Vol. 173, No. 8JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2590-25990021-9193/91/082590-10$02.00/0Copyright © 1991, American Society for Microbiology

The Bacillus subtilis hemAXCDBL Gene Cluster, WhichEncodes Enzymes of the Biosynthetic Pathway from

Glutamate to Uroporphyrinogen IIIMATS HANSSON, LARS RUTBERG, INGRID SCHRODER, AND LARS HEDERSTEDT*Department of Microbiology, University of Lund, Solvegatan 21, S-223 62 Lund, Sweden

Received 16 November 1990/Accepted 11 February 1991

We have recently reported (M. Petricek, L. Rutberg, I. Schroder, and L. Hederstedt, J. Bacteriol. 172:2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemAproposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the Cs pathway for 5-aminolevulinicacid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encodinghydroxymethylbilane synthase. In the present communication, we report the sequences and identities of threeadditional hem genes located immediately downstream of hemC, namely, hemD encoding uroporphyrinogen IHsynthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-l-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for thesynthesis of uroporphyrinogen HI from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shownto be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create agrowth requirement for ALA or heme, indicating that formation of ALA from glutamate-l-semialdehyde canoccur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B.subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that nofurther genes in heme synthesis are part of the proposed hem operon.

Heme, chlorophyll, and corrinoids such as vitamin B12 aremetal-containing tetrapyrrole derivatives constituting pros-thetic groups in respiratory chain complexes, light-harvest-ing complexes, catalases, and peroxidases and are thusessential components of most organisms.

Tetrapyrroles are synthesized from 5-aminolevulinic acid(ALA), which in turn is formed via either of two majorpathways. In the C4 pathway, ALA is formed by thecondensation of glycine and succinyl coenzyme A, a reac-tion catalyzed by ALA synthase (EC 2.3.1.37). This pathwayis found in animals (37), yeast cells (55), and some bacteria,e.g., rhizobia (38, 52). In the C5 pathway, ALA is formedfrom the carbon skeleton of glutamate (27). The C5 pathwayinvolves at least three enzymatic reactions (Fig. 1). In thefirst step, a tRNAGJU is charged with glutamate, which issubsequently reduced by a NAD(P)H-dependent glutamyl-tRNA reductase to form glutamate-1-semialdehyde (GSA).ALA is then formed by isomerization of GSA catalyzed byGSA 2,1-aminotransferase (EC 5.4.3.8). The C5 pathway ispresent in higher plants (7, 56), algae (49), and severalbacteria, e.g., Escherichia coli (5, 43), Salmonella typhimu-rium (16), and Bacillus subtilis (43, 44).

Little is known about the organization and regulation ofgenes encoding enzymes which are involved in the biosyn-thetic pathway from glutamate to the tetrapyrrole uropor-phyrinogen III (UroIll) in bacteria. B. subtilis mutants withhemA mutations are ALA auxotrophs (3, 29), whereasmutations in hemB, hemC, or hemD affect later steps inUroIII synthesis and the corresponding mutants requireheme for growth (8, 9). The hemABCD genes are clustered at2450 on the B. subtilis chromosomal genetic map (45). Wehave recently cloned the hemA region of the B. subtilischromosome (44). Sequence analysis revealed that hemA

* Corresponding author.

probably encodes the NAD(P)H:glutamyl-tRNA reductaseof the C5 pathway for ALA synthesis. This conclusion wasbased on the high level of sequence similarity between thepredicted E. coli and B. subtilis HemA proteins (44) and onthe fact that E. coli HemA mutants are defective in theNAD(P)H:glutamyl-tRNA reductase of the C. pathway (5).Immediately downstream of B. subtilis hemA, two moregenes were found which seem to be part of the sametranscription unit as hemA, namely, hemX encoding a hy-drophobic protein of unknown function and hemC encodinghydroxymethylbilane synthase (EC 4.3.1.8).The close linkage between hemC, hemB, and hemD in

transformation crosses (8, 39) and the observation thatdeletion of a promoter in front of hemA results in hemeauxotrophy (44) and loss of porphobilinogen (PBG) synthase(EC 4.2.1.24) activity (this work) suggested to us that hemBand hemD are also parts of a hem operon. The aim of thiswork was to determine the numbers and identities of hemgenes located downstream of hemC.Three more hem genes were found, hemD encoding

UroIllI synthase (EC 4.2.1.75), hemB encoding PBG syn-thase, and hemL encoding GSA 2,1-aminotransferase. Adeletion-substitution analysis of hemL and regions immedi-ately downstream of this gene demonstrated that no furthergenes essential for heme synthesis are part of this hemoperon. Hence, the B. subtilis hemAXCDBL gene clusterrepresents an operon encoding enzymes required for thesynthesis of UrollI from glutamyl-tRNA.

MATERIALS AND METHODS

Bacterial strains and plasmids. Bacterial strains and plas-mids used are listed in Table 1.Media. B. subtilis strains were kept on tryptose blood agar

base (TBAB; Difco); the minimal medium of Spizizen (51) orLB (36) was used for liquid cultures. E. coli strains were

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B. SUBTILIS hemAXCDBL GENE CLUSTER 2591

6

hmnmf

C0O-tRNACHNH2

X IF1I

I* hemACOOH

tRNA-Glu 2

CHC

CHN

ICOC

HMB

hemC \

PBG

hemB /4

co

ICFthemLh

iLt

IQ ALA

*H

GSA

FIG. 1. Pathway of UrollI biosynthesis in B. subtilis. The sixsteps are catalyzed by the following enzymes: 1, glutamyl-tRNAsynthetase; 2, NAD(P)H:glutamyl-tRNA reductase; 3, GSA 2,1-aminotransferase; 4, PBG synthase; 5, hydroxymethylbilane (HMB)synthase; 6, UrollI synthase. The designations of correspondinggenes in B. subtilis are given. Glu, Glutamate.

grown in LB or on LA plates (36). The following antibioticsand concentrations were used: ampicillin (LA, 50 mg/liter;LB, 120 mg/liter), chloramphenicol (B. subtilis, 3 mg/liter; E.coli, 10 mg/liter), and tetracycline (15 mg/liter). ALA andrequired amino acids were used at 10 mg/liter. Media con-taining hemin (2.5 mg/liter) also contained cysteine (25mg/liter) and bovine serum albumin (fraction V; SigmaChemical Co.) (500 mg/liter). Stock solutions of hemin(Sigma Chemical Co.) were prepared as described previ-ously (44).Competent cells. Competent E. coli cells were prepared as

described by Mandel and Higa (35). B. subtilis was grown tocompetence as described by Arwert and Venema (4).

General DNA techniques and enzymes. Large-scale prepa-rations of plasmid DNA from E. coli were done by themethod of Ish-Horowicz and Burke (25). Small-scale prepa-rations of plasmid DNA from E. coli XL1-Blue and E. coliMM294 were done by the boiling method (36) and themethod of Kieser (28), respectively. Southern blot analysisof chromosomal DNA was done with a nonradioactive DNAlabeling and detection kit from Boehringer GmbH (Mann-heim, Germany). General DNA techniques were as de-scribed by Maniatis et al. (36). Restriction endonucleasesand T4 DNA ligase were from Boehringer or New EnglandBioLabs (Beverly, Mass.). Exonuclease III and mung beannuclease were from Bethesda Research Laboratories (Gaith-ersburg, Md.), and RNase was from Worthington Biochem-ical Corp. (Freehold, N.J.).

Deletion of DNA using exonuclease III. Exonuclease IIItreatment followed by mung bean nuclease treatment wasused to create unidirectional nested deletions in cloned DNAfragments as described in a protocol from Stratagene (La

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant properties Reference or source

StrainsB. subtilis 3G18 trpC2 met ade G. VenemaB. subtilis 1A589 trpC2 hemBI BGSCaB. subtilis 1A590 trpC2 hemC33 BGSCB. subtilis 1A591 trpC2 hemDJ I BGSCB. subtilis 3G18A401 trpC2 met ade AhemA401 cat; lacks the promoter and the first 519 bp of hemA, 44

requires heminB. subtilis 3G18A401R ALA requiring pseudorevertant of B. subtilis 3G18zA401 44B. subtilis 3G18AL17 trpC2 met ade AhemL17 cat This workE. coli XL1-Blue recAl endAl gyrA96 thi hsdRJ7 supE44 relAl (lac) [F' proAB lacq lacZAM15 Stratagene

TnJO(Tetr)]E. coli JM83 ara rpsL A(lac-proAB) F80 lacZAM15 59E. coli MM294 endAl thi pro hsdRJ7 supE44 L.-O. HedenE. coli SASZ31 hemD31 derivative of SAS245 12

PlasmidspBluescript II KS(-) bla lacZ' StratagenepHV32 bla cat tet 42pUC18 bla lacZ' 59pLUP212 bla cat; pHV32 derivative with the hemA region (Fig. 2) This workpLUX3201 bla; Clal deletion derivative of pLUP212 (Fig. 2) This workpLUX1 bla; EcoRI fragment of pLUX3201 cloned in pBluescript II KS(-) This workpLUX2 bla; EcoRI fragment of pLUX3201 cloned in pBluescript II KS(-) (fragment in This work

orientation opposite that of pLUX1pLUX207 bla hemD'BL; exonuclease III derivative of pLUX2 (contains bp 3902 to 6634) This workpLUX209 bla hemL; exonuclease III derivative of pLUX2 (contains bp 5052 to 6634) This workpLUX3202 bla cat; pHV32 derivative (Fig. 10) This workpLUX3203 bla cat tet; pHV32 derivative (Fig. 10) This workpLUX3204 bla cat tet; pHV32 derivative (Fig. 10) This workpAhemL17 bla cat; cat of pHV32 cloned in HindlIl and EcoRV sites of pLUX207 (Fig. 10) This work

a Bacillus Genetic Stock Center, Ohio State University, Columbus.

UrolllCOOH

CHNH2cI-CF2CH2COOH

Glu

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2592 HANSSON ET AL.

Clal

CalIFIG. 2. Physical map of pLUP212 and pLUX3201. pLUX3201 was generated from pLUP212 by deletion of two ClaI fragments. Arrows

indicate the location and direction of transcription of genes.

Jolla, Calif.). Digestion with exonuclease III was carried outat 31°C, and one aliquot was removed every minute. Aftermung bean nuclease treatment, 1 ,ug of DNA from eachaliquot was run on a 0.8% agarose gel. Distinct DNA bandswere excised from the gel, purified with Geneclean (BiolOl,La Jolla, Calif.), incubated with T4 DNA ligase, and trans-formed into E. coli XL1-Blue with selection for ampicillinresistance.

Construction of B. subtUis insertion and deletion-substitu-tion mutants. Insertion mutants were constructed by trans-forming B. subtilis 3G18 to chloramphenicol resistance withpHV32 derivatives carrying different DNA fragments fromthe hemA region (42) (see Fig. 10).A hemL deletion-substitution mutant was constructed in

the following way. The cat gene ofpHV32 was isolated on aHindIII-EcoRV fragment and used to substitute the hemL427-bp internal HindIII-EcoRV fragment in pLUX209 (Table1) to give plasmid pAhemL15. A 2,230-bp HindIII-EcoRI

pux1pLUX

-hmCh hwt

E S S EV EV H H EV CE

1000 bp

pLUX22pLUX22

pLUX24

FIG. 3. Restriction map of the cloned EcoRI fragment present inpLUXi and pLUX2. The EcoRI fragment in pLUX2 is oriented sothat the hem genes can be transcribed from the lac promoter of thevector pBluescript II KS(-). The exonuclease III deletion plasmids,pLUX21 to pLUX24, were used to map the hem mutation in B.subtilis 1A589 (hemBI) and 1A591 (hemDlJ). The plasmids containhem DNA starting at the positions indicated: pLUX21, bp 3398;pLUX22, bp 3673; pLUX23, bp 4150; and pLUX24, bp 4312. Thenucleotide sequence strategy used is indicated by arrows in thelower part of the figure. The sequences of hemC and the first part ofhemD have been reported previously (44). Restriction site abbrevi-ations: C, ClaI; E, EcoRI; EV, EcoRV; H, Hindlll; S, SphI.

fragment (including the cat gene and 780 bp of the end ofhemL) from pAhemL15 was cloned in HindIII-EcoRI-di-gested pLUX207 (Table 1) to give plasmid pAhemL17 (seeFig. 10). B. subtilis 3G18 was then transformed to chloram-phenicol resistance with pAhemL17 linearized with ScaI. Inthe resulting mutant, B. subtilis 3G18AL17, 427 bp of hemL(bp 5450 to 5877 in Fig. 4) has been replaced by the cat gene.The structure of the deletion-substitution mutant was con-firmed by Southern blots (data not shown).DNA sequence analysis. Nucleotide sequences were deter-

mined by the dideoxy chain termination method (47), usingmodified T7 DNA polymerase (Sequenase version I or II;U.S. Biochemical Corp., Cleveland, Ohio) and [a-35S]dATP(Amersham). The template was plasmid DNA isolated fromE. coli XL1-Blue by the boiling method. A synthetic oligo-nucleotide complementary to bp 825 to 841 of pBluescript IIKS(-) was used as a primer. The GCG Sequence SoftwarePackage (13) was used for analysis of nucleotide sequencedata. Amino acid sequence alignments were done by usingthe GAP program (13).Enzyme activity measurements. To determine PBG syn-

thase activity in B. subtilis, the cells were grown aerobicallyat 37°C in 100 ml of LB-hemin medium. The cells wereharvested when entering stationary growth phase andwashed once in 50 mM Na-phosphate buffer, pH 6.4. Thepellets were resuspended in 1 ml of Na-phosphate buffercontaining 5 mM dithiothreitol, 0.25 mg oflysozyme, and 0.1mg of DNase I (Sigma Chemical Co.), incubated for 15 minat 37°C, and then sonicated while chilled on ice. The lysatewas centrifuged at 5,000 x g for 10 min at 4°C, and PBGsynthase activity in the supernatant was measured by theprocedure of Li et al. (32), using 40 ,ul of cell extract perassay.To determine GSA 2,1-aminotransferase activity in E. coli

JM83 carrying different plasmids, the cells were grownovernight at 37°C in 100 ml of LB containing 50 ,ug ofampicillin per ml. The cells were harvested, washed, sus-pended in 1 ml of 0.1 M Tricine-NaOH buffer (pH 9.0)containing 0.3 M glycerol, 25 mM MgCl2, and 1 mM dithio-threitol and sonicated while chilled on ice. The resultinglysate was centrifuged at 5,000 x g for 10 min at 4°C toremove cell debris. GSA 2,1-aminotransferase in the super-

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B. SUBTILIS hemAXCDBL GENE CLUSTER 2593

EcoRI3022 GAATTCCTTGAGCCTGAGCGCTGTTTGCC185 E F L E P E R C L P

The terminal part of heC3082 TGCCGAGAATCGGATGAAGAGCTGTTGGC205 C R E S D E E L L A

3142 AAACGGACTGTCTTAGCGGAACGTGCTTTI225 K R T V L A E R A F

'TGCTGTGGGGCAGGGAGCCCTGGCGATTGAGA V G Q G A L A I E

'GTTGTTTTCTCAGTTTACAGATGAATATACL F S Q F T D E Y T

TTTAAACGCGATGGAGGGCGGCTGCCAGGTL N A N E G G C Q V

3202 CCGATCGCGGGCTACTCCGTGTTAAATGGACAGGATGAAAT1GAAATGACAGGTCTTGTC245 P I A G Y S V L N G Q D E I E N T G L V

3262 GCTTCACCTGAGAAlACCGTCACCGGAAACGATCCGGAGGAA265 A S P D G K I I F K E T V T G N D P E E

3322 GTAGGAAAGCGCTGTGCCGCTCTTATGGCTGACAAAGGAGCAAAAGATTTAATTGATCGT285 V G K R C A A L N A D K G A K D L I D R

3382 GTAMCGGGG1CSTTGACGAGGATGGAAAATGATTTTCCGTTGAAAGGAAAAACAGTGCT305 V K R E L D E D G K *

1 heD M E N D F P L K G K T V L3442 TGTCACCCGGAATAAGGCACAGGCAGCATCATTTCAGCAAAAAGTGGAGGCGCTTGGCGG

14 V T R N K A Q A A S F Q Q K V E A L G G

3502 TAAAGCGGTTTTAACCTCTTTGATTACGTTTCGCCGCGCTTTGCCGAATGATGTTGCGGA34 K A V L T S L I T F R R A L P N D V A E

3562 ACAGGTAACCGCGCCAGGCTGGCTTGTTTTTACAAGTGTGAACGGGGC54 Q V R E D L A A P G W L V F T S V N G A

3622 AGACTTCTTITITTCTTATCTGAAGGAAATCAGCTTATTCTCCCTGCGCATAAAAAAT74 D F F F S Y L K E N Q L I L P A H K K I

3682 ACCCGCGCGCCGTTTAAAAATGCATAACGTATCGGTTGATGT94 A A V G E K T A R R L K N H N V S V D V

SphI3742 GATGCCACAGGAGTATATTCTGAAAGTG AGCCA CATGCTGAACC114 N P Q E Y I A E Q L A D A L K Q H A E P

3802 GGGGGAGACCATTACCGTGATGAAAGGGAATTTGTCACGTGATGTGATAAAACAAGAGCT134 G E T I T V N K G N L S R D V I K Q E L

3862 TGTCCCGCTCGGTITTGAAGTAAAGGAATGGGTYCTCTAC0AAACGAT1CCGGATGAAGA154 V P L G F E V K E W V L Y E T I P D E E

3922 CTTTGACTATGTAACATTTAC174 G I E A L K D A A G Q Y S F D Y V T F T

3982 GAGTTCATCAACCGTACATACGTTTATGCATGTCTTGGGAGAAGAATTAAAAAGTGGAA194 S S S T V H T F N H V L G E E L K K W K

4042 GGCGAATGGGACGGCCTGTATCAGCATTGGGCCTTTAACAAATGATGCCCTTCTGACGTA214 A N G T A C I S I G P L T N D A L L T Y

SphI4102 CGGCATCACATCGCATACGCCTGATACATTTACAATAGATGGCATGCTTGAGTTAATGTG234 G I T S H T P D T F T I D G N L E L N C

4162 CAGCAT0TCAGA0AAGAATATGATTAAATAGACACCGCCGCCTG254 S N S R E E E R I *

1 heB N S Q S F N R H R R L4222 CGGA T AAAATGGTAAAGGAAACACGTTTGCATCCATCAGATTTT

12 R T S K A N R E N V K E T R L H P S D F

4282 CCCT32 I Y P I F V V E G L E G K K A V P S N P

4342 GATGTTCACCATGTAT ATA _T-G GTAA GCTGGTCAAACTG52 D V H H V S L D L L K D E V A E L V K L

4402 GGCATTCAATCTGTTATCGTGTT= CGGCCCATATGArG AACACAA72 G I Q S V I V F G I P E E K D D C G T Q

4462 GCGTACCATGATCACGGAATTGTCCAAAAAGCCATCACAGAAATTAAGAACACTTCCCT92 A Y H D H G I V Q K A I T E I K E H F P

4522 GAAATGGTTGTTGTCGCTGACACGTGCCTGTGCGAATATACAGACCACGGCCATTGCGGA112 E N V V V A D T C L C E Y T D H G H C G

4582 CTTGTCAAAGACGGAGTC TTCTCAATOATGAATGGCTGGAGCTTTTGGCGCAGACAGCT132 L V K D G V I L N D E S L E L L A Q T A

EcoRV4642 GTCAGCCAAGCGAAAGCAGGTGCGGATATCATTGCGCCATCAAACATGATGGACGGATTT152 V S Q A K A G A D I I A P S N N N D G F

4702 GTTACAGTGATCAAGAGAAGCACTTGATAAAGAAGGATTCGTCAATATTCCCATCATGTCT172 V T V I R E A L D K E G F V N I P I N S

4762 TACGCTGTTAAATATTCAAGTGAGTTTTACGGTCCGTTCCGTGATGCAGCAAACAGCACA192 Y A V K Y S S E F Y G P F R D A A N S T

4822 CCGCAATTCGGAGACCGCAAAACATATCAGATGGACCCTGCCAACCGTATGGAGGCACTC212 P Q F G D R K T Y Q N D P A N R N E A L

3081204

3141224

3201244

3261264

3321284

3381304

344131413350133

356153

362173

368193

3741113

3801133

3861153

3921173

3981193

4041213

4101233

4161253

422126211428131

434151

440171

446191

4521111

4581131

4641151

4701171

4761191

4821211

4881231

4882232

4942252

5002272

5062292

5122312

51821

524218

530238

536258

542278

548298

5542118

5602138

5662158

5722178

5782198

5842218

5902238

5962258

6022278

6082298

6142318

6202338

6262358

6322378

6382398

6442418

6502

6562

6622

CGCGAAGCACAATCAGATGTTGAGGAAGGCGCGGACTTTTTGATTGTCAAACCTTCGCTTR E A Q S D V E E G A D F L I V K P S L

EcoRVTCTTATATGGATATCATGCGTGACGTAAAAAATGAGTTTACTTTGCCGCTCGTCGCTTATS Y N D

AATGTAAGCGGN V S G

AAAGAAATTGIK E I V

ACGTATCATGCT Y H A

AOTO'AGGCAGhe"

TCATGCCGGGCN P G

TTTYTATGGAGF MG E

ACTACGTCTTGY V L

GCCTCAAAAAAL K K

AACTGGCTAAGL A K

CCGGAACAGAGG T E

AGATTTTAAAAI L K

GTTCAGOO=S G V

AAAACACCATCN T I

TCGGTGAAGACG E D

CCGC-%CAGAJP Q E

I Ft D

TCGGCGTAACXG V T

A Y G

AAGCTGGTACA G T

AGCTGACACCL T P

TTTCAAAAACS 1C T

G F F

TGAAGCTGTTR L F

CACAATTCGAQ F E

TCCAGGCAGCQ AA

I N R D V K N E F

;AGAGTATTCAATGGTGAAGGCTGCAGCE Y S N V K A A A

rGTTGGAAATTTTGACAAGCATGAAGCGL E I L T S N K R

:GAAAGACGCAGCGAAATGGCTTGCGGAK D A A K W L A E

;ATGAGAAGCTATGAAAAATCAAAAACGN R S Y E K S K T

:GGTGTGAACAGTCCCGTTCGCGCATT7G V N S P V R A F

;CGCGGAAAAGGCTCGAAAATCTTTGATR G K G S K I F D

;TCATGGGGGCCTTTAATTTTAGGGCATS W G P L I L G H

HindIIIkGTGGCTGAATACGGGACAAGCTTTGG3V A E Y G T S F G

;CTCGTCATTGATCGTGTGCCATCTGTAL V I D R V P S V

3GCTACAATGAGTGCCCTCCGTTTGGCAA T N S A L R L A

STTTGAGGGCTGCTACCACGGACACGGCF E G C Y H G H G

rGCCACTCTCGGTCTGCCTGACAGCCCGA T L G L P D S P

CACCGTTCCGTACAATGATTTAGAAAGIT V P Y N D L E S

CATTGCGGGAGTCATTGTAGAGCCAGTII A G V I V E P V

EcoRVGGmTTCCTTCAGGGTCTGCGTGATATCG F L Q G L R D I

TGAAGTGATGACTGGCTTCCGGGTCGAIE V M T G F R v D

GCCTGATCTGACTTGTTTAGGAAAAGTlP D L T C L G K V

CGGAAAGGCAGAAATCATGGAGCAGATCG K A E I lI E Q I

ATTGTCAGGCAACCCGCTTGCGATGACL S G N P L A M T

TGAATCCTACAAGAATTTCATCAAAAAJE S Y K N F I R K

CGCCGGGGCTCATGGCATTCCCGATACA G A H G I P H T

CTTTACAAACGAACCAGTCATCAATPTAF T N E P V I N Y

CGCAAGAT GAAA S Y Y K G 11 A N

AGGTCTTTTCCTCTCAACGGCCCATACG L F L S T A H T

P0A0AAAGTATTT0CT0AATCA0CCO4KVFAGE ISR

T L P L V A Y

CGCAGAACGGCTGGATCAAAGAAQ N G W I K E

GCGCGGGTGCCGACCTGATTATTA G A p L I I

AGTAATTTTATTCAGTTGACAC

GCTGTrTAAAGAAGCGCAAAAACA F K E A Q K L

TAAATCGGTAGACATGGACCCGAK S V D M D P I

TATTGACGGGAATGAATATATTGI D G N E Y I D

TACAAATGACCGCGTCGTAGAAAT N D R V V E S

TGCTCCGACTGAAGTAGAAAATGA P T E V E N E

AGAAATTGTACGAATGGTAAGCTE I V R N V S S

AAGGGGCTATACGGGCCGCAACAR G Y T G R N K

CGATTCTCTCTTGATTAAAGCTGD S L L I K A G

GGGGGTGCCTGAAGGCATTGCGAG V P E G I A K

HindIIITGTAAAGCTTGCTTTCCAGCAATV K L A F Q Q F

1Y;CCGGAAATATGGGTGTTGTTCA G N 11 G V V P

CACTGAGCAGTACGGCTCCCTGCT E Q Y G S L L

,TTATAACTGCGCTCAAGGCTACTY N C A Q G Y F

'AATCGGGGCGGGACTTCCTGTCGI G G G L P V G

'CGCTCCAAGCGGTCCGATCTATCA P S G P I Y Q

GGCTGGCTTAGAGACATTGAAACA G L E T L K Q

_G~ACAATGGAA0A0AG D R L E E G I

FGATAACACGAATCAT:C1TGAT

E T A K S S D L

['GAAGGGGTATTCCTTCCGCCATE G V F L P P S

: TGAAGATATTGAAAACACAAD E D I E N T I

;CAGATAAGAGTGAAAACCGGTATR *

CAAGGACTCCTTGTGCCGGTICGTGCTCTCCCACTCATATTTTCTCCAGTTCATAC

ClaICAAAATCATCGAT 6634

4941251

5001271

5061291

5121311

5181324

524117

530137

536157

542177

548197

5541117

5601137

5661157

5721177

5781197

5841217

5901237

5961257

6021277

6081297

6141317

6201337

6261357

6321377

6381397

6441417

6501430

6561

6621

FIG. 4. Nucleotide sequence of hemC (the terminal half), hemD, hemB, and hemL and the derived amino acid sequences of thecorresponding polypeptides. The numbering of base pairs follows that of Petricek et al. (44). Possible ribosome binding sites are underlined,and an inverted repeat 6 bp after the TAA stop codon of hemL is overlined. Errors in the previously published sequence (44) at positions 3773(C to G) and 3774 (G to C) have been corrected.

natant was assayed by utilizing chemically synthesized GSA phoresis (PAGE) (41), followed by autoradiography of the(19) as described by Houghton et al. (24), except that the gel when required. Protein was determined by the method ofcofactors pyridoxal 5'-phosphate and pyridoxamine 5'-phos- Lowry et al. (34), using bovine serum albumin as thephate were not added. standard.Other methods. In vitro transcription-translation was done Nucleotide sequence accession number. The complete nu-

with E. coli S30 extracts, using a kit from Amersham and cleotide sequence of hemAXCDBL has been submitted toL-[35S]methionine (Amersham). Polypeptides were analyzed the EMBL, GenBank, and DDBJ data bases under accessionby sodium dodecyl sulfate (SDS)-polyacrylamide gel electro- number M57676.

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TABLE 2. PBG synthase activity in different B. subtilis strains

Strain PBG synthaseactivity'

3G18 (wild type) .................................. 5.5 ± 1.3 (n = 6)1A589 (hemBl) .................... .............. 0.21A589 Hem' (transformant)b ......................... 4.03G18A401 (promoter and part of hemA

deleted) .............. .................... 0.13G18A401R (ALA-requiring pseudorevertant

of 3G18A401) ................................. 13.1

Activity is given as nanomoles of PBG per milligram of protein and 1.5 h.The standard deviation and number of times the experiment was repeated isshown for B. subtilis 3G18.

b Obtained by transformation with pLUX23.

RESULTS AND DISCUSSION

Cloning of the hemC downstream region. The B. subtilishemA region was previously cloned by Petricek et al. (44)using plasmid pHV32. Plasmid pLUP212 is a pHV32 deriv-ative which carries B. subtilis hemA, hemX, and hemC on aca. 20-kb DNA fragment. pLUP212 DNA was cleaved withClaI to give pLUX3201 (Fig. 2). The EcoRI fragment ofpLUX3201 containing the terminal part of hemC, 3.3 kb ofB. subtilis DNA downstream of hemC, and 24 bp of pHV32DNA (Fig. 2) was cloned in the EcoRI site of pBluescript IIKS(-). Two ampicillin-resistant E. coli XL1-Blue transfor-mants containing plasmids with the EcoRI fragment insertedin the two possible orientations were selected for furtheranalysis. These plasmids were designated pLUX1 andpLUX2 (Fig. 3). Exonuclease III treatment of both plasmidswas used to generate unidirectional nested deletions in theEcoRI fragment. KpnI and XhoI sites in pBluescript IIKS(-) were used to create 3' and 5' overhangs, respectively,before the DNA was treated with exonuclease III. Theresulting deletion plasmids were used for DNA sequenceanalysis, for transformation of B. subtilis Hem mutants, andfor analysis of gene products.

Nucleotide sequence analysis. Recently we have reportedthe nucleotide sequences of hemA, hemX (a gene withunknown function previously designated ORF2), hemC, and

1 2 3

46 kDa---

41 kDa--_

32 kDa -

FIG. 5. In vitro transcription-translation analysis of the geneproducts of hemB and hemL. The resulting [35S]methionine-labeledpolypeptides were separated in a SDS-10 to 15% polyacrylamidegradient gel and visualized by autoradiography. Lanes: 1, pLUX23containing hemB and hemL; 2, pLUX209 containing hemL; 3,pBluescript II KS(-). The 32-kDa polypeptide is the bla geneproduct.

the start of what appeared to be hemD (44). To determine thecomplete sequence of hemD and additional downstreamgenes, 3,613 bp of B. subtilis DNA present in pLUX1 andpLUX2 were sequenced (Fig. 3 and 4). Three complete openreading frames were found and designated hemD, hemB, andhemL. hemD starts with an ATG codon at position 3404 andends with a TGA stop codon at position 4190. The hemDgene overlaps the end of hemC by 11 nucleotides. A possibleribosome binding site, AaAcgGGAGcT, is located 9 nucle-otides upstream of the translation start codon of hemD. Thestop codon of hemD and the ATG start codon of hemB atposition 4189 overlap. A possible ribosome binding site,AGAgAGGAaGaGA, is found 5 bp upstream of the start ofhemB. hemB ends with a TAA stop codon at position 5161.The hemL ATG start codon is at position 5193, and the TAAstop codon is at 6483. A possible ribosome binding site,GAcAGGAGtTGA, is present 6 bp upstream of the startcodon of hemL. This extends the previous hemAXC se-quence to a hemAXCDBL sequence consisting of 6,634 bp.

Identification of hemD. On the basis of the followingfindings, the hemD gene of B. subtilis was identified asencoding UrollI synthase.

(i) E. coli K-12 SASZ31 (hemD31) is deficient in UroIllsynthase (12). This strain was transformed with plasmidscontaining B. subtilis DNA with different parts of the hemgene cluster, pLUX3201 (hemAXCDBL), pLUX2 (hem-C'DBL), pLUX23 (hemBL), and pLUX209 (hemL). Selec-tion was for ampicillin resistance and heme prototrophy.Only pLUX3201 and pLUX2 complemented the E. colihemD mutation, i.e., only transformants obtained with theseplasmids grew well on LA-ampicillin plates without hemin.

(ii) B. subtilis HemD mutants have been isolated as heminauxotrophs defective in UrollI synthase (39). The mutationin B. subtilis 1A591 (hemDJIJ) was mapped by transformingthis strain to hemin prototrophy with various plasmidscontaining different parts of the cloned DNA. This mappedthe mutation to the proximal part of hemD or possibly to thedistal part of hemC, i.e., plasmid pLUX21 rescued thehemDJJ mutation, whereas pLUX22 did not (Fig. 3).

(iii) The B. subtilis hemD gene can encode a protein of 29kDa as deduced from the nucleotide sequence, and this sizeis in the same range as that of Urolll synthases purified fromE. coli (26 kDa) (2), Euglena gracilis (31 kDa) (23), and ratliver (28 kDa) (50). In these organisms, the enzyme is amonomer. Using an E. coli in vitro transcription-translationsystem and different plasmid constructs, a polypeptide en-coded by hemD could not be detected.

(iv) The deduced amino acid sequence of B. subtilis hemDshowed 25 and 21% identity to the sequence of UrolIlsynthase from E. coli (1, 26, 48) and from humans (53),respectively (data not shown). The sequence identity be-tween E. coli and human Urolll synthase is 20%. Thesesimilarities are of borderline significance (14), which isnotable since the derived amino acid sequences of other hemgenes from different organisms generally show a high degreeof similarity (see for example Fig. 6 and 9).Chemical modification studies of UrolIl synthase from E.

gracilis suggest that arginine residues are essential for theactivity of the enzyme (23). The arginine at position 146 inthe B. subtilis Urolll synthase polypeptide appears to beconserved in the E. coli and human enzymes.

Identification of hemB. The hemB gene was concluded toencode PBG synthase on the basis of the complementationof HemB mutants, the size of the gene product, and aminoacid sequence similarities.

B. subtilis 1A589 (hemBI) is deficient in PBG synthase

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B. SUBTILIS hemAXCDBL GENE CLUSTER 2595

MHTAEFLETE PT.EISSVLA GGYN.HPL.L RQWQSERQLT.....*...M..QPQSVLH SGY.FHPL.L RAWQT............H.. QSVLH SGY. FHPL.L RAWQTTPSTV... ...............MT DLIQRPR.RL RKSPALPEF..................MS QSFNRHR.RL RTSKANEEMV

* *

LPNINRIGVNLPGVARYGVKLPGVARYGVNMPGVMRIPEKMPDVHRVSLD*

RLKDYLRPLV AKGLRSVILFRLEINRPLV ECEGLRCVLIFQLEELRPLV EAGLRCVLIF}} IERIA KAGIRSVNTFLLKDEVAELV KLGIQSVIVF* * * *

X. ..N MaIFPLFISD.ATTTLNAS NLIYPIFVTDSAT. NLIYPIFVTD..EETTLSLSI DLVLPIFVEEK. .ETRLHPS DFIYPIFVVE

* *

GVPLIPGTKD PVGTAADDPAGVP. SRVPKD ERGSAADSEEGVP. SRVPKD EQGSAADSEDGISHH. .T. D ETGSDA&REDGIPE. .E.KD DCGTQAYHDH** * *

REYFPELYII CDVCLCEYTS HGHCVLYDD GTINRMSVS RLAAVAVNYARKTFPNLLVA CDI*LCPYTS HGHC*LLSEN GAFRAEESRQ RLAEVALAYARRTFPTLLVA CDVWLCPYTS HGHaLLSEN GAFLAE$5RQ RLAEVALAYAXQTVPEMIVM SDTCFCEYTS HGHCVLCEH G.VDNDATLE NLGKQAVVAAKEHFPEkvVV ADTLCEYTD HGHC.LVKD. GVILNDESLE LLAQTAVSQA

* *I1* * ** ****1* * * * *I-----------J

DMIDGRIRDI KRGLINANIA HKTFVISYAA KFSGNLYGPF1MNDGRVEAI REALMAHGLG NRVSVNSYSA KFASCFYGPFDINDGRVEAI XAALLRHGLG NRVSVMSYSA KFASCFYGPFAAMDGQVQAI RQALDAAGFK D.TAIKSYST KFASSFYGPFNNODGFVTVI REALDKEGFV N.IPINSYAV KYSSEFYGPF

** * * ** * ****

GDRKCYQLPP AGRGLARRAL ERDMSEGADGGDRRCYQLPP GARGLALRAV DRDVREGADNGDRRCYQLPP GARGLALRAV ARDIQEGADIGDRKSYQNNP MNPREAIRES LLDEAQGADCGDRRTYQMDP ^ANREALREA QSDVEEGADF*** ** * * * * ***

SDEYANLHAA AEKGVVDLKT IAFESHQGFLSGEFAKLWHG AQAGAFDLKA AVLEAMTAFRSGEFAMLWHG AKAGAFDLRT AVLESMTAFRSGEYAMIKFA ALAGAIDEEK VVLESLGSIKSGEYSMVKAA AQNGWIKEKE IVLEILTSMK* * * * * *

____-_

IEVKRPSiPYLI)VKPGNPYL

ILVPSIYNI *** I *

RAGARIIITYRAGADIIITYRAGADIIITYRAGADLIFSYRAGADLIITY**** * *

DIMRDASEICDIVREVKDKHDNVQEVXDRHDIVRELRER.DIMRDVKNEF*

LAPEFL. . DWYTPQLL..QWFAPQLL..KWFALDLAEKKI

RDAACSAPSNRDAAKSSPAFRDAAQSSPAFREAAGSALK.RDAANSTPQF* ** *

KDLPICAYHVPDLPLAVYHVPELPLAVYQVTELPIGAYQVT. LPLVAYNV

** * *

LDEEN 342LREE. 330LKEE. 330LR... 324

FIG. 6. Alignment of the predicted amino acid sequences of yeast (yea) (40), human (hum) (57), rat (11), E. coli (Eco) (31), and B. subtilis(Bsu) PBG synthase. Positions with identical amino acid residues in all five polypeptides are marked by asterisks. The upper box indicatesthe tentative zinc binding region (57), and the lower box indicates the sequence containing the lysine residue in the active site (18). Thesequences are from references 11, 31, 40, 57 and this work.

(Table 2) and has an absolute growth requirement for hemin(8, 9). By transforming this mutant with plasmids containingdifferent parts of cloned DNA and selecting for heminprototrophy, the hemBI mutation was mapped to the prox-imal part ofhemB or possibly to the distal part of hemD, i.e.,plasmid pLUX23 transformed the mutant, whereas pLUX24did not (Fig. 3). Hemin prototrophic transformants obtainedby transforming strain 1A589 (hemBI) with pLUX23 showedwild-type levels of PBG synthase activity (Table 2), and thisactivity was completely inhibited by 10 mM levulinic acid, a

competitive inhibitor for PBG synthase.hemB from B. subtilis encodes a polypeptide of 36.2 kDa,

as deduced from the nucleotide sequence, and was found toencode a polypeptide of 41 kDa in an E. coli in vitrotranscription-translation system (Fig. 5). Human and bovineliver PBG synthase are homooctamers, with subunits of 35kDa (54, 57). In Spinacia oleracea, the enzyme is a homo-hexamer of 50-kDa subunits (33).The deduced amino acid sequence of B. subtilis hemB is

very similar to the sequence of PBG synthase from human(57), rat (11), yeast (40) and E. coli (15, 31) (Fig. 6). Thehighest degree of identity (48%) was found between the E.coli and B. subtilis enzymes. In human PBG synthase, thelysine residue in the sequence MetValLysProGlyMet hasbeen identified to be in the active site (18). This Lys isproposed to bind covalently to the substrate, ALA. In B.subtilis, the corresponding sequence IleValLysProSerLeu is

found. The three residues, ValLysPro, are conserved in allPBG synthases analyzed so far (Fig. 6). Tsukamoto et al.(54) have shown the importance of one zinc atom, twocysteine residues, and two histidine residues for the activityof bovine liver PBG synthase and have suggested that thezinc binding region is at the active site. The tentative zincbinding region of the enzyme constitutes a highly conservedregion (Fig. 6), and Gibbs and Jordan (18) have proposedthat this zinc region plays a structural rather than a catalyt-ical role. Wetmur et al. (57) found that the zinc bindingregion of human PBG synthase shows similarities to theconsensus sequence of the zinc-chelating domain in zincfingers (10, 30) and suggested Cys-119, Cys-122, His-129,and Cys-132 as zinc ligands, but they also considered Cys-124 and His-131 possible ligands. The Cys corresponding toCys-119 in human PBG synthase is not conserved in E. coliand B. subtilis. In the bacterial enzymes, the correspondingamino acid residue is a Ser or an Ala (Fig. 6). Ala cannotfunction as a ligand to zinc. It thus seems probable that in B.subtilis PBG synthase Cys-120, Cys-122, His-127, and His-129 or Cys-130 chelate the zinc. These five residues are

conserved in all the PBG synthases shown in Fig. 6. How-ever, it has yet to be confirmed that B. subtilis PBG synthasealso contains zinc.hemL encodes GSA 2,1-aminotransferase. A comparison of

the predicted amino acid sequence of the hemL gene productwith the sequences predicted for GSA 2,1-aminotransferase

yeahumratEcoBsu

NPDDFTEIDSVPDDIQPITSVPDDVQPIASEIDDYKAVEAGLEGKKRAVPS

GPVIQGIKFISPAIEALSPTTEAVRLLGLVARMSRICGIVQKAITEI

KAGAHCVAPSKAGCQVVAPSXAGCQVVAPSAAGADFIAPSKAGADIIAPS** ***

4939393939

10998989696

169158158155155

229218218213214

289278278272273

HAKD..AAKW LAE.. 324

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2596 HANSSON ET AL.

1 2 345

46~~~~~~~~~~..kDa. .. ...4c

mi

FIG. 7. Expression ofB. subtilis GSA 2,1-aminotransferase froma plasmid carrying hemL in E. coli JM83, The hemL gene product isindicated and has an apparent mass of 46 kDa. Cell extracts wereprepared as described in Materials and Methods for enzyme activitymeasurements. Supernatants were obtained by centrifugation at48,000 x g for 30 min at 4C. Twenty-five micrograms of protein wasadded to each lane. The 10%o (wt/vol) gel is shown after electropho-resis stained for protein with Coomassie brilliant blue. The plasmidscarried in E. coli JM83 were as follows. Lane 1, pBluescript II

KS(-) (total lysate); lane 2, pLUX209 (hemL) (total lysate); lane 3,pBluescript II KS(-) (supernatant); lane 4, pLUX209 (supernatant);lane 5, protein standards: 3-galactosidase (130 kDa), bovine serumalbumin (68 kDa), catalase (57.5 kDa), fumarase (48 kDa) andcarbonic anhydrase (29 kDa).

from barley (20), Synechococcus species (21), and E. coli(the popC gene product [21]) shows 39o identity between allfour proteins (see Fig. 9). GSA 2,1-aminotransferase purifiedfrom barley and the cyanobacterium Synechococcus strainPCC 6301 consists of two identical 46-kDa subunits and of a

single 46-kDa subunit, respectively (22). The B. subtilishemL gene is predicted to code for a 46.1-kDa polypeptide.A polypeptide of this size was also found in an E. coli in vitrotranscription-translation system using B. subtilis hemL DNA(Fig. 5). Furthermore, a soluble 46-kDa polypeptide wasfound in large amounts in E. coli JM83 containing hemL ona high-copy-number plasmid (pLUX209) (Fig. 7).

Extracts of E. coli JM83/pLUX209 contained GSA 2,1-aminotransferase activity which was inhibited by gabaculin(3-amino-2,3-dihydroxybenzoic acid) (Fig. 8). Such ami-notransferase activity could not be detected in extracts of E.coli JM83 containing only the plasmid vector (Fig. 8). Thesensitivity to gabaculin has also been shown in B. subtilisextracts by O'Neill et al. (43), which suggests that theenzyme uses pyridoxamine 5'-phosphate as a cofactor. Fromsequence comparisons to other aminotransferases, the lysineresidue that binds the cofactor has tentatively been identifiedin the amino acid sequences for GSA 2,1-aminotransferasefrom barley, Synechococcus species, and E. coli (21). Acorresponding lysine is also found in the B. subtilis GSA2,1-aminotransferase at position 268. The sequence compar-

isons and the enzyme activity measurements demonstratethat the product of B. subtilis hemL is GSA 2,1-aminotrans-ferase.

E. coli mutants that require ALA for growth carry muta-tions in one of two loci, hemA (homologous to B. subtilishemA) or popC (6, 46, 58). The predicted amino acid

WAVELENGTH (nm)

FIG. 8. GSA 2,1-aminotransferase activity in extracts of E. coliJM83 carrying pBluescript II KS(-) or pLUX209 (hemL). Absorp-tion spectra of ALA-derived pyrrole after reaction with Ehrlich'sreagent are shown. Spectrum 1, JM83/pBluescript II KS(-); 2,JM83/pBluescript II KS(-) in the presence of gabaculine; 3, JM83/pLUX209; 4, JM83/pLUX209 in the presence of gabaculine. Fivehundred micrograms of protein was used in each assay, and gabac-uline was added to a final concentration of 10 ,uM. Spontaneousformation ofALA from GSA was analyzed by omitting cell extractsfrom the assay mixture. The resulting spectrum overlapped withspectrum 2.

sequence ofpopC shows 54% identity to that of B. subtilishemL (Fig. 9). Also, in S. typhimurium there are at least twoloci involved in ALA synthesis, hemA and hemL (16, 17a).The nucleotide sequence of S. typhimurium hemL was veryrecently reported (17) and is homologous to B. subtilis hemLand E. coli popC.The mutations causing ALA auxotrophy in B. subtilis that

have been analyzed map at hemA (44, 45). It was therefore ofinterest to analyze the effect of a hemL mutation on growthand heme synthesis. A 427-bp HindIII-EcoRV fragmentwhich covers an internal part of hemL was deleted from thechromosome and replaced by a cat gene (Fig. 10). Thedeletion was constructed and confirmed as described inMaterials and Methods. The deletion removes the sequencecorresponding to amino acid residues 87 to 128 of hemL andis. expected to inactivate GSA 2,1-aminotransferase. Thestrain with the deletion in hemL, 3G18AL17, was found togrow on minimal glucose medium without ALA, but ALAstimulated growth. Hence, it can be concluded that an intacthemL gene is not essential for ALA synthesis in B. subtilis.Possibly, GSA is spontaneously converted to other productsincluding ALA at neutral pH or there is more than oneprotein with GSA 2,1-aminotransferase activity in B. sub-tilis.A hemAXCDBL operon? Biosynthetic pathways in B. sub-

tilis are characterized by clustered and sometimes overlap-ping genes organized as operons (60, 61). The hemAXCDBLcluster consists of six closely spaced genes of which hemDoverlaps with hemC and hemB overlaps with hemD (Fig. 4and 10). This gene cluster is preceded by a putative promoterlocated approximately 70 bp upstream ofhemA (unpublishedS1 nuclease mRNA mapping data). Deletion ofthis promoterand the first 519 bp of hemA from the B. subtilis chromosomeand substitution of this fragment with the cat gene of plasmidpHV32 (strain 3G18A401) results in an absolute growth

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bar AVSIDEKAYT VQKSEEIFNA AKELMPGGVN SPVRAFKSVG GQPIVFDSVK GSHMWDVDGNsyn .. LVTSSPFK TIKSDEIFAA AQKLMPGGVS SPVRAFKSVG GQPIVFDRVK DAYAWDVDGNEco .......... MRKSENLYQA ARELIPGGVN SPVRAFTGVG GTPLFIEKAD GAYLYDVDGKBsu ....... MRS YEKSKTAFKE AQKIMPGGVN SPVRAFKSVD NDPIFMERGK GSKIFDIDGN

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

PAIIGHADDK VNAALIETLKPAICGHAHPE VIEALKVAMEPMVLGHNHPA IRNAVIEAAEPLILGHTNDR VVESLKKVAE* **

KGTSFGAPCA LENVLAQMVIKGTSFGAPCA LENVLAEMVIRGLSFGAPTE MEVKMAQLVTYGTSFGAPTE VENELAKLVI* ***** * * *

FVNSGTEACH GALRLVRAFT GREKILKFEG CYHGHADSFLFVNSGTEACM AVLRLMRAYT GRDKIIKFEG CYHGHADNFLMVNSGTEATM SAIRLARGFT GRDKIIKFEG CYHGHADCLLMVSSGTEATM SALRLARGYT GRNKILKFEG CYHGHGDSLL* ***** * ** * * ** ******* ****** *

VKAGSGVATLVKAGSGVATLVKAGSGALTLIKAGSGVATL**.*** **

SAVPSIEMVRDAVPSIEMVRELVPTMDMVRDRVPSVEIVR

** **

GLPDSPGVPKGLPDSPGVPKGQPNSPGVPAGLPDSPGVPE* * *****

GATVGTLTAP YNDADAVKKL FEDNKGEIAA VFLEPVVGNA GFIPPQPAFL NALREVTKQDSTTANTLTAP YNDLEAVKAL FAENPGEIAG VILEPIVGNS GFIVPDAGFL EGLREITLEHDFAKYTLTCT YNDLASVRAA FEQYPQEIAC IIVEPVAGNM NCVPPLPEFL PGLRALCDEFGIAKNTITVP YNDLESVKLA FQQFGEDIAG VIVEPVAGNM GVVPPQEGFL QGLRDITEQY

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

TPFRLAYGGATGFRIAYGGVTGFRVALAGATGFRVDYNCA* **

NPLAMTAGIHNPLAMTAGIK

NPIAMAAGFANPLAMTAGLE** ** **

GG.PVHNFDDEG.PVHNYEDDAESVTCYQDNE.PVINYET

*

DIEKTVEAAE KVLRWI...DIDATLAAAR TVMSAL...DINNTIDAAR RVFAKL...DIENTIQAAE KVFAEISRR** * ** *

QEYFGITPDV TTLGKIIGGG LPVGAYGGRK DIMEMVAPAGQEKFGVTPDL TTLGKIIGGG LPVGAYGGKR EIMQLVAPAGQDYYGVVPDL TCLGKIIGGG MPVGAFGGRR DVMDALAPTGQGYFGVTPDL TCLGKVIGGG LPVGAYGGKA EIMEQIAPSG* * ** * *** ** * **** ** * ** *

TLKRLMEPGTTLELLRQPGTCLNEVAQPGVTLKQL.TPES* *

AKKSDTAKFGAKKSDLQKFSVMACDVERFKAKSSDLKLFA

* *

YEYLDKVTGEYEYLDQITKRHETLDELTTRYKNFIKKGDR

LVRGILDVGA KTGHEMCGGHLSDGLLAIAQ ETGHAACGGQLAEGLLEAAE EAGIPLVVNHLEEGISKTAG AHGIPHTFNR* * *

RFHRGMLEG VYLAPSQFEARFHRGMLEQG IYLAPSQFEARFFHO(DEG VYLAPSAFEASYYKGMANEG VFLPPSQFEG

* * * ****

GFTSLAHTTQGFTSLAHTEEGFMSVAHSMELFLSTAHTDE* * **

120118110113

180178170173

240238230233

300298290293

360358350352

419417410411

435433426430

FIG. 9. Alignment of the predicted amino acid sequences of GSA 2,1-aminotransferase of barley (bar) (20), Synechococcus strain PCC6301 (syn) (21), E. coli (Eco) (21), and B. subtilis (Bsu). Positions with identical amino acid residues in all four polypeptides are marked byasterisks. The amino-terminal sequence of the barley protein is that of the processed polypeptide (22).

requirement for hemin. This result would not be expected ifonly hemA were affected by the deletion (44). Thus, theheme biosynthetic pathway in mutant 3G18A401 is blockedin one or more steps after synthesis ofALA. This conclusionis strengthened by the finding that 3G18A401 lacks PBGsynthase (Table 2) which is encoded by hemB. Pseudorever-

tants of 3G18A401 which grow on ALA can be isolated, andthese revertants show higher PBG synthase activities thanthat of the wild type (Table 2). These pseudorevertants mostlikely carry a mutationally activated promoter(s) allowingtranscription of genes downstream of hemA (44). This pro-moter(s) is possibly located within the inserted pHV32

lO00bpI

00 p

pLUX3202pLUX3204pLUX3203

pAhemLI7

E S

H H

H C

H EV C

I r- cat I

FIG. 10. B. subtilis hemAXCDBL gene cluster. DNA fragments present in different plasmids used for gene disruption or deletion-substitution are presented in the lower part of the figure. cat is the chloramphenicol resistance gene of pHV32. Arrows indicate direction oftranscription. Restriction site abbreviations: C, ClaI; E, EcoRI; EV, EcoRV; H, Hindlll; S, SphI.

VOL. 173, 1991 B. SUBTILIS hemAXCDBL GENE CLUSTER 2597

EYIDYVGSWGRYIDYVGTWGAYIDYVGSWGEYIDYVLSWG***** **

60585053

GALLVFDEVMDALLVFDEVMGALLIIDEVMGSLLIFDEVM

** ****

PMYQAGTLSGPVYQAGTLSGPVYQAGTLSGPIYQAGTLSG* ********

IRGMFGFFFAVSGMFGFFFTVGGMFGIFFTAGSMIGFFFT

* * **

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DNA. The higher PBG synthase activity found in the pseu-dorevertants compared with that of wild-type B. subtilissupports the conclusion that this promoter(s) is differentfrom that of hemA. These and previous (44) experimentaldata suggest that hemA, hemX, hemC, hemD, hemB, andprobably hemL are all transcribed from a promoter upstreamof hemA.The hemL gene is followed by an inverted repeat (Fig. 4)

which may function in transcriptional termination of a he-mAXCDBL operon. To test whether additional hem genesare located downstream of hemL and which belong to thesame transcriptional unit as hemL, the following gene dis-ruption experiments were done. A DNA fragment from thehemCD region, an internal region of hemL, and the end ofhemL were each inserted into plasmid pHV32 (Fig. 10). Thisplasmid contains a cat gene but cannot replicate autono-mously in B. subtilis. Transformation of B. subtilis 3G18with pHV32 containing the above B. subtilis chromosomalDNA fragments and selecting for chloramphenicol resis-tance will result in transformants in which pHV32 hasintegrated into the chromosome via homologous recombina-tion at the inserted fragments. The integrated plasmid willcause disruption of transcription at the site of insertion. Ifthe fragment inserted into pHV32 were internal to an openreading frame, it would also disrupt translation and lead to adefective gene product. As expected, integration of pHV32using the hemCD fragment resulted in chloramphenicol-resistant transformants which require hemin for growth.However, the transformants with pHV32 integrated into ordownstream of hemL grew without hemin or ALA. The factthat pHV32 was integrated into the chromosome at theexpected site in the hemL transformants was confirmed bythe close linkage observed between hemC33 and the cat gene(more than 90% in transformation crosses).These results confirm that hemL is not essential for growth

of B. subtilis. They also indicate that there is no hem geneessential for growth downstream of hemL that is part of theproposed hemAXCDBL operon.

Conclusion. The proposed hemA operon is expected togive rise to a transcript of about 6,400 nucleotides. Theamount ofhemA mRNA present in exponentially growing B.subtilis cells is less than 1/10 of that of the odhAB transcriptencoding the El and E2 subunits of the 2-oxoglutaratedehydrogenase complex (unpublished experiments). Prelim-inary Northern blots using a hemA-specific probe have failedto demonstrate the presence of a hemA operon transcript ofthe expected size. This failure may reflect the low amount ofhemA mRNA in the cells.

In E. coli, hemC and hemD are closely linked and mayform an operon (1, 6, 26, 48), whereas hemA, hemB, andpopC are located at different positions on the chromosome(6). Our present demonstration of a close linkage in B.subtilis between the genes encoding enzymes required forsynthesis of UroIII from glutamyl-tRNA lends further sup-port to the notion that genes encoding enzymes of a commonbiosynthetic pathway generally have a more compact orga-nization in B. subtilis than in E. coli and other entericbacteria (60).

ACKNOWLEDGMENTS

We are grateful to B. Grimm and S. Gough for valuable discus-sions and for providing sequence information prior to publication, toC. G. Kannangara for the kind gift of GSA, to M. Petricek for initialhelp with the cloning, and to A. Sasarman for providing strainSASZ31.

This work was supported by the Swedish Medical ResearchCouncil, the Swedish Natural Science Research Council, and Emiloch Wera Cornells Stiftelse.

REFERENCES1. Alefounder, P. R., C. Abell, and A. R. Battersby. 1988. The

sequence of hemC, hemD and two additional E. coli genes.Nucleic Acids Res. 16:9871.

2. Alwan, A. F., B. I. A. Mgbeje, and P. M. Jordan. 1989.Purification and properties of uroporphyrinogen III synthase(co-synthase) from an overproducing recombinant strain ofEscherichia coli K-12. Biochem. J. 264:397-402.

3. Andersson, T. J., and G. Ivanovics. 1967. Isolation and somecharacteristics of hemin dependent mutants of Bacillus subtilis.J. Gen. Microbiol. 49:31-40.

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