JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4893-4896 Vol. 173, No. 15 0021-9193/91/154893-04$02.00/0 Copyright © 1991, American Society for Microbiology Primary Structure, Expression in , and Properties of S-Adenosyl-L-Methionine:Uroporphyrinogen III Methyltransferase from Bacillus megaterium C. ROBIN,lt F. BLANCHE,2 L. CAUCHOIS,1 B. CAMERON,1 M. COUDER,2 AND J. CROUZETl* Unite de Biologie Moleculaire, Institut des Biotechnologies,' and De'partement de Chimie Analytique,2 Rhone-Poulenc Rorer S.A., Centre de Recherche de Vitry-Alfortville, 13 Quai Jules Guesde B.P. 14, 94 403 Vitry sur Seine Cedex, France Received 11 March 1991/Accepted 21 May 1991 A BacUlus megatenium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen Im methyltrans- ferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Esche- richia coli, partially purified, and shown to exhibit, like P. denirficans SUMT, substrate inhibition at uroporphyrinogen m concentrations above 0.5 ,uM, suggesting a common regulation for aerobic cobalamin- producing organisms.

S-Adenosyl-L-methionine (SAM):uroporphyrinogen III tion (carried by strain B5548 [5]) for cysteine auxotrophy on (urogen III) methyltransferase (SUMT), a key in the M9 medium (15). The 4-kb fragment was reduced to 2 kb coenzyme B12 pathway, is the first enzyme to occur after the through a partial Sau3AI digestion of pGX3512 and cloning branching of the cobalamin and heme pathways. It catalyzes into the BamHI site of pUC129 (13). One recombinant the transfer of two methyl groups from SAM to urogen III at plasmid, named pXL1804, still complementing strain B5548 C-2 and C-7 to give precorrin-2, a common intermediate in was obtained (Fig. 1). An internal XbaI-SspI 1,005-bp frag- corrinoids, siroheme, and F430 synthesis (9, 14). Two en- ment was sequenced on both strands (Fig. 1) by using the zymes having SUMT activity have already been studied. techniques of Sanger et al. (19) and Henikoff (11). An open The Pseudomonas denitrificans enzyme is encoded by the reading frame extending from position 119 (ATG) to 835 cobA gene, and P. denitrificans mutants deficient in SUMT (TGA) was found (Fig. 2). The deduced sequence of amino activity have a cobalamin-minus phenotype (1). The P. acids (238 residues) corresponded to a polypeptide of Mr denitrificans enzyme is a homodimer of Mr 2x 29,200 with a 25,800 showing 43.5% strict homology to P. denitrificans low turnover number and an activity that exhibits a substrate CobA protein (5) (Fig. 3). Since the encoded protein was inhibition phenomenon at urogen III concentrations above 2 homologous to P. denitrificans CobA protein and had a ,uM (1). The Escherichia coli cysG product is involved in the molecular weight quite similar to it (25,800 compared with synthesis of siroheme and has a molecular weight of 49,900 29,200) the gene for this protein was named cobA. The (17). This protein has been found to catalyze the same 1.3-kb XbaI fragment from pXL1804 carrying the cobA gene activity as the P. denitrificans CobA protein (20). Both when in both into the XbaI proteins show more than 40%o strict homology on most of the (Fig. 1), subcloned orientations CobA primary sequence (5). However, the CysG protein has site of pUC129, did not allow the complementation of mutant an NH2-terminal domain (more than 200 amino acids) whose B5548, indicating that expression of the CobA protein was function has been tentatively proposed (5) to be associated not sufficient to complement the cysG mutation. This sug- with the transformation of precorrin-2 into siroheme (involv- gests that at least another gene carried by pXL1804 insert is ing oxidation and iron chelation). We are interested in necessary for siroheme synthesis. One possibility is that understanding the regulation of the cobalamin pathway and such a protein is homologous to the NH2-terminal domain of in improving the productivity of strains used for cobalamin the E. coli CysG protein and/or allows the conversion of production. with SUMT activity are of great precorrin-2 into siroheme. interest, since their activities, in competition with that of A plasmid that enables the expression of the CobA protein urogen III decarboxylase, determine the metabolic flux was constructed. Twenty cycles of a polymerase chain entering the cobalamin pathway rather than the heme path- reaction with the pXL1804 sequence using the primers 5'- way. We have chosen to study the SUMT structural gene GGCCGAATTCAIAI(GGGAAAGTATAT-3' and 5'-GG from Bacillus megaterium, a strict aerobe that produces high CCGAGCTCTACGCGGCTGCC-3' were carried out (18). levels of cobalamins (8) and in which the genetics of cobal- The primers have on their sequences, as underlined, NdeI amin synthesis have been studied (4). and SstI restriction sites, respectively, and their 3' se- We used plasmid pGX3512 (the generous gift of R. N. quences correspond respectively to the first five codons of Brey [3]), which corresponds to pBR322 (2) with a 4-kb the studied gene and a sequence downstream of the cobA Sau3AI B. megaterium fragment inserted into the BamHI gene on the noncoding strand (at position 940 to 951 on the site. This plasmid complemented the E. coli cysG44 muta- sequence). A 780-bp polymerase chain reaction generated cobA-containing fragment was obtained, digested by NdeI and SstI, and then cloned on an expression vector, pXL694 * Corresponding author. (7) (Fig. 1). In this construction, named pXL1805, the cobA t Present address: Dk6partement de Biotechnologie, Institut de initiation codon is 9 bp downstream of the ribosome-binding Recherche Jouveinal, 94265 Fresnes COdex, France. site from the X cII gene, which is under the control of the E. 4893 4894 NOTES J. BACTERIOL.

Snal PAti EcoRI

Polymerase chain reaction with 2 ollgonucleotldes f purificatlon of a 850 bp fragment wBamHl Ndel and SstI digestlons /eparate digestions of pXL694 by EcoRI-Ndel and EcoRI-SstJ Purification of the 120 bp EcoRI-Ndel I ligation and and the 3.1 kb Sstl-Ndel fragments transformatlon Into MC 1060 EoeRI Nd.l Hindill Human cDNA Bacillus megaterium cobA gene S S R B S o~~~obA Terminator region of the E. coil rrnB operon Awp

pXL18O5 4.1 kb

ORI rrnBTlT2 Sstl HhdIil

Nd.l FIG. 1. Construction of plasmids pXL1804 and pXL1805. The numbers under the XbaI and SspI sites in the inserted fragment of pXL1804 correspond to the distance from the pUC129 XbaI site. Ptrp, E. coli Ptrp promoter; RBS, A clI ribosome-; Amp, ampicillin resistance gene; ori, origin of replication. VOL. 173, 1991 NOTES 4895

aatatttagctagccgaaaatttatctaaatgacagcggactttgtcctgtgtctaattc 60 1 2 3 4 aaattaaaatgtgggagcgctgcgagacgtggaaatgatggaagggtgaaaggcattc 118 ATG GGG AAA GTA TAT CTA GTC GGT GCA GGA CCG GGA GAT CCA GAT 163 97-4 M G K V Y L V G A G P G D P D t TTA ATT ACA TTA AAA GGG TTG AAA GCA ATT CAG CAA GCA GAT GTT 208 66.6 L I T L K G L K A I Q Q A D V ATC TTA TAT GAT CGT TTA GTG AAT AAG GAC CTG CTG GAG TAT GCT 253 I L Y D R L V N K D L L E Y A 42.7 AAA AGT GAT GCA GAT ATC ATT TAC TGC GGA AAG CTT CCG AAC TAC 298 K S D A D I I Y C G K L P N Y 31 CAT ACC CTC AAG CAA GAA ACA ATC AAC AAC TTT TTA GTG AAA TTC 343 _a H T L K Q E T I N N F L V K F GCT AAA AAA GGA AAA ATT GTA ACG CGC TTA AAG GGC GGA GAT CCA 388 A K K G K I V T R L K G G D P 21 5 - TTT GTT TTC GGA CGC GGA GGG GAA GAA GCA GAA GCT CTC GTG CAG 433 14-4 F V F G R G G E E A E A L V Q CAG GGC ATT TCA TmT GAA ATT Gr CCA GGA ATT A TCA GGT ATC 478 Q G I S F E I V P G I T S G I FIG. 4. Sodium dodecyl sulfate-polyacrylamide gel electropho- GCA GCC GCT GCT TAT GCA GGA ATC CCT GTC ACT CAC CGG GAA TAC 523 R E Y resis of soluble bacterial extracts stained with Coomassie blue. The A A A A Y A G I P V T H position of B. megaterium SUMT (30 kDa) is indicated by an arrow. AGC GCA AGC TTT GCT TTT GTA GCA GGC CAT CGT AAA GAT AGC AAG 568 Lanes: 1, pUC13 in E. coli B supplemented with tryptophan; 2, S A S F A F V A G H R K D S K pUC13 not supplemented; 3 and 4, same as lanes 1 and 2, respec- CAT GAT GCA ATC AAA TGG GAT AGC CTA GCC AAA GGT GTA GAT ACT 613 tively, with E. coli B(pXL1805). The cells were grown for 6 h to an H D A I K W D S L A K G V D T A600 of about 2 in M9 medium (15) supplemented when required for CTT GCA ATT TAT ATG GGA GTG CGG AAC TTA CCG TAC ATT TGC CAA 658 the repression of the Ptrp promoter with 40 ,ug of L-tryptophan per L A I Y M G V R N L P Y I C Q ml. Cell extracts were prepared as previously described (7). CAG TTA ATG AAA CAC GGA AAA ACT TCA GCT ACG CCA ATT GCA TTG 703 Q L M K H G K T S A T P I A L

ATAlknrsrskt TfVrsP GAAA-lr-l- 'rli TGmsr_rr rr1i-G-1 1--IfrGAW t'rsl1A1rfli--lC4- ACTr 1rmiklrlGT61bAACT flP21GA rzrACT _15'7AQ * I H W G T C A D Q R T V T G T coli Ptrp promoter (7). cobA gene expression was studied in as described The shown CTT GGT ACA ATT GTT GAT ATT GTT AM GAA CAA CAA ATT GAA AAT 793 E.. coli..B (16) previously . (7). results, L G T I V D I V K E E Q I E N in Fig. 4, demonstrated a specific high-level expression of a CCA AGT ATG Arr ATC GTT OCT CAA GTT GTA AAT T TCA TGA ccg 838 Mr 30,000 protein, in agreement with the CobA expected P S M I I V G E V V N F S * molecular weight, corresponding to at least 10% of the total attaSattggtttgagaagactggacagcattactattcgtatgcacaagaagcttacta 898 protein. SUMT activity of the expressed protein in strain B5548 was assayed at 0.5 ,uM urogen III as originally aagaggttgattatatggatgcagttttatatgtttgccatggcagccgcgtaaaagaag 958 described (1). In crude cell extracts of induced cultures of gcgctgatcaggcagttgcttttattgaaaggtgtaaaaaatctaga 1005 strain B5548(pXL1805), SUMT activity was 21 nmol h-' mg FIG. 2. sequence of the cobA gene and predicted of protein-' compared with 9.9 and 0.1 nmol h-1 mg of prrimary sequence of the encoded polypeptide. Noncoding DNA is protein-1 for the uninduced and control [E. coli B(pUC13) represented by lowercase letters. The location of a possible ribo- (21)] cultures, respectively. sc)me-binding site (10) is denoted by a line below the sequence. * The enzyme was purified about 10-fold from a 1-liter sti.op codon of the cobA gene. culture of B5548(pXL1805). The cells were suspended in 10 ml of 0.1 M Tris hydrochloride (pH 7.7)-i mM dithiothrei- tol-20o (wt/vol) glycerol (buffer A) and sonicated at 4°C for 90 min. Cell debris was separated by centrifugation at 50,000 x a 10 20 30 40 50 60 70 g for 1 h, and the supernatant was passed through KVYLVGAGPGDPDLITLKGLKAIQQADVILYDRLVNKDLLEYAKSDADIIYCGKLPNYHTLKQETINNF GSVWLVGAGPGDLA L CL LKPSPKQRDISLR 20 30 40 50 60 70 80 80 90 100 110 120 130 140 1 2 LVKFAKKGIIVTRLKGGDPFVFGRGGEEAEALVQQGISFEIVPGITSGIAAAAYAGIPVTHREYSASFAF

LVEIARAGWIRLKGGDPFVFGRGEEALTLVEHQVPFRIVPGITAGIGGLAYAGIPVTHREVNHAVTF 97-4 90 100 110 120 130 140 150 150 160 170 180 190 200 210 VAGHRnDS-KHDAIKNDSLAK.GVDTLAIY8 M;PYICQ0IKHGTSATPIALIHWGTCADQRTVTGT 66f6 LTGHDSSGLVPDRINWQGIASGSPVIVNMfMIGAITANLIAGGRSPDEPVAFVCNAATPQOAVLETT 160 170 180 190 200 210 220 427 220 230 LGTVIVKEEQIENPSM[IIVGEWNF LARAEADVAAAGLEPPAIVVVGEVVRL 230 240 250 FIG. 3. Alignments of P. denitrificans and B. megaterium CobA proteins from residues 15 to 251 (bottom sequence) and 2 to 237 (top sequence), respectively. The alignments were performed by using 21.5 the program of Kanehisa (12). B. megaterium CobA and P. denitri- ficans CobA sequence similarities are indicated. =, same amino FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electropho- acid; -, amino acids belonging to the same group (hydroxyl or small resis of SUMT performed with a 12.5 homogeneous Phastgel on a aliphatic, A, G, S, and T; acid and acid amide, N, D, E, and Q; PhastSystem instrument (Pharmacia). The gel was stained with basic, H, R, and K; aliphatic, M, I, L, and V; aromatic, F, Y, and silver. Lane 1, partially purified SUMT (100 ng); lane B, molecular W). Horizontal bars show a gap in the alignment. weight markers (in thousands) (50 to 100 ng per band). 4896 NOTES J. BACTERIOL.

1 OC )- L-methionine:uroporphyrinogen III methyltransferase from I\ Pseudomonas denitrificans. J. Bacteriol. 171:4222-4231. A 2. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. 8C A* \ Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977. A Construction and characterisation of new cloning vehicules. II. C) 0 A multipurpose cloning system. Gene 2:95-113. 6C A 3. Brey, R. N. Unpublished data.

-J 4. Brey, R. N., C. D. B. Banner, and J. B. Wolf. 1986. Cloning of 4C A multiple genes involved with cobalamin (vitamin B12) biosyn- z thesis in Bacillus megaterium. J. Bacteriol. 167:623-630. 5. Crouzet, J., L. Cauchois, F. Blanche, L. Debussche, D. Thibaut, 2C M.-C. Rouyez, S. Rigault, J.-F. Mayaux, and B. Cameron. 1990. Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilo- base DNA fragment containing five cob genes and identification 0) 4 6 9 1 0 of structural genes encoding S-adenosyl-L-methionine:uropor- 02') 468 phyrinogen III methyltransferase and cobyrinic acid a,c-diam- [UROPORPHYRINOGEN III M ide synthase. J. Bacteriol. 172:5968-5979. 6. Debussche, L., D. Thibaut, B. Cameron, J. Crouzet, and F. FIG. 6 Dependence of initial velocity of reaction catalyzed by Blanche. 1990. Purification and characterization of cobyrinic B. megaterium SUMT (arbitrary units) or urogen III concentration, acid a,c-diamide synthase from Pseudomonas denitrificans. J. performed as described elsewhere (1). SAM concentration was 50 Bacteriol. 172:6239-6244. FLM. 7. Denefle, P., S. Kovarik, J.-D. Guiton, T. Cartwright, and J.-F. Mayaux. 1987. Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of DEAE-5 3ephadex A 25 column (250 ,il of swollen gel) previ- the product into its active form. Gene 56:61-70. ously equilibrated with buffer A. A sample of this crude cell 8. Garibaldi, J. A., K. IlUchi, J. C. Lewis, and J. McGinnis. extract (250 R,u 5 mg of protein) was loaded on a Mono Q HR December 1951. U.S. patent 2,576,932. 5/5 colurmnmn(Pamca(Pharmacia) equilibratedqiirtdwtwith bufferufrAA. Proteinsrtis 9. intermediateGilles, H., andin theR. K.biosynthesisThauer. 1983.of theUroporphyrinogennickel-containing F430III, anin were eluited at 1 ml min-' with a 30-ml 0 to 0.5 M linear Methanobacterium thermeautotrophicum. Eur. J. Biochem. gradient of potassium chloride in buffer A. Fractions con- 135:109-112. taining S3UMT activity were pooled and concentrated to 200 10. Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B. Swefilius- ,ul with a Centricon 10 microconcentrator (Amicon). The Singer, and G. Stormo. 1981. Translation initiation in procary- protein solution was fractionated on a Bio-Sil SEC 250 otes. Annu. Rev. Microbiol. 35:365-403. column (Bio-Rad) eluted with buffer A containing 0.1 M 11. Henikoff, S. 1984. Unidirectional digestion with III potassium chloride. The most purified preparation showed a creates targeted breakpoints for DNA sequencing. Gene 28:351- 359. major b;and (-90%) with Mr 30,000 as judged by sodium 12. Kanehisa, M. 1984. Use of statistical criteria for screening dodecyl sulfate-polyacrylamidesulfate.polyacrylamidgel electroelectrophoresiss (.(Fig. 5) potential homologies in nucleic acids sequences. Nucleic Acids and exhiibited a specific activity of 215 nmol h' mg at 0.5 Res. 12:203-215. pLM urolgen III and 50 ,uM SAM. When analyzed by gel 13. Keen, N. T., S. Tamaki, D. Kobayashi, and D. Trollinger. 1988. filtration on a Bio-Sil SEC 250 column eluted at 0.5 ml min-' Improved broad host range plasmids for DNA cloning in gram- with 0.1 M potassium chloride in buffer A, this purified negative bacteria. Gene 70:191-197. preparat-ion showed a single peak coeluting with SUMT 14. Leeper, F. J. 1989. The biosynthesis of porphyrins, chlorophylls activity -at Mr 32,000 as estimated by the computer software and vitamin B12. Nat. Prod. Rep. 6:171-203. GPC6/7C)00 (Perkin-Elmer). These results suggest a mono- 15. Miller, J. H. 1972. Experiments in molecular genetics. Cold meric stiructure. The enzyme was inhibited by urogen III at Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 16. Monod, J., and E. Wollman. 1947. Inhibition de la croissance et concentirations abovabove 0.5 ,wM (Fig. 6) wiThwith aptea patternsimilarsimilar de l'adaptation enzymatique chez les bacteries infectees par le to that chbservedatirvedwith P. denitrficans(Fnigrif. SUMT (1), indicating bacteriophage. Ann. Inst. Pasteur 73:937-956. that sucKh inhibition might be a common feature among 17. Peakman, T., J. Crouzet, J.-F. Mayaux, S. Busby, S. Mohan, R. aerobic cobalamin-producing4 bacteria. Nicholson, and J. Cole. Nucleotide sequence, organisation and NuclecAtide sequence accession number. The nucleotide structural analysis of the products of the nirB to cysG region of sequenc4e shown in Fig. 2 has been submitted to GenBank the E. coli . Eur. J. Biochem. 191:315-323. under ac,cession number M62881. 18. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer- We exr press our gratitude to J. Lunel and J.-F. Mayaux for their directed enzymatic amplification of DNA with a thermostable support d[uring this work. We thank the CITI2 (Centre de Traitement DNA polymerase. Science 239:487-491. Interuniv4ersitaire d'Informatique a Orientation Biomedicale, Paris, 19. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- France) fSor nucleic and protein sequence analysis programs. We ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. thank R. N. Brey and Genex for providing pGX3512. USA 74:5463-5467. This w'ork was supported by both Rh6ne-Poulenc Rorer and 20. Warren, M., C. A. Roessner, P. J. Santander, and A. I. Scott. Rh6ne-Po ulenc S.A. 1990. The Escherichia coli cysG encodes S-adenosylmethio- nine-dependent uroporphyrinogen III methylase. Biochem. J. 265:725-729. REFERENCES 21. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved 1. Blanche, F., L. Debussche, D. Thibaut, J. Crouzet, and B. M13 phage cloning vectors and host strains: nucleotide se- Came-ron. 1989. Purification and characterization of S-adenosyl- quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.