Vol. 174, No. 24

JOURNAL OF BACrERIOLOGY, Dec. 1992, p. 8081-8093

0021-9193/92/248081-13$02.00/0 Copyright © 1992, American Society for Microbiology

Cloning and Characterization of the Bacillus subtilis hemEHY Gene Cluster, Which Encodes Protoheme IX Biosynthetic Enzymes MATS HANSSON* AND LARS HEDERSTEDT Department of Microbiology, University of Lund, Solvegatan 21, S-223 62 Lund, Sweden Received 30 July 1992/Accepted 16 October 1992

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen Im, map at 940 on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30%o sequence identity to penicillin-binding proteins IA of Escherichia coli, Streptococcus pneumoniae, and Streptococcus onus and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen Ill oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.

2440 on the chromosomal map. We have previously characterized this locus, which contains the hemAXCDBL operon (16, 36). Genes necessary in the conversion of uroporphyrinogen III to protoheme IX map at 94°. Mutations located at 940 have been correlated with different enzyme defects by analyzing the type of porphyrin the mutant accumulates (3). The accumulated porphyrin was thought to be the autooxidized form of the substrate of the mutated enzyme. Therefore, it was proposed that B. subtilis 1A592 (hemE64) was deficient in uroporphyrinogen III decarboxylase, since it accumulated uroporphyrin III. Strain 1A593 (hemF180) was deficient in coproporphyrinogen III oxidase, since it accumulated coproporphyrin III, and 1A594 (hemG321) was deficient in ferrochelatase, since it accumulated protoporphyrin IX (this mutation should then properly be named hemH321 according to the nomenclature used in Fig. 1) (Table 1) (3). In order to better understand the enzymes of the protoheme IX biosynthetic pathway in bacteria and to analyze ihe organization of the genes required for the operation of the pathway in B. subtilis, we have cloned and identified B. subtilis genes involved in the late steps of protoheme IX biosynthesis. Three hem genes were found located at 940 on the chromosomal map. The genes were identified as hemE, encoding uroporphyrinogen III decarboxylase, hemH, encoding ferrochelatase, and hemY, essential for protoheme IX biosynthesis and probably encoding a protein with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities.

Uroporphyrinogen III is the first closed-ring tetrapyrrole intermediate in the biosynthetic pathways for all cyclic tetrapyrroles, such as protoheme IX (heme), chlorophyll, siroheme, and vitamin B12. Protoheme IX is the prosthetic group of, e.g., b-type cytochromes and hemoglobin, and its biosynthesis from uroporphyrinogen III is achieved in four enzymatic steps (Fig. 1). In the first step the four acetyl side groups of uroporphyrinogen III are decarboxylated, resulting in coproporphyrinogen III. The propionyl groups on pyrrole rings A and B of coproporphyrinogen III are then oxidatively decarboxylated to vinyl groups, yielding protoporphyrinogen IX. The next step is a six-electron oxidation converting protoporphyrinogen IX into protoporphyrin IX. In the last step ferrous iron is inserted into protoporphyrin IX, giving protoheme IX. The enzymes catalyzing these four steps are uroporphyrinogen III decarboxylase (EC 4.1.1.37) (encoded by hemE), coproporphyrinogen III oxidase (EC 1.3.3.3) (hemF), protoporphyrinogen IX oxidase (EC 1.3.3.4) (hemG), and ferrochelatase (EC 4.99.1.1) (hemH), respectively (9). Our knowledge about these biosynthetic enzymes and the corresponding genes is incomplete and has been obtained mainly from studies with eucaryotic systems. For example, there is no published sequence data on hemG from any organism and hemH is the only procaryotic gene for an enzyme of the uroporphyrinogen III-to-protoheme IX pathway that has been reported to have been cloned and sequenced (12, 30, 32). Bacillus subtilis mutants auxotrophic for protoheme IX have been isolated (3). The mutations are located in two sites on the B. subtilis chromosomal genetic map (29, 37). A cluster of genes required for the biosynthesis of uroporphyrinogen III from glutamyl-tRNA via 5-aminolevulinic acid is located at *

MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids used are described in Table 1. Media and enzymes. Tryptose blood agar base (Difco), LB (42), and Spizizen minimal medium (47) were used as growth

Corresponding author. 8081

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HANSSON AND HEDERSTEDT

J. BACTERIOL.

hemE

Uroporphyrinogen III

Coproporphyrinogen III

Protoporphyrinogen IX A

(D

= acetyl =

propionyl

9 = methyl @ = vinyl

Protoheme IX

Protoporphyrin IX

FIG. 1. Pathway of protoheme IX biosynthesis from uroporphyrinogen III. The name of the bacterial gene corresponding to each enzyme activity is indicated on the reaction arrows.

media. The minimal medium was supplemented with required amino acids at 10 mg/liter. The following antibiotics were used: ampicillin (agar plates, 50 mg/liter; LB, 100 mg/liter), chloramphenicol (B. subtilis, 3 mg/liter; Eschenchia coli MM294, 12.5 mg/liter; E. coli JM109, 20 mg/liter), phleomycin (0.5 mg/liter), and tetracycline (15 mg/liter). Media containing 2.5 mg of hemin (Sigma Chemical Co.) per liter also contained 500 mg of bovine serum albumin (BSA) (fraction V; Sigma) per liter. Stock solutions of hemin were prepared as described by McConville and Charles (28) except that Tween 20 was used instead of Tween 80. Restriction endonuclease FspI was from New England Biolabs, Inc., and Bspl286I was from Amersham. Other restriction endonucleases, T4 DNA ligase, exonclease III, and mung bean nuclease were from Boehringer GmbH (Mannheim, Germany). RNase was from Worthington Biochemical Corp. (Freehold, N.J.). DNA techniques. General DNA techniques were as described by Sambrook et al. (42). Large-scale preparations of plasmid DNA were made as described by Ish-Horowicz and Burke (18). Small-scale preparations of E. coli and B. subtilis plasmid DNA were made by the boiling method (42) and by the Magic Minipreps DNA purification system (Promega), respectively. An initial step, lysozyme treatment (1 mg/ml, 37°C, 30 min), was added to the protocol when plasmid DNA was prepared from B. subtilis. B. subtilis chromosomal DNA was extracted as described by Marmur (26). Southern blot analysis of chromosomal DNA was performed with a nonradioactive DNA labeling and detection kit from Boehringer. Competent E. coli cells were prepared as described by Mandel and Higa (25). B. subtilis was grown to competence as described by Arwert and Venema (1). DNA sequence analysis. Unidirectional deletions in DNA

fragments cloned in pBluescript II KS(-), pBluescript II SK (-), and pUC18 were generated by using exonuclease III as previously described (16). DNA nucleotide sequences were determined by the dideoxy chain termination method (43), using modified T7 DNA polymerase (Sequenase version II; U.S. Biochemical Corp., Cleveland, Ohio) and [a-35S]dATP (Amersham). Plasmid DNA for sequencing was isolated from E. coli XL1-Blue by the boiling method (42). Alignments of deduced amino acid sequences were done by using the TFASTA, GAP, and PILEUP programs of the GCG Sequence Software Package (10). Northern (RNA) blot analysis. B. subtilis strains were inoculated to an optical density at 600 nm of 0.1 in Spizizen minimal medium (47) supplemented with 0.05% (wt/vol) casein amino acids and, when required, with specific amino acids, hemin, BSA, and chloramphenicol and grown at 37°C on a rotary shaker (200 rpm) to early exponential phase (optical density at 600 nm of 0.5 to 0.6). RNA was extracted as described by Resnekov et al. (38). Twenty microliters (corresponding to 12 to 18 ,g of total RNA) was run on a 1% agarose gel containing 6% (vol/vol) formamide. Capillary blotting to Hybond-N filters (Amersham) and hybridization were performed as described in a protocol from Amersham. DNA probes were labeled by using a random priming kit obtained from Boehringer and [ac- 3P]dCI? (Amersham). RNA molecular weight markers were obtained from Bethesda Research Laboratories (Gaithersburg, Md.). All solutions were treated with diethylpyrocarbonate (Sigma) before use to inactivate nucleases. Construction of B. subtilis deletion-substitution mutants. Plasmids pLUGA2 and pLUGA7 are pBluescript II KS(-) derivatives used to create two B. subtilis deletion-substitution mutants in which a chromosomal DNA fragment is

VOL. 174, 1992

B. SUBTILIS hemEHY GENES

8083

TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains B. subtilis 3G18 B. subtilis 1A592 B. subtilis 1A593 B. subtilis 1A594 B. subtilis 3G18A2 B. subtilis 3G18A7 E. coli XL1-Blue

Relevant properties

E. coli JM109

trpC2 met ade trpC2 hemE64 trpC2 hemF180 trpC2 hemG321 trpC2 met ade AhemY ble trpC2 met ade AhemEHY ble supE44 hsdR17 recAl endA1 gyrA96 thi reL41 1ac(F' proAB+ lacl lacZAM15 TnlO[Tet']) recA1 supE44 endA1 hsdR17gyrA96 reL41 thi A(lac-proAB)(F' traD36proAB+ lacIq

E. coli MM294

lacZAM15) supE44 hsdR endA1 thi

Plasmids pHP13 pHV32 pAMB22

pBLE1 pBluescript II KS(-) pBluescript II SK(-) pUC18 pUC19 pLUG1301 pLUG1310 pLUG1316 pLUG101 pLUG128 pLUG130 pLUG131 pLUG181 pLUG186 pLUG185 pLUG3201 pLUG3202 pLUGAl pLUGA2 pLUGA7 pLUG18el pLUG18e2 a

or Reference source

G. Venema BGSCa BGSC BGSC This work This work Stratagene 51

L.-O. Heden

cat ery lacZ' bla cat tet cat xylE bla ble; ble of pUBilO cloned in pUC18 bla lacZ' bla lacZ' bla lacZ' bla lacZ' cat ery; pHP13 derivative with the B. subtilis hemEHY region (Fig. 2) cat ery; SphI deletion derivative of pLUG1301 cat ery; PvuI deletion derivative of pLUG1301 bla; 2.7-kb EcoRI fragment of pLUG1301 cloned in pBluescript II KS(-) (Fig. 3A) bla; 2.7-kb EcoRI fragment of pLUG1301 cloned in pBluescript II SK(-) (Fig. 3A) bla; 2.1-kb EcoRI-FspI (PvuI) fragment of pLUG1316 cloned in pBluescript II KS(-) (Fig. 3A) bla; 2.1-kb Sall-FspI (PvuI) fragment of pLUG1316 cloned in pBluescript II SK(-) (Fig. 3A) bla; 2.4-kb HindIII (Sau3AI)-SphI fragment of pLUG1310 cloned in pUC18 (Fig. 3A) bla; 2.4-kb Sall (Sau3AI-SphI) fragment of pLUG181 cloned in pUC18 (Fig. 3A) bla; 2.8-kb SphI fragment of pLUG1301 cloned in pUC18 (Fig. 3A) bla cat; PvuII-XhoI (PvuI) fragment of pLUG130 cloned in pHV32 cat tet; Bsp1286I-HindIII fragment of pLUG131 cloned in pHV32 bla ble; ble of pBLE1 cloned in EcoRI and SphlI sites of pLUG130 bla ble; EcoRI fragment of pLUG185 cloned in pLUGAl (Fig. 3B) bla ble; EcoRI (Sau3AI-EcoRI) fragment of pLUG186 cloned in pLUGAl (Fig. 3B) bla; hemE cloned downstream the lac promoter of pUC18 bla; hemH cloned downstream the lac promoter of pUC18

15 34 54 11 Stratagene Stratagene 51 51 This work This work This work This work This work This work

This work This work This work This work This work This work This work This work This work This work This work

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

replaced by the phleomycin resistance gene, ble. In plasmid pLUGA2 the ble gene, isolated from plasmid pBLE1 (Table 1), is flanked by an EcoRI fragment (bp 1786 to 4444) and a SphI-PvuI fragment (bp 5223 to 6624) (see Fig. 3B). In plasmid pLUGA7 the ble gene is flanked by a Sau3AI-EcoRI fragment (bp 1 to 1786) and a SphI-PvuI fragment (bp 5223 to 6624) (see Fig. 3B). B. subtilis 3G18 was transformed to phleomycin resistance with pLUGA2 and pLIJGA7 linearized with ScaI (cleaves in the bla gene of the vector). In the resulting mutants, B. subtilis 3G18A2 and 3G18A7, 780 bp of the hemY sequence (bp 4444 to 5223) and 3,737 bp of the hemEHY sequence (bp 1786 to 5223), respectively, have been replaced by the ble gene. The deletion-substitution mutants were confirmed by Southern blot analysis (data not shown). Enzyme activity measurements. Catechol-2,3-dioxygenase activity in B. subtilis containing the xylE reporter gene was determined with extracts from cells grown on a rotary shaker (200 rpm) at 37°C overnight in 20 ml of LB medium containing chloramphenicol. The cells were harvested by centrifugation, washed once with 0.1 M K phosphate buffer, pH 7.2,

suspended in 0.8 ml of 0.1 M K phosphate buffer, pH 7.2, containing 80 ,ug of lysozyme, incubated for 20 min at 37°C, and then sonicated while chilled on ice. The lysate was centrifuged at 48,000 x g for 30 min at 4°C, and catechol2,3-dioxygenase activity at 30°C in the supernatant was measured as described by Sala-Trepat and Evans (41). The extinction coefficient of 2-hydroxymuconic semialdehyde at pH 8.0 is 33.9 mM-1 cm-' (2). Ferrochelatase activity in E. coli JM109 containing different plasmids was determined with extracts from cells grown on a rotary shaker (200 rpm) at 37°C overnight in 25 ml of LB medium containing ampicillin. The cells were centrifuged down at 5,000 x g for 5 min, washed once with 50 mM Tris-HCl, pH 7.4, suspended in 1 ml of 50 mM Tris-HCl, pH 7.4, and disrupted by sonication while chilled on ice. The lysate was centrifuged at 100,000 x g for 30 min at 4°C. The pellet was washed once with 50 mM Tris-HCl, pH 7.4, and suspended in 100 ,ul of 50 mM Tris-HCl, pH 7.4. Both the supernatant (cytoplasmic) and the particulate (membrane) fractions were analyzed for ferrochelatase activity. Incorporation of iron into protoporphyrin IX was measured essentially as described by Dailey

8084

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HANSSON AND HEDERSTEDT

(8), using the following constituents in a 4-ml reaction mixture: 70 mM Tris-HCl, pH 7.6, 10 ,uM protoporphyrin IX, 0.10 mM Fe(II)SO4, 3 mM dithiothreitol, 0.3 mg of Tween 20 per ml, and 0.5 mg of bacterial protein. The reaction mixture was incubated in the dark at 37°C. An aliquot was removed after 5 min and mixed with an equal volume of 0.15 M NaOH-4.2 M pyridine solution to stop the reaction. The protoheme IX formed was determined as the pyridine hemochromogen by using the difference (dithionite reduced minus ferricyanide oxidized) extinction coefficient 24 mM 1 cm-' (556 nm minus 540 nm) (46). Incorporation of zinc into protoporphyrin IX was determined essentially as described by Camadro and Labbe (7), but palmitic acid was omitted. The reaction was monitored spectrofluorometrically at room temperature by measuring the rate of protoporphyrin IX disappearance. The excitation wavelength was 410 nm, and a 550-nm emission light cutoff filter was used. Other methods. In vitro coupled transcription-translation was done with a kit from Amersham, using L-[35S]methionine (1,000 Ci/mol) (Amersham). Proteins were analyzed by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) (33). Protein concentrations were determined by the method of Lowry et al. (23), using BSA as a standard. Nucleotide sequence accession number. The nucleotide sequence shown in Fig. 4 has been deposited in the EMBL, GenBank, and DDBJ data bases under accession number M97208. RESULTS AND DISCUSSION Cloning. B. subtilis hem genes located at 940 on the genetic map were cloned by complementation of B. subtilis mutants defective in late steps of protoheme IX biosynthesis. Plasmid pHP13 (Table 1) was used as the cloning vector. In B. subtilis, pHP13 is a low-copy-number plasmid providing resistance to chloramphenicol and erythromycin, while in E. coli it is a high-copy-number plasmid conferring resistance to chloramphenicol. B. subtilis 3G18 chromosomal DNA was partially cleaved with Sau3AI and size fractionated on a sucrose gradient. Fragments of 5 to 15 kb were ligated to BamHI-cleaved pHP13 (fragment-to-vector ratio, 4 to 1) and used to transform B. subtilis 1A592 (hemE64) and 1A593 (hemF180). Transformed cells were plated on tryptose blood agar base (T) plates to select for heme prototrophy and on T plates containing hemin, BSA, and chloramphenicol (THBC) to select for chloramphenicol resistance. Transformants were subsequently tested for growth on T plates containing chloramphenicol (TC), i.e., to test for combined heme prototrophy and chloramphenicol resistance. Transformation of B. subtilis 1A592 (hemE64) and 1A593 (hemF180) with the ligation mixture and selection on THBC plates resulted in 1,226 and 1,365 transformants, respectively. Two and three of these, respectively, grew on TC plates. However, these five transformants contained pHP13 without a chromosomal DNA fragment. Selection of transformants on T plates gave 1,415 and 1,748 transformants, respectively. Five transformants of each strain grew on TC plates. One transformant of each strain harbored a plasmid containing a B. subtilis chromosomal DNA fragment. These plasmids were named pLUG1301 and pLUG1307, respectively. The three B. subtilis mutant strains 1A592 (hemE64), 1A593 (hemF180), and 1A594 (hemG321) were transformed to heme prototrophy by both pLUG1301 and pLUG1307. This shows that the wild-type alleles of these hem mutations

EcoRI ..:..

Sau3AI/ BamHI

// \

Pvul

Sau3AI/ BamHI EcoRI

FIG. 2. Map of plasmid pLUG1301. Arrows indicate the locations and orientations of genes.

are present on the cloned DNA fragments in pLUG1301 and pLUG1307. Restriction enzyme analysis of pLUG1301 and pLUG1307 showed that the cloned fragments were not identical but were partially overlapping. Plasmid pLUG1301 (Fig. 2) was found to be smaller than pLUG1307 and was therefore used in further work. To locate hem genes on the cloned 12-kb chromosomal fragment of pLUG1301, the different B. subtilis hem mutants were transformed with restriction fragments isolated from agarose gels. The B. subtilis hem mutant strains 1A592 (hemE64), 1A593 (hemFl80), and 1A594 (hemG321) were transformed to heme prototrophy by a 2.7-kb EcoRI fragment (Fig. 2). This EcoRI fragment and flanking fragments were cloned on plasmid vectors in E. coli XL1-Blue (Table 1). Some fragments could not be cloned on plasmids in E. coli. This is in accordance with our finding that plasmids pLUG1301 and pLUG1307 cannot be stably maintained in E. coli; i.e., E. coli MM294, XL1-Blue, and JM109 cannot be transformed to chloramphenicol resistance with these two plasmids. Exonuclease III was used as described before (16) to generate unidirectional nested deletions in the cloned fragments, and the resulting plasmids were used for DNA sequence analysis and for mapping of the B. subtilis hemE64, hemF180, and hemG321 mutations. DNA sequence analysis. The nucleotide sequence of a 6,624-bp fragment was determined from both strands (Fig. 3A and 3C). Six genes were found in the sequence, and they were namedponA, hemE, hemH, hemY, ORFA, and ORFB. Our rationale for these gene designations is given below. The sequence begins withponA, the start of which is not present in pLUG1301 (Fig. 2 and 4). There are two possible ATG translational start codons for hemE. The second of these is likely to be the start codon since, preceding it by 5 bp, there is a reasonable ribosome binding site. For hemH there are also two possible ATG start codons. N-terminal sequence analysis of the B. subtilis hemH gene product expressed in E. coli has shown that the GTG codon at bp 3038 is used as a translational start codon (unpublished results). The hemY gene also has two possible ATG start codons. Two possible ribosome binding sites precede the second potential start codon by 6 and 3 bp. The hemY gene is followed by ORFA and ORFB. ORFB continues past the end of the determined DNA sequence.

B. SUBTILIS hemEHY GENES

VOL. 174, 1992

ponA'

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Complementation analysis. To make sure that the cloned hem genes are functional, we transformed the deletion mutant B. subtilis 3G18A7 with plasmid pLUG1301. B. subtilis 3G18A7 is a mutant in which we have deleted hemE, hemH, and hemY and replaced these genes with a ble gene (Fig. 3B). This strain shows an absolute hemin requirement for growth, and it was transformed to heme prototrophy by pLUG1301. The plasmid could subsequently be reisolated from the transformants. These results demonstrate that the cloned hemEHY genes in pLUG1301 are expressed in B. subtilis and encode functional gene products. Mapping ofB. subtilis hem mutations. Mutants defective in early protoheme IX biosynthesis, i.e., blocked in a step prior to uroporphyrinogen III, cannot synthesize siroheme, which is a prosthetic group of assimilatory sulfite reductase. Reduced sulfur, e.g., cysteine, is therefore required in addition to hemin for growth of such mutants. The B. subtilis mutants 1A592 (hemE64), 1A593 (hemF180), and 1A594 (hemG321) can all grow without a source of reduced sulfur in the medium, which indicates that the protoheme IX biosynthetic pathway in these mutants is blocked at a step after uroporphyrinogen III. The primary defect in any of these three mutants has not been directly analyzed, i.e., by enzyme activity measurements. The phenotypes of the mutants were determined by Berek et al. (3) from the types of porphyrins accumulated. B. subtilis 1A592 (hemE64) was reported to accumulate uroporphyrin III, suggesting that this mutant is defective in uroporphyrinogen III decarboxylase (Fig. 1). B. subtilis 1A593 (hemF180) seems to be defective in coproporphyrinogen III oxidase since it accumulates coproporphyrin III. B. subtilis 1A594 (hemG321) was proposed to be defective in ferroche-

latase because of accumulation of protoporphyrin IX. (The of hemG321 would in that case be hemH321.) The fact that porphyrinogens are rapidly auto-oxidized to porphyrins complicates the interpretation of accumulation data. For example, it is difficult to discriminate between the phenotypes of a hemG and a hemH mutant since they primarily accumulate protoporphyrinogen IX and protoporphyrin IX, respectively. This is also illustrated by the properties of a Saccharomyces cerevisiae protoporphyrinogen IX oxidase-defective mutant that was found to accumulate and excrete protoporphyrin IX although it contained a functional ferrochelatase on the basis of in vitro enzyme activity measurements (7). We mapped the locations of the hemE64, hemF180, and hemG321 mutations (Fig. 3A) by transforming strains 1A592, 1A593, and 1A594 to heme prototrophy with plasmids comprising pBluescript or pUC18 and exonuclease III-generated fragments of the B. subtilis hemEHY gene cluster. The hemE64 mutation was found to be located in the proximal part of hemE or just upstream of hemE (this mutation was marker rescued by bp 1786 to 2150; Fig. 4). The hemF180 mutation was found to be located in the very terminal part of hemH or in the proximal part of hemY (this mutation was marker rescued by bp 3791 to 4202). The hemG321 mutation was mapped to an internal part of the hemY gene (this mutation was marker rescued by bp 4043 to 4444). Does the hemEHY gene cluster constitute an operon? In B. subtilis it is often found that genes encoding enzymes of the same biosynthetic pathway are arranged in one operon (53). It was therefore of interest to analyze the transcriptional organization of the sequenced genes. RNA was isolated from three B. subtilis strains, 3G18/pLUG1301, 3G18, and 3G18A7, proper name

HANSSON AND HEDERSTEDT

8086

J. BACTERIOL.

Sau3AI 1 1 111 38

GTACACGTGCAATITTCGCCTAAACTTTAAAAAAAATAATGGTATTGAGGTAACAG I T Q Q L A K N I F L T H D K T F L R K T K E V I I A I N L E R D Y S K D The terminal part of ponA

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1430 477

1431 478

GAAACGCTTTCCAGCAAAGTAGATGAGCGTGGTAGCGCAAATAACGTAAATAACGG E TCGH VF T K P KN V KE LE S P IE L K P V KT L TA D Y TF K AA

1540 513

1541 514

GGTITATTGATAGGGCCCAAGTACGCGAAAATTTTACAGCGGGAAGTCTATGTGGG C L F T I E L K W D A Q E D D R A V Y R I Y V N K D G E E T L L D S V E C

1650 550

1651 551

GAGGACAGATCTAGCATATrGGGGCTTAATTCTAATCCGCAAGGAGGAGACATCTr K C S Y E I P Y A N L F S G A S Y K I V P Y N T Q T K R E G E G T D Y V Q EcoRI AGCAATTTCTTATGATGCATTTACTGGTCTTTTCATGCAACAGAATTAAGCAAAAG P K L F S S *

1760 587

1761 588

T

A

S

T

G

Y

H

D

G

L

G

A

K

T

G

T

S

S

Y

T

V

G

1870 593

1871 1

ACT7GTCGAgA

TQATCGTATACAAAGTACAAGTTAAGTCGGGGAAGGACCCCTGT M S K R E T F N E T F L K A A R C E K A D H T P V W hemE

1980 26

1981 27

GGAAGGCACGGGTAACGATCGAGTAGAAGAGATTTAACCCTACCACTGGGAUCCAA Y M R Q A G R S Q P E Y R K L K E K Y C L F E I T H Q P E L C A Y V T R

2090 62

2091 63

CTCGTGGATCGGCAGTCACTrTAGTTAGCCGTCGCACGGGAGTAACAACGACGCTT L P V E Q Y G V D A A I L Y K D I M T P L P 5 I C V D V E I K N C I C P V

2200 99

2201 100

GTGTACATCGCCGCGC7rAAATGCAATACGACGAGCCTCGCTAAGTAATCTTATGC I D Q P I R S L A D I E K L G Q I D P E Q D V P Y V L E T I K L L V N E Q

2310 136

2311 137

AGTACTCGTACGTCCGTCCTTAGTGAGTTTACAGCGCGCAAATCAAACAACTCTTC L N V P L I G F S G A P F T L A S Y M I E C C P 5 K N Y N K T K A F M Y

2420 172 2530 209

2531 210

AGAGCGTCTGACGTAGCAGTGCAAGTACTTCTAACCGTGGCGCCAACATAACTGTC S M P D A W N L L M S K L A D M I I V Y V K A Q I E A G A K A I Q I F D 5 SacI GTGTGCCTGACGCGTAAACTCTAACGGTACGACTTAACCCAGAATTCGTACTTTGG W V C A L N Q A D Y R T Y I K P V M N R I F S E L A K E N V P L I M F G V

2640 246

2641 247

TTGGAGCTTGAGGTGCTACrCCCAGTTGGTGCGAATGCTGTAGCGTAAGATATAAA G A S H L A G D W H D L P L D V V G L D W R L G I D E A R S K G I T K T

2750 282

2751 283

GTCGGACTGCCTCTTGTGGCTGAGCTGGAAACAGAAATGTAGTTGGCGCGTCTTCA V Q C N L D P 5 I L L A P W E V I E Q K T K E I *L D Q G M E S D C F I F N

2860 319

2861 320

TCTGCTGGATCTAGCGCCAGTIAAACGCGATGCAGAATAAACAAATGTATTCTACT L G H G V F P D V S P E V L K K L T A F V H E Y S Q N K K M G Q Y 5 -

2970 353

2971 1

ACTTGACTAGTTCGCAAGTGACGTATAAG

TAAATATGAGAAGGCTCCTAGCTCCA homE M S R K K M C L L V M A Y C T

3080 15

3081 16

CGCTTAGAAGTTGAGTTAAAAACGAAGAAACTACTAAGTCAATGAGCGTCAGGTGC P Y K E E D I E R Y Y T H I R R C R K P E P E H L Q 0 L K D R Y E A I C

3190 51

3191 52

GGATCCGTGCAACCGAACGCCTACGACGATGAGATCGAGGTAGTAACTTTGATAGA C I S P L A Q I T E Q Q A H N L E Q H L N E I Q D E I T F K A Y I C L K H

3300 88

3301 89

TACACGTATAGCCGACGGTCTAGTGATCGACGCGACTCGCCGATCCAGTACTCGCT H C Y

3410 125

3411 126

AACACCCAAAG(GTAAATGCGTACATCTATGAGTTAGTACGATGTAAATGTGCGTG

3520 161

3521 162

AGAAAAGGCAGCGGAGAGGAAGATCCTGACGAAAC7CGAAATAAATw

GACGACGA 0 P Y P 0

3630 198

3631 199

CCGTCTATGCAATATCGAGGAGGTCGAAGTTGCGCAGGAGACCCTACTGCCGCCAG C C C C 0 C 0 V

3740 235

3741 236

TTAGTTAAGGTTTCACAAGCACACTTTTTTCTTGGTGCCGTATAAGGTTTAATATT 0 0 C C 0 0 0

3850 271

2421 173

SphI

I

P

E

N

K

K

E

Q

R

T

L

Q

F

A

Y

H

S

S

T

A

E

M

A

R

D

E

K

A

E

L

E

I

A

P

K

L

0

I

F

K

E

E

L

E

A

V

Q

C

L

R

E

A

E

H

E

D

K

C

E

L

N

K

T

A

A

Q

A

T

I

L

M

V

Y

T

I

F

S

I

E

S

E

S

W

P

V

0

L

S

Q

E

E

V

P

P

E

P

S

F

A

L

V

Y

W

H

V

V

I

S

A

S

A

Y

V

E

V

V

Y

V

A

H

K

K

F

I

N

A

F

H

T

V

K

T

T

S

E

Y

F

P

P

L

F

E

V

S

W

C W

L

V

V

Q

0

L

Y

S

R

V

P

N

Y

VOL. 174, 1992 3851 272 3961

309

B. SUBTILIS hemEHY GENES

E CK VV T DD .hmY

G R*

GA SY Y RPE MP NA KP E FI

I

DGK KH V VI

M S

I G GG

3960

DA LA TV V LKK L

308

4070

I1K

TGL AA A FY ME KE

I

29

4071 AAAGACGCC7ACGCC7TGGCATCAATGCGAATCGCGCAAAAGCAACTGAGGGCGC 30

4181

67

E

K

N

L

L

P

E

L

T

L

E

V

A

S

R

P

G

V

G

K

Q

I

T

V

K

K

D

G

I

Y

I

E

R

G

P

D

4180 5

CATTTC7GAACGAAAGAAAAGCGCCCCGCAGCT'IOTTAAAGACTTAGGTCTMAGCAiT CT TCAACAATGCGACCGGGCAATCCTATGTGC'TGTAAACCGCACC LV K DL GL EHL L VN NA TG Q SY VL VN RT

FLE R KK SA PQ

4291 103 L H P MP KG A V MGI P TKI A PF V ST G LFS L SG KA RAA MD F EcoRI 4401 140 SalI 4511 177 4621 213 Q G S G Q Q L T A K K Q G Q F Q T L S T G L Q T L V E E I E K Q L K L T K

4731GGTTAAGAAAGACAATACAACGTTGTTCCCACGAACGTCACTACGTCGATGGCGC

250 4841 287 4951 323

V Y K G T K V T K L S H S G S C Y S L E L D N G V T L

D A D S V I

V T A P

102

.4400 139

4510 176 4620

212

4730 249

4840 286

4950 322

V Q MEH EG TG F VI

5060 359 5170

H KA AA G ML

K TL

S EL P AI

SH LKN MH S T SV AN VA L GF

SR N SD FAI

LR A YVG KA G DES

P EG S

T A CT WT NK K WP HAA

I V DLS

I I NI

D ND

PE G

V LE DL K KV MN

I NG EP EMT C VT R WHE SM PQ YH VG H KQ RI

397

KE

L RE A LA

5281 TTCTTCGTTTT7AAGGTCTCAG77GCTCCATCTGTAGAAG7CGGCGCCCTCTTTT 5

A

Y

P

G

V

Y

M

T

G

A

S

F

E

G

V

G

I

P

D

C

I

D

Q

G

K

A

V

A

S

D

L

A

T

Y

L

F

5391 CGTAACCGTTTGGAGT7TG7TCAGCAAT7TAAAGTGATACGAGT7AAATGAAGAT 470 5 * 5501 ATATAAG AAG'AT GGIATCAGCAAACAAAGTAT7GGCGTCAGCTCCCGTGGAA 1

ORFA

M S

I D

R

K

K

L I

L EA

K

A T

S F

T

Q

5611AGGCAATGTTGCCAATGGAGGGAGGACTTTCCAGAACGTACAA

24 5721

66

4290

CCTAGGCGGGAGTMGGTCTCATCCTIAAAAGATCCTCTGAAGCCTAGTCCAGCC

5061 360 5171 433

8087

A T T M D

61

L V A K

L A N V G K G T

K E M KQ KA DEA ME

P S

I

Y

T F F K N K E E L F D E

LP FH EN VH RA L FAI

I

F

G

Y

K

ATCTT

F T T L L

L EF R KTH Q

396

5280 432 5390 469 5500 470 5610 23 5720 60

5830 96

5831 CGCATAATTCAAAICGGTGTCAGCG7AGAIACAAA'GACACAICGCTTTAAGAGTG 97 L T I K I F Q E N A E I G T M A V Q E V I Q K M E R S I L S Y I K S K I E 5941 AAGGTAAGGCCACACGGGCCGGTAACT7TAGTAG7TTTGGTATTGCGGAACAACC 134 D G I K S G A I K P C D P E L T A F V M L K L Y I A L I F D W E K Q H P P 6051 CCTAAAAAATGGGCGTGATTTTGCAGAITACACAAA7CTCCTGAGAAGCATTT7A 171 L D K E T I A G L L E L Y V V K G L S A N * 6161 GGCGTTMT9QCTAGTACCAAGATATGAGCTGACATAAATTGTICACTGCTTGTGC 1 ORFB MN TI R SQ W KD I V TS KK L L I P I1 AI L F VP

28

6271 29

CCTrTAACGGATTAAGTATGACGAGCCGTACGTCTTGTTGIACAGCAGACAGAGAG L I Y SG VF L KAY W DPY G T VDQ LP VV VV NQ D K GAT YE G

6380 64

6381 65

E

GAACTAACGGrGTTGCAGATAGAATAACTG7VGAT CATACCACACTAAGCTrrAC K L Q I G D D L V K E L K D N N N F D W H F S N D L D Q S L K D L L N Q

6490 101 6600 138

6491 102 K YY L V VE I PE DF SK N AS PvuIU 6601 CTAACTATGTAGGGGCAACGATCG 6624 139 N Y V G A T I 145

T V L DKN

PK KL

D LK Y HT N AGS

5940

133 6050 170 6160

191 6270

FIG. 4. Nucleotide sequences of ponA (the terminal part), hemE, hemH, hemY, ORFA, and ORFB (the proximal part) and the derived amino acid sequences of the corresponding polypeptides. Possible ribosome binding sites, i.e., base pairs complementary to B. subtilis 16S rRNA, are underlined or overlined.

analyzed in Northern blot experiments. Three different probes were used: a 519-bp DraI-HindIII hemE fragment, a 779-bp EcoRI-SphI hemY fragment, and a 1,054-bp SspI-SspI fragment containing all of ORFA and 307 bp of ORFB. The hemE and hemY probes hybridized with RNA and

DNA

isolated from B.

subtilis 3G18/pLUG1301

and 3G18 but,

as

expected, not with RNA from the hemEH-Y deletion strain, 3G18A7 (Fig. 5 and data not shown). The RNA recognized by the two hem probes appeared partly degraded, but as anticipated the presence of pLUG13O1 increased the relative amounts of hybridizing hem RNA. The size of the largest hybridizing RNA was 3.7 kb, which is about the size

expected for a polycistronic hemEHY mRNA. The ORFAORFB probe also hybridized to RNA of the hemEHY deletion strain (results not shown), and the size of this RNA was about 0.8 kb. The size of an ORFA transcript would be about 0.7 kb. These results indicate that the hemE, hemH, and hemY genes are cotranscribed and that ORFA is in a separate transcriptional unit, but this has to be confirmed by more detailed transcriptional analysis. The finding of an apparent polycistronic hemEH-Y transcript prompted us to search for a promoter region upstream of hemE. For this purpose, pAMB22 (Table 1) was used as a promoter-searching plasmid. pAMB22 provides resistance

8088

HANSSON AND HEDERSTEDT

J. BACTERIOL.

activity, suggesting that the hemEHY promoter is located within approximately start codon (Fig. 6). 100 bp from the hemE translational

hemE probe

1

2 3

ORFA and ORFB are not essential for heme synthesis. The

- 9.5 kb = 7.5 kb

3 .7 kb

4.4 kb

--- - >

jt ?

23S rRNA -. 16S rRNA

- 2.4 kb

>

1.4 kb

0.24 kb

FIG. 5. Northern blot analysis for hem mRNA. RNA from B. subtilis 3G18/pLUG1301 (12 ,ug) (lane 1), 3G18 (18 ,ug) (lane 2), and 3G18A&7 (18 ,ug) (lane 3) was probed with an internal hemE DNA fragment. The largest hybridizing RNA was 3.7 kb, which is about the size expected for a hemEHY mRNA. to chloramphenicol and contains a xylE reporter gene, encoding catechol-2,3-dioxygenase, which converts catechol into yellow 2-hydroxymuconic semialdehyde. There is no promoter in front of xylE in pAMB22, but upstream of the xylE gene there is a cloning cassette. In this cassette various fragments from the hemE upstream region were cloned, and expression of xylE was analyzed in B. subtilis 3G18. A 640-bp EcoRI-SphI fragment was sufficient to give full XylE

predicted ORFA gene product is a polypeptide of 191 amino acid residues. Only part of ORFB was sequenced. This part corresponds to the first 145 amino acid residues of the putative ORFB polypeptide. We have not found any sequence similarities in the proteins deduced from ORFA and

ORFB and other sequences available in the GenBank/EMBL

data banks.

The Northern blot analysis data indicate that ORFA and ORFB are on transcriptional units separate from hemEHY (Fig. 5 and data not shown). Although ORFA and ORFB are not part of a hemEHY operon, it cannot be excluded that these genes are involved in heme biosynthesis. To determine if ORFA and ORFB are essential for protoheme IX biosynthesis, these two genes were disrupted by integration of plasmid pHV32 (Table 1). pHV32 contains a cat gene but cannot replicate autonomously in B. subtilis. Transformation of B. subtilis with pHV32 containing B. subtilis chromosomal fragments and selection for chloramphenicol resistance will result in transformants in which pHV32 has integrated into the chromosome via homologous recombination at the inserted fragment. Provided that the fragment used for integration is internal to the gene, integration will lead to gene disruption. A 218-bp fragment internal to ORFA (bp 5787 to 6005) (Fig. 4) was ligated to pHV32 which had been cleaved by PstI and HindIII. This plasmid was named pLUG3202. A 290-bp fragment internal to the proximal part of ORFB (bp 6333 to 6623) (Fig. 4) was ligated to pHV32 which had been cleaved by EcoRV and SalI. This plasmid was named pLUG3201. B. subtilis 3G18 was transformed to chloramphenicol resistance with pLUG3202 and pLUG3201.

Relative promoter

DNA fragment cloned in front of xylE

B.subtilis 3G18/ pLUGp3

ponA'

hemE'

pLUGp4

B.subtilis 3G18/

B.subtilis 3G181 pAMB22

ponA

98

hemE'

B.subtilis 3G18/

pLUGp2

activity (%)

4

No inserted fragment

r

100

2

65 kDa. Uroporphyrinogen m decarboxylase is encodeDd by hemE. B. subtilis hemE encodes a polypeptide of 40 ,347 Da, as deduced- from the nucleotide sequence, usin, g the ATG codon at position 1905. A 40.0-kDa polypeptii de was also found to be expressed from the B. subtilis hem]E gene in an E. coli in vitro transcription-translation system (Fig. 7). On the basis of amino acid sequence comparisons, HemE was clearly identified as uroporphyrinogen III dec,arboxylase.

8089

Ferrochelatase is encoded by hemH. Ferrochelatase genes have been sequenced from S. cerevisiae (14, 22), mice (4, 48), humans (31), Bradyrhizobium japonicum (12), and E. coli (visA) (30, 32). In Fig. 9, the deduced amino acid sequence of the B. subtilis hemH gene product is aligned with those of these other ferrochelatases. The eucaryotic sequences exhibit a high degree of similarity (48% identity). Those of the gram-negative bacteria, the B. japonicum hemH and E. coli visA products, are 43% identical. The B. subtilis HemH protein shows the highest degree of similarity to the mouse ferrochelatase polypeptide (28% identity). This degree of similarity is of low significance and is not alone sufficient to determine that B. subtilis hemH encodes ferrochelatase. Enzyme activity measurements were used to demonstrate that the B. subtilis hemH gene encodes ferrochelatase. To this end we expressed the gene from plasmid pLUG18e2 in E. coli JM109 and analyzed cell extracts. Plasmid pLUG18e2 is pUC18 containing the terminal part of hemE (from bp 2923), all of hemH, and the proximal part of hemY (to bp 4444). The hemH gene in pLUG18e2 is oriented so that it can be transcribed from the lac promoter of the vector. A 40.5-kDa polypeptide was found to be expressed from the B. subtilis hemH gene (Fig. 7). The expected mass for HemH is 35,348 Da, as derived from the DNA sequence. This discrepancy of about 5 kDa may be explained by an aberrant mobility of the HemH protein in SDS-PAGE because of a negative net charge of -22 for HemH. Such a phenomenon has been observed, e.g., for glutamyl-tRNA synthetase, which has a net charge of -20 (5). Yeast ferrochelatase can catalyze the incorporation of Fe2" as well as Zn2+ into protoporphyrin IX (7). We found high amounts of both of these activities in crude soluble cell extracts of E. coli JM109/pLUG18e2 but not in membranes or control extracts from E. coli JM109/pUC18. The specific activity (Fe2"), determined as described in Materials and Methods, of the E. coli JM109/pLUG18e2 cell extract was 29 nmol of protoheme formed per mg of protein, whereas that of E. coli JM109/pUC18 was less than 1.2 nmol of protoheme formed per mg of protein. As expected for a ferrochelatase, the activity of E. coli JM109/pLUG18e2 was dependent on the presence of protoporphyrin IX and ferrous ion. Incorporation of zinc was monitored by measuring the disappearance of protoporphyrin IX, i.e., by a decrease in fluorescence of the reaction mixture (Fig. 10). The zinc chelatase assay corroborated the results obtained with Fe2". A low zinc chelatase activity was observed in cell extracts of E. coli JM109/pUC18. This activity was less than 2% of that present in cell extracts from E. coli JM109/pLUG18e2 and may have originated from the endogenous E. coli ferrochelatase. (This low activity is not apparent at the protein concentration used in the experiment of Fig. 10.) Some chelatase activity was also found in JM109/pLUG18e2 if Zn2+ was not added to the reaction mixture, and this could have been due to contaminating Zn2+ originating from the

8090

J. BAcTERIOL.

HANSSON AND HEDERSTEDT hum rat yea Bsu Syn

MEANGLGPQG FPELKNDTFL RAAWGEETDY ... NGLGLQN FPELKNDTFL RAAWGEETDY FPAPKNDLIL RAAKGEKVER .MGN .MS KRETFNETFL KRARGEKADH ... .MVASSS LPR ..... LL RAARGEVLDR .......

........

*

*

RSPEACCELT RSPEACCELT RDAEIASEIT HQPELCAYVT ETPELAIEIS

LQPLRRFP.. LEPVRRFP.. IQPVRRYRGL RLPVEQYG.. LQPFRAFKP.

*

*.

*

VASELGYVFQ VASELGYVFQ VLKELDWAFK PEQDVPYVLE ..... --

----. ... ....

AKRWLYQRPQ AKRWLYQKPV AKQWINMYPE TKAFMYSMPD -

.

.....

*

*

*

*****

.......

MTPLPSIGVD VEIKNGIGPV IDQPIRSLAD GRV.PLIGFA

DALVPYLVGQ HALVPYLIGQ DVAVEFLSQQ DMIIVYVKAQ .......

-

.**

*

*

VVAGAQALQL VAAGAQALQL VVAGAQILQV IEAGAKAIQI -

..........

*

.........

-

......

GKTVTLQGNL GKTVTLQGEL KNRVTLQGNL GITKTVQGNL *

**

*

DPCALYASEE DPCALYASEE DPGVMYGSKE DPSILLAPWE **

VDAVHKHSRL LRQN... LDAVHKHSRL LRQN... LEECHRIGSK TAFVHEYSQN KKMGQYS *------

..........

*

EIGQLVKQML EIGRLVQQML VITKKVKQMI VIEQKTKEIL

*

.......

**

117 114

112 109 79

*

176 173 172 166

.

236 233 232 226

.......

........

*

*

FESHAGHLGP QLFNKFALPY FESHAGHLGS ELFSKFALPY FESWGGELSS VDFDEFSLPY FDSWVGALNQ ADYRTYIKPV

IRDVAKQVKA RLREAG.LAP VPMIIFAKDG HFALEELAQA GYEVVGLDWT IRDVAKRVKA GLQKAG.LTR MPMIIFAKDG HFALEELAQA GYEVVGLDWT LRQIAERVPK RLQELGIMEQ IPMIVFAKGS WYALDKLCCS GFDVVSLDWS .EN MNRIFSELAK VPLIMFGVGA SHLAGDWHDL PLDVVGLDWR *

*

GRVPLIGFAG APWTLMTYMV EGGGSSTMAQ GRVPLIGFAG APWTLMTYMV EGGSFKTMAQ GEVPLFGFCG GPWTLMVYMT EGGGSRLFRF LNVPLIGFSG APFTLASYMI EGGPSKNYNK ..........

59 56 52 51 51

*

LWVPQALGME VTMVPSKGPS FPEPLREEQD LVVPQALAME VTMVPGKGPS FPEPLREERD LVIPQAMGIIR VEMLEGKGPH FPEPLRNPED

**

AITLTRQRLA AITLTRQQLA AITMTRIKLD TIKLL.VNEQ

.

ASHQLLRILT ASHKLLGILT LSHKLLQKIT AWNLLMSKLA -

......

.

*

LDAAIIFSDI LDAAIIFSDI IDAAIIFSDI VDAAILYKDI .DGVILFSDI

*

LEALRDPEV. LERLRDPAA. LQTVLDYKVD IEKL..GQID

**

AA.QDFFSTC AA.QDFFSTC NN.RDFFQTC EKY.GLFEIT PPVWMMRQAG RYMKVYRDLR DKYPGFRERS

TPVWCMRQAG RYLPEFRETR TPVWCMRQAG RYLPEFRETR PPCWIMRQAG RYLPEYHEVK TPVWYMRQAG RSQPEYRKLK

***

VAPKKARECV VAPKKAREPV WDPREAVKIN LGIDEAR.SK

295 292 292 277

*

DDFGPH..RY NDFGPQ..RY EAFGGGKSRY DQ.GMESDGF

IANLGHGLYP IANLGHGLYP IVNFGHGTHP IFNLGHGVFP

DMDPEHVGAF DMDPEHVGAF FMDPDVIKFF DVSPEVLKKL

..........

..........

..........

353 350 352 336

*

367 364 362 353

.......

FIG. 8. Alignment of the predicted amino acid sequences of uroporphyrinogen III decarboxylase of humans (hum) (40), rats (39), S. cerevisiae (yea) (13), B. subtils (Bsu), and Synechococcus sp. strain PCC7942 (Syn) (20). For Synechococcus sp. strain PCC7942, only part of the sequence is known. Positions with identical amino acid residues in all five polypeptides (four after the end of the known Synechococcus sequence) are marked by asterisks.

cell extract itself and/or the buffer chemicals (Fig. 10, trace B). In all eucaryotes examined, ferrochelatase has been found to be an intrinsic mitochondrial membrane protein (9). Ferrochelatase has also been reported to be membrane bound in bacteria (9). A hydropathy plot of the B. subtilis ferrochelatase polypeptide does not indicate any obvious transmembrane segments (results not shown). The ferrochelatase expressed from B. subtilis hemH in E. coli JM109 was found predominantly in the soluble cell fraction and migrated as a soluble protein in gel filtration experiments (unpublished results). Does hemY encode a bifunctional protein? Comparisons of the sequence of the deduced polypeptide product of hemY and sequences available in the GenBank/EMBL data bases did not reveal any similarities. The search included the coproporphyrinogen III oxidase of S. cerevisiae, which is

the only example of a hemF sequence (52). It should be noted that protoporphyrinogen IX oxidase (hemG) sequence data are completely lacking. Data for mutants unambiguously show that the B. subtilis hemYgene is essential for protoheme IX synthesis. Both the previously isolated hemG321 mutation (3), located within the hemY gene (Fig. 3A), and a deletion of the major part of hemYprepared in this study (Fig. 3B) result in blocked heme synthesis. This block is in an enzymatic step after the formation of uroporphyrinogen III, because mutants defective in any of the hemEHY genes can synthesize siroheme; i.e., they do not require reduced sulfur in the growth medium. The finding of an apparent hemEHYmRNA further supports the conclusion that the hemY gene product has a role in the uroporphyrinogen III-to-protoheme IX biosynthetic pathway. Genes for the enzymes of the glutamyl-tRNA-to-uropor-

B. SUBTILIS hemEHY GENES

VOL. 174, 1992

Bja Eco

hum mou yea Bsu

MSTAAPNETT QPTVRSGQKR VGVLLVNLGT MRQTK TGILLANLGT .. GAKP QVQPQKRKPK TGILMLNMGG ...... TTKP QAQPERRKPK TGILMLNMGG ...... NAQKR S P . TGIVLMNMGG .. M S RKK MGLLVMAYGT .. .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

*

KVVLNGIILR WPLLRGVILP QNKLAPFIAK QNKLAPFIAK QKTIAKYIAK QD........

*

PDTADAPGVR VYLKEFLSDA RVIEDQGLVW PDAPTPEAVK RYLKQFLSDR RVVDTSRLLW PE..TLGDVH DFLLRLFLDQ DLMTLP..I. PE..TLGEVQ DFLQRLFLDR DLMTLP..I. PS..KVEETY DFLYQLFADN DLIPISAKY. PY..KEEDIE RYYTHI..RR GRKPEPEML.

QKIWNNEKNE SPLKTITRSQ SAKLAAALSD ASVWMEG..G SPLMVYSRQQ QQALAQRLPE RRI... .GGG SPIKIWTSKQ GEGMVKLLDE RRI .... GGG SPIKMWTSKQ GEGMVKLLDE FRTPKIEKQY REI.... GGG SPIRKWSEYQ ATEVCKILDK .LKDRY EAI.... GGI SPLAQITEQQ AHNLEQHLNE .....

GMRPHLAV.P EHVDHIVVLP DGLERAIAFT DGLERAIAFT DGVKKAVAFS DGITEAVSIV

*

RV..TPPYYE SF..IRDYAD KWSTIDRWPT KWSTIDRWPT SWSVIDRWPT TITSVESWYD

ATVCDEVFRV GAVWDELARI GSSLNAIYRY GSSLNAIYRY GSSINELWRQ LAPHFSTFSV QSYNK.. .RA

LYPQYSASTS LYPQFSCSTV QYPQYSCSTT QYPQYSCSTT QYPHFSYSTT *

DEAYIEALAV NHDYINALAN HHLLIQCFAD HPLLIQCFAD NEGLIKAFSE EPKFVTYWVD

SIETHLATLP SVRASFAKHG HILKELDHFP HILKELNHFP NITKKLQEFP RVKETYASMP

*

FKPE.. .LIV .EPD... LLL LEKRSEVVIL EEKRSEVVIL QPVRDKVVLL EDERENAMLI

ARRLDASKLL ALGMAPEKVM RLEY.CNPYR KLGY.PNPYR KLKF.KNPYR GAGV.SE.YA

LTFQSRF.GN MTFQSRF.GR LVWQSKV.GP LVWQSKV.GP LVWQSQV.GP VGWQSEGNTP

*

*

*

EIAQENAEIF EIAEQNREVF ELDIEYSQVL ELNIEYSQVL EIDLG.. .VI DNDYECKVVT

KHNGG.ETFS LGAGG.KKYE AKECGVENIR AQKCGAENIR GESEYKDKFK DDIGA..SYY

AIPCLNDSEP YIPALNATPE RAESLNGNPL RAESLNGNPL RCESLNGNQT RPEMPNAKPE

*

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*

.

.

.

.

.

.

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.

*

.

.

.

.

.

. ..

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Q.

LARLRAQPTL LARKRSIPGI YNQVGRKPTM

YNEVGQKPTM IKALDSERSI KEEAEKLGGL

175 156 165 163 160

139

ASFHGMPKSY LSYHGIPQRY FSAHSLPMSV FSAHSLPMSV FSAHSLPMDV VSAHSLPEKI *

VDKGDPYQEH ADEGDDYPQR VNRGDPYPQE VNRGDPYPQE VNTGDAYPAE KEFGDPYPDQ **

*

230 210 225 223 220 199

*

TMERL.AKEG TLKML.GEKG SIKGLCE.RG AIKGLCE.RG .IAEFLG.PK LTRDLFEQKG

VRRIAVVTPG VGHIQVMCPG RKNILLVPIA RKNILLVPIA VDGLMFIPIA YQAFVYVPVG

288 268 282 280 276 257

GMDVIRTLVL HIEMMANLVA FSKALADLVH FSKALADLVH FIEGMADLVK FIDALATVVL

RELQGWI... AYR.......

344 320 342 340 333 310

**

**

FAADCLETLE FAADCLETLE FTSDHIETLY FTSDHIETLY FTSDHIETLH FVADHLEVLY

DEWLQPYTDK EPWLMPYTDE MPWLGPQTDE VPWLGPQTDE KPWLGAQTAE DPWLGPDVQD

LSPNTAPHKY LSPATAPHKY TCPETAPHKP I.QDEITFKA

118 98 105 103 100 82

*

*

CIATTEALRA CRTTTRELAS VSATVQKVME VGATVHKVME VAATVYNIMQ LHESAKLIAE

RDHVVVDW.. ...MPVAL..

*

*

**

IKSGIDALIG LESAVDELLA TEEAIEEMER TEEAIEEMER YVAFRYAKPL TAETYKQMLK YIGLKHIEPF IEDAVAEMHK

60 45 49 49 44 40

*

QRPRSKALDY LRSPRVAKLY RRTPKIQEQY RRTPKIQE..

..AMRYGNPS ..GMSYGSPS YIGFRYVHPL YIGFRYVHPL

8091

SHIQSNELCS SHIQSNKLCS SHLQSNQLYS KKLGR.....

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KQLTLSCPLC VNPVCRETKS FFTSQQL.. TQLSLNCPLC VNPVCRKTKS FFTSQQL.. NQLPLDFALG KSNDPVKDLS LVFGNHEST

369 367 362

FIG. 9. Alignment of the predicted amino acid sequences of B. japonicum (Bja) (12), E. coli (Eco) (30, 32), human (hum) (31), murine (mou) (4, 48), S. cerevisiae (yea) (14, 22), and B. subtilis (Bsu) ferrochelatase. The amino-terminal sequences of the eucaryotic proteins are those of the processed polypeptides. Positions with identical residues in all six polypeptides are marked by asterisks.

phyrinogen III biosynthetic pathway in B. subtilis are organized in the hemAXCDBL operon. All the genes of this operon, except hemX, which is not essential for heme synthesis, have been identified and shown to account for the enzymes known to be required for the synthesis of uroporphyrinogen III from glutamyl-tRNA (16, 35, 36, 45). It would seem logical that the genes for the enzymes catalyzing the synthesis of protoheme IX from uroporphyrinogen III (Fig. 1) also constitute an operon. Four enzyme activities are required for these steps, but only three proteins seem to be encoded by the hemEHY gene cluster. Thus, the hemY gene product may be a bifunctional protein with both copropor-

phyrinogen III oxidase and protoporphyrinogen IX oxidase activity. These two enzymatic reactions are sequential oxidations in the protoheme IX biosynthetic pathway. The possibility of HemY being a HemF-HemG enzyme could explain apparent inconsistencies between the mapped locations of the B. subtilis hemF180 and hemG321 mutations and the resulting phenotypes. The hemF180 mutation seems to result in a defective coproporphyrinogen III oxidase, since mutant cells accumulate coproporphyrin IX. Assuming the presence of a functional ferrochelatase in such mutants, the hemF180 mutation would be located in hemY or affect the expression of hemY (see Fig. 3A for hemF180 mapping

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genes for uroporphyrinogen III synthesis and protoheme IX synthesis organized in separate transcriptional units since

A

B 0.1I rfi

C

D E I

0

I

I

1 2 Time (minutes)

FIG. 10. Ferfochelatase enzyme activity measurements. Incorporation of Zn2+ into protoporphyrin IX by soluble cell extracts of E. coli JM109 carrying pLUG18e2 (pUC18 containing B. subtilis hemH) or pUC18. The incorporation of Zn2+ was monitored by measuring the decrease in protoporphyrin IX concentration, i.e., the decrease in fluorescense of the reaction mixture. The following amounts of protein (in milligrams) of E. coli JM109/pLUG18e2 (traces B to E) and E. coli JM109/pUC18 (trace A) were added to the 2.5-ml reaction mixture: A, 0.33; B, 0.31 (Zn2+ omitted); C, 0.16; D, 0.31; E, 0.62. Trace D corresponds to a specific activity of 0.39 nmol/min/mg of soluble cell protein.

data). The hemG321 mutation is definitely located in hemY (Fig. 3A) but confers, in comparison with that conferred by hemF180, a somewhat different phenotype; it causes accumulation of coproporphyrin III and protoporphyrin IX (3), suggesting a defect in protoporphyrinogen IX oxidase. The hemG321 mutation may block the protoporphyrinogen IX oxidase (HemG) activity of the putative HemF-HemG protein, leaving residual coproporphyrinogen III oxidase activity of the mutant enzyme. The S. cerevisiae coproporphyrinogen III oxidase consists of 328 amino acid residues, as predicted from the nucleotide sequence. The B. subtilis HemY polypeptide is 470 or 472 amino acid residues long, depending on which ATG is used for translation initiation. This larger size may also indicate that B. subtilis HemY is a HemF-HemG bifunctional protein. Conclusion. Results from this work and previous studies (16, 36) are consistent with the conclusion that all the enzymes necessary for protoheme IX synthesis from glutamyl-tRNA in B. subtilis may be encoded by the hemAX CDBL and hemEHY gene clusters. However, the possibility of additional loci encoding coproporphyrinogen III oxidase and/or protoporphyrinogen IX oxidase cannot be excluded until the catalytic potential of the HemY protein has been analyzed. The gram-positive bacterium Staphylococcus aureus also appears to have a compact organization of hem genes. Genetic loci required for protoheme IX synthesis are tightly linked on the S. aureus chromosome (49). The hem genes of gram-negative bacteria in contrast are scattered on the chromosome, with the exception of the hemCD operon of E. coli (19, 44) and Salmonella typhimuium (50) and a possible hemACD operon in Chlorobium vibrioforme (24). Uroporphyrinogen III and protoheme IX are intermediates positioned at branch points in the synthetic pathway for various types of hemes; i.e., in B. subtilis, siroheme is synthesized from uroporphyrinogen III, whereas heme a, heme c, and heme d are synthesized from protoheme IX (17). For regulatory purposes it seems advantageous to have the

this would allow independent transcriptional control of siroheme and protoheme IX biosynthesis. Very little is known about the regulation of heme synthesis in bacteria, but 5-aminolevulinic acid, uroporphyrinogen III, and protoheme IX are likely to be key intermediates with respect to regulation. The two cloned and sequenced hem gene clusters are valuable tools for studies of the regulation of tetrapyrrole synthesis in B. subtilis. ACKNOWLEDGMENTS We are grateful to Karin Tsiobanelis for technical assistance. This work was supported by the Swedish Natural Science Research Council, the Swedish Medical Research Council, and Emil och Wera Cornells Stiftelse. REFERENCES 1. Arwert, F., and G. Venema. 1973. Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Mol. Gen. Genet. 123:185-198. 2. Bayly, R. C., S. Dagley, and D. T. Gibson. 1966. The metabolism of cresols by species of Pseudomonas. Biochem. J. 101:293301. 3. Berek, I., A. Miczak, I. Kiss, G. Ivanovics, and I. Durk6. 1975. Genetic and biochemical analysis of haemin dependent mutants of Bacillus subtilis. Acta Microbiol. Acad. Sci. Hung. 22:157167. 4. Brenner, D. A., and F. Fraiser. 1991. Cloning of murine ferrochelatase. Proc. Natl. Acad. Sci. USA 88:849-853. 5. Breton, R., D. Watson, M. Yaguchi, and J. Lapointe. 1990. Glutamyl-tRNA synthetases of Bacillus subtilis 168T and Bacillus stearothermophilus. J. Biol. Chem. 265:18248-18255. 6. Broome-Smith, J. K., A. Edelman, S. Yousif, and B. G. Spratt. 1985. The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding proteins 1A and 1B of Escherichia coli K12. Eur. J. Biochem. 147:437-446. 7. Camadro, J.-M., and P. Labbe. 1988. Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 263:11675-11682. 8. Dailey, H. A. 1977. Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii. J. Bacteriol. 132:302-307. 9. Dailey, H. A. 1990. Conversion of coproporphyrinogen to protoheme in higher eukaryotes and bacteria: terminal three enzymes, p. 123-161. In H. A. Dailey (ed.), Biosynthesis of heme and chlorophylls. McGraw-Hill Publishing Co., New York. 10. Devereux, J., P. Heberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 11. Friden, H., and L. Hederstedt. 1990. Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate:quinone oxidoreductase (complex II). Mol. Microbiol. 4:1045-1056. 12. Frustaci, J. M., and M. R. O'Brian. 1992. Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene. J. Bacteriol. 174:4223-4229. 13. Garey, J. R., R. Labbe-Bois, A. Chelstowska, J. Rytka, L. Harrison, J. Kushner, and P. Labbe. 1992. Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. Eur. J. Biochem.

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Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes.

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on...
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