APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.1.426–433.2001

Jan. 2001, p. 426–433 Vol. 67, No. 1

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Lactobacillus plantarum Genesfor b-Ketoacyl-Acyl Carrier Protein Synthase III (fabH) and

Acetyl Coenzyme A Carboxylase (accBCDA), Which AreEssential for Fatty Acid Biosynthesis

PORNPIMON KIATPAPAN, HAJIME KOBAYASHI, MAKI SAKAGUCHI, HISAYO ONO,MITSUO YAMASHITA, YOSHINOBU KANEKO, AND YOSHIKATSU MUROOKA*

Department of Biotechnology, Graduate School of Engineering, Yamada-oka, Suita, Osaka 565-0871, Japan

Received 23 June 2000/Accepted 5 October 2000

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first stepin the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified sixpotential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotidesequence analysis suggested that fabH encoded b-ketoacyl-acyl carrier protein synthase III, that the accB, accC,accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the b and a subunits ofcarboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes wasdifferent from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coliaccB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. Thepredicted products of all five genes were confirmed by using the T7 expression system in E. coli. The geneproduct of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the fivegenes in L. plantarum were regulated polycistronically in an acc operon.

Strains of Lactobacillus plantarum form a group of industri-ally important lactic acid bacteria that are widely used as start-ers to stimulate malolactic fermentation in wine and lactic acidfermentation in meat and vegetables. We previously isolated L.plantarum from a fermented fish and rice food that is producedin the Philippines, and one of the strains, L137, which canhydrolyze starch and contained 15 plasmids, was studied indetail (32). Using a small plasmid, pLTK2, we developed ahost-vector system for L. plantarum (15). In general, growth oflactic acid bacteria requires various amino acids, vitamins (in-cluding biotin), and lipids or fatty acids, and growth is stimu-lated in the presence of Tween 80. Although lactic acid bacte-ria are industrially important gram-positive bacteria, themechanisms involved in lipid biosynthesis in these bacteriahave not been well characterized. To our knowledge, onlygenes for biotin carboxylase in Lactococcus lactis have beensequenced (accession no. X76191). Recent studies suggest thatfatty acids might act as signaling molecules that are importantfor cellular differentiation in gram-positive bacteria (1, 43).Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) is an en-zyme that is essential for the first step in biosynthesis of fattyacids, and this enzyme belongs to the group of carboxylasesthat use biotin as a cofactor and bicarbonate as a source of thecarboxyl group. ACC catalyzes the addition of CO2 to acetyl-CoA to generate malonyl-CoA. The ACC of both eukaryoticand prokaryotic organisms, such as Escherichia coli (17, 22, 23),Bacillus (27), Pseudomonas (4), Mycobacterium (30), Coryne-bacterium (14), and Anabaena sp. (9) strains, have been stud-

ied. In each of these organisms, the organization of the genesfor each subunit of ACC is different even though the aminoacid sequence around the active sites of each ACC is wellconserved. We have been interested in the genes that areinvolved in fatty acid biosynthesis in L. plantarum, and previ-ously we have described cloning of the accC gene for a subunitof biotin carboxylase (P. Kiatpapan, H. Kobayashi, M. Sakagu-chi, H. Ono, Y. Kaneko, and Y. Murooka, Abstr. IX Congr.Bacteriol. Appl. Microbiol., p. 62, 1999).

In this report, we describe the complete nucleotide se-quences, organization, and details of expression of the genesfor b-ketoacyl acyl carrier protein (ACP) synthase III andACC in L. plantarum.

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and culture conditions. Fragments of thechromosomal DNA of L. plantarum L137 (32) were cloned in pT7Blue (Nova-gen, Madison, Wis.), pUC18/19 (49), pBluescript II KS1 (Stratagene, La Jolla,Calif.), and pTWV228 (Takara Shuzo Co. Ltd., Shiga, Japan). In T7 expressionexperiments, we used pVEX11 which had been provided by Y. Yamada(Yamaguchi University, Yamaguchi, Japan). L. plantarum L137 (32) and E. coliJM109 (49) and BL21(DE3) (39) were used as host strains. Lactic acid bacteriawere grown at 30°C in De Man-Rogosa-Sharpe medium (Difco Laboratories,Detroit, Mich.). E. coli was grown in Luria-Bertani medium (39), M9 medium(39), or YT broth (39) at 37°C. E. coli L8 (11) containing a temperature-sensitivelesion in the biotin carboxyl carrier protein (BCCP), accB22(Ts) [fabE22(Ts)],and E. coli LA1-6 (24), which contains a temperature-sensitive lesion in the bsubunit of the carboxyltransferase, accD6(Ts) fab-6(Ts) (E. coli Genetic StockCenter, Yale University, New Haven, Conn.), were cultured at 30°C.

Restriction enzymes and T4 DNA ligase, obtained from Takara Shuzo Co.Ltd. or from Toyobo Co. Ltd. (Osaka, Japan), were used as suggested by thesuppliers. [35S]methionine was purchased from Amersham Pharmacia Biotech(Buckinghamshire, United Kingdom). Reagent grade chemicals were obtainedfrom Nacalai Tesque Inc. (Kyoto, Japan) or Sigma Chemical Co. (St. Louis,Mo.).

Manipulation of DNA. Chromosomal DNA was prepared from L. plantarumas described previously (15). Preparation of plasmid DNA and genetic manipu-

* Corresponding author. Mailing address: Department of Biotech-nology, Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7416. Fax:81-6-6879-7418. E-mail: murooka@bio.eng.osaka-u.ac.jp.

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lations of L. plantarum were performed as described by Kaneko et al. (15).Plasmid DNA was transferred to L. plantarum by electroporation with a GenePulser (Bio-Rad Laboratories, Hercules, Calif.). Preparation of DNA and ge-netic manipulation of E. coli were performed by standard methods (39).

Cloning of the acc genes. Degenerate oligonucleotide primers BC1 (59-CA[T/C]CCIGGITA[T/C]GGITT[T/C][T/C]TIGC-39) and BC2 (59-CICC[A/G]TG[T/C]TCIAC[T/C]TGIA[A/G]IC-39) were designed by reference to the conservedamino acid sequences AIHPGYGFLA/SENAD/NFA and YFM/IEMNTRI/VQVEH, respectively. PCR amplification was performed with 2.5 U of Taqpolymerase. The parameters used for PCR were as follows: 30 cycles of dena-turation at 94°C for 30 s, annealing at 45°C for 45 s, and elongation at 72°C for60 s after an initial 5-min denaturation step; and a final 10-min extension step.The PCR product was purified from an agarose gel and ligated to the pT7Bluevector. DNA-DNA hybridization was performed by using the standard protocol(42) with a digoxigenin (DIG) chemiluminescence detection kit (Roche Diag-nostics GmbH, Mannheim, Germany). The DNA probe was labeled with DIG-High Prime (Roche Diagnostics) by using a DIG labeling kit from Boehringer(Mannheim, Germany).

DNA sequencing. Nucleotide sequences of both strands were determined bythe dideoxy chain termination method (40) with ABI PRISM dye terminatorcycle sequencing Ready Reaction kits (PE Biosystems) and an AutoRead 1000sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Gaps in se-quences were eliminated by using customized oligonucleotide primers. Se-quences were assembled and analyzed by using the GENETYX-MAC program,version 8 (Software Development, Tokyo, Japan). A homology search was car-ried out with the BLAST (Basic Local Alignment Search Tool) program (2) byusing the DNA Data Bank of Japan (DDBJ) database.

Synthesis of products of genes from L. plantarum in E. coli. The fragmentcontaining fabH and accBCDA was amplified by PCR with pACC as the tem-plate. Forward primer 59-CATATGATGCCAACTTATAC-39 and reverseprimer 59-GAATTCGAAGCGCATCCG-39 were used to create an NdeI restric-tion site at the initiation codon of fabH and an EcoRI site downstream of theaccA termination codon. The PCR was performed in an automated temperature-controlled system (PC700; Astec Co., Fukuoka, Japan). The 100-ml reactionmixture contained 10 ng of template, 50 pmol of each primer, 1 U of Taqpolymerase, and each deoxynucleoside triphosphate at a concentration of 0.2mM in 10 mM Tris-HCl buffer (pH 8.3). A 40-ml layer of mineral oil was used toprevent evaporation. The sample was first incubated at 95°C for 5 min and thensubjected to 30 cycles of denaturation (20 s at 95°C), annealing (60 s at 60°C),and extension (90 s at 72°C). The PCR product was eluted from an agarose gelafter electrophoresis with a GFX PCR DNA and Gel Band purification kit(Amersham Pharmacia Biotech) and cloned into the pT7Blue vector. The NdeI-EcoRI fragment was isolated from the resulting plasmid and subcloned intopVEX-11 expression vector that had been digested with NdeI and EcoRI. Theresulting plasmid, pT7ACC, which included a 4.6-kb fragment, was used totransform E. coli BL21(DE3). Ampicillin-resistant colonies were selected, andcells from individual colonies were picked and cultured in M9 medium supple-mented with glucose (0.4%) and amino acids (100 mg/ml each, except for me-thionine) until the absorbance at 600 nm (optical density at 600 nm [OD600])reached 0.7. Synthesis of T7 RNA polymerase was induced by 30 min of incu-bation with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), and then cellswere treated with rifampin (200 mg/ml) for 30 min. Cellular proteins were labeledfor 10 min with [35S]methionine (37 Bq/ml). Cell pellets were washed with 10mM HEPES buffer (pH 7.4), and proteins were separated by sodium dodecylsulfate (SDS)–12.5% polyacrylamide gel electrophoresis (PAGE) (20). The gelwas fixed and dried overnight, and then labeled proteins were visualized byautoradiography.

Construction of pTACO-1. The promoter region and the 59 end of the openreading frame (ORF) of fabH (nucleotides [nt] 664 to 960) were amplified byPCR with primers 59-TCTAGAGGTGTTTGCGCAGAAAGTCC-39 (forwardprimer) and 59-GATCCAACATATTGTCCCATGGCG-39 (reverse primer).The amplified fragment obtained after PCR was cloned to the pT7Blue vector toobtain pTPac. The gene for cholesterol oxidase (choA9) from pCO117 (29) wasamplified by PCR by using primers 59-GCATGACTGCACAACAGC-39 and59-CGACTAGTTGGTGCGTTCCTTC-39 as the forward and reverse primers,respectively, and a SpeI site was generated at the 39 end of the gene. An NdeIrestriction site at the 59 end was generated after the amplified fragment wassubcloned into the pT7Blue vector. The NdeI-SpeI fragment containing thechoA9 gene was ligated into plasmid pTPac that had been digested with NdeI andSpeI to generate pTACO-1. E. coli cells carrying plasmid pTACO-1 were usedfor preparation of total RNA.

Preparation of RNA. The methods used for preparation of RNA from E. coliand primer extension were based on the method described by Kashima et al.

(16), with some modifications. Cells were grown in 40 ml of 23 YT broth andincubated until the OD600 reached 0.7. Cells were harvested and suspended in 1ml of lysis buffer, which contained 0.5% SDS, 20 mM sodium acetate (pH 5.5),and 1 mM EDTA. The lysate was extracted for 5 min at 65°C with 1 ml of phenolthat had been saturated with 20 mM sodium acetate (pH 5.5). After centrifuga-tion, the aqueous phase was extracted once with an equal volume of a mixture ofchloroform and isoamyl alcohol (24:1, vol/vol). The RNA was precipitated in 3volumes of absolute ethanol. The RNA pellet was treated with 20 U of DNase(RNase-free) and 25 U of RNase inhibitor in 50 ml of distilled water at 37°C for15 min. Total RNA from L. plantarum L137 was prepared with a Fast RNA Bluekit (Bio 101, Inc., Calif.) by using a 40-ml culture with an OD600 of 0.4.

Northern blot analysis. L137 RNA (approximately 20 mg) was electrophoreti-cally separated on 1.2% (wt/vol) agarose gels containing 0.66 M formaldehyde.After electrophoresis, the gels were treated with 0.05 N NaOH for 30 min. TheRNA was transferred to a nylon membrane (Hybond-N; Amersham) by standardmethods (39). Specific DNA fragments used as hybridization probes were labeledwith DIG-High Prime (Boehringer Mannheim, Indianapolis, Ind.). The DraI-NheI and NruI-HindIII DNA fragments (Fig. 1) were used as probes that werespecific for fabH and accC, respectively. Prehybridization was performed at 42°Cfor 2 h in hybridization buffer containing 53 SSC (13 SSC is 0.15 M NaCl plus0.015 M sodium citrate), 2% blocking solution, 0.1% lauryl sarcosine, 7.0% SDS,and 50% formamide in 50 mM phosphate buffer. The probe was heated to 95°Cand then added to the prehybridization mixture at a final concentration of about100 ng/ml. Hybridization was continued at 42°C overnight. The blots werewashed once with 23 SSC–0.1% SDS for 10 min at room temperature and twicewith 0.13 SSC–0.1% SDS for 20 min at 55°C. The probes were visualized onMXJB-1 film (Eastman Kodak, Rochester, N.Y.) by using a DIG-High PrimeDNA labeling and detection starter kit II (Boehringer Mannheim) according tothe manufacturer’s instructions along with the chemiluminescence substrateCSPD.

Primer extension analysis. The primer extension reaction was performed asfollows. A 0.2-pmol portion of fluorescein isothiocyanate (FITC)-labeled oligo-nucleotide primer for L. plantarum (59-FITC-ATTATCAACAACGCGTCCCG-39) or for E. coli (59-FITC-ACAGATGCTGTTGTGCAGTC-39) was mixed with30 mg of RNA in 20 ml (total volume) of distilled water, and then the mixture washeated at 60°C for 1.5 h. After slow cooling to room temperature for 2 h, 6.4 mlof 53 transcriptase buffer, 5.0 ml of a mixture containing each deoxynucleosidetriphosphate at a concentration of 2.5 mM, and 100 U of reverse transcriptase(Promega, Madison, Wis.) were added. After incubation at 37°C for 1 h and thenat 42°C for 30 min, the transcript was precipitated in ethanol, redissolved in 10to 20 ml of formamide dye (39), and then denatured by boiling for 2 min. Theinitiation site of transcription was determined by electrophoresis of the productof primer extension on a 6% polyacrylamide gel that contained 8 M urea next to

FIG. 1. Restriction map and organization of the manB, fabH, accB,accC, accD, and accA genes in L. plantarum. The six ORFs identifiedare indicated by arrows. The arrow above the map indicates the majortranscriptional start site identified. T, potential terminator site. Theprobes used for Northern blot analysis are indicated below the map bythick lines. Potential transcripts (from the start to the terminators) areindicated below the probes by thin lines. Selected restriction sites areindicated. Restriction site abbreviations: B, BalI; D, DraI; E, EcoRI;H, HindIII; Nh, NheI; Nu, NruI; S, SalI. aa, amino acids.

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the products of DNA sequencing reactions generated with the same primer andthe ALF DNA sequencing system (Amersham Pharmacia Biotech).

Complementation test and biotinylation of AccB. A DNA fragment containingthe fabH and accBCDA genes was amplified by PCR performed with synthesizedoligonucleotide primers in order to generate SacI and EcoRV sites at the 59 and39 ends, respectively. The PCR fragment was subcloned into plasmid pTWV228digested with SacI and HincII and gave rise to pWACC. Plasmid pWACC wastransferred to E. coli L8 [accB22(Ts)] and E. coli LA1-6 [accD6(Ts)]. Thetransformants obtained were grown at 42°C to confirm that complementation ofE. coli mutant strains L8 and LA1-6 by L. plantarum accB and accD, respectively,occurred. A biotinylation experiment was performed by using E. coli carryingpWACC. Cells were grown in Luria-Bertani medium until the OD600 reached0.6, IPTG was added to a final concentration of 1 mM, and the cells were grownfor 2 h. The cultured cells were harvested and lysed by boiling them for 2 to 3 minin sample buffer (39), and the proteins were separated by SDS-PAGE (ready gel;10 to 20% polyacrylamide; Bio-Rad) and electrophoretically transferred to anitrocellulose membrane. Biotinylating protein was detected by using streptavi-din conjugated with alkaline phosphatase (Bethesda Research Laboratories Inc.)as described previously (4).

Nucleotide sequence accession number. The nucleotide sequence reportedhere has been deposited in the DDBJ database under accession no. AB025973.

RESULTS

Isolation and cloning of acc genes. To clone the genes thatencode subunits of ACC, we designed oligonucleotides for useas primers by using the conserved sequences in biotin carbox-ylase (accC) genes of various organisms (4, 9, 17, 22, 27). Anapproximately 600-bp amplified product was obtained whenthe genomic DNA of L. plantarum L137 was used as the tem-plate. Sequence analysis confirmed that this product encoded aputative protein that was highly homologous to the biotin car-boxylase subunit of ACC from various organisms. Since theorganizations or orders of the genes for subunits of ACC differmarkedly in different prokaryotes, we attempted to isolate theentire complement of ACC genes from L. plantarum L137.Southern hybridization analysis performed with the DIG-la-beled 600-bp fragment as the probe and chromosomal DNAthat had been digested with EcoRI, BamHI, SalI, or XhoIyielded a 5.5-kbp EcoRI fragment which was cloned intopBluescript KS1 to generate plasmid pACC. Plasmid pACCwas used for analysis of nucleotide sequences.

Nucleotide sequence upstream and downstream of the accCgene. Analysis of the 5.5-kbp sequence revealed six potentialORFs which were encoded on the same DNA strand (DDBJaccession no. AB025973). A database search identified theseORFs as homologs of the following genes in E. coli: manB,fabH, accB, accC, accD, and accA. Therefore, we tentativelygave the same designations to the genes from L. plantarum inthat order (Fig. 1). Each of the ORFs had an ATG initiationcodon, as well as a sequence with reasonable homology to theconsensus ribosome-binding site of E. coli (41) and Lactoba-cillus (46).

ORFs upstream of the accC gene. The first ORF encoded aputative protein containing 172 amino acids. There were highlevels of homology at the amino acid level (61 to 67%) betweenthis ORF and the manB gene that encodes the phosphotrans-ferase IIB component of the phosphotransferase system (PTS)in E. coli (5) and Bacillus subtilis (18) and enzyme element IIB (manB) of the mannose PTS in Lactobacillus curvatus (47).The manB gene was followed by a flanking region at nucleo-tides 756 to 918 and then by the fabH gene.

The putative fabH gene encoded a 34.0-kDa protein thatexhibited significant homology (56 to 61%) to FabH from E.

coli (45), B. subtilis (18), and Streptomyces glaucescens (44). TheFabH protein corresponds to b-ketoacyl ACP synthase III,which catalyzes the first condensation reaction in the biosyn-thesis of fatty acids (10, 26). The consensus amino acid se-quence AACAGF at the active site of the condensing enzymewas found in the FabH protein of L. plantarum at amino acids112 to 117 (accession no. AB025973).

The third ORF encoded a putative 133-amino-acid proteinthat was 54 to 64% homologous to BCCP (encoded by accB),one of the subunits of ACC in E. coli (22), Pseudomonasaeruginosa (4), B. subtilis (27), and Anabaena sp. strainPCC7120 (9). The lysine residue that serves as a biotin-bindingsite in the biotin-dependent carboxylase in B. subtilis (27) wasalso found in AccB of L. plantarum at position 96 (accessionno. AB025973). The amino acid sequence surrounding thebiotin-binding site was MKLF and was identical to that in B.subtilis AccB.

Downstream of the accB gene of L. plantarum, we found theaccC gene, which encoded a homolog of biotin carboxylase.This gene was immediately downstream of the terminationcodon of the accB gene. The deduced amino acid sequence ofAccC was 79 to 84% homologous to the sequences of E. coliand B. subtilis. The amino acid sequence included a predictedATP-binding site, GGGGKG, located at positions 161 to 166,with strong similarity to similar sequences in other organisms(4, 9, 22, 27). Several palindromic sequences, which served astranscriptional terminators, were found in the 39 regions ofaccB and accC.

ORFs downstream of the accC gene. We found two moreORFs downstream of the accC gene of L. plantarum. Theamino acid compositions and amino acid sequences deducedfrom these two ORFs were very similar to those of the b anda subunits of the carboxyltransferase of E. coli, which areencoded by accD and accA. One base of the ORF of theputative accD sequence overlapped with the last codon of theaccC gene. The putative accA gene of L. plantarum encoded aprotein containing 257 amino acids. The amino acid residues atthe predicted carboxybiotin-binding site (GGARMQE) in theb subunit of the carboxyltransferase and the putative acyl-CoA-binding site (SGGAL) in the a subunit were identical tothose in E. coli (23) and B. subtilis (21). However, the genes forthese two subunits in E. coli are located at sites on the E. colichromosome different from the sites on the chromosome of L.plantarum. The gene for the a subunit is located downstreamof the polC gene (4.3 min on the genetic map of E. coli),whereas the gene for the b subunit is located within an ORFwith an unknown function designated dedB and usg (50 min onthe genetic map of E. coli [23]). By contrast, the putative accDand accA genes of L. plantarum were located downstream offabH, accB, and accC and overlapped. The accA gene of B.subtilis is also located downstream of the accD (or yttI) gene(21).

The 39 nontranslated region of the accA gene of L. planta-rum was followed by a palindromic structure that was borderedby five A residues. This region should be able to form a 20-bpimperfect stem-loop structure with a calculated DG value of118.3 kJ/mol (25°C). These features are typical of a rho-inde-pendent signal for termination of transcription in E. coli (34).

Expression of the fabH, accB, accC, accD, and accA genes ofL. plantarum. To confirm expression of proteins from the fabH-

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accBCDA gene cluster of L. plantarum L137, we subcloned thefive ORFs into an expression vector, pVEX11, under control ofthe T7 promoter to generate pT7ACC. We transferredpT7ACC into E. coli BL21(DE3) and analyzed the gene prod-ucts by SDS-PAGE. The estimated molecular masses of theproteins encoded by the fabH, accB, accC, accD, and accAgenes were 35, 14, 47, 28, and 27 kDa, respectively. Five[35S]methionine-labeled proteins with molecular masses of 47,34, 27, 25, and 17 kDa were recognized after SDS-PAGE (Fig.2). The molecular mass of each protein, as estimated from itsmobility during SDS-PAGE, was identical to that calculatedfrom the deduced amino acid sequence, with one exception.AccB yielded a slightly larger molecular mass after SDS-PAGEthan the molecular mass calculated from the amino acid se-quence. Thus, in E. coli, the fabH, accB, accC, accD, and accAgenes from L. plantarum were overexpressed from a singleoperon under control of the T7 promoter.

Transcription of fabH and accBCDA. To confirm that co-transcription of the fabH and accBCDA genes occurred in L.plantarum, RNA blot hybridization was studied. Hybridizationwas performed by using logarithmically growing strain L137cells with either a probe specific for fabH or a probe specific foraccC (Fig. 1). Autoradiography results revealed that at leastthree species of RNA were transcribed. Transcripts that were4.3, 2.7, and 1.3 kb long (Fig. 3) were observed; the 4.3-kbtranscript hybridized to the two probes, and its size was iden-tical to that of the theoretical transcript from the site of initi-ation of transcription of mRNA for fabH to a predicted ter-mination site for accA which is located 12 bp downstream fromthe TTA termination codon of accA (Fig. 1). The 2.7-kb tran-script bound to the two probes, whereas the 1.3-kb transcriptbound to the fabH probe but not to the accC probe, indicatingthat the 2.7- and 1.3-kb transcripts may start at the initiation

site in the fabH gene and may stop at the predicted terminationsites in the accC and accB genes (Fig. 1), respectively, althoughtheir functions are unknown.

Analysis of the promoter region upstream of the fabH gene.Since the genes fabH and accBCDA genes were separated fromthe first ORF (manB), we searched for a potential promoter-like sequence that resembled the 235 and 210 sequences of E.coli and lactobacilli (7). A 235 sequence (TTGACG) from nt854 to 859 and two potential 210 sequences from nt 876 to 881(TAAAAT) and from nt 878 to 882 (AAAATT) were verysimilar to 235 and 210 sequences. A 235 sequence (TTTTAT) from nt 867 to 872 and a 210 sequence (TCGAAA) fromnt 890 to 895 were similar to the sequences of promoters inlactobacilli (8). To identify the site of initiation of transcriptionin the putative promoter region, we performed a primer ex-tension analysis with total RNA prepared from L. plantarumL137. We identified the initiation site by comparing the dataobtained from nucleotide sequencing of the products of theprimer extension reaction with the results of a thymidine ter-minating reaction (Fig. 4Bb). The 11 site guanine residue waslocated 12 bp upstream of the initiation codon of fabH foundin L. plantarum (nt 907, accession no. AB025973). This site islocated at the site of a possible ribosome-binding site and 1 bpupstream, as reported for the rec operon of Streptococcus pneu-moniae (33). We also performed a primer extension reactionanalysis with RNA from E. coli(pTACO-1) and loaded theFITC-labeled primer together with the products of a cytosineterminating reaction. The 11 site adenine residue, which was31 bp upstream of the initiation codon of fabH (nt 888, acces-sion no. AB025973), was identified as the site of initiation oftranscription (Fig. 4Bc). Differences between the sites of ini-

FIG. 2. Expression of products of the L. plantarum fabH, accB,accC, accD, and accA genes under the T7 promoter. Proteins wereseparated by electrophoresis on an SDS-polyacrylamide gel, and[35S]methionine-labeled proteins were visualized by autoradiography.Cells were prepared from strain BL21 carrying pVEX11 (control)(lanes 1 and 2) or pT7ACC (lanes 3 and 4). Lanes 1 and 3, no IPTGinduction; lanes 2 and 4, IPTG induction. The arrowheads indicate thepositions of the expression products obtained. The correspondinggenes coding for the proteins are indicated. The numbers on the leftindicate the molecular masses of the standards.

FIG. 3. Northern blot analysis of transcripts from the fabH-accB-CDA regions. Total RNA was isolated from L. plantarum L137. Twoprobes specific for fabH (DraI-NheI DNA fragment in fabH) and accC(NruI-HindIII DNA fragment in accC) were used. The sizes of mRNAspecies that hybridized with the probes (arrows) are indicated. Thepositions of size standards are indicated on the left.

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FIG. 4. Mapping of the 59 end of fabH by primer extension analysis by using a 59-end FITC-labeled primer. See text for details. (A) Primerextensions with RNA from L. plantarum (a) and RNA from E. coli that harbored pTACO-1 (b). The arrows indicate the transcript determined

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tiation of transcription in E. coli and lactic acid bacteria havebeen reported for several operons (3, 8, 33).

The promoter upstream of the fabH gene was able to me-diate expression of a reporter enzyme, cholesterol oxidase (3U/mg of protein), in L. plantarum that harbored a plasmid inwhich the promoter was fused to the choA9 gene (data notshown). Together, our results indicate that fabH and accBCDAis an operon in L. plantarum, which we designated the accoperon.

Functional analysis of accB and accD by complementationand biotinylation experiments. We tested the ability of the L.plantarum accB and accD genes to complement E. coli L8[accB22(Ts)] (11) and E. coli LA1-6 [accD6(Ts)] (24). Bothmutants are viable at 30°C but not at higher temperatures.Plasmid pWACC carrying the fabH-accBCDA genes was trans-ferred to E. coli L8 and LA1-6 at 30°C. The transformants wereincubated at 42°C. The transformants of strains L8 and LA1-6carrying pWACC could grow at 42°C, but the strains withoutthe plasmid could not. These results indicated that the L.plantarum accB and accD genes were able to complement theBCCP in E. coli L8 and the b subunit of carboxyltransferase inE. coli LA1-6, respectively.

Furthermore, biotinylation of the gene product from L.plantarum accB was tested by performing a Western blot anal-ysis with streptavidin conjugated with alkaline phosphatase.Two biotin-containing proteins were detected in E. coli carry-ing plasmid pWACC as bands at 14 and 17 kDa (Fig. 5, lane 5).The size of the 14-kDa protein corresponded to the size of L.plantarum BCCP estimated from the amino acid sequence.The 17-kDa protein is the E. coli biotin-binding BCCP. IPTGinduction was observed only in the biotin-binding BCCP of L.plantarum (Fig. 5, lane 6). However, a low level of inductionwas observed due to the low copy number of plasmidpTWV228 used in construction of pWACC. When cells pre-pared from E. coli L8 grown at 30, 37, and 42°C were used, thebiotinylating protein was observed only at 30°C, and this pro-tein was 17 kDa long (Fig. 5, lanes 2 through 4). These resultssuggest that the L. plantarum BCCP has the biotinylation func-tion in E. coli.

DISCUSSION

ACC, the first enzyme in the fatty acid biosynthetic pathway,has been extensively studied by gene cloning and enzyme char-acterization in E. coli. The first reaction consists of two half-reactions: carboxylation of biotin which is bound to BCCP withbicarbonate, which is catalyzed by biotin carboxylase; and sub-sequent transfer of the carboxyl group from carboxybiotin toacetyl-CoA to obtain malonyl-CoA, which is catalyzed by car-boxyltransferase. Moreover, it has been reported that the firststep in the elongation of fatty acid chains is catalyzed by b-ke-toacyl ACP synthase III, which is encoded by fabH (6, 10, 26).

Since the first enzymatic step in a metabolic pathway is often

rate limiting, we attempted to isolate and characterize thegenes for ACC in a lactic acid bacterium. In this study, wecloned and characterized the homologs of the manB, fabH,accB, accC, accD, and accA genes in L. plantarum.

The enzymes of the PTS in L. curvatus are encoded by fourgenes, manABCD. These proteins are similar to the PTS trans-porters EIIA, EIIB, EIIC, and EIID of the mannose class, andit has been proposed that EIIB, encoded by manB, might playa regulatory role in L. curvatus (47). However, the manB genein L. plantarum seems to be organized differently than themanB gene in L. curvatus. The arrangements of genes involvedin phosphorylation of sugar or PTSs are different in differentspecies.

The fabH and accBCDA genes appear to form a singleoperon, although we found three sizes of transcripts in L.plantarum. In E. coli (22), B. subtilis (27), and P. aeruginosa (4),the accB and accC genes are transcribed together. The accDand accA genes in E. coli are located at different positions (4.3and 50 min, respectively) (23), whereas in B. subtilis the accAand accD (yttI) genes are located in the rrnB-dnaB region (21).In P. aeruginosa, the accA and accD genes are neither imme-diately upstream nor immediately downstream of the accBCoperon (4). The fabH gene encodes b-ketoacyl ACP synthaseIII, which catalyzes the condensation of acetyl-CoA with ma-lonyl-ACP to initiate chain elongation in the biosynthesis offatty acids. This gene is located in the plsX-fab gene cluster inE. coli (50). A study of the regulation of expression of the fabgene cluster in E. coli suggested that a promoter in the plsXcoding sequence might allow read-through of downstream fabgenes (35). Full-length and short transcripts were generateddepending on the amount of ACP, a small protein containingfewer than 90 amino acids that plays a key role in lipid bio-synthesis and is encoded by the acpP gene. The fabH gene inStreptomyces glaucescens is located in the fab gene cluster, inwhich the genes are arranged in the order fabD, fabH, fabC,fabB, which implies that there is a functional connection be-tween the metabolism of fatty acids and biosynthesis ofpolyketide (44). In E. coli and Salmonella typhimurium, thefabH gene is organized in a cluster with the g30k, rmpF, andplsX genes and other fab genes (namely, fabD, fabG, acpP, andfabF) (31, 36, 45). In B. subtilis, the two putative fabH genes,fabH1 and fabH2 (equivalent to yjaX and yhfB), are also lo-cated outside the fab cluster (18, 28). The fabH1 (yjaX), yjaY,and fabH2 (yhfB) genes encode the subunits of 3-oxoacyl ACPsynthase. In P. aeruginosa, the fabH gene is also not part of themajor cluster of fab genes (19). Unlike the fabH gene in Strep-tomyces, E. coli, and B. subtilis, the fabH gene of L. plantarumis not located in the same transcript as the acpP or plsX gene.It seems likely that the promoter upstream of the fabH gene inL. plantarum is a major promoter responsible for transcriptionof the fabH and accBCDA genes together, although we foundthree sizes of transcripts. In the T7 RNA polymerase expres-sion system, the L. plantarum fabH-accBCDA genes are co-

by primer extension. 11 indicates the site of initiation of transcription. (B) Chromatography of the products of sequencing reactions. Green, blue,black, and red lines indicate the products of adenine, cytosine, guanine, and thymine sequencing reactions, respectively. The arrow indicates thesite of initiation of transcription (11) in the primer extension reaction. Reaction products were loaded together with the products of thymine (red)(b) or cytosine (blue) (c) sequencing reactions. (a) DNA sequence used as a control; (b) RNA prepared from L. plantarum L137; (c) RNA preparedfrom E. coli that harbored pTACO-1.

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transcribed as a single transcript (Fig. 2). The T7 RNA poly-merase may read through two potential terminators. TheNorthern blot analysis in which the fabH and accC probes wereused suggested that the transcripts started from the fabH pro-moter seem to stop at the termination signals of accB, accC,and accA (Fig. 3). Such heterologous transcripts of one operonhave been found in gram-positive bacteria (12, 13, 48). How-ever, we have not ruled out the possibility that there might bea minor promoter(s) located within the acc gene cluster.

The complementation test showed that the L. plantarumBCCP containing the Met-Lys-Leu biotinylation motif wasable to complement the accB mutation in E. coli. The highlyconserved MKM motif for biotinylation was studied previouslyin E. coli, and no biotinylation of MAK or KAM mutants wasobserved (37). Only the lysine residue in its native position inthe hairpin turn, KKM or MKK, was found to be biotinylated.Our results, together with the biotinylation test results, alsosupport this conclusion. The L. plantarum BCCP, which con-tains the MKL motif, was biotinylated in E. coli even thoughthe L. plantarum BCCP lacks Thr-94, which was suggested toform a protruding polypeptide “thumb” (38). The putative L.plantarum accD gene was also able to complement the accDmutation in E. coli.

In bacteria, fatty acids are primarily the precursors of phos-pholipids rather than storage fuel, and thus ACC activity iscoordinated with cell growth and division (1, 25, 26). Therequirement for biotin and lipids in most lactobacilli suggeststhat expression of the acc operon might play an important rolein growth of the bacteria. Lactic acid bacteria living under fattyconditions, such as meat or milk, might be expected to lose theability to synthesize lipids or fatty acids. However, the bacteriaretain these genes in an operon, and the genes are efficientlyexpressed to allow synthesis of fatty acids upon addition ofbiotin. The differences in gene organization and coregulationof expression of ACC subunits among microorganisms might

reflect differences in control of growth under various environ-mental conditions.

ACKNOWLEDGMENTS

We thank Y. Yamada of Yamaguchi University for providingpVEX11 and Mary Berlyn of the E. coli Genetic Stock Center forproviding E. coli mutant strains L8 and LA1-6.

This work was supported in part by Monbusho, Japan (Kiban B;grant 10556019). P.K. was supported by the Ronpaku Program of theJapan Society for the Promotion of Science.

REFERENCES

1. Allen, S. D., J. A. Siders, M. J. Riddell, J. A. Fill, and W. S. Wegener. 1995.Cellular fatty acid analysis in the differentiation of Clostridium in the clinicalmicrobiology laboratory. Clin. Infect. Dis. Suppl. 2:198–201.

2. Altschul, S. F., F. Stephen, T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang,W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic Acids Res. 25:3389–3402.

3. Araya-Kojima, T., N. Ishibashi, S. Shimamura, K. Tanaka, and H. Taka-hashi. 1995. Identification and molecular analysis of Lactococcus lactis rpoDoperon. Biosci. Biotechnol. Biochem. 59:73–77.

4. Best, E. A., and V. C. Knauf. 1993. Organization and nucleotide sequences ofthe genes encoding the biotin carboxyl carrier protein and biotin carboxylaseprotein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase. J. Bac-teriol. 175:6881–6889.

5. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M.Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor,N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y.Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science277:1453–1474.

6. Choi, K. H., R. J. Heath, and C. O. Rock. 2000. b-Ketoacyl-acyl carrierprotein synthase III (FabH) is a determining factor in branched-chain fattyacid biosynthesis. J. Bacteriol. 182:365–370.

7. Djordjevic, G., B. Bojovic, A. Banina, and L. Topissirovic. 1994. Cloning ofpromoter-like sequences from Lactobacillus paracasei subsp. paracasei CG11and their expression in Escherichia coli, Lactococcus lactis, and Lactobacillusreuteri. Can. J. Microbiol. 40:1043–1050.

8. Fremaux, C., C. Ahn, and T. R. Klaenhammer. 1993. Molecular analysis ofthe lactacin F operon. Appl. Environ. Microbiol. 59:3906–3915.

9. Gornicki, P., L. A. Scappino, and R. Haselkorn. 1993. Genes for two subunitsof acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotincarboxylase and biotin carboxyl carrier protein. J. Bacteriol. 175:5268–5272.

10. Han, L., S. Lobo, and K. A. Reynolds. 1998. Characterization of b-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role ininitiation of fatty acid biosynthesis. J. Bacteriol. 180:4481–4486.

11. Harder, M. E., I. R. Beacham, J. E. Cronan, Jr., K. Beacham, J. L. Honeger,and D. F. Silbert. 1972. Temperature-sensitive mutants of Escherichia colirequiring saturated and unsaturated fatty acid for growth: isolation andproperties. Proc. Natl. Acad. Sci. USA 69:3105–3109.

12. Hopwood, D. A., M. J. Bibb, K. F. Chater, G. R. Janssen, F. Malpartida, andC. P. Smith. 1986. Regulation of gene expression in antibiotic-producingStreptomyces, p. 251–276. In I. R. Booth and C. F. Higgins (ed.), Regulationof gene expression—25 years on. SGM, Cambridge, United Kingdom.

13. Horii, M., T. Ishizaki, S.-Y. Paik, T. Manome, and Y. Murooka. 1990. Anoperon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp. J. Bacteriol. 172:3644–3653.

14. Jager, W., P. G. Peter-Wendisch, J. Kalinowski, and A. Puhler. 1996. ACorynebacterium glutamicum gene encoding a two-domain protein similar tobiotin carboxylases and biotin-carboxyl-carrier proteins. Arch. Microbiol.166:76–82.

15. Kaneko, Y., H. Kobayashi, P. Kiatpapan, T. Nishimoto, R. Napitupulu, H.Ono, and Y. Murooka. 2000. Development of a host-vector system for Lac-tobacillus plantarum L137 isolated from a traditional fermented food pro-duced in the Philippines. J. Biosci. Bioeng. 89:62–67.

16. Kashima, Y., Y. Nakajima, T. Nakano, K. Tayama, Y. Koizumi, S. Udaka,and F. Yanagida. 1999. Cloning and characterization of ethanol-regulatedesterase genes in Acetobacter pasteurianus. J. Biosci. Bioeng. 87:19–27.

17. Kondo, H., K. Shiratsuchi, T. Yoshimoto, T. Masuda, A. Kitazono, D. Tsuru,M. Anai, M. Sekiguchi, and T. Tanabe. 1991. Acetyl-CoA carboxylase fromEscherichia coli: gene organization and nucleotide sequence of the biotincarboxylase subunit. Proc. Natl. Acad. Sci. USA 88:9730–9733.

18. Kunst, F., N. Ogasawara, I. Moszer, et al. 1997. The complete genomesequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256.

19. Kutchma, A. J., T. T. Hoang, and H. P. Schweizer. 1999. Characterization ofa Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification ofacyl carrier protein (ACP) and malonyl-coenzymeA:ACP transacylase(FabD). J. Bacteriol. 181:5498–5504.

FIG. 5. Western blot analysis of biotinylation of BCCP of L. plan-tarum L137 in E. coli. Lane 1 contained protein molecular mass stan-dards. E. coli mutant strain L8 was grown at 30°C (lane 2), 37°C (lane3), or 42°C (lane 4). E. coli carrying plasmid pWACC without IPTGinduction (lane 5) or with IPTG induction (lane 6) was also included.The positions of the streptavidin-binding biotinylating BCCP in E. coliand in L. plantarum are indicated by arrows. The numbers on the leftindicate the molecular masses of the standards.

432 KIATPAPAN ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 13

Nov

embe

r 20

21 b

y 87

.110

.98.

104.

20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

21. Lapidus, A., N. Galleron, A. Sorokin, and S. D. Ehrlich. 1997. Sequencingand functional annotation of the Bacillus subtilis genes in 200 kb rrnB-dnaBregion. Microbiology 143:3431–3441.

22. Li, S. J., and J. E. Cronan, Jr. 1992. The gene encoding the biotin carbox-ylase subunit of Escherichia coli acetyl-coA carboxylase. J. Biol. Chem. 267:855–863.

23. Li, S. J., and J. E. Cronan, Jr. 1992. The genes encoding the two carboxy-ltransferase subunits of Escherichia coli acetyl-CoA carboxylase. J. Biol.Chem. 267:16841–16847.

24. Li, S. J., C. O. Rock, and J. E. Cronan, Jr. 1992. The dedB (usg) open readingframe of Escherichia coli encodes a subunit of acetyl-coenzyme A carboxy-lase. J. Bacteriol. 174:5755–5757.

25. Li, S. J., and J. E. Cronan, Jr. 1993. Growth rate regulation of Escherichiacoli acetyl coenzyme A carboxylase, which catalyzes the first step of lipidbiosynthesis. J. Bacteriol. 175:332–340.

26. Magnuson, K., S. Jackowski, C. O. Rock, and J. E. Cronan. 1993. Regulationof fatty acid biosynthesis in Escherichia coli. Microbiol. Rev. 57:522–542.

27. Marini, P., S. J. Li, D. Gardiol, J. E. Cronan, and D. de Mendoza. 1995. Thegenes encoding the biotin carboxyl carrier protein and biotin carboxylasesubunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzymeof fatty acid synthesis. J. Bacteriol. 177:7003–7006.

28. Morbidoni, H. R., D. de Mendoza, and J. E. Cronan, Jr. 1996. Bacillus subtilisacyl carrier protein is encoded in a cluster of lipid biosynthesis genes. J.Bacteriol. 178:4794–4800.

29. Nomura, N., K. P. Choi, M. Yamashita, H. Yamamoto, and Y. Murooka.1995. Genetic modification of the Streptomyces cholesterol oxidase gene forexpression in Escherichia coli and development of promoter-probe vectorsfor use in enteric bacteria. J. Ferment. Bioeng. 79:410–416.

30. Norman, E., K. A. De Smet, N. G. Stoker, C. Ratledge, P. R. Wheeler, andJ. W. Dale. 1994. Lipid synthesis in mycobacteria: characterization of thebiotin carboxyl carrier protein genes from Mycobacterium leprae and M.tuberculosis. J. Bacteriol. 176:2525–2531.

31. Oh, W., and T. J. Larson. 1992. Physical locations of genes in the me(ams)-rpk PlsX-fab/plsX-fab region of the Escherichia coli K-12 chromosome. J.Bacteriol. 174:7873–7874.

32. Olympia, M., H. Fukuda, H. Ono, Y. Kaneko, and M. Takano. 1995. Char-acterization of starch hydrolyzing lactic acid bacteria isolated from a fer-mented fish and rice food, Burong Isda, and its amylolytic enzyme. J. Fer-ment. Bioeng. 80:193–197.

33. Pearce, B. J., A. M. Naughton, E. A. Campbel, and H. R. Masure. 1995. Therec locus, a competence-induced operon in Streptococcus pneumoniae. J.Bacteriol. 177:86–93.

34. Platt, T. 1986. Transcription termination and regulation of gene expression.Annu. Rev. Biochem. 55:339–372.

35. Podkovyrov, S. M., and T. J. Larson. 1996. Identification of promoter andstringent regulation of transcription of the fabH, fabD and fabG genesencoding fatty acid biosynthetic enzymes of Escherichia coli. Nucleic AcidsRes. 24:1747–1752.

36. Rawlings, M., and J. E. Cronan. 1992. The gene encoding Escherichia coliacyl carrier protein lies within a cluster of fatty acid biosynthetic genes.J. Biol. Chem. 267:5751–5754.

37. Reche, P., Y. L. Li, C. Fuller, K. Eichhorn, and R. N. Perham. 1998. Selec-tivity of post-translational modification in biotinylated proteins: the carboxycarrier protein of the acetyl-CoA carboxylase of Escherichia coli. Biochem. J.329:589–596.

38. Roberts, E. L., N. Shu, M. J. Howard, R. W. Broadhurst, A. Chapman-Smith,J. C. Wallace, T. Morris, J. E. Cronan, Jr., and R. N. Perham. 1999. Solutionstructures of apo and holo biotinyl domains from acetyl coenzyme A car-boxylase of Escherichia coli determined by triple-resonance nuclear magneticresonance spectroscopy. Biochemistry 38:5045–5053.

39. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

40. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

41. Shine, J., and L. Dalgarno. 1974. The 3-terminal sequence of Escherichia coli16S ribosomal RNA: complementary to nonsense triplets and ribosomebinding sites. Proc. Natl. Acad. Sci. USA 71:1342–1346.

42. Southern, E. M. 1975. Detection of specific sequences among DNA fragmentseparated by gel electrophoresis. J. Mol. Biol. 98:503–517.

43. Stuible, H. P., C. Wagner, I. Andreou, G. Huter, J. Haselmann, and E.Schweizer. 1996. Identification and functional differentiation of two type Ifatty acid synthases in Brevibacterium ammoniagenes. J. Bacteriol. 178:4787–4793.

44. Summer, R. G., A. Ari, B. Shen, W. A. Wessel, and A. R. Hutchinson. 1995.Malonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomycesglaucescens: a possible link between fatty acid and polyketide biosynthesis.Biochemistry 34:9389–9402.

45. Tsay, J.-T., W. Oh, T. J. Larson, S. Jackowski, and C. O. Rock. 1992.Isolation and characterization of the b-ketoacyl-acyl carrier protein synthaseIII gene (fabH) from Escherichia coli K-12. J. Biol. Chem. 267:6807–6814.

46. Van de Guchte, M., J. Kok, and G. Venema. 1992. Gene expression inLactococcus lactis. FEMS Microbiol. Rev. 88:73–92.

47. Veyrat, A., M. J. Gorsalbes, and G. Perez-Martinez. 1996. Lactobacilluscurvatus has a glucose transport system homologous to the mannose familyof phosphoenolpyruvate-dependent phosphotransferase systems. Microbiol-ogy 142:3469–3477.

48. Westpheling, J. M., and M. Brawner. 1989. Two transcribing activities areinvolved in expression of the Streptomyces galactose operon. J. Bacteriol.171:1355–1361.

49. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phagecloning vectors and host strains: nucleotide sequences of M13 mp18 andpUC19 vectors. Gene 33:103–119.

50. Zhang, Y., and J. E. Cronan, Jr. 1998. Transcriptional analysis of essentialgenes of the Escherichia coli fatty acid biosynthesis gene cluster by functionalreplacement with the analogous Salmonella typhimurium gene cluster. J.Bacteriol. 180:3295–3303.

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