PhaQ in B. megaterium, a 490-bpDNA fragment carrying the Shine-Dalgano sequence plus the phaQ gene andflanked by EcoRI and HindIII sites was amplified by PCR and cloned betweenthe EcoRI and HindIII sites of pHY300PLK (Takara Shuzo Co. Ltd., Kyoto,Japan) to generate plasmid pGS1056. The promoter of the tetracycline resis-tance gene present in pHY300PLK can thus drive the expression of phaQ in B.megaterium.

To construct plasmid pGS253, a 380-bp DNA fragment carrying the codingsequence of bm3P1 (12) and flanked by BamHI and HindIII sites was amplifiedby PCR and was cloned between the BamHI and HindIII sites of pQE30 (QiagenInc.). To construct plasmid pGS1041, a 443-bp DNA fragment carrying thecoding sequence of phaQ and flanked by BamHI and HindIII sites was amplifiedby PCR and cloned between the BamHI and HindIII sites of pQE30. Thesequence of phaQ was verified by DNA sequencing. Plasmid pGS1041F54S,which contains a single-base mutation (T to C at nucleotide position �161relative to the phaQ translational start site) in the phaQ gene, was serendipi-tously obtained during the course of construction of a pQE30-based phaQ-overexpressing plasmid. To construct plasmid pGS1144, the above-mentioned443-bp DNA fragment carrying the coding sequence of phaQ and flanked byBamHI and HindIII sites was cloned between the BamHI and HindIII sites ofpMAL-c2 (New England Biolabs, Inc.) to generate a maltose-binding protein(MalE)-PhaQ fusion protein.

Construction of B. megaterium mutant strains with protein-GFP fusions. Plas-mid pSG1151-based integrative plasmids pGS1042, pGS1071, and pGS1111 wereindividually introduced into B. megaterium cells. Integration was achieved by aCampbell-like single-crossover recombination. Resistance to chloramphenicolwas used to select the mutant strains. The correctness of each integrant wasverified by PCR and Southern blot analysis.

Overproduction and purification of His-tagged Bm3P1, BscR, PhaQ, andPhaQ(F54S) proteins. E. coli JM109 cells bearing plasmid pGS253, pGS482,pGS1041, or pGS1041F54S were grown in LB medium. After the absorbance at600 nm reached 0.5, IPTG (isopropyl-�-D-thiogalactopyranoside) was added at afinal concentration of 0.3 mM, and incubation was continued for 2 h. After

harvesting cells by centrifugation and disrupting resuspended cells by sonicationon ice, the disrupted cells were subjected to centrifugation at 15,000 � g for 10min. Purification of His-tagged proteins from the supernatants by Ni-nitrilotri-acetic acid affinity column was performed according to the instructions of thematrix manufacturer (Qiagen Inc.).

Overproduction and purification of the MalE-PhaQ fusion protein. Cellgrowth, induction by IPTG, cell disruption, and centrifugation were performedwith the same procedure as that described above. Purification of the MalE-PhaQfusion protein by amylose column was carried out according to the instructionsof the matrix manufacturer (New England Biolabs, Inc.). After elution theMalE-PhaQ fusion protein was concentrated by using the Centricon-10 concen-trator (Amicon, Inc.). In order to cleave the MalE from PhaQ, 100 �g ofMalE-PhaQ was incubated with 1 �g of factor Xa protease (New EnglandBiolabs, Inc.) at 4°C for 2 days. The progress of cleavage was checked by sodiumdodecyl sulfate–13 polyacrylamide gel electrophoresis (SDS–13% PAGE). Thereleased PhaQ protein was used without further purification.

DNase I footprinting analysis. About 4 ng of 32P-end-labeled DNA fragmentcontaining the phaQ promoter region was incubated with the PhaQ protein (1.5to 12 ng). DNase I footprinting assays were performed exactly as describedpreviously (3).

Preparation of artificial PHB granules. Native PHB granules were isolatedfrom B. megaterium cells as described previously (19). Approximately 2 mg ofisolated native PHB granules were then incubated with 50 �g of trypsin at 37°Cin 500 �l of a reaction mixture containing 10 mM Tris-HCl (pH 7.4) for 1 h.Trypsin was then inactivated by treatment with 4 mM phenylmethylsulfonylfluoride and by heating to 70°C for 10 min. After centrifugation the pellets wereexhaustively washed with 10 mM Tris-HCl (pH 7.4), resuspended in TE buffer(10 mM Tris-HCl and 1 mM EDTA), and stored at 4°C.

Rebinding of PhaQ to artificial PHB granules in vitro. Purified His-taggedPhaQ (100 ng) or His-tagged Bm3P1 (120 ng) was incubated with artificial PHBgranules (100 �g) in a final volume of 25 �l of reaction mixture containing 10mM Tris-HCl (pH 7.4). After incubation for 30 min the reaction mixtures wereseparated into pellets and supernatants by centrifugation. The PHB granulespresent in pellets were washed twice with 10 mM Tris-HCl buffer and were

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Descriptiona Reference or sourceb

E. coliJM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi �(lac-proAB) F�[traD36 proAB� lacIq lacZ� M15] Takara

B. megateriumATCC 11561 Wild-type strain ATCCBM647 ATCC 11561 derivative; phaR::pGS1042 This studyBM679 ATCC 11561 derivative; phaP::pGS1071 This studyBM695 ATCC 11561 derivative; phaC::pGS1111 This study

PlasmidpLC4 Promoter probe vector with xylE as the reporter gene; Apr Cmr 22pQE30 Expression vector for producing His-tagged proteins; Apr QiagenpMAL-c2 Expression vector for producing fusions of maltose-binding protein; Apr New England BiolabspSG1151 GFP tagging vector; Apr Cmr 11pHY300PLK Shuttle vector; Apr Tcr TakarapGS253 pQE30 carrying the bm3P1 gene This studypGS482 pQE30 carrying the bscR gene 10pGS1041 pQE30 carrying the wild-type phaQ gene This studypGS1041F54S pQE30 carrying the mutated phaQ gene (F54S substitution) This studypGS1042 pSG1151 derivative; PhaR-GFP in-frame fusion plasmid; phaR (aa 104 to 199)::gfp This studypGS1056 pHY300PLK with an insert containing the Shine-Dalgano sequence plus the phaQ gene This studypGS1060 pLC4 carrying the phaQ promoter region This studypGS1071 pSG1151 derivative; PhaP-GFP in-frame fusion plasmid; phaP (aa 1 to 100)::gfp This studypGS1108 pLC4 carrying the phaQ-phaP intergenic region This studypGS1111 pSG1151 derivative; phaC disruptive plasmid; PhaC-GFP in-frame fusion plasmid; phaC (aa 59 to 157)::gfp This studypGS1120 pLC4 carrying the phaQ coding region This studypGS1132 pLC4 carrying the phaQ coding region and the phaQ-phaP intergenic region This studypGS1142 pLC4 carrying the promoter region and the coding region of phaQ This studypGS1143 pLC4 carrying the promoter region and the mutated coding region of phaQ This studypGS1144 pMAL-c2 carrying the phaQ gene This studypGS1151 pLC4 carrying the mutated regulatory region and the wild-type coding region of phaQ This study

a Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Tcr, tetracycline resistant. aa, amino acids.b ATCC, American Type Culture Collection.

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resuspended in the denaturing sample buffer. Proteins were analyzed in anSDS–12% polyacrylamide gel and were stained with Coomassie brilliant blue.

Other methods. Transformation of B. megaterium cells was achieved by theprotoplast method (2). An established method was used for spectrophotometricmeasurement of XylE (catechol 2,3-dioxygenase) activity (22). The procedure ofLaemmli (9) was used for SDS-PAGE. Western blotting was performed asdescribed previously (26). Gel mobility shift assays were carried out according tothe method of Fried and Crothers (5). Protein concentrations were determinedby the bicinchoninic acid protein assay method according to the instructions ofthe manufacturer (Pierce Biotechnology, Inc.) with bovine serum albumin as thestandard.

RESULTS AND DISCUSSION

Analysis of the regulatory role of PhaQ. The phaQ gene ofB. megaterium is located immediately upstream of the phasin-encoding phaP gene (17). PhaQ of B. megaterium does not

exhibit significant amino acid similarity to PhaR of R. eutrophaor P. denitrificans or to other proteins in the data banks withknown function. To determine whether PhaQ plays a regula-tory role, a series of plasmid pLC4 (22)-derived constructswere made with promoterless xylE as a reporter gene (Fig. 1A)and were introduced into B. megaterium cells. As shown in Fig.1B, the presence of phaQ in plasmid pGS1142 led to a muchlower XylE-specific activity than deletion of phaQ in plasmidpGS1060, raising the possibility that phaQ might encode anegative regulator.

During the course of construction of a PhaQ-overproducingplasmid in E. coli (see below), a mutant form of phaQ thatcontains a single-base mutation (T to C at nucleotide position�161 relative to the phaQ translational start site) was seren-dipitously generated by PCR amplification of the phaQ gene.This led to an F54S replacement in PhaQ. While carrying outgel mobility shift assays (see below) we found that the wild-type PhaQ had specific DNA-binding ability in vitro. However,when PhaQ carried an F54S mutation its DNA-binding abilitywas abolished. To test if this would occur in vivo, a single-basesubstitution (T to C at nucleotide position �161 relative to thephaQ translational start site) was introduced into the codingregion of phaQ (leading to an F54S replacement) to generateplasmid pGS1143 (Fig. 1A). B. megaterium cells carrying plas-mid pGS1143 showed sixfold higher XylE-specific activity thanthose carrying plasmid pGS1142 (Fig. 1B). These results sug-gested that phaQ might encode a regulatory protein that neg-atively autoregulates its own expression.

To further confirm the phaQ autoregulation, a binary-vectorsystem in B. megaterium cells was used to examine the effect ofPhaQ produced from the low-copy plasmid pGS1056 on ex-pression of the phaQ promoter region-xylE transcriptional fu-sion from the high-copy plasmid pGS1060 (Fig. 1A). TheXylE-specific activity obtained from B. megaterium cells carry-ing compatible plasmids pGS1056 and pGS1060 was 88 � 8mU/mg of protein, whereas the XylE-specific activity in B.megaterium cells carrying compatible plasmid pGS1060 and thelow-copy control vector pHY300PLK was 236 � 19 mU/mg ofprotein. This result is in agreement with the notion that phaQexpression is subject to negative autoregulation.

phaQ and phaP constitute a bicistronic operon. phaQ andphaP are transcribed in an orientation opposite to that of thephaRBC operon (17) (Fig. 1A). Sequence analysis revealed notypical -independent transcription terminator within the168-bp phaQ-phaP intergenic region. Northern blot analysiswas used to examine whether phaQ and phaP genes could becotranscribed. It was found that both a phaQ-specific probeand a phaP-specific probe could hybridize to a 1.2-kb transcriptthat is sufficient to span the phaQ and phaP genes (data notshown), suggesting that these two genes can be cotranscribed.Reverse transcriptase PCR also confirmed the operonic orga-nization of these two genes (data not shown). The phaP-spe-cific probe, but not the phaQ-specific probe, also hybridized toa 0.7-kb transcript whose length is sufficient to span the phaPgene. To investigate whether a promoter is present in thephaQ-phaP intergenic region, we constructed plasmid pGS1108(Fig. 1A). The XylE-specific activity obtained from B. megate-rium cells carrying plasmid pGS1108 suggested that no pro-moter exists within the phaQ-phaP intergenic region (Fig. 1B).To exclude the possibility that a promoter might be present in

FIG. 1. Schematic representation of the plasmid constructs andtheir XylE-specific activities in B. megaterium. (A) The number aboveeach line denotes base position relative to the transcriptional initiationsite of phaQ (17). The solid bar below the phaQ promoter regionrepresents a 19-bp imperfect inverted repeat (positions �18 to �1). Atwo-base substitution within the 19-bp inverted repeat (AC to GT atpositions �6 and �5) or a single-base change in the phaQ codingregion (T to C, leading to an F54S replacement in PhaQ) is repre-sented by a cross. Each pLC4-derived plasmid contains an xylE re-porter gene. (B) XylE-specific activities of B. megaterium cells carryingthe above plasmids and grown in LB medium. Each value representsthe mean of at least four determinations. Each error bar indicates thestandard error of the mean.

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the phaQ coding region or span the boundary between thephaQ coding region and the phaQ-phaP intergenic region, wealso constructed plasmids pGS1120 and pGS1132 (Fig. 1A). Itwas found that no promoter activity could be detected withinthese regions (Fig. 1B). Taken together, these results suggestthat the 0.7-kb transcript is most likely to be a processingproduct derived from the 1.2-kb transcript.

It is generally thought that the phaP-encoded phasin is anabundant protein in PHA-producing bacteria, whereas thelevel of a transcriptional regulator like PhaQ is supposed to below in bacteria. Should phaQP be derepressed, a posttranscrip-tional processing event occurring in the phaQP cotranscriptcould selectively degrade the phaQ transcript and leave thephaP transcript intact, thus allowing differential syntheses ofthese two proteins. It is tempting to speculate that the longphaQ-phaP intergenic region might provide a signal for theprocessing event to occur.

Negative regulation of phaP expression by PhaQ. To test theeffect of PhaQ on phaP expression, we constructed the B. meg-aterium strain BM679 in which the N-terminal 100 amino acidsof PhaP were translationally fused to a green fluorescent pro-tein (GFP) encoded by the integrative plasmid pSG1151 (11).Whole-cell extracts prepared from B. megaterium strain BM679carrying the above-mentioned PhaQ-overproducing plasmidpGS1056 or control vector pHY300PLK were subjected toSDS–13% PAGE (Fig. 2A, lanes 1 and 2) and Western blottingwith anti-GFP antibody as the probe (Fig. 2A, lanes 3 and 4).A protein band corresponding to a translational fusion be-tween the truncated PhaP and GFP could be detected by anti-GFP antibody in B. megaterium strain BM679 bearing the con-trol vector pHY300PLK (Fig. 2A, lane 3). However, the band

intensity of the same fusion protein from B. megaterium strainBM679 carrying the PhaQ-overproducing plasmid pGS1056was much weaker (Fig. 2A, lane 4), indicating that PhaQ cannegatively regulate phaP expression.

To further investigate the possible effect of PhaQ on phaRexpression, we constructed the B. megaterium strain BM647 inwhich PhaR was translationally fused to GFP. Whole-cell ex-tracts prepared from B. megaterium strain BM647 carrying thePhaQ-overproducing plasmid pGS1056 or the control vectorpHY300PLK were subjected to SDS–11% PAGE (Fig. 2B,lanes 1 and 2) and Western blotting with anti-GFP antibody asthe probe (Fig. 2B, lanes 3 and 4). In contrast to the repressiveeffect of PhaQ on phaP expression, PhaQ had no apparenteffect on phaR expression (Fig. 2B, lanes 3 and 4).

Interaction of PhaQ with the phaQ promoter region in vitro.Because the above results revealed that PhaQ could autoregu-late its own expression in vivo, it was tempting to explore wheth-er PhaQ could bind to the phaQ regulatory region in vitro. Tofacilitate purification we constructed plasmid pGS1144, whichcould overproduce a maltose-binding protein (MalE)-PhaQ fu-sion protein. This MalE-PhaQ fusion protein was purified fromthe crude extract of E. coli cells carrying pGS1144 by affinitychromatography on an amylose column. The PhaQ proteinreleased from cleavage of purified MalE-PhaQ fusion proteinwith factor Xa protease was used without further purification.

To determine whether PhaQ was able to bind DNA se-quence specifically, gel mobility shift assays with two PCR-amplified DNA fragments end-labeled with 32P were per-formed. One was a 0.15-kb fragment containing the phaQpromoter region (�105 to �39 relative to the transcriptionalinitiation site of phaQ). The other was a 0.16-kb fragmentcontaining the bscR promoter region (10). The results showedthat PhaQ could retard the DNA fragment containing thephaQ promoter region (Fig. 3A, lanes 7 to 10) but not thecontrol DNA fragment at the assay concentrations (Fig. 3A,lanes 2 to 5). No such retardation was detected when purifiedMalE–�-galactosidase fusion protein was used at similarconcentrations (data not shown). It is noteworthy that, in con-trast to the purified wild-type PhaQ protein, purified PhaQprotein containing an F54S replacement did not exhibit DNA-binding ability in gel mobility shifts assays under similar assayconditions (data not shown), suggesting that the native PhaQprotein is indeed a DNA-binding protein.

We next carried out DNase I footprinting analysis to definethe binding site(s) for PhaQ in the phaQ promoter region.When the lower strand (template strand) of phaQ promoter-containing DNA was end labeled with 32P, the PhaQ proteinprotected two regions from DNase I cleavage. The longer onecorresponds to the sequence �19 to �4 (relative to the tran-scriptional initiation site of phaQ), which covers a 19-bp im-perfect inverted repeat (�18 to �1) (Fig. 4). The shorter onecorresponds to the sequence �34 to �32. When the upperstrand (non-template strand) of phaQ promoter-containingDNA was end labeled with 32P, the regions were protected bythe PhaQ protein span �25 to �3 and �35 to �31 (Fig. 4).These protected regions overlap with the �35 and �10 regionsof the �A-like promoter of phaQ, suggesting that PhaQ prob-ably acts by interfering with RNA polymerase binding.

To further confirm that PhaQ could interact specifically withthe 19-bp inverted repeat, a double-stranded oligonucleotide

FIG. 2. Western blot analyses of effects of PhaQ overproduction onexpression of phaP and phaR genes. (A) Whole-cell extracts wereprepared from B. megaterium strain BM679 (bearing a chromosomalPhaP-GFP fusion) transformed with the PhaQ-overproducing plasmidpGS1056 (lanes 2 and 4) or the control vector pHY300PLK (lanes 1and 3). Equal amounts of total proteins were separated in SDS–13%polyacrylamide gels. Bands were visualized with Coomassie brilliantblue staining (lanes 1 and 2). Western blotting was performed on aduplicate gel with anti-GFP antibody as the probe (lanes 3 and 4).Lane M, molecular mass markers. (B) Whole-cell extracts were pre-pared from the B. megaterium strain BM647 (bearing a chromosomalPhaR-GFP fusion) transformed with the PhaQ-overproducing plasmidpGS1056 (lanes 2 and 4) or the control vector pHY300PLK (lanes 1and 3). Equal amounts of total proteins were separated in SDS–11%polyacrylamide gels. Bands were visualized with Coomassie brilliantblue staining (lanes 1 and 2). Western blotting was performed on aduplicate gel with anti-GFP antibody as the probe (lanes 3 and 4).

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containing the 19-bp inverted repeat (oligo I) and a double-stranded oligonucleotide containing a four-base mutation inthe 19-bp inverted repeat (oligo II) (Fig. 3B) were used asprobes in gel mobility shift assays. The result showed thatPhaQ was capable of binding to oligo I but not to oligo II,suggesting that the wild-type 19-bp inverted repeat is a bindingsite for PhaQ.

Effect of mutations in the 19-bp inverted repeat on expres-sion of the phaQ-xylE transcriptional fusion in vivo. To exam-ine the effect of mutations in the 19-bp inverted repeat onexpression of the phaQ-xylE transcriptional fusion in vivo, atwo-base substitution (AC to GT at positions �6 and �5) wasintroduced into the 19-bp inverted repeat to generate plasmidpGS1151 (Fig. 1A). As shown in Fig. 1B, B. megaterium cells car-rying plasmid pGS1151 exhibited approximately 2.4-fold high-er XylE-specific activity than cells bearing plasmid pGS1142,suggesting that the 19-bp inverted repeat contributes, at leastin part, to the control of phaQ expression in vivo.

Effect of artificial PHB granules on formation of PhaQ-DNAcomplex. We next used purified PhaQ protein and a DNAfragment containing the phaQ promoter region (positions �105to �39) end labeled with 32P for gel mobility shift assays todetermine the effect of artificial PHB granules on formation of

FIG. 3. Gel mobility shift assays of the DNA-binding ability ofPhaQ and the effects of artificial PHB granules on formation of PhaQ-DNA complex and BscR-DNA complex. (A) A 0.16-kb DNA fragmentcontaining the B. subtilis bscR promoter region (10) (lanes 1 to 5) anda 0.15-kb DNA fragment containing the B. megaterium phaQ promoterregion (positions �105 to �39) (lanes 6 to 10) were used as probes.About 2 ng of 32P-labeled DNA probe was used in each reactionmixture (final volume, 20 �l). Lanes 1 and 6, DNA probe alone; lanes2 to 5 and 7 to 10, DNA probe plus increasing amounts of PhaQ (3, 6,12, and 24 ng, respectively). Samples were run in an 8% native poly-acrylamide gel. (B) Double-stranded oligo I containing an imperfectinverted repeat (positions �18 to �1) in the phaQ promoter regionused as the probe in lanes 6 to 10. The sequence of its upper strand isshown at the bottom of the panels. Double-stranded oligo II contain-ing a four-base mutation in the inverted repeat (shown at the bottomof the panels) was used as the probe in lanes 1 to 5. About 0.3 ng of32P-labeled DNA probe was used in each reaction mixture (final vol-ume, 20 �l). Lanes 1 and 6, DNA probe alone; lanes 2 to 5 and 7 to 10,DNA probe plus increasing amounts of PhaQ (1.5, 3, 6, and 12 ng,respectively). Samples were run in an 8% native polyacrylamide gel.(C) The above-mentioned 0.16-kb DNA fragment containing the bscRpromoter region was used as the probe in lanes 1 to 4. The above-mentioned 0.15-kb DNA fragment containing the phaQ promoter re-gion was used as the probe in lanes 5 to 8. Purified His-tagged BscR(12 ng) or PhaQ (8 ng) was incubated with 2 ng of 32P-labeled DNAprobe in the absence or presence of artificial PHB granules in a finalvolume of 20 �l of reaction mixture. Lanes 1 and 5, DNA probe alone;lane 2, DNA probe plus BscR; lanes 3 and 4, DNA probe plus BscR inthe presence of 15 and 30 �g of PHB, respectively; lane 6, DNA probeplus PhaQ; lanes 7 and 8, DNA probe plus PhaQ in the presence of 15and 30 �g of PHB, respectively.

FIG. 4. DNase I footprinting analysis of PhaQ binding to the phaQpromoter region. (A) A 0.4-kb SmaI-HindIII DNA fragment contain-ing the phaQ promoter region (positions �356 to �39) and labeledwith 32P at its HindIII site was incubated in the absence or presence ofthe PhaQ protein. Lanes 1 and 6, no PhaQ protein; lanes 2 to 5contained 1.5, 3, 6, and 12 ng of the PhaQ protein, respectively. (B) A0.36-kb BamHI-EcoRI DNA fragment containing the phaQ promoterregion (positions �105 to � 249) and labeled with 32P at its BamHIsite was incubated in the absence or presence of the PhaQ protein.Lanes 1 and 6, no PhaQ protein; lanes 2 to 5 contained 1.5, 3, 6, and12 ng of the PhaQ protein, respectively. The numbers on the leftindicate the positions of bases relative to the transcriptional initiationsite of phaQ. Solid brackets on the right denote the protected regions.

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PhaQ-DNA complex in vitro. Artificial PHB granules wereprepared as described in Materials and Methods. As a control,the effect of artificial PHB granules on the formation of BscR-DNA complex was also examined. The bscR gene has beenpreviously demonstrated to encode a repressor that negativelyregulates the transcription of the bscR-CYP102A3 operon ofBacillus subtilis (10). The results showed that the intensity ofthe shifted band representing PhaQ-DNA complex graduallydecreased as the concentrations of artificial PHB granulesgradually increased (Fig. 3C, lanes 6 to 8), whereas artificialPHB granules at the same range of concentrations did notinterfere with the formation of BscR-DNA complex (Fig. 3C,lanes 2 to 4), suggesting that artificial PHB granules can spe-cifically inhibit the formation of PhaQ-DNA complex.

Rebinding of PhaQ to artificial PHB granules in vitro. Wenext examined if PhaQ could bind artificial PHB granulesdirectly. For comparison, we constructed two pQE30-basedplasmids (pGS1041 and pGS253) that could overproduce His-tagged PhaQ protein and His-tagged Bm3P1 protein in E. coli,respectively. The Bm3P1 protein of B. megaterium is a solubleprotein of 122 amino acids (12). After incubation of artificialPHB granules with either purified His-tagged PhaQ protein orpurified His-tagged Bm3P1 protein, the reaction mixtures wereseparated into pellets and supernatants by centrifugation. Pro-teins were analyzed in an SDS–12% polyacrylamide gel. Theresult showed that the His-tagged PhaQ protein exhibited highaffinity for artificial PHB granules, whereas the His-taggedBm3P1 protein displayed no affinity for artificial PHB granulesunder similar assay conditions (Fig. 5). Results from Fig. 3Cand 5 suggest that inhibition of formation of PhaQ-DNA com-plex by artificial PHB granules is probably through direct in-teraction between PHB and the PhaQ protein and raised the

possibility that PhaQ might sense the presence of nascent PHBin vivo.

Effect of PHB synthesis on phaQ expression in B. megate-rium. To explore whether phaQ expression would respond tothe synthesis of PHB in vivo, we constructed the phaC disrup-tion mutant BM695 as described in Materials and Methods.The correct disruption was verified by PCR and Southern blotanalysis. The PHB-negative phenotype of the phaC disruptionmutant was verified by Nile Blue A staining (18) (data notshown). Plasmid pGS1142 (Fig. 1A), which contains the xylEreporter gene preceded by both the promoter region and thecoding region of phaQ, was introduced into the wild-type B.megaterium and the phaC disruption mutant. As shown in Fig.6, XylE-specific activities at various time points in the phaCmutant bearing plasmid pGS1142 were very low. For the wild-type B. megaterium carrying plasmid pGS1142, there was atime lag of several hours before dramatic increases in XylE-specific activity were observed. This correlates well with pre-vious observations that there was a lag phase for PHB accu-mulation in B. megaterium, after which PHB synthesis occurredat an accelerating rate (19). Taken together, these results sug-gest that PhaQ can sense the onset of PHB synthesis in vivoand is a PHB-responsive repressor.

In this study we have provided evidence that the B. megate-rium PhaQ protein plays a role in regulation of phaP expres-sion as well as autoregulation. Our data also indicate thatPhaQ is able to bind artificial PHB granules as well as DNA invitro and sense the presence of PHB in vivo. These findingssuggest that PhaQ is a PHB-responsive repressor, and PHBcan act as an inducer for phaP expression in a PhaQ-mediatedregulatory system.

By using different programs based on the method of Doddand Egan (4), no typical helix-turn-helix DNA-binding motif(20) could be detected in PhaQ. Moreover, PhaQ of B. mega-

FIG. 5. Rebinding of PhaQ to artificial PHB granules. PurifiedHis-tagged PhaQ (100 ng) (lanes 1 to 3) or His-tagged Bm3P1 (120 ng)(lanes 4 to 6) was incubated with artificial PHB granules (100 �g) for30 min. The reaction mixtures were then separated into pellets andsupernatants by centrifugation. Proteins were analyzed in a SDS–12%polyacrylamide gel and stained with Coomassie brilliant blue. Resultsare for total input protein before incubation (T), protein in superna-tant after centrifugation (S), and granule-associated protein after cen-trifugation (G). Lane M, molecular size markers.

FIG. 6. Effect of PHB synthesis on phaQ expression in B. megate-rium. Plasmid pGS1142 (Fig. 1A) was introduced into wild-type B.megaterium cells (circles and diamonds) and the phaC disruption mu-tant BM695 (squares and triangles). Overnight cultures were diluted100-fold in fresh LB medium, and samples were taken at the indicatedtimes to determine the absorbance at 600 nm (diamonds and triangles)and XylE-specific activity (circles and squares). OD600, optical densityat 600 nm.

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terium does not exhibit significant amino acid sequence simi-larity to any protein with a known function in the data banks.Although the regulatory protein PhaR of R. eutropha showssimilarity to PhaR of P. denitrificans at its N-terminal part, theN-terminal portion of PhaQ of B. megaterium does not showsignificant similarity to that of PhaR of either R. eutropha or P.denitrificans. The sequence of the cis-acting element for PhaQis also quite different from the corresponding sequences ofcis-acting elements for PhaR of P. denitrificans (15) and PhaRof R. eutropha (21). The size of PhaQ (146 amino acids) is alsosmaller than that of PhaR of P. denitrificans (195 amino acids)or PhaR of R. eutropha (183 amino acids). Nevertheless, PhaQof B. megaterium shows high amino acid sequence similaritywith the hypothetical PhaQ of Bacillus anthracis (57.5% iden-tity and 63.0% similarity) (23) and with the hypothetical PhaQof Bacillus cereus (56.2% identity and 61.6% similarity) (7).These results suggest that PhaQ represents a new class oftranscriptional regulator. Further investigation of the crystalstructure of PhaQ and identification of a novel DNA-bindingmotif of PhaQ are under way.

ACKNOWLEDGMENT

This research was supported by grant NSC 90-2311-B-010-003 fromthe National Science Council of the Republic of China.

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