Vol. 172, No. 5

JOURNAL OF BACTERIOLOGY, May 1990, p. 2250-2258

0021-9193/90/052250-09$02.00/0

Cloning and Characterization of the hemA region of the Bacillus subtilis Chromosome MIROSLAV PETRICEK,t LARS RUTBERG, INGRID SCHRODER, AND LARS HEDERSTEDT* Department of Microbiology, University of Lund, Solvegatan 21, S-223 62 Lund, Sweden Received 26 October 1989/Accepted 29 January 1990 A 3.8-kilobase DNA fragment from Bacilus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteineto-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coi HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. colt hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen m cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.

hemB (14, 30), hemC (65), and hemD (25, 55) have been determined. The derived amino acid sequences are very similar to those of the corresponding genes from humans (hemB [71] and hemC [51]) and yeasts (HEM2, i.e., hemB [44]), confirming the conserved nature of the heme biosynthetic pathway from ALA. In B. subtilis, mutations causing ALA or heme auxotrophy have been mapped at two loci. Four genes, called hemA, hemB, hemC, and hemD, are located at 2450 on the B. subtilis chromosomal map (8, 28, 43). HemA mutants are ALA auxotrophs, and hemA has been suggested to code for ALA synthase (1, 8). All other B. subtilis Hem mutants are heme auxotrophs. On the basis of physiological studies and measurements of enzyme activities, hemB, hemC, and hemD are suggested to code for porphobilinogen synthase (EC 4.2.1.24), porphobilinogen deaminase (EC 4.3.1.8), and uroporphyrinogen III cosynthase (EC 4.2.1.75), respectively. Three other B. subtilis hem genes have been mapped at 90°. They are proposed to code for uroporphyrinogen III decarboxylase (EC 4.1.1.37), coproporphyrinogen III oxidase (EC 1.3.3.3), and ferrochelatase (EC 4.99.1.1) (8). No mutants defective in protoporphyrinogen oxidase (EC 1.3.3.4) have been reported. In mammals, it has been proposed that ALA synthase is the rate-limiting enzyme in heme synthesis and is subjected to feedback inhibition by heme. Recent experiments indicate, however, that heme affects the rate of synthesis of ALA synthase rather than its activity (40). Virtually nothing is known about the control of hem genes in bacteria. B. subtilis is an aerobic bacterium with an absolute dependence on heme for growth (23). The amounts and types of cytochromes made by B. subtilis vary (67) according to growth conditions, suggesting a regulatory coupling between apocytochrome and heme synthesis. In yeasts, heme is a cofactor in transcriptional control of several apocytochrome genes (22, 44). To better understand the mechanism of heme synthesis and its regulation in B. subtilis, we have begun to isolate genes involved in heme synthesis. In this report, we describe the cloning of hemA and adjacent regions of the B. subtilis

Heme is the prosthetic group of cytochromes and thus serves an essential function in electron transport. The first step leading to heme is synthesis of 5-aminolevulinic acid (ALA). The biosynthetic pathway from ALA to heme involves seven different enzymatic steps and seems to be highly conserved in animals, plants, and bacteria (20). ALA is synthesized by either of two major pathways. In the C4 pathway, ALA is formed by condensation of glycine and succinyl coenzyme A. This reaction is catalyzed by ALA synthase (EC 2.3.1.37), which is found in animals (40), yeasts (68), and some bacteria (e.g., rhizobia) (41, 63). In the C5 pathway, ALA is formed from glutamate, starting with a charged tRNAGlU and involving an NAD(P)H-dependent reductase and a glutamate-l-semialdehyde 2,1-aminomutase (EC 5.4.3.8) (26). The C5 pathway is found in plants (7, 26) and several bacteria (3, 4, 19, 47, 48). In bacteria, the organization of genes involved in heme synthesis has been studied most extensively in Escherichia coli (5), Salmonella typhimurium (52), and Bacillus subtilis (49). In E. coli, genes hemB to hemH code for the seven enzymes needed to make heme from ALA (5). hemC and hemD map at 85 min and form a uro operon (55). hemG maps close to the uro operon at 86 min, whereas hemB, hemE, hemF, and hemH all map at different positions on the chromosome (5), indicating that they each represent separate transcription units. Mutations to ALA auxotropy in E. coli have been mapped at two separate loci, hemA (57) and popC (50); mutations in S. typhimurium have been mapped at hemA (56) and hemL (16). hemA has been cloned from both of these enteric bacteria, in which they seem to form an operon with the gene for release factor 1 (15, 31). The derived amino acid sequences for these two HemA proteins are very similar. The E. coli hemA has recently been shown to encode the NAD(P)H:glutamyl-tRNA reductase of the ALA C5 pathway (3). The nucleotide sequences of E. coli *

Corresponding author.

t Present address: Department of General Microbiology, Institute of Microbiology, Czechoslovak Academy of Sciences, Videnska 1083, 14220 Prague, Czechoslovakia. 2250

hemA REGION OF THE B. SUBTILIS CHROMOSOME

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TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains B. subtilis 168 3G18 BR95 KAll LUll 1A440 E. coli MM294 AN344 JM83 JM103 GM161 Plasmids pHV32 pUC18 pUC19 pBR322 pBLE1 pLUP402 pLUP502

pLUP5O5 pLUP507 pLUP509 pLUP510 pLUP512 pLUP513

pLUP515 a

Description

Source or reference

Our collection G. Venema J. Spizizen

Prototroph trpC2 met ade trpC2 pheA ilvCI trpC2 met hemA20 trpC2 ilvCI hemA20 trpC2 hemAl

BR95 transformed with KAll DNA Bacillus Genetic Stock Center

thi pro hsdR supE4 pro leu hemA ara rpsL A(lac-proAB) thi rpsL sbcB15 endA hspR4 supE A(lac-proAB) dam

L.-O. Heden 34 74 74 E. Holmgren

Apr Tcr Cmr Apr Apr Apr Tcr Apr Pmr; ble of pUB110a cloned in the SmaI site of pUC18 The hemA4 0.6-kb HindIII-PstI (Fig. 1A) fragment was cloned in M13mpl8. This vector was then cleaved with BamHI and HindIII, and the resulting hemAcontaining fragment was cloned in pBR322 to give pLUP402. SalI fragment of pLUP211 (Fig. 1A) cloned in pUC18 PstI-BgI fragment of pLUP211 cloned in pUC18 SphI-BcIl fragment of pLUP211 cloned in pUC19 SphI-MluI fragment of pLUP211 cloned in pUC19 pLUP507 deleted for the hemA internal Sall fragment SphI-PstI fragment in pLUP509 replaced by EcoRI-PstI fragment from pLUP402 EcoRV-EcoRI fragment from pLUP330 (Fig. 1A) cloned in PUC19 pLUP513 deleted for the 0.6-kb Sall-MluI fragment

46 74 74 9 H. Friden This work This work This work This work This work This work This work This work This work

See reference 35.

chromosome, including hemC, encoding porphobilinogen deaminase. The predicted amino acid sequence of the HemA protein from B. subtilis has a high degree of similarity to those of the E. coli and S. typhimurium HemA proteins, suggesting that also in B. subtilis ALA is synthesized from glutamate. Recently, O'Neill and co-workers (48) have shown that ALA can be formed from glutamate in extracts from B. subtilis. MATERIALS AND METHODS Bacterial strains and plasmids. The bacteria and plasmids used are described in Table 1. Media and growth of bacteria. B. subtilis strains were kept on tryptose blood agar base (Difco Laboratories) plates. Spizizen minimal medium (61) was used, with required amino acids added at 25 mg liter-1. To media containing hemin (2.5 mg liter-1), cysteine (20 mg liter-1) and 0.5% bovine serum albumin fraction V (Sigma Chemical Co.) were also added. Hemin stock solutions were prepared as described previously (42) except that Tween 20 was used instead of Tween 80. E. coli strains were kept on LA plates (38). For preparation of chromosomal or plasmid DNA, the bacteria were grown in LB broth. Antibiotics were used at the following concentrations (milligrams per liter): ampicillin, 50; chloramphenicol, 12.5 (E. colt) or 3 (B. subtilis); and phleomycin, 10 (E. colt) or 0.2 (B. subtilis). Competent B. subtilis and E. coli were prepared as previously described (2, 37). Competent cultures were kept in 10% glycerol at -70°C until used. DNA techniques. Large- and small-scale preparations of

plasmid DNA were done as described in Ish-Horowicz and Burke (24) and Kieser (27), respectively. B. subtilis chromosomal DNA was extracted by the procedure of Marmur (39). General DNA techniques were as described by Maniatis et al. (38). Southern blot analyses were done with digoxigeninlabeled probe DNA, using a kit obtained from Boehringer GmbH. DNA sequence analysis was done by the dideoxychain termination method (53), using modified T7 DNA polymerase (Sequenase; U.S. Biochemical Corp.) and [a35S]dATP on single-stranded M13mpl8 and -mpl9 (74) DNA or on plasmid DNA. Computer analysis of the nucleotide sequences obtained was performed with the GSG sequence software package, version 5.3 (12). Restriction endonucleases, T4 DNA ligase, and E. coli DNA polymerase Klenow fragment were from Boehringer or New England BioLabs, Inc. Construction of B. subilis deletion-substitution mutants. Mutants with deletions in the hemA region were constructed by transforming strain 3G18 with linearized pHV32 derivatives containing the cat gene of pHV32 or the ble gene of pBLE1 flanked by B. subtilis chromosomal DNA. The principle has been described previously (18). The restriction sites in the chromosomal DNA (Fig. 1) used in construction of the deletion-substitution mutants (see Fig. 7) were as follows: A401, HindIII and PstI; A404, HindIII and Bcll; A407, EcoRI and PvuI; A410, MluI and PvuI; and A411, NsiI and HindIII. Other methods. In vitro coupled transcription-translation was done with a kit from Amersham Corp., using [35S] methionine. Proteins were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)

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Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. T...
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