OF BACTERIOLOGY, Apr. 1991, p. 2590-2599 0021-9193/91/082590-10$02.00/0 Copyright © 1991, American Society for Microbiology

JOURNAL

Vol. 173, No. 8

The Bacillus subtilis hemAXCDBL Gene Cluster, Which Encodes Enzymes of the Biosynthetic Pathway from Glutamate to Uroporphyrinogen III MATS 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 hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the Cs pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstream of hemC, namely, hemD encoding uroporphyrinogen IH synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-l-semialdehyde 2,1aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen HI from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-l-semialdehyde can occur 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 no further genes in heme synthesis are part of the proposed hem operon.

Heme, chlorophyll, and corrinoids such as vitamin B12 are metal-containing tetrapyrrole derivatives constituting prosthetic groups in respiratory chain complexes, light-harvesting complexes, catalases, and peroxidases and are thus essential components of most organisms. Tetrapyrroles are synthesized from 5-aminolevulinic acid (ALA), which in turn is formed via either of two major pathways. In the C4 pathway, ALA is formed by the condensation of glycine and succinyl coenzyme A, a reaction catalyzed by ALA synthase (EC 2.3.1.37). This pathway is found in animals (37), yeast cells (55), and some bacteria, e.g., rhizobia (38, 52). In the C5 pathway, ALA is formed from the carbon skeleton of glutamate (27). The C5 pathway involves at least three enzymatic reactions (Fig. 1). In the first step, a tRNAGJU is charged with glutamate, which is subsequently reduced by a NAD(P)H-dependent glutamyltRNA reductase to form glutamate-1-semialdehyde (GSA). ALA is then formed by isomerization of GSA catalyzed by GSA 2,1-aminotransferase (EC 5.4.3.8). The C5 pathway is present in higher plants (7, 56), algae (49), and several bacteria, e.g., Escherichia coli (5, 43), Salmonella typhimurium (16), and Bacillus subtilis (43, 44). Little is known about the organization and regulation of genes encoding enzymes which are involved in the biosynthetic pathway from glutamate to the tetrapyrrole uroporphyrinogen III (UroIll) in bacteria. B. subtilis mutants with hemA mutations are ALA auxotrophs (3, 29), whereas mutations in hemB, hemC, or hemD affect later steps in UroIII synthesis and the corresponding mutants require heme for growth (8, 9). The hemABCD genes are clustered at 2450 on the B. subtilis chromosomal genetic map (45). We have recently cloned the hemA region of the B. subtilis chromosome (44). Sequence analysis revealed that hemA *

probably encodes the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for ALA synthesis. This conclusion was based on the high level of sequence similarity between the predicted E. coli and B. subtilis HemA proteins (44) and on the fact that E. coli HemA mutants are defective in the NAD(P)H:glutamyl-tRNA reductase of the C. pathway (5). Immediately downstream of B. subtilis hemA, two more genes were found which seem to be part of the same transcription unit as hemA, namely, hemX encoding a hydrophobic protein of unknown function and hemC encoding hydroxymethylbilane synthase (EC 4.3.1.8). The close linkage between hemC, hemB, and hemD in transformation crosses (8, 39) and the observation that deletion of a promoter in front of hemA results in heme auxotrophy (44) and loss of porphobilinogen (PBG) synthase (EC 4.2.1.24) activity (this work) suggested to us that hemB and hemD are also parts of a hem operon. The aim of this work was to determine the numbers and identities of hem genes located downstream of hemC. Three more hem genes were found, hemD encoding UroIllI synthase (EC 4.2.1.75), hemB encoding PBG synthase, and hemL encoding GSA 2,1-aminotransferase. A deletion-substitution analysis of hemL and regions immediately downstream of this gene demonstrated that no further genes essential for heme synthesis are part of this hem operon. Hence, the B. subtilis hemAXCDBL gene cluster represents an operon encoding enzymes required for the synthesis of UrollI from glutamyl-tRNA. MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids 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) or LB (36) was used for liquid cultures. E. coli strains were

Corresponding author. 2590

B. SUBTILIS hemAXCDBL GENE CLUSTER

VOL. 173, 1991

6

grown in LB or on LA plates (36). The following antibiotics and 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 and required amino acids were used at 10 mg/liter. Media containing hemin (2.5 mg/liter) also contained cysteine (25 mg/liter) and bovine serum albumin (fraction V; Sigma Chemical Co.) (500 mg/liter). Stock solutions of hemin (Sigma Chemical Co.) were prepared as described previ-

hmnmf lert I IL

HMB

hemC

Urolll

\

COOH

CHNH2

cI-CF2

PBG

CH2 COOH hemB

Glu C0O-tRNA CHNH2

ICFt CHC

1I

I*

/4

co

X IF CHN

hemL

h

iLt

hemA

COOH

tRNA-Glu

COC

2

I

OIQ

2591

ALA

*H

GSA

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

ously (44). Competent cells. Competent E. coli cells were prepared as described by Mandel and Higa (35). B. subtilis was grown to competence as described by Arwert and Venema (4). General DNA techniques and enzymes. Large-scale preparations of plasmid DNA from E. coli were done by the method of Ish-Horowicz and Burke (25). Small-scale preparations of plasmid DNA from E. coli XL1-Blue and E. coli MM294 were done by the boiling method (36) and the method of Kieser (28), respectively. Southern blot analysis of chromosomal DNA was done with a nonradioactive DNA labeling and detection kit from Boehringer GmbH (Mannheim, Germany). General DNA techniques were as described by Maniatis et al. (36). Restriction endonucleases and T4 DNA ligase were from Boehringer or New England BioLabs (Beverly, Mass.). Exonuclease III and mung bean nuclease were from Bethesda Research Laboratories (Gaithersburg, Md.), and RNase was from Worthington Biochemical Corp. (Freehold, N.J.). Deletion of DNA using exonuclease III. Exonuclease III treatment followed by mung bean nuclease treatment was used to create unidirectional nested deletions in cloned DNA fragments as described in a protocol from Stratagene (La

TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains B. subtilis B. subtilis B. subtilis B. subtilis B. subtilis

3G18 1A589 1A590 1A591 3G18A401

B. subtilis 3G18A401R B. subtilis 3G18AL17 E. coli XL1-Blue

E. coli JM83 E. coli MM294 E. coli SASZ31

Plasmids pBluescript II KS(-) pHV32 pUC18 pLUP212 pLUX3201 pLUX1 pLUX2 pLUX207 pLUX209 pLUX3202 pLUX3203 pLUX3204

pAhemL17

Relevant properties

trpC2 met ade trpC2 hemBI trpC2 hemC33 trpC2 hemDJ I trpC2 met ade AhemA401 cat; lacks the promoter and the first 519 bp of hemA, requires hemin ALA requiring pseudorevertant of B. subtilis 3G18zA401 trpC2 met ade AhemL17 cat recAl endAl gyrA96 thi hsdRJ7 supE44 relAl (lac) [F' proAB lacq lacZAM15

TnJO(Tetr)] ara rpsL A(lac-proAB) F80 lacZAM15 endAl thi pro hsdRJ7 supE44 hemD31 derivative of SAS245 bla lacZ' bla cat tet bla lacZ' bla cat; pHV32 derivative with the hemA region (Fig. 2) bla; Clal deletion derivative of pLUP212 (Fig. 2) bla; EcoRI fragment of pLUX3201 cloned in pBluescript II KS(-) bla; EcoRI fragment of pLUX3201 cloned in pBluescript II KS(-) (fragment in orientation opposite that of pLUX1 bla hemD'BL; exonuclease III derivative of pLUX2 (contains bp 3902 to 6634) bla hemL; exonuclease III derivative of pLUX2 (contains bp 5052 to 6634) bla cat; pHV32 derivative (Fig. 10) bla cat tet; pHV32 derivative (Fig. 10) bla cat tet; pHV32 derivative (Fig. 10) bla cat; cat of pHV32 cloned in HindlIl and EcoRV sites of pLUX207 (Fig. 10)

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

Reference or source

G. Venema BGSCa BGSC BGSC 44 44 This work

Stratagene 59 L.-O. Heden 12

Stratagene 42 59 This This This This

work work work work

This This This This This This

work work work work work work

J. BACTERIOL.

HANSSON ET AL.

2592

Clal

CalI FIG. 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 out at 31°C, and one aliquot was removed every minute. After mung bean nuclease treatment, 1 ,ug of DNA from each aliquot was run on a 0.8% agarose gel. Distinct DNA bands were excised from the gel, purified with Geneclean (BiolOl, La Jolla, Calif.), incubated with T4 DNA ligase, and transformed into E. coli XL1-Blue with selection for ampicillin resistance. Construction of B. subtUis insertion and deletion-substitution mutants. Insertion mutants were constructed by transforming B. subtilis 3G18 to chloramphenicol resistance with pHV32 derivatives carrying different DNA fragments from the hemA region (42) (see Fig. 10). A hemL deletion-substitution mutant was constructed in the following way. The cat gene of pHV32 was isolated on a HindIII-EcoRV fragment and used to substitute the hemL 427-bp internal HindIII-EcoRV fragment in pLUX209 (Table 1) to give plasmid pAhemL15. A 2,230-bp HindIII-EcoRI - h hmC

pux1 pLUX

E

S

S

hwt EV

EV

H

H EV

CE

1000 bp

pLUX22 pLUX22

pLUX24

FIG. 3. Restriction map of the cloned EcoRI fragment present in pLUXi and pLUX2. The EcoRI fragment in pLUX2 is oriented so that the hem genes can be transcribed from the lac promoter of the vector 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 contain hem DNA starting at the positions indicated: pLUX21, bp 3398; pLUX22, bp 3673; pLUX23, bp 4150; and pLUX24, bp 4312. The nucleotide sequence strategy used is indicated by arrows in the lower part of the figure. The sequences of hemC and the first part of hemD have been reported previously (44). Restriction site abbreviations: C, ClaI; E, EcoRI; EV, EcoRV; H, Hindlll; S, SphI.

fragment (including the cat gene and 780 bp of the end of hemL) from pAhemL15 was cloned in HindIII-EcoRI-digested pLUX207 (Table 1) to give plasmid pAhemL17 (see Fig. 10). B. subtilis 3G18 was then transformed to chloramphenicol resistance with pAhemL17 linearized with ScaI. In the 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 confirmed by Southern blots (data not shown). DNA sequence analysis. Nucleotide sequences were determined by the dideoxy chain termination method (47), using modified 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 from E. coli XL1-Blue by the boiling method. A synthetic oligonucleotide complementary to bp 825 to 841 of pBluescript II KS(-) was used as a primer. The GCG Sequence Software Package (13) was used for analysis of nucleotide sequence data. Amino acid sequence alignments were done by using the GAP program (13). Enzyme activity measurements. To determine PBG synthase activity in B. subtilis, the cells were grown aerobically at 37°C in 100 ml of LB-hemin medium. The cells were harvested when entering stationary growth phase and washed once in 50 mM Na-phosphate buffer, pH 6.4. The pellets were resuspended in 1 ml of Na-phosphate buffer containing 5 mM dithiothreitol, 0.25 mg of lysozyme, and 0.1 mg of DNase I (Sigma Chemical Co.), incubated for 15 min at 37°C, and then sonicated while chilled on ice. The lysate was centrifuged at 5,000 x g for 10 min at 4°C, and PBG synthase activity in the supernatant was measured by the procedure of Li et al. (32), using 40 ,ul of cell extract per assay. To determine GSA 2,1-aminotransferase activity in E. coli JM83 carrying different plasmids, the cells were grown overnight at 37°C in 100 ml of LB containing 50 ,ug of ampicillin per ml. The cells were harvested, washed, suspended in 1 ml of 0.1 M Tricine-NaOH buffer (pH 9.0) containing 0.3 M glycerol, 25 mM MgCl2, and 1 mM dithiothreitol and sonicated while chilled on ice. The resulting lysate was centrifuged at 5,000 x g for 10 min at 4°C to remove cell debris. GSA 2,1-aminotransferase in the super-

VOL. 173, 1991

B. SUBTILIS hemAXCDBL GENE CLUSTER

3082 205

EcoRI GAATTCCTTGAGCCTGAGCGCTGTTTGCC 'TGCTGTGGGGCAGGGAGCCCTGGCGATTGAG E F L E P E R C L P A V G Q G A L A I E The terminal part of heC TGCCGAGAATCGGATGAAGAGCTGTTGGC 'GTTGTTTTCTCAGTTTACAGATGAATATAC C R E S D E E L L A L F S Q F T D E Y T

3142 225

3022 185

3081 204

4882 232

2593

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

4941 251

EcoRV

3141 224

4942 252

....... -1 ----------------- ---------TCTTATATGGATATCATGCGTGACGTAAAAAATGAGTTTACTTTGCCGCTCGTCGCTTAT S Y N D I N R D V K N E F T L P L V A Y

5001 271

AAACGGACTGTCTTAGCGGAACGTGCTTTI nTTTAAACGCGATGGAGGGCGGCTGCCAGGTrT K R T V L A E R A F L N A N E G G C Q V

3201 244

5002 272

AATGTAAGCGG ;AGAGTATTCAATGGTGAAGGCTGCAGCCGCAGAACGGCTGGATCAAAGAA N V S G E Y S N V K A A A Q N G W I K E

5061 291

3202 245

CCGATCGCGGGCTACTCCGTGTTAAATGGACAGGATGAAAT1GAAATGACAGGTCTTGTC

3261 264

5062 292

AAAGAAATTGIrGTTGGAAATTTTGACAAGCATGAAGCGGCGCGGGTGCCGACCTGATTATT K E I V L E I L T S N K R A G A p L I I

5121 311

3262 265

GCTTCACCTGAGAAlACCGTCACCGGAAACGATCCGGAGGAA

3321 284

5122 312

ACGTATCATGC:GAAAGACGCAGCGAAATGGCTTGCGGAAGTAATTTTATTCAGTTGACAC T Y H A K D A A K W L A E

5181 324

3381 304

5182 1

AOTO'AGGCAG;ATGAGAAGCTATGAAAAATCAAAAACG GCTGTrTAAAGAAGCGCAAAAAC N R S Y E K S K T A F K E A Q K L he"

5241 17

3441 314 13 3501 33

5242 18

TCATGCCGGGC:GGTGTGAACAGTCCCGTTCGCGCATT7TAAATCGGTAGACATGGACCCGA

I

5301 37

5302 38

TTTYTATGGAG;CGCGGAAAAGGCTCGAAAATCTTTGATTATTGACGGGAATGAATATATTG F MG E R G K G S K I F D I D G N E Y I D

5361 57

3561 53

5362 58

5421 77

3621 73

5422 78

ACTACGTCTTG;TCATGGGGGCCTTTAATTTTAGGGCATTACAAATGACCGCGTCGTAGAAA Y V L S W G P L I L G H T N D R V V E S HindIII GCCTCAAAAAAkGTGGCTGAATACGGGACAAGCTTTGG3 TGCTCCGACTGAAGTAGAAAATG L K K V A E Y G T S F G A P T E V E N E

3681 93

5482 98

AACTGGCTAAG;CTCGTCATTGATCGTGTGCCATCTGTAAGAAATTGTACGAATGGTAAGCT L A K L V I D R V P S V E I V R N V S S

5541 117

3741 113

5542 118

CCGGAACAGAG3GCTACAATGAGTGCCCTCCGTTTGGCAAAGGGGCTATACGGGCCGCAACA G T E A T N S A L R L A R G Y T G R N K

5601 137

3801 133

5602 138

AGATTTTAAAASTTTGAGGGCTGCTACCACGGACACGGCCGATTCTCTCTTGATTAAAGCTG I L K F E G C Y H G H G D S L L I K A G

5661 157

3861 153

5662 158

5721 177

3921 173

5722 178

GTTCAGOO= rGCCACTCTCGGTCTGCCTGACAGCCCGGGGGGTGCCTGAAGGCATTGCGA S G V A T L G L P D S P G V P E G I A K HindIII AAAACACCATCCACCGTTCCGTACAATGATTTAGAAAGITGTAAAGCTTGCTTTCCAGCAAT N T I T V P Y N D L E S V K L A F Q Q F

3981 193

5782 198

5841 217

4041 213

5842 218

TCGGTGAAGACCATTGCGGGAGTCATTGTAGAGCCAGTI1Y;CCGGAAATATGGGTGTTGTTC G E D I A G V I V E P V A G N 11 G V V P EcoRV CCGC-%CAGAJ GGmTTCCTTCAGGGTCTGCGTGATATC CACTGAGCAGTACGGCTCCCTGC P Q E G F L Q G L R D I T E Q Y G S L L

4101 233

5902 238

TGAAGTGATGACTGGCTTCCGGGTCGAI,TTATAACTGCGCTCAAGGCTACT E V M T G F R v D Y N C A Q G Y F

5961 257

4161 253

5962 258

TCGGCGTAACXGCCTGATCTGACTTGTTTAGGAAAAGTl'AATCGGGGCGGGACTTCCTGTCG G V T P D L T C L G K V I G G G L P V G

6021 277

4221 262 11 4281 31

6022 278

CGGAAAGGCAGAAATCATGGAGCAGATC'CGCTCCAAGCGGTCCGATCTATC G K A E I lI E Q I A P S G P I Y Q

6081 297

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

298

AAGCTGGTACCI ATTGTCAGGCAACCCGCTTGCGATGAC GGCTGGCTTAGAGACATTGAAAC A G T L S G N P L A M T A G L E T L K Q

6141 317

CCCT

4341

51

6142 318

AGCTGACACCCTTGAATCCTACAAGAATTTCATCAAAAAJ _G~ACAATGGAA0A0A L T P E S Y K N F I R K G D R L E E G I

6201 337

4401

71

6202 338

TTTCAAAAACccCGCCGGGGCTCATGGCATTCCCGATAC S 1C T A G A H G I P H T FGATAACACGAATCAT:C1TGAT

6261 357

4461 91

6262

4521 111

6322 378

TGAAGCTGTTTzCGCAAGAT

S

6381 397

4581 131

6382 398

CACAATTCGA AGGTCTTTTCCTCTCAACGGCCCATAC : TGAAGATATTGAAAACACAA Q F E G L F L S T A H T D E D I E N T I

6441 417

4641 151

6442

418

TCCAGGCAGCCTP0A0AAAGTATTT0CT0AATCA0CCO4 ;CAGATAAGAGTGAAAACCGGTAT Q AA KVFAGE ISR R *

6501 430

4701

6502

CAAGGACTCCTTGTGCCGGTICGTGCTCTCCCACTCATATTTTCTCCAGTTCATAC

6561

3322 285

3382 305 1 3442 14

A

I

P

S

A

G

P

Y

D

S

K

G

L

V

N

I

I

Q

G

K

F

D

E

E

T

I T

V

N

E G

T

N

L

G

E

P

D

V E

GTAGGAAAGCGCTGTGCCGCTCTTATGGCTGACAAAGGAGCAAAAGATTTAATTGATCGT V

K

G

R

C

A

A

N

L

K

D

A

A

G

K

D

L

I

D

R

GTAMCGGGG1CSTTGACGAGGATGGAAAATGATTTTCCGTTGAAAGGAAAAACAGTGCT K

G K * M E N D F P L K G K T V L TGTCACCCGGAATAAGGCACAGGCAGCATCATTTCAGCAAAAAGTGGAGGCGCTTGGCGG V T R N K A Q A A S F Q Q K V E A L G G

V

R

E

L

D

E

D

heD

3502 34

TAAAGCGGTTTTAACCTCTTTGATTACGTTTCGCCGCGCTTTGCCGAATGATGTTGCGGA

3562 54

ACAGGTAACCGCGCCAGGCTGGCTTGTTTTTACAAGTGTGAACGGGGC

3622 74

AGACTTCTTITITTCTTATCTGAAGGAAATCAGCTTATTCTCCCTGCGCATAAAAAAT

3682 94 3742

114

K Q D

A

V

V

R

F

F

L E

F

T D S

L

S L

I

A

Y

A

K

L

T P

E

F G

N

R W

Q

R L

L

A V

I

L

P

F

T

L

P

N S

A

D V

H

V N

K

A G

K

E A I

ACCCGCGCGCCGTTTAAAAATGCATAACGTATCGGTTGATGT A

A

V

G

E

K

T

A

R

R

L

K

GATGCCACAGGAGTATATTCTGAAAGTG N

Q

P

E

Y

I

A

E

Q

L

A

N

N

H

V

AGCCA D

A

L

K

S

V

D

V

SphI CATGCTGAACC Q

H

A

E

P

3802 134

GGGGGAGACCATTACCGTGATGAAAGGGAATTTGTCACGTGATGTGATAAAACAAGAGCT

3862 154

TGTCCCGCTCGGTITTGAAGTAAAGGAATGGGTYCTCTAC0AAACGAT1CCGGATGAAGA

G

E

T

I

T

N

V

K

G

L

N

S

R

V

P

L

G

F

E

V

K

E

W

V

L

Y

G

I

E

A

L

K

D

A

A

G

Q

Y

S

3922 174

D

V

E

T

I I

K

Q

P

D

E E

L E

CTTTGACTATGTAACATTTAC F

D

Y

T

V

F

T

3982 194

GAGTTCATCAACCGTACATACGTTTATGCATGTCTTGGGAGAAGAATTAAAAAGTGGAA

4042

GGCGAATGGGACGGCCTGTATCAGCATTGGGCCTTTAACAAATGATGCCCTTCTGACGTA A N G T A C I S I G P L T N D A L L T Y SphI CGGCATCACATCGCATACGCCTGATACATTTACAATAGATGGCATGCTTGAGTTAATGTG

214 4102 234 4162 254 1 4222 12

S

S

S

I

G

T

T

V

S

H

T

H

T

F

P

N

H

T

D

L

V

F

G

D

I

T

E

E

L

N

G

K

K

E

L

W

K

N

L

C

CAGCAT0TCAGA0AAGAATATGATTAAATAGACACCGCCGCCTG S

N

CGGA R T

S

R

T S

K

E

E

E

I

R

heB

* N S

Q

S

F N

4282 32

I

4342 52

A GATGTTCACCATGTAT TA D V H H V S L D

4402 72

GGCATTCAATCTGTTATCGTGTT= G I Q S V I V F G I

4462 92

GCGTACCATGATCACGGAATTGTCCAAAAAGCCATCACAGAAATTAAGAACACTTCCCT

4522 112

GAAATGGTTGTTGTCGCTGACACGTGCCTGTGCGAATATACAGACCACGGCCATTGCGGA

4582 132

CTTGTCAAAGACGGAGTC

4642 152

V

A

E

L

Y

Y

N

V

P

H V

K

I

D

V

D

F

H V

G

V

G

A

V

E

V

I

V

D

I

T

G

L

E

_T-G L

Q C

L

K L

K

G

K

K

A

GTAA D

E

V

V

P

S

N

P

GCTGGTCAAACTG

A

E

L

V

K

L

TGArG AACACAA CGGCCCATA P A C

E I E

E T Y

K E T

D I D

D K H

C E G

G

H H

T F C

Q P G

TTCTCAATOATGAATGGCTGGAGCTTTTGGCGCAGACAGCT L

N D E EcoRV

S

D

A

L

E

L

L

A

Q

T

A

GTCAGCCAAGCGAAAGCAGGTGCGGATATCATTGCGCCATCAAACATGATGGACGGATTT S

Q

A

K

A

G

A

I

I

P

S

N

N

N

D

G

F

4702 172

GTTACAGTGATCAAGAGAAGCACTTGATAAAGAAGGATTCGTCAATATTCCCATCATGTCT

4762 192

TACGCTGTTAAATATTCAAGTGAGTTTTACGGTCCGTTCCGTGATGCAGCAAACAGCACA

4822

CCGCAATTCGGAGACCGCAAAACATATCAGATGGACCCTGCCAACCGTATGGAGGCACTC

212

V

Y

P

T

A

Q

V V

F

I

K

G

R Y D

E S R

A S

K

L E T

D

F Y

K Y Q

E G

N

G

P D

F F P

V R A

N D N

I A

R

P A

N

I N E

N S A

S T L

6082

358

171

N

I

A

P

Ft

Y

G

D

G

G

V

N

R

F L

F F

F A

T S

N Y

V

R

A

F

K

S

V

D M

D

P

CAAAATCATCGAT

E Y

P

V

K

G

I

N

Y

E T A K

S

S

D

L

GAA ['GAAGGGGTATTCCTTCCGCCAT 11

A

N

E

G

V

F

L

P

P

5481 97

5781 197

5901 237

6321 377

6621

ClaI 6622

P

CTTTACAAACGAACCAGTCATCAATPTA G

6562

4761 191

S

6634

4821 211 4881 231

FIG. 4. Nucleotide sequence of hemC (the terminal half), hemD, hemB, and hemL and the derived amino acid sequences of the corresponding 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 (19) as described by Houghton et al. (24), except that the

cofactors pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate were not added. Other methods. In vitro transcription-translation was done with E. coli S30 extracts, using a kit from Amersham and L-[35S]methionine (Amersham). Polypeptides were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-

phoresis (PAGE) (41), followed by autoradiography of the gel when required. Protein was determined by the method of Lowry et al. (34), using bovine serum albumin as the standard. Nucleotide sequence accession number. The complete nucleotide sequence of hemAXCDBL has been submitted to the EMBL, GenBank, and DDBJ data bases under accession number M57676.

2594

HANSSON ET AL.

J. BACTERIOL.

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

Strain

3G18 (wild type) .................................. 5.5 ± 1.3 (n = 6) 1A589 (hemBl) .................... .............. 0.2 1A589 Hem' (transformant)b ......................... 4.0 3G18A401 (promoter and part of hemA .................... 0.1 deleted) .............. 3G18A401R (ALA-requiring pseudorevertant 13.1 of 3G18A401) ................................. 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 is

shown for B. subtilis 3G18. b Obtained by transformation with pLUX23.

RESULTS AND DISCUSSION Cloning of the hemC downstream region. The B. subtilis hemA region was previously cloned by Petricek et al. (44) using plasmid pHV32. Plasmid pLUP212 is a pHV32 derivative which carries B. subtilis hemA, hemX, and hemC on a ca. 20-kb DNA fragment. pLUP212 DNA was cleaved with ClaI to give pLUX3201 (Fig. 2). The EcoRI fragment of pLUX3201 containing the terminal part of hemC, 3.3 kb of B. subtilis DNA downstream of hemC, and 24 bp of pHV32 DNA (Fig. 2) was cloned in the EcoRI site of pBluescript II KS(-). Two ampicillin-resistant E. coli XL1-Blue transformants containing plasmids with the EcoRI fragment inserted in the two possible orientations were selected for further analysis. These plasmids were designated pLUX1 and pLUX2 (Fig. 3). Exonuclease III treatment of both plasmids was used to generate unidirectional nested deletions in the EcoRI fragment. KpnI and XhoI sites in pBluescript II KS(-) were used to create 3' and 5' overhangs, respectively, before the DNA was treated with exonuclease III. The resulting deletion plasmids were used for DNA sequence analysis, for transformation of B. subtilis Hem mutants, and for analysis of gene products. Nucleotide sequence analysis. Recently we have reported the nucleotide sequences of hemA, hemX (a gene with unknown function previously designated ORF2), hemC, and 1 2

3

46 kDa--41 kDa--_

32 kDa

-

FIG. 5. In vitro transcription-translation analysis of the gene products of hemB and hemL. The resulting [35S]methionine-labeled polypeptides were separated in a SDS-10 to 15% polyacrylamide gradient gel and visualized by autoradiography. Lanes: 1, pLUX23 containing hemB and hemL; 2, pLUX209 containing hemL; 3, pBluescript II KS(-). The 32-kDa polypeptide is the bla gene product.

the start of what appeared to be hemD (44). To determine the complete sequence of hemD and additional downstream genes, 3,613 bp of B. subtilis DNA present in pLUX1 and pLUX2 were sequenced (Fig. 3 and 4). Three complete open reading frames were found and designated hemD, hemB, and hemL. hemD starts with an ATG codon at position 3404 and ends with a TGA stop codon at position 4190. The hemD gene overlaps the end of hemC by 11 nucleotides. A possible ribosome binding site, AaAcgGGAGcT, is located 9 nucleotides upstream of the translation start codon of hemD. The stop codon of hemD and the ATG start codon of hemB at position 4189 overlap. A possible ribosome binding site, AGAgAGGAaGaGA, is found 5 bp upstream of the start of hemB. hemB ends with a TAA stop codon at position 5161. The hemL ATG start codon is at position 5193, and the TAA stop codon is at 6483. A possible ribosome binding site, GAcAGGAGtTGA, is present 6 bp upstream of the start codon of hemL. This extends the previous hemAXC sequence to a hemAXCDBL sequence consisting of 6,634 bp. Identification of hemD. On the basis of the following findings, the hemD gene of B. subtilis was identified as

encoding UrollI synthase. (i) E. coli K-12 SASZ31 (hemD31) is deficient in UroIll synthase (12). This strain was transformed with plasmids containing B. subtilis DNA with different parts of the hem gene cluster, pLUX3201 (hemAXCDBL), pLUX2 (hemC'DBL), pLUX23 (hemBL), and pLUX209 (hemL). Selection was for ampicillin resistance and heme prototrophy. Only pLUX3201 and pLUX2 complemented the E. coli hemD mutation, i.e., only transformants obtained with these plasmids grew well on LA-ampicillin plates without hemin. (ii) B. subtilis HemD mutants have been isolated as hemin auxotrophs defective in UrollI synthase (39). The mutation in B. subtilis 1A591 (hemDJIJ) was mapped by transforming this strain to hemin prototrophy with various plasmids containing different parts of the cloned DNA. This mapped the mutation to the proximal part of hemD or possibly to the distal part of hemC, i.e., plasmid pLUX21 rescued the hemDJJ mutation, whereas pLUX22 did not (Fig. 3). (iii) The B. subtilis hemD gene can encode a protein of 29 kDa as deduced from the nucleotide sequence, and this size is in the same range as that of Urolll synthases purified from E. coli (26 kDa) (2), Euglena gracilis (31 kDa) (23), and rat liver (28 kDa) (50). In these organisms, the enzyme is a monomer. Using an E. coli in vitro transcription-translation system and different plasmid constructs, a polypeptide encoded by hemD could not be detected. (iv) The deduced amino acid sequence of B. subtilis hemD showed 25 and 21% identity to the sequence of UrolIl synthase from E. coli (1, 26, 48) and from humans (53), respectively (data not shown). The sequence identity between E. coli and human Urolll synthase is 20%. These similarities are of borderline significance (14), which is notable since the derived amino acid sequences of other hem genes from different organisms generally show a high degree of similarity (see for example Fig. 6 and 9). Chemical modification studies of UrolIl synthase from E. gracilis suggest that arginine residues are essential for the activity of the enzyme (23). The arginine at position 146 in the B. subtilis Urolll synthase polypeptide appears to be conserved in the E. coli and human enzymes. Identification of hemB. The hemB gene was concluded to encode PBG synthase on the basis of the complementation of HemB mutants, the size of the gene product, and amino acid sequence similarities. B. subtilis 1A589 (hemBI) is deficient in PBG synthase

B. SUBTILIS hemAXCDBL GENE CLUSTER

VOL. 173, 1991 yea hum rat Eco Bsu

N MHTAEFLETE PT.EISSVLA GGYN. HPL.L RQWQSERQLT X. .ATTTLNAS .....*... M..QPQSVLH SGY.FHPL.L RAWQT. H.. QSVLH SGY. FHPL.L RAWQTTPSTV SAT . ........... ....... MT DLIQRPR.RL RKSPALPEF ..EETTLSLSI .......... MS QSFNRHR.RL RTSKANEEMV K. .ETRLHPS ..

........

...

........

MaIFPLFISD NLIYPIFVTD NLIYPIFVTD DLVLPIFVEE DFIYPIFVVE

* *

NPDDFTEIDS VPDDIQPITS VPDDVQPIAS EIDDYKAVEA

LPNINRIGVN LPGVARYGVK LPGVARYGVN MPGVMRIPEK GLEGKKRAVPS MPDVHRVSLD

REYFPELYII RKTFPNLLVA RRTFPTLLVA XQTVPEMIVM KEHFPEkvVV *

KAGAHCVAPS KAGCQVVAPS XAGCQVVAPS AAGADFIAPS KAGADIIAPS **

AKGLRSVILF ECEGLRCVLIF EAGLRCVLIF KAGIRSVNTF KLGIQSVIVF *

**

GVPLIPGTKD GVP. SRVPKD GVP. SRVPKD GISHH. .T. D GIPE. .E.KD

*

**

CDVCLCEYTS HGHCVLYDD HGHC*LLSEN CDI*LCPYTS CDVWLCPYTS HGHa LLSEN SDTCFCEYTS HGHCVLCEH

GTINRMSVS GAFRAEESRQ GAFLAE$5RQ G.VDNDATLE

PVGTAADDPA ERGSAADSEE EQGSAADSED

ETGSDA&RED DCGTQAYHDH *

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

*

RLAAVAVNYA RLAEVALAYA RLAEVALAYA NLGKQAVVAA

*

*

KFSGNLYGPF KFASCFYGPF KFASCFYGPF KFASSFYGPF KYSSEFYGPF *

**

*

109 98 98 96 96

*

ADTLCEYTD HGHC.LVKD. GVILNDESLE LLAQTAVSQA

DMIDGRIRDI KRGLINANIA HKTFVISYAA 1MNDGRVEAI REALMAHGLG NRVSVNSYSA DINDGRVEAI XAALLRHGLG NRVSVMSYSA AAMDGQVQAI RQALDAAGFK D.TAIKSYST NNODGFVTVI REALDKEGFV N.IPINSYAV

***

49 39 39 39 39

* *

*

*

*

GPVIQGIKFI SPAIEAL SPTTEAVRLL GLVARMSRIC GIVQKAITEI

RLKDYLRPLV RLEINRPLV QLEELRPLV IERIA }} LLKDEVAELV

2595

*

169 158 158 155 155

*

RDAACSAPSN RDAAKSSPAF RDAAQSSPAF REAAGSALK. RDAANSTPQF

229 218 218 213 214

**** * ** *

____-_

GDRKCYQLPP GDRRCYQLPP GDRRCYQLPP GDRKSYQNNP GDRRTYQMDP ***

**

AGRGLARRAL GARGLALRAV GARGLALRAV MNPREAIRES ^ANREALREA

*

* *

SDEYANLHAA SGEFAKLWHG SGEFAMLWHG SGEYAMIKFA

AEKGVVDLKT AQAGAFDLKA AKAGAFDLRT ALAGAIDEEK

* *

*

ERDMSEGADG IEVKRPSiPYL DIMRDASEIC KDLPICAYHV DRDVREGADN I)VKPGNPYL DIVREVKDKH PDLPLAVYHV ARDIQEGADI DNVQEVXDRH PELPLAVYQV LLDEAQGADC DIVRELRER. TELPIGAYQV QSDVEEGADF ILVPSIYN DIMRDVKNEF T. LPLVAYNV *

***

I ***

I *

*

**

IAFESHQGFL RAGARIIITY LAPEFL. . DW LDEEN AVLEAMTAFR RAGADIIITY YTPQLL..QW LREE. AVLESMTAFR RAGADIIITY FAPQLL..KW LKEE. VVLESLGSIK RAGADLIFSY FALDLAEKKI LR... SGEYSMVKAA AQNGWIKEKE IVLEILTSMK RAGADLIITY HAKD..AAKW LAE.. *

*

*

****

*

289 278 278 272 273

* *

342 330 330 324 324

*

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 indicates the tentative zinc binding region (57), and the lower box indicates the sequence containing the lysine residue in the active site (18). The sequences 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 containing different parts of cloned DNA and selecting for hemin prototrophy, the hemBI mutation was mapped to the proximal part of hemB or possibly to the distal part of hemD, i.e., plasmid pLUX23 transformed the mutant, whereas pLUX24 did not (Fig. 3). Hemin prototrophic transformants obtained by transforming strain 1A589 (hemBI) with pLUX23 showed wild-type levels of PBG synthase activity (Table 2), and this activity 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 to encode a polypeptide of 41 kDa in an E. coli in vitro transcription-translation system (Fig. 5). Human and bovine liver PBG synthase are homooctamers, with subunits of 35 kDa (54, 57). In Spinacia oleracea, the enzyme is a homohexamer 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). The highest degree of identity (48%) was found between the E. coli and B. subtilis enzymes. In human PBG synthase, the lysine residue in the sequence MetValLysProGlyMet has been identified to be in the active site (18). This Lys is proposed to bind covalently to the substrate, ALA. In B. subtilis, the corresponding sequence IleValLysProSerLeu is

found. The three residues, ValLysPro, are conserved in all PBG synthases analyzed so far (Fig. 6). Tsukamoto et al. (54) have shown the importance of one zinc atom, two cysteine residues, and two histidine residues for the activity of bovine liver PBG synthase and have suggested that the zinc binding region is at the active site. The tentative zinc binding region of the enzyme constitutes a highly conserved region (Fig. 6), and Gibbs and Jordan (18) have proposed that this zinc region plays a structural rather than a catalytical role. Wetmur et al. (57) found that the zinc binding region of human PBG synthase shows similarities to the consensus sequence of the zinc-chelating domain in zinc fingers (10, 30) and suggested Cys-119, Cys-122, His-129, and Cys-132 as zinc ligands, but they also considered Cys124 and His-131 possible ligands. The Cys corresponding to Cys-119 in human PBG synthase is not conserved in E. coli and B. subtilis. In the bacterial enzymes, the corresponding amino acid residue is a Ser or an Ala (Fig. 6). Ala cannot function as a ligand to zinc. It thus seems probable that in B. subtilis PBG synthase Cys-120, Cys-122, His-127, and His129 or Cys-130 chelate the zinc. These five residues are conserved in all the PBG synthases shown in Fig. 6. However, it has yet to be confirmed that B. subtilis PBG synthase also contains zinc. hemL encodes GSA 2,1-aminotransferase. A comparison of the predicted amino acid sequence of the hemL gene product with the sequences predicted for GSA 2,1-aminotransferase

2596

HANSSON ET AL.

J. BACTERIOL.

1 2 345

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

mi c

WAVELENGTH (nm)

FIG. 7. Expression of B. subtilis GSA 2,1-aminotransferase from carrying hemL in E. coli JM83, The hemL gene product is indicated and has an apparent mass of 46 kDa. Cell extracts were prepared as described in Materials and Methods for enzyme activity measurements. Supernatants were obtained by centrifugation at 48,000 x g for 30 min at 4C. Twenty-five micrograms of protein was added to each lane. The 10%o (wt/vol) gel is shown after electrophoresis stained for protein with Coomassie brilliant blue. The plasmids carried 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 serum albumin (68 kDa), catalase (57.5 kDa), fumarase (48 kDa) and carbonic anhydrase (29 kDa). a plasmid

from barley (20), Synechococcus species (21), and E. coli (the popC gene product [21]) shows 39o identity between all four proteins (see Fig. 9). GSA 2,1-aminotransferase purified from barley and the cyanobacterium Synechococcus strain PCC 6301 consists of two identical 46-kDa subunits and of a single 46-kDa subunit, respectively (22). The B. subtilis hemL gene is predicted to code for a 46.1-kDa polypeptide. A polypeptide of this size was also found in an E. coli in vitro transcription-translation system using B. subtilis hemL DNA (Fig. 5). Furthermore, a soluble 46-kDa polypeptide was found in large amounts in E. coli JM83 containing hemL on a high-copy-number plasmid (pLUX209) (Fig. 7). Extracts of E. coli JM83/pLUX209 contained GSA 2,1aminotransferase activity which was inhibited by gabaculin (3-amino-2,3-dihydroxybenzoic acid) (Fig. 8). Such aminotransferase activity could not be detected in extracts of E. coli JM83 containing only the plasmid vector (Fig. 8). The sensitivity to gabaculin has also been shown in B. subtilis extracts by O'Neill et al. (43), which suggests that the enzyme uses pyridoxamine 5'-phosphate as a cofactor. From sequence comparisons to other aminotransferases, the lysine residue that binds the cofactor has tentatively been identified in the amino acid sequences for GSA 2,1-aminotransferase from barley, Synechococcus species, and E. coli (21). A corresponding lysine is also found in the B. subtilis GSA 2,1-aminotransferase at position 268. The sequence comparisons and the enzyme activity measurements demonstrate that the product of B. subtilis hemL is GSA 2,1-aminotransferase. E. coli mutants that require ALA for growth carry mutations in one of two loci, hemA (homologous to B. subtilis hemA) or popC (6, 46, 58). The predicted amino acid

FIG. 8. GSA 2,1-aminotransferase activity in extracts of E. coli JM83 carrying pBluescript II KS(-) or pLUX209 (hemL). Absorption spectra of ALA-derived pyrrole after reaction with Ehrlich's reagent 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. Five hundred micrograms of protein was used in each assay, and gabaculine was added to a final concentration of 10 ,uM. Spontaneous formation of ALA from GSA was analyzed by omitting cell extracts from the assay mixture. The resulting spectrum overlapped with spectrum 2.

sequence of popC shows 54% identity to that of B. subtilis hemL (Fig. 9). Also, in S. typhimurium there are at least two loci involved in ALA synthesis, hemA and hemL (16, 17a). The nucleotide sequence of S. typhimurium hemL was very recently reported (17) and is homologous to B. subtilis hemL and E. coli popC. The mutations causing ALA auxotrophy in B. subtilis that have been analyzed map at hemA (44, 45). It was therefore of interest to analyze the effect of a hemL mutation on growth and heme synthesis. A 427-bp HindIII-EcoRV fragment which covers an internal part of hemL was deleted from the chromosome and replaced by a cat gene (Fig. 10). The deletion was constructed and confirmed as described in Materials and Methods. The deletion removes the sequence corresponding to amino acid residues 87 to 128 of hemL and is. expected to inactivate GSA 2,1-aminotransferase. The strain with the deletion in hemL, 3G18AL17, was found to grow on minimal glucose medium without ALA, but ALA stimulated growth. Hence, it can be concluded that an intact hemL gene is not essential for ALA synthesis in B. subtilis. Possibly, GSA is spontaneously converted to other products including ALA at neutral pH or there is more than one protein with GSA 2,1-aminotransferase activity in B. subtilis. A hemAXCDBL operon? Biosynthetic pathways in B. subtilis are characterized by clustered and sometimes overlapping genes organized as operons (60, 61). The hemAXCDBL cluster consists of six closely spaced genes of which hemD overlaps with hemC and hemB overlaps with hemD (Fig. 4 and 10). This gene cluster is preceded by a putative promoter located approximately 70 bp upstream of hemA (unpublished S1 nuclease mRNA mapping data). Deletion ofthis promoter and the first 519 bp of hemA from the B. subtilis chromosome and substitution of this fragment with the cat gene of plasmid pHV32 (strain 3G18A401) results in an absolute growth

B. SUBTILIS hemAXCDBL GENE CLUSTER

VOL. 173, 1991

AVSIDEKAYT VQKSEEIFNA AKELMPGGVN SPVRAFKSVG .. LVTSSPFK TIKSDEIFAA AQKLMPGGVS SPVRAFKSVG .......... MRKSENLYQA ARELIPGGVN SPVRAFTGVG Bsu ....... MRS YEKSKTAFKE AQKIMPGGVN SPVRAFKSVD

bar syn Eco

**

*

* ****

******

** *

FVNSGTEACH GALRLVRAFT FVNSGTEACM AVLRLMRAYT MVNSGTEATM SAIRLARGFT MVSSGTEATM SALRLARGYT ** *

* ***** *

GATVGTLTAP STTANTLTAP DFAKYTLTCT GIAKNTITVP * *

GREKILKFEG GRDKIIKFEG GRDKIIKFEG GRNKILKFEG FEDNKGEIAA FAENPGEIAG FEQYPQEIAC FQQFGEDIAG

***

*

*

**

**

LENVLAQMVI LENVLAEMVI MEVKMAQLVT VENELAKLVI *

SAVPSIEMVR DAVPSIEMVR ELVPTMDMVR DRVPSVEIVR

**.***

GLPDSPGVPK

GLPDSPGVPK GQPNSPGVPA GLPDSPGVPE

*

**

*

**

*

*

**

**** **

* *** ** *

*

PIYQAGTLSG NPLAMTAGLE TLKQL.TPES YKNFIKKGDR LEEGISKTAG AHGIPHTFNR **

IRGMFGFFFA VSGMFGFFFT VGGMFGIFFT AGSMIGFFFT

GG.PVHNFDD EG.PVHNYED DAESVTCYQD NE.PVINYET

* * **

**

**

*

**

*

*

AKKSDTAKFG AKKSDLQKFS VMACDVERFK AKSSDLKLFA

*

*

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

*

*

180 178 170 173

300 298 290 293

** *

PMYQAGTLSG NPLAMTAGIH TLKRLMEPGT YEYLDKVTGE LVRGILDVGA KTGHEMCGGH PVYQAGTLSG NPLAMTAGIK TLELLRQPGT YEYLDQITKR LSDGLLAIAQ ETGHAACGGQ PVYQAGTLSG NPIAMAAGFA CLNEVAQPGV HETLDELTTR LAEGLLEAAE EAGIPLVVNH * ********

113

**

GSLLIFDEVM TGFRVDYNCA QGYFGVTPDL TCLGKVIGGG LPVGAYGGKA EIMEQIAPSG ****

110

NALREVTKQD 240 EGLREITLEH 238 PGLRALCDEF 230 QGLRDITEQY 233

GALLVFDEVM TPFRLAYGGA QEYFGITPDV TTLGKIIGGG LPVGAYGGRK DIMEMVAPAG DALLVFDEVM TGFRIAYGGV QEKFGVTPDL TTLGKIIGGG LPVGAYGGKR EIMQLVAPAG GALLIIDEVM TGFRVALAGA QDYYGVVPDL TCLGKIIGGG MPVGAFGGRR DVMDALAPTG **

118

* * *****

**

GFIPPQPAFL GFIVPDAGFL NCVPPLPEFL GVVPPQEGFL

**

120

**

**

*

VKAGSGVATL VKAGSGVATL VKAGSGALTL IKAGSGVATL

*

VFLEPVVGNA VILEPIVGNS IIVEPVAGNM VIVEPVAGNM **

50 53

*

CYHGHADSFL CYHGHADNFL CYHGHADCLL CYHGHGDSLL

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

YNDADAVKKL YNDLEAVKAL YNDLASVRAA YNDLESVKLA

GTPLFIEKAD GAYLYDVDGK NDPIFMERGK GSKIFDIDGN *

* *****

**

60 58

*

EYIDYVGSWG PAIIGHADDK VNAALIETLK KGTSFGAPCA RYIDYVGTWG PAICGHAHPE VIEALKVAME KGTSFGAPCA AYIDYVGSWG PMVLGHNHPA IRNAVIEAAE RGLSFGAPTE EYIDYVLSWG PLILGHTNDR VVESLKKVAE YGTSFGAPTE *****

GQPIVFDSVK GSHMWDVDGN GQPIVFDRVK DAYAWDVDGN

360 358 350 352

*

*

RFHRGMLEG VYLAPSQFEA GFTSLAHTTQ

419

RFHRGMLEQG IYLAPSQFEA GFTSLAHTEE RFFHO(DEG VYLAPSAFEA GFMSVAHSME SYYKGMANEG VFLPPSQFEG LFLSTAHTDE

417 410 411

*

*

2597

* ****

*

*

**

435 433 426 430

*

FIG. 9. Alignment of the predicted amino acid sequences of GSA 2,1-aminotransferase of barley (bar) (20), Synechococcus strain PCC 6301 (syn) (21), E. coli (Eco) (21), and B. subtilis (Bsu). Positions with identical amino acid residues in all four polypeptides are marked by asterisks. The amino-terminal sequence of the barley protein is that of the processed polypeptide (22).

requirement for hemin. This result would not be expected if only hemA were affected by the deletion (44). Thus, the heme biosynthetic pathway in mutant 3G18A401 is blocked in one or more steps after synthesis of ALA. This conclusion is strengthened by the finding that 3G18A401 lacks PBG synthase (Table 2) which is encoded by hemB. Pseudorever-

tants of 3G18A401 which grow on ALA can be isolated, and these revertants show higher PBG synthase activities than that of the wild type (Table 2). These pseudorevertants most

likely carry a mutationally activated promoter(s) allowing transcription of genes downstream of hemA (44). This promoter(s) is possibly located within the inserted pHV32

I

pLUX3202 pLUX3204 pLUX3203

E

00 p lO00bp

S

H

H C

H

H

pAhemLI7 I

r-

C

EV

cat

I

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

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DNA. The higher PBG synthase activity found in the pseudorevertants compared with that of wild-type B. subtilis supports the conclusion that this promoter(s) is different from that of hemA. These and previous (44) experimental data suggest that hemA, hemX, hemC, hemD, hemB, and probably hemL are all transcribed from a promoter upstream of hemA. The hemL gene is followed by an inverted repeat (Fig. 4) which may function in transcriptional termination of a hemAXCDBL operon. To test whether additional hem genes are located downstream of hemL and which belong to the same transcriptional unit as hemL, the following gene disruption experiments were done. A DNA fragment from the hemCD region, an internal region of hemL, and the end of hemL were each inserted into plasmid pHV32 (Fig. 10). This plasmid contains a cat gene but cannot replicate autonomously in B. subtilis. Transformation of B. subtilis 3G18 with pHV32 containing the above B. subtilis chromosomal DNA fragments and selecting for chloramphenicol resistance will result in transformants in which pHV32 has integrated into the chromosome via homologous recombination at the inserted fragments. The integrated plasmid will cause disruption of transcription at the site of insertion. If the fragment inserted into pHV32 were internal to an open reading frame, it would also disrupt translation and lead to a defective gene product. As expected, integration of pHV32 using the hemCD fragment resulted in chloramphenicolresistant transformants which require hemin for growth. However, the transformants with pHV32 integrated into or downstream of hemL grew without hemin or ALA. The fact that pHV32 was integrated into the chromosome at the expected site in the hemL transformants was confirmed by the 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 gene essential for growth downstream of hemL that is part of the proposed hemAXCDBL operon. Conclusion. The proposed hemA operon is expected to give rise to a transcript of about 6,400 nucleotides. The amount of hemA mRNA present in exponentially growing B. subtilis cells is less than 1/10 of that of the odhAB transcript encoding the El and E2 subunits of the 2-oxoglutarate dehydrogenase complex (unpublished experiments). Preliminary Northern blots using a hemA-specific probe have failed to demonstrate the presence of a hemA operon transcript of the expected size. This failure may reflect the low amount of hemA mRNA in the cells. In E. coli, hemC and hemD are closely linked and may form an operon (1, 6, 26, 48), whereas hemA, hemB, and popC 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 for synthesis of UroIII from glutamyl-tRNA lends further support to the notion that genes encoding enzymes of a common biosynthetic pathway generally have a more compact organization in B. subtilis than in E. coli and other enteric bacteria (60). ACKNOWLEDGMENTS We are grateful to B. Grimm and S. Gough for valuable discussions and for providing sequence information prior to publication, to C. G. Kannangara for the kind gift of GSA, to M. Petricek for initial help with the cloning, and to A. Sasarman for providing strain SASZ31.

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