Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter

Mol Gen Genet (1991) 226:250-256 002689259100099N

© Springer-Verlag 1991

Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter M. Drolet and A. Sasarman

Department of Microbiology and Immunology, Universit~ de Montr6al, Montr6al, Qu6bec, Canada, H3C 3J7

Received August 31, 1990 / November 16, 1990

Summary. The hemA gene of Agrobacterium radiobacter ATCC4718 was identified by hybridization with a hemA probe from Rhizobium meliloti and cloned by complementation of a hemA mutant of Escherichia coIi K12. E. coli hemA transformants carrying the hemA gene of Agrobacterium showed 6-aminolevulinic acid synthetase (6-ALAS) activity in vitro. The hemA gene was carried on a 4.4 kb EcoRI fragment which could be reduced to a 2.6 kb EcoRI-SstI fragment without affecting its complementing or 6-ALAS activity. The sequence of the hemA gene showed an open reading frame of 1215 nucleotides, which could code for a protein of 44361 Da. This is very close to the molecular weight of the HemA protein obtained using an in vitro coupled transcription- translation system (45000 Da). Comparison of amino acid sequences of the 6-ALAS of A. radiobacter and Bradyrhizobium japonicum showed strong homology between the two enzymes; less, but still significant, homology was observed when A. radiobacter and human 6- ALAS were compared. Primer extension experiments en- abled us to identify two promoters for the hemA gene of A. radiobacter. One of these promoters shows some similarity to the first promoter of the hemA gene of R. meliloti.

Key words: cS-Aminolevulinic acid synthetase (6-ALAS) - hemA gene - Agrobacterium radiobacter - Tetrapyr- roles - Vitamin B12

Introduction

6-Aminolevulinic acid (6-ALA) is the first intermediate of tetrapyrrole synthesis in all organisms. Heine, chlorophyll, and vitamin B12, which is produced only by some microbes, are all derivatives of this pathway. 6-ALA is synthesized either in three steps, via a five-carbon (5C) pathway from the intact carbon skeleton of glutamate (Castelfranco and Beale 1983) or in one step, by the condensation of glycine with succinyl-CoA (Granick and Beale 1978).

Offprint requests to .' A. Sasarman

In plants (Castelfranco and Beale 1983), algae (Mayer et al. 1987), cyanobacteria, methanogenic bacteria (Av- issar et al. 1989) and some purple bacteria, Escheriehia coli (Li et al. 1989a), probably Salmonella typhimurium (Elliot 1989) and Bacillus subtilis (Petricek et al. 1990), and many others (Avissar et al. 1989), 6-ALA is synthesized from glutamate. The hemA gene of E. coli K12 was the first gene of this pathway to be cloned and sequenced (Drolet et al. 1989; Li et al. 1989b; Verkamp and Chelm 1989). As shown by Avissar and Beale (1989), this gene codes for glutamyl-tRNA dehydrogenase, the second enzyme of the 5C pathway.

In animals, yeast, fungi and other bacteria the hemA gene encodes 6-aminolevulinic synthetase (6-ALAS) which catalyzes the condensation of glycine with succinyl-CoA to form 6-ALA. Several hemA genes of this 4C pathway have been cloned and sequenced, among them the hemA gene of Rhizobium meliloti (Leong et al. 1982; 1985), Bradyrhizobium japonicum (Robertson McClung et al. 1987) and Rhodobacter eapsulatus (Biel et al. 1988; Hornberger et al. 1990). In R. sphaeroides two genes re- sponsible for 6-ALA synthesis (hemA and hemT) have been isolated Tai et al. 1988). In eucaryotic organisms, several hemA genes have been cloned and sequenced, namely the hemA genes of Saecharomyces cerevisiae (Ar- rese et al. 1983; Bard and Ingolia 1984; Urban-Grimal et al. 1984; 1986), chicken liver (Borthwick et al. 1984; 1985; Maguire etal. 1986; Pirola etal. 1984), mouse liver and anemic spleen (Schoenhaut and Curtis 1986), rat (Srivastava et al. 1988), and human (Bawden et al. 1987). Comparison of amino acid sequences of the various cS-ALAS revealed close homology between them.

The regulation of the tetrapyrrole pathway has been studied mainly in the photosynthetic bacterium, R. sphaeroides (for a review see Lascelles 1975). In this bacterium, heine (Burnham and Lascelles 1963) and Mg- protoporphyrin (Yubisui and Yoneyama 1972) inhibit the activity of 6-ALAS, while trisulfides activate this enzyme (Sandy et al. 1975). It seems likely that the main regulatory mechanisms of the tetrapyrrole pathway in most bacteria act at the level of 6-ALA synthesis (Las- celles 1975), but we know very little about them.

Agrobacterium radiobacter ATCC 4718 is a good pro- ducer of vitamin B12, as shown in our laboratory (un-

published). The presence of the cobalamin pathway in this organism allows the study of the relationship between the regulation of vitamin B12 and of heme synthesis, without the interference of bacteriochlorophyll synthesis, as in Rhodobacter. In order to study this relationship, we decided to clone the hemA gene of A. radiobacter and to determine its nucleotide sequence. As a preliminary step to the study of the regulation of the hemA gene of A. radiobacter, we also identified two promoters for this gene.

Materials and methods

Bacterial strains and plasmids. The bacterial strains and plasmids used in the present study are listed in Table 1.

Media. Strains of E. coli K12 were routinely maintained on brain heart infusion agar supplemented, when re- quired, with appropriate antibiotics (10 gg/ml tetracycline or 50 pg/ml ampicillin), or with 6-ALA at 50 btg/ml. Strains of A. radiobacter and R. meliloti were routinely maintained on NZ-amine (Miller 1972) or LB media. YT medium supplemented with 0.1 mM isopropyl fi-I)- thiogalactopyranoside (IPTG) and 0.004% X-Gal (5- bromo-4-chloro-indolyl-~-D-galactopyranoside), was used for cloning with M13mp18 and M13mp19 vectors.

Enzymes and chemicals. Restriction endonucleases and DNA modification enzymes were purchased from Beth- esda Research Laboratories (Gaithersburg, Md.), Phar- macia PL Biochemicals, and New England Biolabs (Bev- erly, Mass.). Various chemicals used in cloning, DNA

251

sequencing, and other molecular biology experiments were from Sigma (St. Louis, Mo.), Bethesda Research Laboratories, and Boehringer Mannheim Biochemicals (Indianapolis, Ind.). L-[3SS]methionine was purchased from Amersham International (Arlington Heights, Ill.), and ~-[32p]nucleotide triphosphates were from ICN Pharmaceuticals (Irvine, Calif.).

DNA isolation. Isolation of chromosomal DNA of A. radiobacter and R. meliloti was performed as described by Silhavy et al. (1984). Large scale isolation of plasmid DNA was performed by the alkaline lysis method (Man- iatis et al. 1982). For rapid analysis of recombinant plas- raids, the method of Holmes and Quigley (1981) was used.

DNA cloning. Restriction digests, ligations and transfor- mations were done essentially as described by Maniatis et al. (1982).

Hybridization. Southern blot experiments were performed essentially as described by Maniatis et al. (1982). For high stringency conditions, the last wash in 0.1 x SSC and 0.1% SDS was performed at 55 ° C for 30 min and under low stringency this wash was done at 37 ° C for 10 min.

Catalase test. The qualitative catalase tests were performed as previously described by Sasarman etal. (1970).

f -ALAS assay. Bacteria (E. coli strains) were grown aer- obically in brain heart infusion broth to an O.D. of

Table 1. Bacterial strains and plasmids Relevant characteristics Source

Strains

Eseherichia coli SASX475 SAS2025 SAS2026 SAS2027 Rhizobium meliloti Agrobacterium radiobacter

Plasmids

pUC18 pUC19 pSAS1421

pSASI423

pSAS1424

pSAS1425

pSAS1426

hemA, recA, Tc r SASX475/pSAS1424, HemA + SASX475/pSAS1425, HemA + SASX475/pSAS1426, HemA + Prototroph

Prototroph

Apr ap r NheI-- Sau3A HemA + fragment from E. coli K12,

inserted in pUC18 Sall HemA + fragment from R. meliloti,

inserted in pBR322 EcoRI HemA + fragment from A. radiobacter,

inserted in pUC19 SstI-EcoRI HemA ÷ fragment from A. radiobacter,

inserted in pUC 19 Sau3A HemA + fragment (1.4 kb) from A. radiobacter, inserted in pUCJ 8

This laboratory This work This work This work ATCC 10310

ATCC 4718

Drolet et al. (1989)

This work

This work

This work

This work

Tc, tetracycline; Ap, ampicillin

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0.6 at 600 nm. a-ALAS activity in sonic extracts of bacteria was assayed by the procedure of Burnham (1970). Protein content was determined by the biuret method using human serum albumin as standard.

DNA sequencing. Nucleotide sequencing by the dideoxy chain termination method of Sanger et al. (1977) was carried out using phages M13mp18 and M13mp19 and the synthetic 17mer oligonucleotide primer, purchased from Pharmacia PL Biochemicals.

RNA isolation. RNA was extracted and purified from E. coIi K12 and A. radiobacter as described by Aiba et al. (1981).

Primer extension analysis. For the mapping of the 5' end of the hemA mRNA, the following protocol was used. Fifty micrograms of RNA was resuspended in 20 gl of a solution of 20 mM TRIS-HC1 pH 8.3,200 mM NaC1, 0.1 mM EDTA and 1 pmol of an oligonucleotide. After heating for 3 rain at 100°C the samples were cooled slowly to room temperature over a period of 45 rain. The mixture was then precipitated with ethanol and resuspended in 50 mM TRIS-HC1 pH 8.3, 5 mM MgCI2, 5 mM DTT, 50 mM KC1, 50 units RNAguard (from Pharmacia PL), 1.25 gg actinomycin D, 2 mM each ofdATP, dTTP and dGTP, 40 gCi ofc~ [32p]-dCTP and 20 units of AMV reverse transcriptase (Pharmacia PL). After incubation for 60 rain at 45 ° C, 2 mM cold dCTP was added, and incubation continued for 1 h. Fi- nally, 17 gl of a stop solution from the Sequenase kit was added to stop the reaction. We used the same synthetic oligonucleotide (complementary to nucleotides 632-613, see Fig. 2) for primer extension and for DNA sequencing of the corresponding region of the hemA gene.

In vitro plasmid DNA-directed translation. Proteins en- coded by the different recombinant plasmids were detected using an in vitro coupled transcription-translation system of E. coli purchased from Amersham Internation- al. The proteins were separated in 12% acrylamide gels according to Laemmli (1970).

Results

Cloning the hemA gene of A. radiobacter

In order to clone the hemA gene of A. radiobacter, a DNA probe was obtained from the corresponding gene of R. meliloti isolated in our laboratory (unpublished). The probe was hybridized under low stringency to the chromosomal DNA of A. radiobacter digested with either BamHI, PstI, SaII, EcoRI or HindIII. A positive signal was obtained with an EcoRI fragment of about 4 kb and, in the case of the other endonucleases, with fragments larger than 10 kb (results not shown). Since only one hybridization signal was obtained for each of the restriction endonuclease digestions, it was concluded that only one hemA gene was present in A. radiobacter. A large quantity of chromosomal DNA of A. radiobacter

was subsequently digested with EcoRI and following electrophoresis in a low melting temperature agarose gel, the gel region containing DNA of around 4 kb in size was isolated and the DNA was extracted. The DNA was ligated to phosphatase-treated pUC19 and used to transform the E. coli K12 hemA mutant, SASX475. Ap- proximately 1000 ampicillin-resistant transformants were obtained, and analysis of the plasmids carried by 20 of them indicated that about two-thirds contained inserts of 4-5 kb. Only four transformants were able to complement the hemA mutant, as shown by the for- mation of large colonies and production of catalase (heme-deficient mutants of E. coli form tiny colonies on solid media and are not able to produce catalase since heme is the prosthetic group of this enzyme; see Sasarman etal. 1968a, b). The presence of the hemA gene in these transformants was confirmed by determi- nation of a-ALAS activity in cellular extracts (see below).

Analysis of the plasmids carried by the four HemA ÷ transformants showed that all possessed an EcoRI insert of 4.4 kb (Fig. 1) which was oriented in one direction in two plasmids and in the opposite orientation in the other two. The EcoRI insert of one of these, pSAS1424, was extracted from an agarose gel and used as a probe for Southern blot analysis. Hybridization under high stringency was done with chromosomal DNA of A. radiobacter, digested with the same enzymes as used in the first Southern blot experiment with the R. meIiloti probe. The same hybridization patterns were obtained as with the R. meliloti probe, but with stronger signals, as expected for homologous DNA (results not shown).

To determine the location of the hemA gene on the EcoRI fragment, subfragments obtained with either SstI, KpnI or HpaI were recloned into the pUC18 and pUC19 vectors and transformed into SASX475. The smallest

I I I I I I I I I I 0 1 2 3 4 4.4 kb

Hem A + I I

Hem A -+ I I

Hem A- I !

rt-

Fig. 1. Comptementation studies. HemA +, complementation of the hernA mutation by the corresponding fragment, regardless of its orientation with respect to the fl-galactosidase promoter of the pUC vectors; HemA +, complementation of the hemA mutation only when the gene is in the same orientation as the fl-galactosidase promoter of the pUC vectors; HemA-, absence of complementation in both orientations

253

f ragment still able to complemen t the hemA m u t a n t after ~ i so TCGCGATAAT AGC/~TAGCC CGGCTCGGAA GCA'PI'GCCGA TC~GCACC GCCGACACCG 120 2 days i n c u b a t i o n was an EcoRI-SstI insert of 2.6 kb eeGATeGerG eGee~erGe eGeGeeGeGA AeAa"rAeeeG rGATGGeree Aee~T~eA

(Fig. 1), which gave a positive test in bo th or ientat ions . 18o Fur the r subc lon ing experiments with the EcoRI frag- GcGeeGG,,'a eGGCAeeGAT eG~GeGcce a~r~ecGTcr GeGTGeAGe'r TGe~GeGAe240

men t were per formed using par t ia l Sau3A digestion, i~IAGGCCGACGA GCGCAAATAT GATTGCC'I'*I~ ~TCATGAAAC T~CTCCAAC G~IACAATGA300

Two clones were ob ta ined that gave full complementa - GCTCTTCGGA TCGGTAGGCG TCCCATATTT ATCTGCGGCG ACTGCTACCT CCGACCGCCA360 t ion after i ncuba t i on for 1 day and accumula ted por- eAGAT~^ ACAA'I~CGAT GTCG~'I~AGG GAAGCCTCAT ~FI~CCAGGCA ~I~CCTGCGAG

420 phyrins. One of the clones was able to complemen t CGTa~,a~TCGA TTGTGTGCCA GACAACGGCG CGGTGCGAAA AAGGTTCCAA ATCTCGCGGT

P~-~ 480 SASX475 in both orientations, whereas the other, hav- ~TTGATGT GAATCAATTT TGTACTTTTC ATCAGAGTCT ATAAACGGTT TATTGG~ATG

540 ing the shortest insert (1.4 kb), failed to complemen t G'I~C~%.CA ACGGCG~.'I~ GACGCAGACG TGG~'~I~KAGC AGCTCGCa~.G GGTTGACGGT

~..~ 3 A ~ 600 when inserted in the reverse orientation (Fig. 1). This CCCGGTGTCG AGGCAGAAAA ACGATCTGGT CAATTCTGGA GCATGGCATG GACTTCGAGG

result al lowed us to determine the direct ion of t ranscrip- ,~ D F E 660 CATTTTTCAC AACGGAACTG CAAAGCCTGC ATTCTGAGGG ACGCTATCGC GTTTTTGCGG t i o n o f t h e h e m A g e n e o f A , radiobacter as being f rom ^ F F • T E L Q s ,. , s E G ~ Y R v F A

the r igh t -hand EcoRI site (Fig. 1) toward the KpnI and 720 ATATCGAACG CCAGCAGGGC AATTTTCCCC GCGCGACACG GTACAACGCC AATGGCCAAC

BglII sites. This result was conf i rmed subsequent ly by D ~ ~ ~ ~ ~ G ~ ~ ~ ~ A • ~ ~ ~ A ~ G 780

mapp ing the 5' end of the hemA m R N A (see below). GCAAGGACGT AACCGTCTGG TGCTCCAACG ACTATCTGGG CATGGGCCAG AACCCGAAAG

The presence of the hemA gene in t r ans fo rman t s was R K D V T V W e S N D Y L G M G Q N P K 840 by TCATCGAAGC CATGAAAGCC GCTATCGATC ACTGTGGCGC GGGTGCGGGA GGCACCCGGA

c o n f i r m e d t h e d e t e r m i n a t i o n o f g - A L A S activity i n v i E A M K A A I D H C e A e A e G T R

900 cellular extracts as described by B u r n h a m (1970). 6- A~C~GG TACCAACCAT TATCACGTCC TGCTTGAACA GGAACTTGCC GACCTCCATG

A L A S activity, which is absent in E. c o l i K ] 2 as this , ~ s G ~ ~ , ~ ~ v '~ ~ ~ ~ ~ '. A D ~ , 960

bac te r ium uses the g lu tamate pa thway for the biosynthe- GCAAGGAATC GGCGCTGATC TTCACCTCGG GTTATGTGTC AAACTGGGCC ACACTCGGCA sis of cS-ALA, was easy to determine in the hemA m u t a n t ~ ~ z s A ~. ~ ~ • s G V V S N W A ~ Z G 1020

hyb pl CGCTTGGCCA GAAAATTCCG GGCCTGATCA TTTTCTCGGA TGCGCTCAAC CATGCATCGA S A S X 4 7 5 h a r b o u r i n g v a r i o u s hemA + r i d a s m i d s • L G Q K I P e L I I F S D A L N H A S

1080 (Table 2). The higher ~5-ALAS activity observed for TGATCGAAGG CATCCGTTAT GGCCG'I~GCG AGCGGGTTAT CTGGAAACAC AACGATCTCG

SAS2027 is p robab ly due to the fact tha t the enzyme ~ I E G I R Y G R C E R V I W K H N D L 1140

in this s train appears to be synthesized unde r the cont ro l AAGATCTCGA AGCCAAGCTC AAGGCTGCCG ATCCGAACGC GCCGAAGCTG ATCGCTTTCG o f t h e l a c p r o m o t e r o f t h e p U C 1 8 v e c t o r . ~ ~ '" ~ A ~ ,~ K A A D ~ N A ,' ~ ~ ~ A 1200

by AATCCGTTTA TTCCATGGAT GGCGATATCG CGCCGATCAA GGAAATCTGC GATCTGGCCG Sequencing the hemA gene was per formed the di- ~ s v Y N M D G D I A P I K E I C D L A

1260 deoxy chain termination method of Sanger et al. (1977). ACCGTTATGG CGCCATGACC TATCTCGATG AAGTTCATGC CGTCGGCATG TATGGCCCGC

Both s t rands were sequenced f rom the NruI site, which D R Y G A M T Y L D E V H A V G M Y G P 1320

is 587 bp ups t ream f rom the start codon, to the Sau3A GCGGCGGCGG CATTGCCGAG CGCGAAGGTC TCATGGATCG CCTGACGATC ATCG/b~GGAA

site, which is 130 bp downs t r eam f rom the stop codon R G G G I A E R E G L M D R L T I I E G 1380 (1935 ) CGCTCGGCAA GGCCTTCGGC GTTATGGGCG GCTATATTAC CGGGTCCACG GCGGTCTGCG nucleotides. We sequenced along stretch of nucle- T L G K A F G V M U G Y I T G S T A V C

1440 otides ups t ream of the start codon to enable compar i son ACTTCATCCG TT~-FFI'CGCC TCCGGTTTCA TCTTCACGAC GGCCCTGCCG CCGTCGCTCG

w i t h t h e 5 ' region of the hemA gene of R. meliloti and D F I a S F A S e F I F T T A L P P S L 1500

jap a j ( O R F ) CCGCCGGCGC GATCGCCTCG ATCCAGCATT TGAAGGCAAG CCCCTTCGAG CGTGCCCGCC B. onicum. One m o r open reading frame A A G A I A S I Q H L K A S P F E R A R

star t ing f rom nucleot ide 588 and ending at nucleot ide ls6o (Fig 2) ATCAGGACCG GGTGCGCAAG CTGCGCGGCC TTCTCGATGC ACGTGGCATT CCGCATATGG

1802 was identif ied . . The or ien ta t ion of this O R F . ~ D R v ~ K ~ R G ~. ~ D A ~ ~ ~ ~ n

1620 corresponds to the o r ien ta t ion of the t ranscr ip t ion of AeA~eeCAG CCACATCGTG CCTGTCATGG TGGGTGATGC CGCC~GTGC ~TGGATTT

the hemA gene as deduced by the c loning exper iment D N P S H I V P V M V G D A A K C K W I 1680

dig CGGATATCCT GCTCGACAGT CACGGCGTCT ATGTGCAGCC GATCAACTAT CCGACCGTGC with p a r t i a l S a u 3 A estion. The amino acid sequence s D ~ z ~ ~ s H ~ v ~ v e ~ ~ ~ ~ ~ • v of the hemA gene, as deduced f rom the nucleot ide se- 1~40

CGCGCAAGAC CGAGCGCCTG CGCATTACCC CGACGCCGCT GCATAGCGAT GCCGACATCG quence, indicates a protein with a molecular weight of ~ R K T E R L R I T P T P L H S D A D I

1800 44361 Da, which is very close to that found for the c~- AGCATCTCGT CGGCGCGCTG CACCAGCTCT GGTCACATTG TGCACTGGCG CGTGCGGTGG

A L A S o f B . j a p o n i c u m ( 4 4 5 9 9 D a ; R o b e r t s o n M c C l u n g ~ " ~ v G ^ ~ ~ ~ Z W s H e A ~ A ~ A v 1860 et al. 1987). ACGTGAGGCCG~,** CAG~AAGAT AAA.AGAAGAC CGGGCCGCGT "/'~I'ACGCGGCC ~CT'I'T,l~G1920

TGGCGATCAA TTCACGGGCG TGACGACGGG CTTCCTCATC CGCTGCGAAC ACATCTTCCA 1935

TCGTGGCGGC AGATC ~.~3A Table 2. 6-Aminolevulinic acid synthetase (ALAS) activity of var-

ious hemA clones a

Time (rain) cS-ALAS activity (nmoles of 6-ALA formed per mg of protein)

SAS2025 SAS2026 SAS2027 SASX475

0 0 0 0 0 10 12 13 20 0 2O 22 24 40 0 30 32 39 58 0

a J-ALAS activity of cellular extracts in the presence of succinyI- CoA and glycine. See Table i for a description of the strains

Fig. 2. Nucleotide sequence of the hemA gene of Agrobacterium radiobacter and the deduced amino acid sequence of the HemA protein. A potential Shine-Dalgarno sequence is underlined. PI and P2 refer to the two transcriptional start sites mapped by primer extension (see Fig. 3)

Mapping o f the 5'end o f the h e m A m R N A

The 5' ends of the hemA m R N A were mapped by pr imer extension, us ing R N A f rom A. radiobacter A T C C 47] 8 grown in LB medium. Two major bands , cor responding to start sites at nucleotides 569 and 472 (see Fig. 2),

472

P E A T G C A a C D E F G

- 6 8

569

254

Fig. 3. Mapping of the 5' end of the hernA mRNA by primer extension. RNA was extracted from A. radiobacter grown in LB medium. The numbers refer to the position in the nucleotide sequence represented in Fig. 2. PE, primer extension

A. radiobacter GGGTTGAC.. GGTCCCGGTGTCGAGGCAGAAAAACGATCTGG I I I I I I I I I I I I I I I I I I I I I I I I I I

R. melill2ti GGGTTGACCAC TGATC GCT T TGAAGGAAGAAAGGCGA . CAGG

Fig. 4. Alignment of the P1 promoter region of the hemA gene of A. radiobacter and Rhizobium meliloti (Leong et al. 1985)

were obtained (Fig. 3). Analysis of the promoter regions deduced from this primer extension experiment, allowed us to identify a T T G A C sequence (starting at position 532; see Fig. 2), located 31 bp upstream from the first start (569) of transcription (P1). This sequence is very similar to the - 3 5 consensus sequence of the E. coli a70 promoters. In the - 1 0 region of the P1 promoter there is a purine-rich sequence (from nucleotides 550- 564; see Fig. 2) which is also found in the same region of the two promoters of the hemA gene of R. meliloti (Leong et al. 1985). Very weak homology is found between these - 1 0 regions and the consensus - 1 0 region of the E. coli 0-70 promoters. An alignment of the PI promoters of the hemA gene of R. meliloti and A. radiobacter is shown in Fig. 4.

Seven base pairs upstream from the P2 transcription start, a TATAAA sequence is found, which is very similar to the consensus - 10 region of the E. coli ~ 70 promoters. However, the - 3 5 region of the P2 promoter

- 4 5 l>

- 2 4

- 18

Kd

Fig. 5. Identification of the hemA gene product by in vitro DNA- dependent translation. Lane A, pBR322; lane B, pSAS1423; lane C, pSAS1425; lane D, pSAS1426; lane E, pSASI424; lane F, pUC18; lane G, pSAS1421 (for a description of the plasmids, see Table 1). The arrow indicates the HemA protein of A. radiobacter

(TTTTGT) is rather atypical when compared to the consensus - 3 5 region of the same organism.

A promoter activity upstream of the hemA gene of A. radiobacter was detected in E. coli following cloning of the NruI-EcoRV fragment (nucleotides 1-662; see Fig. 2) in the p K O l l promoter probe vector (galK as the reporter gene, McKenney etal . 1981; data not shown).

In vitro protein synthesis directed by the hemA gene

Plasmid DNA from various clones was used in an in vitro prokaryotic DNA-directed translation system fol- lowed by electrophoresis in 12% polyacrylamide gels. As seen in Fig. 5, all hemA + clones from A. radiobacter are able to produce a polypeptide of about 44000 Da, which is close to the molecular weight of 44361 Da deduced from the sequence of the hemA gene. As a molecular weight control, the plasmid pSAS1421 containing the hemA gene of E. coli K12 (Drolet et al. 1989) was also used in this in vitro system (lane G, HemA of E. coli, approximately 46000 Da).

Comparison of amino acid sequences o f 6 - A L A S

A comparison was made between the deduced amino acid sequence of the A. radiobacter 5-ALAS and those published for R. meIiloti (Leong et al. 1985), B. japonicum (Robertson McClung et al. 1987) and human (Baw- den et al. 1987) (Fig. 6). There is 70% identity between A. radiobacter and R. meliloti proteins over the 76 amino

255

1 50 A STFHY DRFFEKY.S_DEKN DDHTYRVFRTVNRRAHI FPMADDY S DS LI TK~Q~SVWCS NDY B . . MDYSQFFNSALDRLHTERRYRVFADLERMAGRFpHAIWH • SPKG. KRDVVIWCSNDY C . . MDFEAFFTTELQS~SEGRYRVFADIERQQ~NFPRATRY. NANGQRKDVTVWCSNDY D MMMDFE S FFKNELDGLHQEGRYRVFADLARI{RGS FPKATRY . TADG . AQEVTVWCSNDY

i00 A LGMSRHPRVCGAVMDTLKQHGAGAGGTR~I SGT S KFHVDLERE LADLHGKDAALLFS S C B LGMGQHPKVVGAMVETATRVGTGAGGTRNIAGTHHPLVQ_~_AELADLHGKEAALLFTS G C LGMGQNPKVl EAMKAAI DHCGAGAGGTRNI S GTNHYHVLLEQE LADLHGKE SALI FTS G D LGMGQC_PIVTEAMKNAI DE

150 A FV_AHDSTLFTLVKMMPGCE I YS DS GDHASMI QGIRNSRVPKYI FRHNDVSHLRELLQRS B YVSN_QTGIAT IAKLI PNCLI LS DELNHS SMIEGIRQSGCERQVFRHNDLADLEALLKAA C YVSNWATLGTLGQKI PGLI_I FS DALNHASMIEGIRYGRCERV_IWKHNDLE DLEAKLKAA

200 A DPSVPKIVAFETVHSVDGAVLPLEE LCDVAHEFGAITFVDEVHAWGLYGARGGGIGDRD B GANRPKLIACE S LYS MDGDVAPLAKI CDLAEKYNAMTYVDEVHAVGMYGPRGGGIAERD C DPNAPKLIAFE SVYSMDGDIAPIKE I CDLADRYGAMTYLDEVHAVGMYGPRGGGIAERE

250 A GVMPKMDI I SGTLGKAFGCVGGY IASTRS LMDTVRS YAAGFI FTTS LPPMLI~GALESV B GVMHRI DI LEGTLAKAFGCLGGY IAANGRI I DAVRS YAPGFI FTTALPPAI CSAATAAI C G LMDRLTI I EGTLGKAFGVMGGYITGSTAVCDF IRS FAS GFI FTTALPPS LAAGA IAS I

300 350 A RI LKSAEGRVV~_ Q~RN~_QMLMDAGLPVVHCPSHI I PVRVADAAKNTEVCDE_LM B KHLK.. T_S SWERERHQDRAARVKAI LNAAGLPVMS S DTHIVPLF I GDAEKCKQAS DLLL C QHLK.. AS PFERARHQDRVRKLRGLLDARG I PHMDNPS HIVPVMVGDAAKCKWI S DILL

40O A S _PJ~NI YVQAINY PTVPRGEE LLRIAPTPHHTPQMMNYFLENLLVTWKQVGLE LKPHS S A B EEHG I YI 0PINYPTVAKGS ERLRI TPS PYHDDGLI DQLAEALLQVWDRLGLPLKQKSLA_ C DSHGVYVQPI NY PTVPRKTERLRI TPTPLH_S DADIEHLVGALHOL_WSHCA_LARAVA*

A ECN B AE*

Fig. 6. Comparison of the deduced amino acid sequences of human (A, amino acids 197-612, Bawden et al. 1987), Bradyrhizobiumja- ponicum (B, Robertson McClung et al. 1987), A. radiobacter (C) and R. meliloti (D, the 76 known amino acids, Leong et al. 1985)

acid sequence published for R. meIiloti. For the same region, the B. japonicum and A. radiobacter proteins show 54% identity and those of R. meliloti and B. japonicum, 53%. When the entire length of the A. radiobacter protein was compared with the B. japonicum protein, we found 60% identity, while the same comparison between the A. radiobacter and human proteins showed 48% amino acid identity. After including in this comparison the highly conserved amino acids as described by Dayhoff et al. (1972), the percentage homology between the HemA proteins of A. radiobacter and B. japonieum increased to 81% and that between the A. radiobacter and human proteins to 71%.

Discussion

A. radiobacter, a close relative of R. meliloti, uses for the synthesis of 6-ALA the C4 (succinate) pathway, in contrast to the C5 (glutamate) pathway used by E. coli. In spite of the presence of different pathways for the synthesis of 6-ALA, the hemA gene of A. radiobacter is able to complement a hemA mutant of E. coil K12. Similar complementation of E. coli hemA mutants has already been described in the case of the hemA gene of R. meliloti (Leong et al. 1982) and of mouse (Schoen- haut and Curtis 1986). The interesting point here is that these cases do not represent a classical genetic complementation, where the complementing cistron is part of the same pathway as the deficient one.

We observed that the complementation of the hemA mutant of E. coli K12 was very slow (2 days) even when

the hemA gene was present on a very high copy number vector (in pUC, more than 200 copies per cell). We also observed that the fragment cloned in pKO11 vector allowed E. coli cells to metabolize galactose very slowly. Consequently, we have to conclude that these hemA promoters are poorly active in E. coli. Moreover, when the hemA gene of A. radiobacter was cloned in the low copy number vector pRK290 and used to transform a hemA mutant of E. coli K]2, only very weak complementation was observed after prolonged incubation (not shown). The same situation was observed with the hemA gene of R. meliloti cloned in the same vector (Leong et al. 1982) and used to transform the same E. coIi hemA mutant. It is of interest to note that the hemA gene of Rhodobacter capsulatus has been shown to be able to complement a hemA mutant of E. coli only when the gene was under the control of the lac promoter of the pGEM vector (Hornberger et al. 1990)

Comparison between the P1 promoters of the hemA genes of R. meliloti and A. radiobacter revealed a high degree of homology. The fact that the unusual " 1 0 regions (purine-rich) of these promoters are similar may reflect the presence of an alternative factor essential for efficient transcription of these hemA genes. The absence of such a factor in E. coli may explain why these hemA gene are so poorly expressed in this bacterium.

Homology studies between the first 76 amino acids of the HemA protein of R. meliloti with those of A. radiobacter and B. japonicum, revealed a higher degree of amino acid sequence identity between R. meliloti and A. radiobacter. This suggests that R. meliloti and A. radiobacter are more closely related than, at least, R. meliloti and B. japonicum. Comparison between the entire length of HemA proteins revealed the presence of strong homology between such evolutionary distant organisms as bacteria and human. This suggests major structural constraints for the activity of 6-ALAS, as suggested by Robertson McClung et al. (1987) from comparisons between 6-ALAS of chicken and B. japonicum.

Acknowledgments. We gratefully acknowledge the valuable help of D. Tardif in maintaining our collection of strains. We thank H. Bergeron for the preparation of figures. This study was supported by grant OGP0036561 from the Natural Sciences and Engineering Research Council of Canada and grant CAFIR-196 from Univer- sit6 de Montr6al to A. Sasarman. M. Drolet was the beneficiary of a studentship from the Medical Research Council of Canada.

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C o m m u n i c a t e d by A. K o n d o r o s i

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Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter