Cloning and Sequencing of the Escherichia coli panB Gene,Which Encodes Ketopantoate Hydroxymethyltransferase,
and Overexpression of the EnzymeCAROL E. JONES,"2 JUDITH M. BROOK,3 DAVID BUCK,3 CHRIS ABELL,2
AND ALISON G. SMITH"*Department ofPlant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, 1 University
Chemical Laboratory, Lensfield Road, Cambridge CB2 JEW,2 and Schering Agrochemicals Ltd,Chesterford Park Research Station, Saffron Walden, Essex, CB10 1XL,3 England
Received 24 September 1992/Accepted 15 January 1993
The panB gene from Escherichia coli, encoding the first enzyme of the pantothenate biosynthesis pathway,ketopantoate hydroxymethyltransferase (KPHMT), has been isolated by functional complementation of apanBmutant strain with an E. coli genomic library. The gene is 792 bp long, encoding a protein of 264 amino acidswith a predicted Mr of 28,179. The identity of the gene product as ketopantoate hydroxymethyltransferase wasconfirmed by purification of the enzyme protein, which was overexpressed approximately 50-fold in the mutantharboring the gene on a high-copy-number plasmid. The N-terminal amino acid sequence of the purifiedprotein was found to be identical to that predicted from the gene sequence, as was its mass, determined byelectrospray mass spectrometry. Upstream of the panB gene is an incomplete open reading frame encoding aprotein of 220 amino acids, which shares sequence similarity to fimbrial precursor proteins from otherbacteria. Northern (RNA) analysis showed that the panB gene is likely to be cotranscribed with at least oneother gene but that this is not the putative fimbrial protein, since no transcripts for this gene could be detected.
Pantothenic acid is the precursor of coenzyme A. It issynthesized by microorganisms and plants but not mammals,which require it as part of their diet. The three-step pathwayto pantothenic acid has been studied in microorganisms(4, 15, 16) and is shown in Fig. 1. The first committed step isthe formation of ketopantoate from ox-ketoisovalerate, cata-lyzed by the enzyme ketopantoate hydroxymethyltrans-ferase (KPHMT; 5,10-methylene-tetrahydrofolate:ot-keto-isovalerate hydroxymethyltransferase, EC 2.1.2.11). KPHMThas been purified to homogeneity from wild-type Escherichiacoli by Teller et al. (28).
Pantothenate-requiring E. coli mutants were first gener-ated in the early 1950s by UV irradiation (15, 16) and indeedwere instrumental in establishing the precise biochemicalpathway of pantothenate synthesis. Thus, thepanB mutant,which is completely lacking in KPHMT, requires ketopan-toic acid, pantoic acid, or pantothenic acid for growth (7).With conjugational crosses and phage transduction, thepanB gene was shown to be closely linked to the genes fortwo other enzymes, pantothenate synthetase and aspartate1-decarboxylase (designatedpanC and panD, respectively),and they have been mapped on the E. coli chromosome at3.1 min (6). In order to study pantothenate synthesis in E.coli more thoroughly, we decided to isolate the panB geneand to characterize its gene product. In this paper wedescribe the isolation and characterization of the gene andthe overexpression and purification of the encoded enzyme.This is the first reported sequence of a gene on the panto-thenate pathway.
Strains and plasmids. E. coli Hfr3000 YA139, a panBmutant derivative of E. coli K-12 (6), was used to isolate thepanB gene by functional complementation. The mutant wasgrown on GB1 minimal medium (13.6 g of KH2PO4 [pH 7.0],2 g of (NH4)2SO4, 4 g of glucose, 0.25 g of MgSO4, 0.25 pugof FeSO4, 5 mg of vitamin B1, and 5 mg of pantothenic acidper liter. E. coli K-12 was used for the generation of an E.coli genomic library and for enzyme assays. The E. colistrain XL1-Blue (Stratagene) was used for the propagation ofplasmids. The vector pBluescript M13 (pKS-) was fromStratagene and was used for all cloning experiments.DNA manipulations. All restriction enzymes were pur-
chased from Boehringer, and digestions were performedunder the conditions recommended by the manufacturer. T4DNA ligase and calf intestinal phosphatase were also fromBoehringer, and plasmid manipulations were carried out bythe method of Sambrook et al. (24). The isolation of DNAfragments for subcloning after electrophoresis in agarose(Bio-Rad) was carried out as previously described (24), as
were transformations in the presence of calcium chloride.Alkaline minipreparations of DNA were prepared by themethod Birnboim and Doly (2). Cesium chloride purifica-tions of DNA were carried out by the method of Sambrooket al. (24).
Generation of unidirectional exonuclease III deletions. Exo-nuclease III deletions of the plasmid pCEJ01 were per-formed with exonuclease III and mung bean nuclease pur-chased from Stratagene in the form of a kit. The reactionswere carried out by a method described in the pBluescriptExo/Mung DNA sequencing system instruction manual.DNA sequence analysis. All the components for DNA
sequence analysis were purchased in the form of a kit fromUnited States Biochemical Corp.; Sequenase 2.0 was usedfor sequencing, and reactions were carried out by meth-
ods recommended by the manufacturer. [a-355]dATP (3,000Ci/mmol) was purchased from Amersham International.Electrophoresis was on buffer gradient gels (1). Analysisof DNA and amino acid sequences were performed withStaden programs (26) and Genetics Computer Group analy-sis software package version 7.0 (8). panB-specific oligonu-cleotide primers used to sequence part of the fragment wereobtained from the Protein and Nucleic Acid ChemistryFacility, Department of Biochemistry, University of Cam-bridge.
Northern (RNA) analysis. Total cellular RNA was pre-pared from log-phase cultures of E. coli strains by themethod of Duncan and Coggins (9) and purified on cesiumchloride gradients to remove contaminating DNA. It wasglyoxylated and electrophoresed on 1.2% agarose gels be-fore being blotted onto Gene Screen Plus nylon filtersessentially as described by Sambrook et al. (24). Blots wereprehybridized for 3 h in 1% sodium dodecyl sulfate (SDS)-1M NaCl-10% dextran sulfate at 60°C and then hybridized atthe same temperature overnight with radiolabelled probe infresh hybridization buffer. Blots were washed twice for 30min in 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate)-1% SDS at 60°C and then autoradiographed at-70°C for 1 to 7 days. Probes were fragments of DNAexcised from agarose gels and radiolabelled with [t-32P]dCTP (3,000 Ci/mmol; Amersham International) by therandom oligonucleotide method of Feinberg and Vogelstein(11).PAGE in the presence of SDS. Polyacrylamide gel electro-
phoresis (PAGE) was carried out in the presence of SDS bythe method of Laemmli (14) with a 15% running gel and a 5%stacking gel.Enzyme assays. Enzyme assays for KPHMT were per-
formed in the reverse direction with ketopantoate as asubstrate and by measuring the amount of formaldehydeproduced with the Nash reagent, as described by Teller et al.(28). Ketopantolactone, used to prepare the substrate keto-pantoate for the enzyme assay, was synthesized by themethod of Ojima et al. (18).
Purification of KPHMT. The purification procedure was
based on that described by Teller et al. (28) and usedidentical buffers and the same method for crude cell lysatepreparation. However, the column matrices used wereslightly different, and the chromatography was performedwith a Pharmacia fast-protein liquid chromatography sys-tem. A 750-ml culture of the E. colipanB mutant containingplasmid pCEJ01 was grown overnight at 37°C, and cells (7 to15 g [wet weight]) were harvested by centrifugation at 8,000x g for 15 min. They were resuspended in 14 ml of buffer A(50 mM KH2PO4 [pH 6.8], 1 mM EDTA, 10 mM 2-mercap-toethanol) and lysed by sonication. After removal of debrisby centrifugation at 15,000 x g for 30 min, the crude lysatewas applied to a DEAE-Sepharose anion-exchange column(28 by 2.6 cm; Pharmacia) equilibrated in buffer A and elutedwith a salt gradient (0.2 to 0.5 M KCl). The fractionscontaining KPHMT activity were concentrated by bringingto 70% ammonium sulfate saturation. The pellet was recov-ered by centrifugation at 15,000 x g for 15 min and resus-pended in 400 RI of 100 mM KH2PO4 (pH 6.8)-i mMEDTA-10 mM 2-mercaptoethanol and dialyzed against thesame buffer. The sample was applied to a Superose 12HR10/30 column (Pharmacia) and eluted in the same buffer.KPHMT-containing fractions were heated to 80°C for 5 min,and denatured proteins were removed by centrifugation.KPHMT remained in the supernatant. Protein concentrationwas determined by the method of Bradford (3).
N-terminal amino acid determination. The N-terminalamino acid sequence of the purified KPHMT was deter-mined by the Protein and Nucleic Acid Chemistry Facility inthe Department of Biochemistry, University of Cambridge.
Electrospray mass spectrometry. Electrospray mass spec-trometry was performed on a VG BioQ mass spectrometer.Samples containing approximately 200 pmol of protein wereapplied in 10 RI of MeOH-H20-acetic acid (50:50:1) at a flowrate of 4 plI/min.
Nucleotide sequence accession number. The sequence ofthe 1.7-kb EcoRI-SalI fragment containing the panB genehas been deposited at EMBL under accession numberX65538.
Isolation of the E. coil panB gene. The E. coli panB genewas isolated by functional complementation of E. coliHFr3000 YA139, which carries thepanB mutation and lacksKPHMT activity (6). Cells of the mutant were made compe-tent, transformed with an E. coli EcoRI genomic plasmidlibrary, and plated out onto minimal media containing ampi-cillin but no pantothenic acid. Twenty-three colonies (of atotal of 1,100 transformants) grew and so presumably hadrestored KPHMT activity. Six were taken for further anal-ysis, and each was shown to contain a plasmid with an insertof 2.5 kbp. Crude lysates of E. coli Hfr3000 YA139 harboringthe clones were assayed for KPHMT activity by the methoddescribed by Teller et al. (28) and showed levels of KPHMTactivity 20- to 100-fold higher than wild-type E. coli K-12(Table 1). One of the isolated plasmids, designated pSAL38,was chosen for further subcloning and sequencing.
Restriction mapping of pSAL38. The restriction map ofpSAL38 is shown in Fig. 2. Four subclones (pCEJ01 topCEJ04) were prepared on the basis of the restriction mapand used to retransform E. coli Hfr3000 YA139. Only clonespCEJ01 and pCEJ02 were able to complement the panBmutation, allowing the mutant to grow in the absence ofpantothenate. Enzyme assays for KPHMT on crude celllysates with all four clones confirmed the functional comple-mentation results (Table 1). The smallest clone, pCEJ01,contained a 1.7-kb EcoRI-SalI insert. This was sequenced inboth directions by the Sanger dideoxy chain terminationmethod (25).
El
S
Sm BYI III
P ~ -
P EV SC
0.5 kb
FIG. 2. Restriction map and subcloning strategy for plasmidpSAL38. pCEJ01 to pCEJ04 were subcloned from pSAL38 intopBluescript. The diagrams also shows a series of nested deletionclones (pCEJ013 to pCEJ020) generated by exonuclease III deletionfrom the EcoRI end of plasmid pCEJ01. El, EcoRI; Sm, SmaI; B,BglI; S, Sall; A, AccI; P. PvuII; EV, EcoRV; Sc, Scal; C, ClaI.
Nucleotide sequence of the panB gene. The nucleotidesequence of the 1.7-kb EcoRI-SalI fragment containing thepanB gene is shown in Fig. 3. Analysis of the sequence withthe STADEN nucleotide interpretation program (version4.1) revealed two open reading frames (ORFs) transcribedfrom the same strand. All other reading frames had multiplestop codons. The first ORF, which is not complete, is 660 bpin length, and the second is 792 bp.A series of eight nested deletions from the EcoRI site of
the 1.7-kb insert of pCEJ01 (Fig. 2) were generated withexonuclease III as described in Materials and Methods.Transformation of Hfr3000 YA139 with these clones sug-gested that thepanB gene lay within the second ORF of 792bp, since deletions which interrupted this ORF did notcomplement the mutation. The orientations of the ORFs aresuch that the gene must be transcribed from its own pro-moter, as the promoters of the ampicillin resistance and lacZgenes in the vector would transcribe the other strand.A putative ribosome binding site (AGGA, overlined on
Figure 3) is situated 7 bp upstream of the ATG start codon ofthepanB gene. A putative promoter sequence (boxed in Fig.3) which starts 35 bp 5' to the initiating ATG codon wasidentified by its similarity to the consensus -35 and -10sequences (13), although clearly its authenticity cannot beconfirmed without functional analysis.Sequence comparison of the first ORF with the PIR28 data
base (11.6.1991) found that there were short stretches (30 to80 amino acids) which had 30 to 50% sequence similaritywith the sequences of fimbrial protein precursors of Kieb-siella pneumoniae, Serratia marcescens, Salmonella typh-imurium, E. coli, and Haemophilis influenzae (12, 17, 22, 29,30), suggesting that this sequence codes for all or part of afimbrial protein precursor.
Northern analysis. In order to determine whether either, orboth, of the ORFs encoded by CEJ01 was part of an operon,Northern analysis was carried out. Figure 4 shows the resultof probing RNA extracted from log-phase cultures of E. coliK-12 and Hfr3000 YA139 with probes internal to ORF1 andthepanB gene (a 475-bp HincII-SmaI fragment and a 530-bpAflIII-PvuII fragment, respectively). It is clear that in bothwild-type and mutant strains, a major band of 1.9 kbp isdetected with thepanB gene-specific probe. Since the codingregion of thepanB gene is less than 800 bp, it is likely that itis cotranscribed with at least one other gene. However, thisis not the putative fimbrial protein encoded by ORF1, sinceon an identical blot, no message is detectable for this gene(Fig. 4). Even when the blot was overexposed (for 7 ratherthan 2 days), no signal was observed, suggesting that ORF1is not expressed in log-phase cultures growing in rich media,supporting previous observations (19). The fact that anidentical message for panB is present in the mutant andwild-type cells, and in approximately the same amount,suggests that the defect carried by Hfr3000 YA139 is likely tobe a point mutation in the coding region.
Purification of KPHMT overexpressed in Hfr3000 YA139cells. In order to confirm the identity of the second ORF asthe panB gene, the protein which it encodes was overex-pressed and purified, and the N-terminal sequence wasdetermined. The construct with clone pCEJ01 in Hfr3000YA139 showed levels of KPHM 50-fold higher than those ofthe wild type in enzyme assays (Table 1). Furthermore,SDS-PAGE analysis of total soluble protein from the mutantharboring plasmid pCEJ01 showed the presence of a prom-inent band of 29,000 Da, which was absent in the mutantalone, or indeed from E. coli K-12 cells (Fig. 5). KPHMTwas purified to homogeneity from the transformed cells by a
1 GAATTCTATACAGATACAAACTTTGATCCAACAGTAACTCAACAGATCAAATTATCCAGCTCATCAAATTATCTGTATTCATTTAAAGCCF Y T D T N F D P T V T Q Q I K L S S S S N Y L Y S F K A
91 TATGGCGCAGGCCAAGGTATAAATGAGCATAGTTATTTTATCAAAATCGATTTCGATCTGCTCAATGTCAAGTTGACTAATCCCACTTGTY G A G Q G I N E H S Y F I K I D F D L L N V K L T N P T C
181 TTTACCGCTATGCTTAGTGGAACTTCTGTGACTGGTTCTACGGTAAAAATGGGTGAATATAGTGCAGAACAAATCAGAAACGGTGCCACAF T A M L S G T S V T G S T V K M G E Y S A E Q I R N G A T
271 CCGGTTCCTTTTGATATTTCACTTCAAAACTGCGTTCGTGTGACTAATATTGAGACAAAATTAGTTTCAACAAAGGTTGGTACTGAAAACP V P F D I S L Q N C V R V T N I E T K L V S T K V G T E N
361 GGGCAACTCCTTGGTAATACTCTCACAGGTAATGACGCAGCAAAAGGAGTCGGTGTACTCATAGAAGGTTTAGCAACTAGTAAAAATCCTG Q L L G N T L T G N D A A K G V G V L I E G L A T S K N P
451 CTAATGACATTGAAACCTAATGATTCAAATTCTGTTTATAAAGATTACGALCCCNAGAGKCAAAGAC AAAADAGGAGAD G ACC
L M T L K P N D S N S V Y K D Y D P R G K D D T T G G V Y P
541 GATCAAGATACAGGTATAACATACCCTCTCCATTTCCAGGCCACGCTACAACAGGATGGAACTATACCAATAGAAGCTGGTGAGTTTAAAD Q D T G I T Y P L H F Q A T L Q Q D G T I P I E A G E F K
631 GCCACCAGTACTTTCCAGGTAACCTACCCTTAATAAGTCCACCGCACCGCCATCATCTGGCGGTGCGGTAATTGATAAATCP3ZCA T S T F Q V T Y P stop
721 ACCAATGTGACTGA:3CCAGGCAACACGACATCATTTATCAGGACACGTTATGAAACCGACCACCATTGCTTCACTTCAGAAATGM K P T T I A S L O K C
811 TAAGCAGGATAAAAAGCGTTTCGCGACCATCACCGCTTACGACTACAGCTTCGCGAAACTATTTGCTGATGAAGGACTTAACGTCATGCT,K O D K K R F A T I T A Y D Y S F A K L F A D E G L N V M L
901 GGTAGGCGATTCGCTGGGCATGACGGTTCAGGGCCACGACTCCACCCTTCCCGTTACCGTCGAGGATATCGCCTATCATACCGCCGCCGTV G D S L G M T V Q G H D S T L P V T V E D I A Y H T A A V
991 ACGTCGCGGCGCGCCAAACTGCCTGCTGTTGGCTGACCTGCCGTTTATGGCGTATGCCACGCCGGAACAAGCCTTTGAAAACGCCGCAACR R G A P N C L L L A D L P F M A Y A T P E Q A F E N A A T
1081 GGTTATGCGTGCCGGTGCCAATATGGTCAAAATTGAGGGCGGTGAGTGGCTGGTCGAAACCGTAAAAATGCTGACCGAACGTGCCGTTCCV M R A G A N M V K I E G G E W L V E T V K M L T E R A V P
1171 TGTATGTGGTCACTTAGGTTTAACCCCACAGTCAGTGAATATTTTCGGTGGCTACAAAGTTCAGGGGCGCGGCGATGAAGCGGGCGATCAV C G H L G L T P Q S V N I F G G Y K V Q G R G D E A G D Q
1261 ACTGCTCAGCGATGCATTAGCCTTAGAAGCCGCTGGGGCACAGCTGCTGGTGCTGGAATGCGTGCCGGTTGAACTGGCAAAACGTATTACL L S D A L A L E A A G A Q L L V L E C V P V E L A K R I T
1351 CGAAGCACTGGCGATCCCGGTTATTGGCATTGGCGCAGGCAACGTCACTGACGGGCAGATCCTCGTGATGCACGACGCCTTCGGCATTACE A L A I P V I G I G A G N V T D G Q I L V M H D A F G I T
1441 CGGCGGTCACATTCCTAAATTCGCTAAAAATTTCCTCGCCGAAACGGGCGACATCCGCGCGGCTGTGCGGCAGTATATGGCTGAAGTGGAG G H I P K F A K N F L A E T G D I R A A V R Q Y M A E V E
1531 GTCCGGCGTTTATCCGGGCGAAGAACACAGTTTCCATTAAGGAGTCACGTTGTGTTAATTATCGAAACCCTGCCGCTGCTGCGTCAGCAAS G V Y P G E E H S F H stop
FIG. 3. Sequence of the 1.7-kbp fragment which complemented the panB mutation. The nucleotide sequence of the 1.7-kb EcoRI-SalIinsert in pCEJ01 is shown with the translated sequence of the putative fimbrial protein precursor (ORF1) and that of the translatedpanB geneshown below. The postulated ribosome binding site forpanB is overlined, and the putative -10 and -35 promoter regions are boxed. Theamino acid sequence determined experimentally from the purified protein is underlined. ¶, EcoRI site; §, Sall site.
four-step procedure, detailed in Materials and Methods,which closely followed the original purification from wild-type E. coli (28). A crude cell lysate was subjected toanion-exchange chromatography, ammonium sulfate precip-itation, gel filtration, and finally, a heat treatment. A 16-foldpurification ofKPHMT was achieved with a 57% recovery ofactivity (Table 2). The purification was followed by SDS-PAGE, and the heat-treated sample appeared as a singleband of 29,000 Da in Coomassie (Fig. 5)- and silver-stainedgels.
Analysis of purified KPHMT. N-terminal sequence analy-sis of the first 15 residues of purified KPHMT confirmed thetranslational start of thepanB gene (underlined in Fig. 3) andthe predicted protein sequence. The purified KPHMT sam-ple was also subjected to electrospray mass spectrometry(10) (data not shown) and was shown to have a subunitmolecular weight of 28,178 + 5 (predicted molecular weight,
28,179). The native Mr was determined by gel filtration on aSuperose 12 HR10/30 column to be 174,000, suggesting thatthe enzyme is a hexamer. The Km value of KPHMT wasdetermined for ketopantoate in the reverse enzyme-cata-lyzed reaction to be 0.15 mM. This compares well with thatof 0.16 mM, determined by Powers and Snell (21).
DISCUSSION
In this paper we report the isolation of the panB geneencoding the pantothenate biosynthesis enzyme KPHMTfrom E. coli by functional complementation of an E. colipanB mutant (strain Hfr3000 YA139) deficient in KPHMT.After transformation of the mutant with a genomic library ofE. coli DNA, a clone containing a 2.5-kb insert was isolated.Transformation of E. coli Hfr3000 YA139 with subclones ofthis initial fragment identified a smaller clone (pCEJ01) with
FIG. 4. Northern analysis of transcripts from the ORF1 andpanB genes. Autoradiograph of a Northern blot of RNA extractedfrom E. coli K-12 (lanes 1 and 3) and Hfr3000 YA139 (lanes 2 and 4)probed with an internal fragment of ORFI (lanes 1 and 2) or panB(lanes 3 and 4).
a 1.7-kb insert which included the entire panB gene. Se-quence analysis revealed two ORFs of 660 and 792 bp. Thesmaller ORF shows sequence similarity to fimbrial proteinprecursor sequences (12, 17, 22, 29, 30). The second ORF of792 nucleotides was confirmed as encoding KPHMT, bytransformation of the E. coli panB mutant with a series ofnested deletions of CEJ01; only those which did not inter-rupt this second ORF complemented the panB mutation.When the amino acid sequence ofpanB was used to screen
the available protein data bases (Swissprot, PIR, and Gen-Bank) with the program FASTA, no similar proteins were
identified. In contrast, when it was compared directly toenzymes known to carry out similar reactions, with tetrahy-drofolate as cofactor, it was found to have some weaksimilarity to a region of serine hydroxymethyltransferasesfrom several bacterial sources (Fig. 6). However, it is notpossible to draw any conclusions about the functional sig-nificance of this, if any, until more is known about thebinding of tetrahydrofolate to these enzymes.KPHMT activity in E. coli Hfr3000 YA139 harboring
plasmid pCEJ01 was 50-fold greater than levels in wild-typeE. coli (Table 1), presumably because of the high copynumber of the pBluescript vector. It was therefore relativelystraightforward to purify KPHMT to homogeneity from therescued mutant. The preparation yielded a single protein of29,000 Da on SDS-PAGE (Fig. 5). N-terminal protein se-
quence analysis of this band confirmed the predicted trans-lational start of the panB gene and the sequence of the first15 residues. Significantly, the sequencing confirmed that thesecond residue is lysine, not tyrosine as had been reportedby Powers and Snell (21).The subunit molecular weight of KPHMT was determined
by electrospray mass spectrometry to be 28,178 + 5. This is
FIG. 5. SDS-PAGE analysis of overexpressed and purifiedKPHMT. Proteins were electrophoresed on a 15% polyacrylamidegel and visualized with Coomassie blue. Lanes: 1, 12.5 ,ug of proteinfrom E. coli K-12; 2, 12.5 ,ug of protein from E. coli Hfr3000YA139/pBluescript; 3, 12.5 pLg of protein from E. coli Hfr3000YA139/pCEJ01; 4, 0.6 ,ug of purified KPHMT; M, marker proteins(bovine serum albumin [66 kDa], ovalbumin [45 kDa], glyceralde-hyde-3-phosphate dehydrogenase [36 kDa], carbonic anhydrase [29kDa], trypsinogen [24 kDa], trypsinogen inhibitor [20 kDa], anda-lactalbumin [14 kDa]).
in agreement with the predicted molecular weight of 28,179and shows that there is no posttranslational modification ofthe enzyme. The native molecular mass of KPHMT wasdetermined by gel filtration to be 174,000 Da, suggesting theenzyme is a hexamer. This is smaller than the values of285,000 Da (gel filtration) and 255,000 Da (sedimentationequilibrium) reported by Teller et al. (28) for the enzymepurified from wild-type E. coli, but the yield they obtainedfor the enzyme was low, and their estimation of the subunitmolecular weight (25,000) was also incorrect.By genetic analysis, the panB, panC, and panD genes
have been found to be closely clustered at 3.1 min of the E.coli K-12 genetic map (6, 7). In the same work, the clockwiseorder of the genes was found to be panB panD panC byphage Pl-mediated three-factor crosses, and the possibilityof apan operon was proposed. This suggestion is lent furthersupport by our finding that the panB transcript is 1.9 kbp
TABLE 2. Recovery and yield obtained during purification ofrecombinant KPHMT from E. coli Hfr3000 YA139
containing plasmid pCEJ01
Sample Total Sp act (mmol % Recovery purification(mg) min m ) r step (fold)
E. col i SHMT GDTVLGMNLAHGGHLTHGSPVS. typhi SHMT GDTVLGMNLAQGGHLTHGSPV
B. japonicum SHMT GDTFMGLDLAAGGHLTHGSPVC. jejuni SHMT GDKILGMDLSHGGHLTHGAKV
FIG. 6. Alignment of KPHMT with bacterial serine hydroxy-methyltransferases (SHMT). A region of E. coli KPHMT (residues44 to 60) is shown aligned with internal sequences of SHMT fromCampylobacter jejuni (5), E. coli (20), S. typhimunum (27), andBradyrhizobium japonicum (23). An asterisk indicates an identicalresidue, and a period indicates a similar one. Gaps (-) have beenintroduced into the KPHMT sequence to improve the alignment.
(Fig. 4), significantly larger than the size of the panB gene.However, since nothing is known about the physical char-acteristics of the proteins encoded by panC or panD (andthus the sizes of the corresponding genes), whether thistranscript could encode all three genes or only two requiresfurther investigation. What can be concluded is that theoperon is strongly expressed in actively growing cultures ofE. coli, which would be expected given the requirement forpantothenate-containing cofactors for lipid synthesis.
ACKNOWLEDGMENTS
We thank the Protein and Nucleic Acid Chemistry Facility(PNACF), Biochemistry Department, University of Cambridge, forcarrying out the N-terminal sequencing of KPHMT and synthesizingoligonucleotide primers and J. E. Dancer, S. G. Foster, J. B.Pillmoor, and K. Wright for their interest in this research. ThePNACF and the VG BIO Q electrospray mass spectrometer are inthe Cambridge Centre for Molecular Recognition.We thank Schering Agrochemicals and the SERC for a CASE
Studentship for C.E.J. We acknowledge the support of the Well-come Trust for the PNACF.
REFERENCES1. Biggen, M. D., T. J. Gibson, and G. F. Hong. 1983. Buffer
gradient gels and 35S label as an aid to rapid DNA sequencedetermination. Proc. Natl. Acad. Sci. USA 80:3963-3965.
2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.
3. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.
4. Brown, G. M., and J. M. Williamson. 1982. Biosynthesis ofriboflavin, folic acid, thiamin and pantothenic acid. Adv. Enzy-mol. 53:345-381.
5. Chan, V. L., and H. L. Bingham. 1991. Complete sequence ofthe Campylobacter jejuni glyA gene encoding serine hydroxy-methyltransferase. Gene 101:51-58.
6. Cronan, J. E., Jr. 1980. ,B-Alanine synthesis in Escherichia coli.J. Bacteriol. 141:1291-1297.
7. Cronan, J. E., Jr., K. J. Littel, and S. Jackowski. 1982. Geneticand biochemical analyses of pantothenate biosynthesis in Esch-erichia coli and Salmonella typhimurium. J. Bacteriol. 149:916-922.
8. Devereux, J., P. Haeberli, and O. Smithies. 1984. Comprehen-sive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.
9. Duncan, K., and J. R. Coggins. 1986. The serC-aroA operon ofE. coli-a mixed function operon encoding enzymes from two
different amino acid biosynthetic pathways. Biochem. J. 234:49-57.
10. Edmonds, S. G., and R. D. Smith. 1990. Electrospray ionisationmass spectrometry. Methods Enzymol. 193:412-431.
11. Feinberg, A. P., and B. Vogelstein. 1983. A technique forradiolabelling DNA restriction endonuclease fragments to highspecific activity. Anal. Biochem. 132:6-13.
12. Gerlach, G. F., S. Clegg, and B. L. Allen. 1989. Identificationand characterization of the gene encoding type 3 and type 1fimbrial adhesions of Kiebsiella pneumoniae. J. Bacteriol. 171:1262-1270.
13. Harley, C. B., and R. P. Reynolds. 1987. Analysis of E. colipromoter sequences. Nucleic Acids Res. 15:2343-2361.
14. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.
15. Maas, W. K., and B. D. Davis. 1950. Pantothenate studies. I.Interference by D-serine and L-aspartic acid with pantothenatesynthesis in Eschenchia coli. J. Bacteriol. 60:733-745.
16. Maas, W. K., and H. J. Vogel. 1953. a-Ketoisovaleric acid, aprecursor of pantothenic acid in Escherichia coli. J. Bacteriol.65:388-393.
17. Mizunoe, Y., Y. Nabeppa, M. Sekiguchi, S. I. Kawabata, T.Moriya, and K. Amako. 1988. Cloning and sequence of the geneencoding the major structural component of mannose-resistantfimbriae of Serratia marcescens. J. Bacteriol. 169:3567-3574.
18. Ojima, I., T. Kogure, and Y. Yoda. 1985. Asymmetric hydroge-nation of ketopantoyl lactone: D-(-)-pantoyl lactone. Org.Synth. 63:18-25.
19. Pearce, W. A., and T. M. Buchann. 1980. Structure and cellmembrane-binding properties of bacterial fimbriae, p. 291-297.In E. H. Beachey (ed.), Bacterial adherence. Chapman andHall, London.
20. Plamann, M. D., L. T. Stauffer, M. L. Urbanowski, and G. V.Stauffer. 1983. Complete nucleotide sequence of the E. coli glyAgene. Nucleic Acids Res. 11:2065-2075.
21. Powers, S. G., and E. E. Snell. 1976. Ketopantoate hydroxy-methyltransferase: physical, catalytic and regulatory properties.J. Biol. Chem. 251:3786-3793.
22. Purcell, B. K., J. Pruckler, and S. Clegg. 1987. Nucleotidesequences of the genes encoding typel fimbrial subunits ofKlebsiella pneumoniae and Salmonella typhimunium. J. Bacte-riol. 169:5831-5834.
23. Rossbach, S., and H. Hennecke. 1991. Identification ofglyA as asymbiotically essential gene in Bradyrhizobium japonicum.Mol. Microbiol. 5:39-47.
24. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.
25. Sanger, F., Nicklen, S. and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.
26. Staden, R. 1987. Computer handling of DNA sequencingprojects, p. 173-217. In M. J. Bishop and C. J. Rawlings (ed.),Nucleic acid and protein sequence analysis: a practical ap-proach. IRL Press, Oxford.
27. Steiert, J. G., M. L. Urbanowski, L. T. Stauffer, and G. V.Stauffer. 1990. Nucleotide sequence of the Salmonella typhimu-rium glyA gene. DNA Sequence 1:107-113.
28. Teller, J. H., S. G. Powers, and E. E. Snell. 1976. Ketopantoatehydroxymethyltransferase: purification and role in pantothenatebiosynthesis. J. Biol. Chem. 251:3780-3785.
29. Van Die, I., and H. Bergmans. 1984. Nucleotide sequence of thegene encoding the f72 fimbrial subunit of a uropathogenicEscherichia coli strain. Gene 32:83-90.
30. van Ham, S. M., F. R. Mooi, M. G. Sindhunata, W. R. Maris,and L. van Alphen. 1989. Cloning and expression in Eschenichiacoli ofHaemophilus influenzae fimbrial genes establishes adher-ence to oropharyngeal epithelial cells. EMBO J. 8:3535-3540.
