Vol. 172, No. 8JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4315-43210021-9193/90/084315-07$02.00/0Copyright C 1990, American Society for Microbiology

Cloning of aldB, Which Encodes a-Acetolactate Decarboxylase,an Exoenzyme from Bacillus brevis

B0RGE DIDERICHSEN,* ULLA WEDSTED, LISBETH HEDEGAARD,BIRGER R. JENSEN, AND CARSTEN SJ0HOLM

Novo Nordisk, DK-2880 Bagsvaerd, Denmark

Received 18 December 1989/Accepted 18 May 1990

A gene for a-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and inBacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flankingregions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptideof 24 or 27 amino acids. A B. subtilis strain harboring the aidB gene on a recombinant plasmid processed andsecreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular.

The main fermentation of beer is followed by a maturationperiod of several weeks, during which a-acetolactate isconverted to diacetyl by a slow, nonenzymatic oxidativedecarboxylation. Subsequently, diacetyl is converted toacetoin catalyzed by diacetyl reductase of the yeast cells stillpresent at this stage (Fig. 1; 11). If a-acetolactate is notproperly removed during the maturation, diacetyl later ap-pears. The quality of beer may as a consequence be reduced,since diacetyl at a level of above 0.1 mg/liter has a negativeeffect on aroma and taste. By adding a-acetolactate decar-boxylase (ALDC) to the fermenting wort, a-acetolactate canbe removed as it is formed, thereby avoiding the formationof diacetyl and eliminating the need for maturation (Fig. 1;12).We have in our laboratories discovered that a Bacillus

brevis strain produces an ALDC which is well suited for usein the wort. To study the properties of this enzyme in moredetail, we have cloned and expressed aldB, the structuralgene for B. brevis ALDC, in Escherichia coli and in Bacillussubtilis. Analyses of the gene and its product show thatALDC from B. brevis is an exoenzyme that can be secretedby way of a proper signal peptide.

MATERIALS AND METHODSBacterial strains and plasmids. The B. brevis donor strain

was a derivative of ATCC 11031. The E. coli strains were:SJ2 lacIqZAM]5 hsdR, a derivative ofE. coli K-12 C600, andSJ6 leuB hsdR, a derivative of E. coli K-12 MC1000 (2). TheB. subtilis strains were DN1885 amyE, a derivative of B.subtilis 168 RUB200 (21), and DN969 and ToC46, protease-deficient derivatives of B. subtilis 168 RUB200 (21) and1A289 of the Bacillus Genetic Stock Center, Columbus,Ohio, respectively. E. coli plasmids are listed in Table 1. AllE. coli strains are derivatives of SJ2 except UW25, which isderived from SJ6. B. subtilis plasmids are listed in Table 2.All B. subtilis strains are derived from DN1885 exceptUW123 and UW214, which are derived from DN969 andToC46, respectively.Media. LB (16) and 2% agar was used for solid media, and

TY medium was used for liquid media. TY contains, per1,000 ml of distilled water: 20 g of Trypticase, 5 g of yeastextract, 6 mg of FeCl2 4H20, 1 mg of MnCl2 - 4H20, and 15mg of MgSO4- 7H20, pH 7.3. ALDC was purified from afermentation of B. brevis on the following medium: 10 g of

* Corresponding author.

yeast extract, 20 g of corn steep liquor, and 50 g of sucroseper 1,000 ml of water, pH 6.2.

Transformation. Competent cells of E. coli and B. subtiliswere prepared and transformed by established procedures(15, 20). E. coli transformants were selected for resistance toampicillin (100 ,ug/ml). B. subtilis transformants were se-lected for resistance to chloramphenicol (6 ,ug/ml).

Manipulations of DNA. Chromosomal DNA was preparedby phenol extraction (20). Plasmid DNA was prepared byalkaline extraction (1). Restriction, ligation, and exonucleasetreatment were with enzymes from New England BioLabs,Inc. Exonuclease III was used for generation of deletions(14). DNA for construction of gene banks and recombinantplasmids was isolated from agarose gels, using DEAE-cellulose paper (7).

Oligonucleotides. Probes, primers, and linkers were ob-tained from New England BioLabs or synthesized on anApplied Biosystems DNA synthesizer and purified beforeuse. On the basis of a partial amino acid sequence of ALDCfrom B. brevis (B. R. Jensen, I. Svendsen, and M. Ottesman,Abstr. Eur. Biotechol. Congr. 1987, p. 393-400), mixture Aof 12 17-mers and mixture B of 32 17-mers were designed asfollows:

target A: GluA: 3'- CTT/Ctarget B: PheB: 5'- TTT/C

Met Ile GlnTAC TAA/G/T GTT/CGlu Phe LysGAA/G TTT/C AAA/G

MetTACAsn

AAT/C

GlyCC-5'ValGT-3

Probes A and B correspond to base pairs (bp) 487 to 471 and777 to 803, respectively, in the nucleotide sequence shown inFig. 4.

Southern blotting analyses. Transfer of DNA was per-formed by electroblotting as described in the instructionmanual for GeneScreen Plus hybridization transfer mem-branes (Dupont, NEN Research Products). Hybridizationswere performed at 37°C with 32P-labeled oligonucleotides(16).DNA sequencing. Sequencing was performed directly on

double-stranded plasmid templates, using the Sequenase kitfrom United States Biochemical Corp. and [35S]dATP fromDupont. Primers were pUC19 sequencing primers fromDupont or synthesized on an Applied Biosystems oligonu-cleotide synthesizer. The protocol was as described in thesequenase booklet except that 2 to 5 ,ug of plasmid DNA in8 ,ul of H20 was denatured by addition of 2 RI of 2 M NaOH,

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4316 DIDERICHSEN ET AL.

0 0

OH

OH

a-Acetolactic

0

0

Diacetvl

Dia(

Acetolactate red'

decarboxylase \

0HX

cetylluctase

OH

Acetoin

FIG. 1. Degradation of a-acetolactate. Efficient enzymatic con-version of acetolactate to acetoin prevents the slow, nonenzymaticformation of diacetyl (11).

neutralized by addition of 3 ,ul of 3 M sodium acetate (pH4.6), and ethanol precipitated before resuspension in waterand addition of primer and sequencing buffer.

Colony hybridizations. Colonies were transferred to What-man 540 paper filters, lysed, and immobilized (8). The filterswere hybridized with 32P-labeled heptadecamer mixtures.Hybridization and washing of the filters were done at 37°C,followed by autoradiography for 4 h.ALDC antiserum. ALDC antiserum was raised in rabbits

against ALDC purified from B. brevis ATCC 11031 (I.Svendsen, B. R. Jensen, and M. Ottesen, Carlsberg Res.Commun., in press).Colony immunoblotting. Nitrocellulose filters were placed

on plates with recombinant colonies at 37°C for at least 30min. The filters were blocked with 2% (wt/vol) Tween 20 inwashing buffer (0.05 M Tris hydrochloride [pH 10], 0.15 MNaCl). They were incubated for 2 h in washing buffercontaining 0.05% (wt/vol) Tween 20 and ALDC antiserum.The filters were washed three times for 10 min in washingbuffer and incubated for 2 h in washing buffer containing0.05% (wt/vol) Tween 20 and anti-rabbit peroxidase-conju-gated antibody (Dakopats). For visualization of antigen-antibody complexes, 3-amino-9-ethylcarbazole was used assubstrate.ALDC assay. Enzymatic activity was assayed by measur-

TABLE 1. E. coli plasmidsa

Plasmid Size Allele Origin

pDN3000 2,732 bp pUC19 + polylinker'pUWl1 5.2 kb aldB+ pUC19 + 2.6 kb from

B. breviscpUW25 4.2 kb aldB25 pDN3000 + 1.5 kb

from pUWllC

aPlasmids pUW88-92 and pUW94-97 were derived from pUW25 by exo-nuclease degradation (see text, Table 3, and Fig. 2). All plasmids are Apr andare derivatives of pUC19.

b The polylinker Qf pDN3000 is the 58-mer KFN602 (5'-AATTGATCAAGCTTTAAATGCATGCTAGCAACGCGGCCGCCAACCTCGAGATCTCATG-3') which in the above orientation was inserted in the EcoRI site ofpUC19. The order of the most important new restriction sites is as follows:BclI HindlIl Dral NsiI SphI NotI Xhol BgllI EcoRI, followed by Sacl KpnI,etc., of the original pUC19 polylinker.

c See text and Fig. 2.

TABLE 2. B. subtilis plasmidsa

Plasmid Size Allele Origin

pPL1385 2,674 bp pDN1050 + polylinkerbpDN2801 2,767 bp pDN1380 + polylinkercpUW100 4.0 kb aldB91 pDN2801 + 1.3 kb from

pUW9ldpUW102 3,943 bp aldB92 pDN2801 + 1.2 kb from

pUW92efpUW104 3,927 bp aldB94 pDN2801 + 1.1 kb from

pUW94dpUW106 3,871 bp aldB97 pDN2801 + 1.0 kb from

pUW97d

a All plasmids are Cmr and are derivatives of pDN1050, a B. subtilis cloningvector composed of the replication region of pUB110 and the chloramphenicolresistance gene of pC194 (5).

b The polylinker was constructed by first replacing the 183-bp BamHI-SphIfragment of pDN1050 (see above) with a 43-bp linker GV462 (5'-GATCCCGGGTACCGCGGCCGATCGGGCCCAGAGCTCTGGCATG-3') to ob-tain pDN2800. Then the 58-mer KFN602 (Table 1) was inserted in the EcoRIsite of pDN2800 in the opposite orientation, as shown in Table 1. The order ofthe most important restriction sites in the linker area is as follows: EcoRIBglII XhoI Notl SphI NsiI Dral Hindlll BclI ClaI HindlIl PstI Sall BamHISmaI KpnI SaclI XmaIII PvuI ApaI SacI SphI.

c See Fig. 2. The polylinker is the Clal HindIl. Sacl SphI fragment ofthe polylinker of pPL1385 (see above), which replaces the 215-bp ClaT-ClaTfragment downstream of the amyM promoter on pDN1380 (6).

d See text.e See Fig. 2.

ing acetoin production in a fixed-time spectrophotometricassay. One unit is the amount of enzyme that produces 1,umol of acetoin per min under the following conditions. Theincubation mixture (400 ,ul) was 50 mM MES [2-(N-mor-pholino) ethanesulfonic acid], 0.5 M NaCl, 0.2% TritonX-100, 5.3 mM R,S-cx-acetolactate (pH 6.0), and enzymeprotein. The reaction was performed at 30°C for 20 min, andthe acetoin was quantified at 522 nm after addition of 4.6 mlof color reagent (1% 1-naphthol-0.1% creatine in 1 N NaOH)and 40 min of color development time. The acetoin concen-tration was calculated by means of a linear acetoin standardcurve from 0 to 80 mg/liter. The amount of acetoin producedby the enzyme was calculated after corrections for sponta-neous decarboxylation and sample color. R,S-a-acetolactatewas prepared from ethyl-2-acetoxy-2-methylacetate by con-trolled saponification at pH 11.5 in the pH stat in order toavoid 1-keto ester cleavage.ALDC purification. Cells were centrifuged (6000 x g, 4°C,

30 min), and the supernatant was dialyzed overnight at 5°Cagainst 5 liters of 0.025 M MES (pH 6.1) with two changes ofbuffer. The retentate was centrifuged (13,000 x g, 40C, 30min), and the supernatant was filtered on 1.2- and 0.45-,utm-pore-size membrane filters. Then 40 ml of filtrate (506U/ml) was passed through Polybuffer Exchanger 94 (16-mmdiameter; 18 cm = 36 ml; Pharmacia) equilibrated with 0.025M MES (pH 6.1) at a flow rate of 1 ml/min. Enzymaticallyactive fractions were pooled (78 ml; 247 U/ml; step yield,95%) and concentrated by ultrafiltration on an AmiconPM-10 membrane to 10 ml (1,380 U/ml; step yield, 72%).Buffer was exchanged on Sephadex G-25 SF (16-mm diam-eter; 36 cm - 72 ml; flow rate, 1 m/min Pharmacia) to 0.025M Tris (pH 8.3). Active fractions were pooled (18 ml; 193U/ml; step yield, 25%) and subjected to chromatofocusing onMono-P HR 5/20 (Pharmacia) at a flow rate of 0.5 ml/min.The column was washed with 0.025 M Tris (pH 8.3) and theneluted with Polybuffer 96 (pH 6.2; 0.0075 mmol/pH unit perml; Pharmacia) with acetic acid. The two most active frac-tions were pooled (2 ml; 1,035 U/ml; step yield, 60%).Polybuffer was removed by gel filtration on Superose 12 HR

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CLONING OF THE aldB GENE OF B. BREVIS 4317

B HmW3 Dral Nail Sphl Nhel NotlEA Xhol eQIBWv*n Xbal

FIG. 2. Maps of plasmids pUW11, pUW25, pDN2801, and pUW102. pUW11: Apr; bla is the beta-lactamase gene of pUC19; P,ac is the lacpromoter of pUC19; A and B are the positions of the oligonucleotide probes (see Materials and Methods); L is the polylinker of pUC19 withthe following important sites: Sacl KpnI SmaI BamHI XbaI Sall PstI SphI. pUW25: Apr Ald+. pDN2801: Cmr; cat is the chloramphenicolacetylase gene of plasmid pC194 of Staphylococcus aureus; the replication origin is from plasmid pUB110 of Staphylococcus aureus; thesequence of the polylinker is given in Table 2. pUW102: Cmr Ald+.

10/30 (flow rate, 0.5 ml/min; 0.2 M ammonium acetate at pH6.0; Pharmacia). A 0.3-ml sample was applied in each run,and fractions of 0.5 ml were collected. The peak fractionsfrom all runs were pooled (251 U/ml; step yield, 53%). Totalyield was 5%.

N-terminal determination. Automatic Edman degradationin a Beckman sequencer was used for determination ofN-terminal amino acids (19).

RESULTS

Cloning of aldB in E. coli. On the basis of reverse transla-tion of a partial amino acid sequence of ALDC from B.brevis (Jensen et al., Abstr. Eur. Biotechnol. Congr. 1987;Svendsen et al., in press), two oligonucleotide probes, A andB, were synthesized (see Materials and Methods).Chromosomal DNA from the B. brevis strain was ana-

lyzed by Southern blotting with radiolabeled probes A andB. A positive signal from a HindIII-HindIII band of approx-imately 2.6 kilobase pairs (kb) was observed with bothprobes, and the corresponding DNA fraction was isolatedfrom an agarose gel and cloned on plasmid pUC19 in E. coli.Approximately 0.6% of the ampicillin-resistant recombinanttransformants were positive in a colony hybridization withprobes A and B. These colonies also reacted in a colonyimmunoblotting assay with B. brevis ALDC antiserum,harbored a 2.6-kb Hindlll-HindIll insert, and producedsignificant amounts of acetoin from acetolactate in the in

vitro ALDC assay (data not shown). Finally, a partial DNAsequence which conformed to the known amino acid se-quence of the ALDC enzyme (Jensen et al., Abstr. Eur.Biotechnol. Congr. 1987) was obtained by the dideoxymethod, using oligonucleotide mixtures A and B as primers.One of the ALDC-positive transformants, strain UWllharboring plasmid pUWll with an insert of 2.6 kb, was keptfor further study.

Analyses of recombinant plasmids. pUWll was mapped byrestriction enzyme analyses, and locations of the regionshomologous with each of the two probes were determined bySouthern blotting to be on either side of the KpnI site at 1.3kb (Fig. 2). Thus, the orientation of the insert was deter-mined.To subclone aldB and to construct a plasmid suitable for

exonuclease degradation of the cloned region, the BamHI-SmaI fragment of pUWll was cloned on pDN3000, a deriv-ative ofpUC19 with a new polylinker. The resulting plasmid,pUW25 of 4.2 kb (Fig. 2), expressed strong ALDC activity inE. coli.

Plasmid pUW25 was cut in the unique NsiI and NotI sitesand subjected to exonuclease degradation, removal of over-hang by Si nuclease, and ligation in the presence of BglIIlinker. A number of plasmids from Ald+ and Ald- transfor-mants (as judged by colony immunoblotting with ALDCantiserum) were analyzed, and the extent of the degradationwas compared with the phenotype (Table 3). The four

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4318 DIDERICHSEN ET AL.

TABLE 3. ALDC yields

ALDC yield (U/mi)aldB allele" Upstream bpb

E. coli B. subtilis

88 410C 4489 360C 4390 237d 6091 230d 74 4492 130d 2 5194 114 1 3795 70d <0.196 58d <0.197 20C <0.1 <0.1

aConstructed by exonuclease degradation of pUW25 in E. coli and trans-ferred on a HindIII-BamHI fragment to plasmid pDN2801 in B. subtilis (seetext).

b Remaining number of nucleotides from B. brevis upstream position 279(Fig. 4) at the second observed N-terminal threonine of the mature enzyme(see text).

' Determined by restriction fragment analysis.d Determined by sequencing (see Fig. 4).

plasmids pUW88 to 91 had a strong Ald+ phenotype similarto that of the parental pUW25. pUW92 and pUW94, on theother hand, were Ald+ but gave significantly reduced yields(Table 3).Cloning of aidE in B. subtilis. The entire 2.6-kb HindIII-

HindIII fragment from pUWll was cloned in B. subtilis onplasmid pPL1385 in both possible orientations. Neitherplasmid, however, gave rise to more than barely detectableALDC levels. If aldB on the same 2.6-kb fragment wasinserted in one orientation on pDN2801 (Fig. 2) behind theamyM promoter (6), ALDC was strongly expressed. In theother orientation, no ALDC activity was detected. It isconcluded that the 2.6-kb fragment from B. brevis does notharbor a promoter that is sufficiently active in B. subtilis togive ALDC activity. Surprisingly, ALDC activity was ex-pressed from pUWli and pUW25 in E. coli despite theabsence of known promoters on pUC19 which could causetranscription of aldB on these plasmids (Fig. 2).From E. coli plasmids pUW91, pUW92, and pUW94, 1.3-,

1.2-, and 1.1-kb HindIII-BamHI fragments, respectively,were cloned on B. subtilis plasmid pDN2801 (Fig. 2). Theresulting plasmids pUW100, pUW102 (Fig. 2), and pUW104(Tables 2 and 3), on which the amyM promoter reads into thealdE gene, all gave high amounts of ALDC activity in B.subtilis (Table 3). The yields of pUW104 were, however,lower than those of the two longer constructs. Subcloningfrom the ALDC-negative E. coli plasmid pUW97 gavepUW106 with a negative phenotype in B. subtilis (Table 3).To test whether upstream or downstream regions on the

HindIII fragment, which was originally cloned in E. coli, hadany influence on the ALDC yield in B. subtilis, B. subtilisplasmids with the entire 2.6-kb insert and pUW102 (with a1.2-kb insert) were compared. Since the ALDC yields weresimilar, the 1.4-kb B. brevis DNA, which was removed bysubcloning on pUW102, has no important effect on theexpression of aldB behind the amyM promoter in B. subtilis.

It should be noted, however, that removal of an upstreamarea present on aidB91 but not on aldB92 significantlyreduced ALDC yields in E. coli without affecting B. subtilisyields (Table 3).DNA sequencing and analyses. Using the dideoxy method

performed directly on double-stranded DNA from either E.coli or B. subtilis, the entire aidB gene including flanking

__________ U___> ------ >

N *********************************** C

<- ---------

FIG. 3. Sequencing strategy. Sequencing of 1,296 bp ofB. brevisDNA as on pUW34 was done by using oligonucleotide primers andthe dideoxy method on double-stranded plasmid DNA (see Materi-als and Methods). The bold line (-- - +---) represents the 1,296 bpshown in Fig. 4; bars (-) represent 25 bp; crosses (+) mark every200 bp. Stars (***) represent the extent of the ALDC proteinincluding the signal peptide (bp 196 to 1049 in Fig. 4). Circles (0)represent the oligonucleotide mixtures A and B (see Materials andMethods) at bp 487 to 471 and bp 777 to 803, respectively. Arrows(----->) represent partial sequences and the direction of the poly-merase sequencing reaction (one bar is 25 bp).

regions has been sequenced. The sequencing strategy isshown in Fig. 3, and the sequence is shown in Fig. 4.From sequence analyses of aidB, the following conclu-

sions were drawn.(i) No obvious promoterlike sequences were identified.

However, the region from bp 41 to 148 (present on aidB9lbut not on aldB92) probably has some promoter activity in E.coli (Fig. 4 and Table 3).

(ii) A typical ribosome-binding site is seen at bp 184 infront of an ATG initiation codon at bp 195 (Fig. 4). Threeamino acids upstream at bp 186 there is a putative GTGinitiation codon, which, however, is not well combined witha potential ribosome-binding site.

(iii) The N-terminal part of a long open reading frame of285 amino acids is initiated by a region that strongly resem-bles a signal peptide. Typical processing sites (according tothe PC/GENE version 5.16 program PSIGNAL fromGENOFIT SA) are found after amino acid 24 or 27.

(iv) The amino acid sequence of the remaining part of theopen reading frame; is in almost complete accordance withthe sequence obtained by amino acid sequencing (Svendsenet al., in press).

(v) The C-terminal sequence predicted in Fig. 4 is inaccordance with the C-terminal sequence ERK found bySvendsen et al. (in press).

(vi) The open reading frame is followed by a typicalterminator hairpin structure of 14 bp starting at bp 1084.

Expression in E. coil. To study the compartmentalizationin E. coli, cultures of strain UW25 harboring plasmid pUW25were fractionated in supernatant, periplasmic fraction, andspheroblasts as described previously (9). More than 90% ofthe ALDC activity (i.e., 4.4 U/ml) in cells grown to anoptical density at 450 nm of 1.7 was found in the periplasmicfraction, suggesting that the putative signal peptide is func-tional in E. coli.

Expression in B. subdis. From the supernatant of B.subtilis UW214 aldBE, an ALDC sample was prepared andcompared by sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) to ALDC purified from B. brevis(Fig. 5). Of the ALDC produced by B. subtilis, 74% wasfound in the supernatant. To test whether this was princi-pally due to cell lysis, samples of supernatant and cells of

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CLONING OF THE aldB GENE OF B. BREVIS 4319

10 20 30 40***' 50

TCGAATATTGCCAATTCCAACTCCAGAGATTCCAGCGATACTCTGTCCAATATTGAAAAA70 80 90 100 110

CAACAAGAATCTTTGCATGAATTAATGGAACAAGTGAAAATTGTACTGGATACCGCACAA

130 140 ***7 160 ***

CAAATCAGAAATAATGTGCAAAACTACAAACTTCGTTAGCAACAAATTTTATATATAAAG

RBS 190 200 210 * 230

GTGGAGTGAAAAACATGAAAAAAAATATCATCACTTCTATCACATCTCTGGCTCTGGTTGM K K N I I T S I T S L A L V

250 260 270 280 290

CCGGGCTGTCTTTGACTGCTTTTGCAGCTACAACGGCTACTGTACCAGCACCACCTGCCAA G L S L T A F A A T T A T V P A P P A

310 320 330 340 350

AGCAGGAATCCAAACCTGCGGTTGCCGCTAATCCGGCACCAAAAAATGTACTGTTTCAATK Q E S K P A V A A N P A P K N V L FQ

370 380 390 400 410I

ACTCAACGATCAATGCACTCATGCTTGGACAGTTTGAAGGGGACTTGACTTTGAAAGACCY S T I N A L M L G Q F E G D L T L K D

430 440 450 460 470

TGAAGCTGCGAGGCGATATGGGGCTTGGTACCATCAATGATCTCGATGGAGAGATGATTCL K L R G D M G L G T I N D L D G E M I

490 500 510 520 530

AGATGGGTACAAAATTCTACCAGATCGACAGCACCGGAAAATTATCGGAGCTGCCAGAAAQM G T K F Y Q I D S T G K L S E L P E

550 560 570 580 590

GTGTGAAAACTCCATAiGCGGTTACTACACATTTCGAGCCGAAAGAAAAAACTACATTAAS V K T P F A V T T H F E P K E K T T L

610 620 630 640 650

CCAATGTGCAAGATTACAATCAATTAACAAAAATGCTTGAGGAGAAATTTGAAAACAAGAT N V Q D Y N Q L T K M L E E K F E N K

670 680 690 700 710

ACGTCTTTTATGCCGTAAAGCTGACCGGTACCTTTAAGATGGTAAAGGCTAGAACAGTTCN V F Y A V K L T G T F K M V K A R T V

730 740 750 760 770

CAAAACAAACCAGACCTTATCCGCAGCTGACTGAAGTAACCAAAAAACAATCCGAGTTTGP K Q T R P Y P Q L T E V T K K Q S E F

790 800 810 820 830

AATTTAAAAATGTTAAGGGAACCCTGATTGGCTTCTATACGCCAAATTATGCAGCAGCCCE F K N V K G T L I G F Y T P N Y A A A

850 860 870 880 890

TGAATGTTCCCGGATTCCATCTCCACTTCATCACAGAGGATAAAACAAGTGGCGGACACGL N V P G F H L H F I T E D K T S G G H

910 920 930 940 950

TATTAAATCTGCAATTTGACAACGCGAATCTGGAAATTTCTCCGATCCATGAGTTTGATGV L N L Q F D N A N L E I S P I H E F D

970 980 990 1000 1010

TACAATTGCCGCACACAGATGATTTTGCCCACTCTGATCTGACACAAGTTACTACTAGCCV Q L P H T D D F A H S D L T Q V T T S

1030 1040 1050 1060 1070

AAGTACACCAAGCTGAGTCAGAAAGAAAATAAAGCAGCCTCATGCGAGAAATTTAGCGAGQV H QA E S E RK -

************** 1100 ************** 1130

GACATGAAAAAGCTGGCCTATTATAAGTGCCAGCu[TTTTTCATATCTAGGAGTCCCCTCCA

1150 1160 1170 1180 1190

GCTTGGACTTCTGCCTGGTAATTGGTGCTCTimCCTGTACCAATAGTGTTTTTCTAATCC

1210 1220 1230 1240 1250

TGAAACTTCAAATCTCTATTTTTGTCTCCAGAAACTCAAGGTACTTTGGGTCATCTTCCA

1270 1280 1290

TAAAGATCGCAATCCCGGCACCCATGTAAGCCCGGG

FIG. 4. DNA sequence of aldB and flanking regions. The sequencing strategy is shown in Fig. 3. ***' shows the first three remaining basepairs after exonuclease degradation of pUW25, where XX specifies the number of the E. coli plasmid and the aldB allele. Thus, ***92 showsthe start of the B. brevis DNA on plasmid pUW92 harboring aldB92. aldB90 starts at bp 42, aldB92 starts at bp 149, aldB94 starts at bp 165,and aldB96 starts at bp 222. RBS at bp 184 is the proposed ribosome-binding site. The stars (***) flanking bp 1100 indicate a 14-bp invertedrepeat and a possible terminator.

B. subtilis UW123 aldB+ were analyzed on SDS-gels (Fig.5). The result shows that ALDC relative to total proteinsoccurred preferentially in the supernatant.

N-terminal region of ALDC. N-terminal preparation wasperformed on a preparation of the B. brevis enzyme (Svend-sen et al., in press). Approximately equal amounts of twoN-terminal sequences were found: TTATVPAPPA andTVPAPPA (corresponding to cleavage sites p and q in Fig.6). The purified enzyme from B. subtilis also revealed twoN-terminal sequences, but in this case identical apart froman N-terminal alanine: ATVPAPPA and TVPAPPA (corre-sponding to cleavage sites y and q in Fig. 6). The ratio of theN-terminal amino acids alanine and threonine was 0.7. Thus,in B. brevis the actual N-terminal amino acids correspond tocleavage at either of the two predicted signal peptide matu-ration sites, followed by removal of an N-terminal alanine.In the case ofB. subtilis, only the larger 27-amino-acid signalpeptide may be cleaved, followed by partial removal of an

N-terminal alanine. These results are summarized in Fig. 6.

DISCUSSION

In this report, cloning, expression, and sequencing ofaldB, the structural gene for an ALDC from Bacillus brevis,have been described.

1 2 3 4 5 6 7

FIG. 5. SDS-PAGE analyses. Comparison of ALDC from B.

brevis and B. subtilis in SDS-PAGE under reducing conditions.

Lanes: 1, Molecular size markers (94, 67, 43, 30, 20.1, and 14.4

kilodaltons; Pharmacia); 2, ALDC from B. subtilis, pool from

Mono-P experiment (see Materials and Methods); 3, ALDC from B.

subtilis, pool from Superose 12 HR 10/30 (See Materials and

Methods); 4 and 5, ALDC from B. brevis 6, pellet from culture

broth of B. subtilis; 7, supenatant from B. subtilis with the same

ALDC activity as the sample in lane 6.

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4320 DIDERICHSEN FT AL.

-24 -10

M K K N I I T S I T S L A L V

-9 -1 +1x p

A G L S L T A F A A T

+6y q

T A T V

+7

P A P p A K Q E S K P A V A A

FIG. 6. N-terminal region of ALDC, showing the first 45 aminoacids of the open reading frame of the aldB gene. The computerprogram (see text) predicts potential signal peptidase processingsites at positions x and y, with a slight preference for x. Cleavage atx corresponds to a signal peptide of only 24 amino acids, whichwould be unusually short for bacilli. N-terminal determination ofALDC purified from B. brevis or B. subtilis showed about equalamounts of enzyme processed at p and q or y and q, respectively.

Analyses of the DNA and amino acid sequence revealedthat the primary translation product of the aldB gene isinitiated by a typical signal peptide of either 24 or 27 aminoacids. The function of this signal is supported by the follow-ing evidence.

(i) Of the ALDC activity produced by B. brevis and B.subtilis harboring the cloned aldB gene, 74% is found in thesupernatant. For B. brevis, more than 80% of the ALDCactivity produced may be found in the supermatant underoptimal conditions for ALDC production. Under conditionsoptimal for growth, however, more than 90% of the activityis cell associated (Carsten Sj0holm. unpublished results).

(ii) The two N-terminal sequences of ALDC from B.subtilis correspond to cleavage at the second predictedcleavage site and subsequent partial removal of an N-terminal alanine. The two N-terminal sequences of extracel-lular ALDC produced by B. brevis conform to the twopredicted processing sites of a typical signal peptide, fol-lowed by removal of an N-terminal alanine residue.

(iii) Expression of the aldB gene is impaired by deletion ofa region including the ribosome-binding site correspondingto the N-terminal methionine and the following eight aminoacids of the signal peptide (aldB96 in Table 3 and Fig. 4).

(iv) No likely combination of ribosome-binding site andinitiation codon is found close to the N-terminal region of themature ALDC.Thus, there is good evidence that ALDC from B. brevis is

an exoenzyme.Two potential signal peptides can be identified in the

primary translation product of aldB. In each case, signalpeptide processing is followed by removal of one N-terminalalanine (or possibly of a TTA tripeptide) to obtain twodifferent mature products both with an N-terminal threonine(Fig. 6). This finding suggests either the presence of aspecific alanyl aminopeptidase in both B. brevis and B.subtilis (as described for B. subtilis [4]) or that the N-terminal alanines in processed ALDC are labile.The biochemical function of ALDC suggests an internal

enzyme with a physiological role associated with valine andleucine metabolism. Godtfredsen et al., however, find thenatural function of ALDC a matter of speculation (10), andD. L. Rathbone (Ph.D. thesis, University of Warwick,Warwick, England, 1987) reports that ALDC from B. brevishas a broad substrate specificity. Thus, the physiologicalrole of ALDC, as well as it extracellular function, is puz-zling.

In 1972, Collins (3) suggested that the presence of ALDCmay cause auxotrophy for valine and panthotenic acid as a

N. brevi;s -25 M K K N I I T S I T S L A L V A G L S L T A F A AE. aerogenes

+1 T T A T V P A P P A K Q E S K P A V A A N P A P K N V I. F Q1 M H S S A C D C F. A S L C E T L R G F S A K H P D S V I Y Q

31 Y S T I N A L M L G Q F E G D L T L K D L K L R G D M G L G.1 T S L M S A L L S G V Y E G D T T I A D L L A H G D F G L G

61 T I N D L D G E M I Q M G T K F Y Q I D S T G K L S E L P E61 T F N E L D G E M I A F S S Q V Y Q L R A D G S A R A A K P

91 S V K T P F A V T T H F E P K E91 E Q K T P F A V M T W F Q P Q Y

K T T L T N V Q D Y N Q L TR K T F D A P V S R Q Q I H

121 K M L E E K F E N K N V F Y A V K L T G T F K M V K A R T V121 D V I D Q Q I P S D N L F C A L R I D G N F R H A H T R T V

151 P K Q T R P Y P Q L T E V T K K Q S E F E F K N V K G T L I151 P R Q T P P Y R A M T D V L D D Q P V F R F N Q R E G V L V

181 G F Y T P N Y A A A L N V P G F H L H F I T E D K T S G G H181 G F R T P Q H M Q G I N V A G Y H E H F I T D D R Q G G G H

** ****** * * * ** * * * *

* * * * * * * *

211 V L N L Q F D N A N L E I S P I H E F D V Q L P H T D D F A211 L L D Y Q L E S G V L T F G E I H K L M I D L P A D S A F L

* * * * * * * *

241 H S D L T Q V T Q S Q V H Q A E S E R K 260 B. brevis241 Q A N L H P S N L D A A I R S V E N 258 E. aerogenes

FIG. 7. Alignment of the amino acid sequences of ALDC ofE. aerogenes (17) and aldB of B. brevis (this work). Eighty-fiveidentical amino acids are marked (*). Note that the E. aerogenesenzyme may include two extra N-terminal methionines (17).

result of diversion of an essential metabolic precursor andthat all microorganisms with ALDC activity therefore wouldrequire panthotenic acid or valine. The fact that ALDC fromB. brevis is a secreted enzyme adds another dimension tothis speculation.Comparison between the aldB protein and the functionally

similar enzyme from Enterobacter aerogenes (17, 18) revealsthat 33% of the amino acids are identically positioned (Fig.7). However, the E. aerogenes enzyme is not preceded by atypical signal peptide, and the authors do not report that theE. aerogenes enzyme is secreted. Cloning of an ALDC fromStreptococcus lactis has also been described (13), but nosequence data or other information suggestive of a secretedenzyme are given.A search for characteristic amino acid combinations, using

the PROSITE program of PC/GENE version 5.16 fromGENOFIT SA, revealed a five-amino-acid sequenceGFHLH (at bp 852 to 866) which hitherto has been identifiedonly in copper-zinc superoxide dismutases that bind thecopper atom at the two histidines. This sequence is notpresent in the E. aerogenes enzyme.The homology between the extracellular and intracellular

enzymes (Fig. 7) raises the question of when and how thesignal peptide was lost (or acquired) during evolution. Fur-thermore, it would be interesting to know whether the E.aerogenes enzyme can be secreted through the membraneby way of an appropriate signal peptide.

ACKNOWLEDGMENTS

We thank Steen J0rgensen, Novo Nordisk, for helpful discussionsand valuable comments on the manuscript. lb Svendsen, Carlsberg

J. BACTF.RIOL.

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CLONING OF THE aldB GENE OF B. BREVIS 4321

Laboratory, Copenhagen, kindly undertook N-terminal determina-tions, and Kjeld and Fanny Norris synthesized the oligonucleotidesused in this work.

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18. Sone, H., T. Fujii, K. Kondo, and J. Tanaka. 1987. Molecularcloning of the gene encoding alpha-acetolactate decarboxylasefrom Enterobacter aerogenes. J. Biotechnol. 5:87-91.

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