nucleotide sequence genetic analysis ofa 13.1-kilobase ... · orf5is a cob gene, and it was...
TRANSCRIPT
Vol. 173, No. 19JOURNAL OF BACTERIOLOGY, OCt. 1991, p. 6074-60870021-9193/91/196074-14$02.00/0Copyright © 1991, American Society for Microbiology
Nucleotide Sequence and Genetic Analysis of a 13.1-Kilobase-PairPseudomonas denitrificans DNA Fragment Containing Five cob
Genes and Identification of Structural Genes Encoding Cob(I)alaminAdenosyltransferase, Cobyric Acid Synthase, and BifunctionalCobinamide Kinase-Cobinamide Phosphate GuanylyltransferaseJ. CROUZET,1* S. LEVY-SCHIL,l B. CAMERON,' L. CAUCHOIS,lt S. RIGAULT,'T M.-C. ROUYEZ,l§
F. BLANCHE,2 L. DEBUSSCHE,2 AND D. THIBAUT2Unite de Biologie Moleculaire, Institut des Biotechnologies,l and Departement Analyse,2 Rh6ne-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 24 July 1991
A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotidesequence and genetic analysis revealed that this fragment contained five different cob genes named cobN tocobQ and cobW. Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cobproteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibitingboth cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified ascob(I)alamin adenosyltransferase. CobN is proposed to play a role in cobalt insertion reactions. Four otheropen reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not leadto a cobalamin-minus phenotype.
The cobalamin biosynthetic pathway probably involves 20to 30 different enzymatic steps (Fig. 1) consisting of (i)formation of uroporphyrinogen III (urogen III), which is thecommon intermediate for the synthesis of hemes, chloro-phylls, cobalamins, F430, and sirohemes; (ii) conversion ofurogen III into cobyrinic acid by three successive methyla-tions at C-2, C-7, and C-20 followed by five other methyla-tions at C-17, C-11, C-1, C-5, and C-15, decarboxylation ofthe acetic acid side chain at C-12, elimination of C-20,NADPH-dependent reduction of the macrocycle, methylmigration from C-11 to C-12, and insertion of cobalt; (iii)formation of cobinamide from cobyrinic acid (Fig. 1) byamidation of the peripheral carboxyl groups a, b, c, d, e, andg and insertion of (R)-l-amino-2-propanol at position f; and(iv) conversion of cobinamide into coenzyme B12 (for re-views on cobalamin synthesis, see references 4, 5, 23, 34,and 40). In addition, the central cobalt atom is adenosylatedto form the coenzyme derivatives. We have described else-where the cloning of Pseudomonas denitrificans genes in-volved in cobalamin synthesis (cob genes) (12) from urogenIII. These genes are grouped into four genomic loci thatcorrespond to four distinct complementation groups (from Ato D). We have reported the nucleotide sequence and theresults of genetic analysis of a 5.4-kb DNA fragment fromcomplementation group C carrying five cob genes (cobA tocobE) elsewhere (16). This fragment is located on the rightend of the complementation group C restriction map (Fig. 2).cobA and cobB are the structural genes for S-adenosyl-L-
* Corresponding author.t Present address: 5 bis rue Pierre Curie, 10150 Pont-Sainte
Marie, France.t Present address: Residence le Gilly, 73200 Gilly sur Isere,
France.§ Present address: INSERM U. 152, Hopital Cochin, 75674 Paris
Cedex 14, France.
methionine:uroporphyrinogen III methyltransferase (9, 16)and cobyrinic acid a,c-diamide synthase (16, 19), respectively(Fig. 1), and the CobC and CobD proteins are supposed toplay a role in converting cobyric acid into cobinamide (16)(Fig. 1). An 8.7-kb fragment from complementation group Acarrying all the genes defined by complementation analysisin this locus has also been investigated in detail (15). Eightcob genes (cobF to cobM) were shown to be implicated inthe conversion of precorrin-2 into cobyrinic acid (Fig. 1 and2). One of them, cobI, has been identified as the structuralgene of S-adenosyl-L-methionine :precorrin-2 methyltrans-ferase (15, 45) (Fig. 1). On the basis of protein sequenceshomologies and S-adenosyl-L-methionine affinity of theCobF protein, the cobF, cobJ, cobL, and cobM genes wereproposed to code for other methyltransferases of the path-way (Fig. 1). These studies have allowed 13 cob genes to beidentified. The aim of our research is to identify all the genesinvolved in cobalamin synthesis and to characterize theactivity of the encoded polypeptides. We report here thegenetic analysis and nucleotide sequence of a 13.1-kb frag-ment from complementation group C. One border of thisfragment corresponds to one end of the previously investi-gated 5.4-kb fragment (16) (Fig. 2). This study combinedwith the previous investigation represents a complete ge-netic and nucleotide sequence analysis of complementationgroup C. In the present report, five cob genes are identified.
MATERIALS AND METHODS
Bacterial strains and plasmids. Bacterial strains and plas-mids used in this study are described in Table 1. For each ofthe constructed plasmids, a specific DNA fragment from thepXL156 or pXL157 insert (12) was cloned into either aderivative of RK2 (pRK290 to generate pXL1560) or aderivative of RSF1010 (i.e., pXL59 to generate pXL221 orpXL435 to generate pXL593, pXL1908, pXL1909, andpXL1938) (see Fig. 4). For pXL1908, XbaI linkers were
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introduced at both ends of the fragment before it was clonedinto pXL435.Media, bacteriological techniques and chemicals, general
methods, and DNA sequencing. Techniques and methodswere carried out as already described (13). The aminoterminus of the CobW protein was determined as followed:the 45,000Mr protein overexpressed in strain SC510 Rif-(pXL593) was electroeluted from a sodium dodecyl sulfate-polyacrylamide gel (2) after electrophoresis of the totalbacterial proteins and was microsequenced as describedelsewhere (19). The same procedure was followed for thedetermination of the NH2 terminus of the 28,000-Mr proteinoverproduced in SC510 Rif(pXL227).
Mutagenesis. Three different mutagenesis strategies wereused to identify the cob genes on the sequenced DNAfragment. First, pXL1560 (pRK290 containing the 9.1-kbEcoRI fragment from pXL156) was mutagenized with TnSSpr as described previously (13). Twenty-seven insertionswere obtained and mapped. These insertions were intro-duced into P. denitrificans SC510 Rif' through homologousrecombination as described previously (13). Southern blotswere carried out in order to check that the transposon hadindeed inserted into the P. denitrificans chromosomethrough homologous recombination. Second, plasmidpXL189 was mutagenized with transposon Tn5 as describedby de Bruijn and Lupski (17) by using X467 to infect LE392harboring pXL189. After plasmid DNA purification, 13 Tn5insertions were identified on the 4.5-kb Sau3AI-ClaI insertof pXL189. Third, a spectinomycin resistance (Sp) cassette(from plasmid pHP45fl) was introduced by marker exchangemutagenesis into the P. denitrificans chromosome withinORF6 to ORF9. For this purpose, the 4.3-kb EcoRI-ClaIfragment containing these open reading frames (ORFs) wassubcloned into EcoRI-linearized pUC13 after the ClaI sitehad been filled in and an EcoRI linker had been added. Thefl_Spr cassette was inserted into the resulting plasmid,pXL332, totally or partially digested at the following sites:SmaI (position 9868, within ORF6), BamHI (position 10664,within ORF7), ClaI (position 11687, within ORF8), and NcoI(position 12474, within ORF9), giving insertions Q2, Q1, Q3,and f14, respectively (see Fig. 4). The EcoRI fragmentscarrying these insertions were subcloned into EcoRI-linear-ized pRK415. The resulting plasmids were then introducedinto SC510 Rif' by bacterial mating. The exconjugants ob-tained were subjected to another biparental mating withMC1060 (pR751), and the presence of pR751 and the Sprcassette were selected for. This selection favored the loss ofpRK415-derived replicons and marker exchange, becausepRK415-derived replicons and pR751 belong to the sameincompatibility group, incP. It was verified by Southern blotthat the strains derived from SC510 Rif' contained a chro-mosomal Spr gene at the expected positions.
Nucleotide sequence accession number. The nucleotidesequence shown in Fig. 3 has been submitted to GenBankunder accession number M62866.
RESULTS
DNA sequence of the 13.1-kb fragment from complementa-tion group C. The sequence of a 13.1-kb fragment from bothpXL156 and pXL157 was determined on both strands (Fig.3). This sequence was bounded by a SstI site at its left endand a BglII site at its right end. Previous complementationanalysis showed that this fragment carries at least three cobgenes (12). There is a 64-bp ClaI-BglII overlap between this
sequence and the previously sequenced 5.4-kb fragmentfrom the same complementation group (16).
This sequence was analyzed by the program of Staden andMcLachlan (42, 43) with a codon usage table obtained fromsequenced Pseudomonas genes, as already described (15).Nine ORFs (named ORF1 to ORF9), characterized by a highcoding probability, were identified (Table 2 and Fig. 4).Seven of them (ORF1 to ORF6 and ORF8) were on the samestrand, while ORF7 and ORF9 were on the other strand (Fig.4). ORF9 had a high coding probability from the BglII site atthe sequence end. The only possible initiation codon next tothe BglII site on the previously determined sequence was 5bp downstream, as shown in Fig. 3. All nine ORFs werepreceded by potential ribosome-binding sequences havingsome homology to the consensus ribosome-binding site ofEscherichia coli (TAAGGAGGTG ... 5 to 9 bp . . . ATG)(41), as shown by the overlined bases in Fig. 3. ORFs 2 and3 presented the characteristics of ORFs that were transla-tionally coupled, since the ORF2 stop codon was 3 bpupstream of the ORF3 ATG (37). The same observationcould be made for ORFs 5 and 6. No sequences exhibitingcharacteristics of a p-independent terminator (38) could bedetected downstream from the stop codons of the ORFs.The mean G+C content of this sequence was 63.6%, whichwas slightly lower than the value for other P. denitrificansfragments already sequenced (65.7%) (15, 16). Direct re-peated sequences (25 bp each) were found at positions 2558and 2586 (Fig. 3). Their significance is unknown. They mightbe involved in the binding of a regulatory protein since theyare positioned 800 bp upstream of ORF2. No region withsignificant homology with the E. coli promoter consensussequences could be found upstream of the ORFs. A region ofdyad symetry was found on the sequence at position 5019between ORF3 and ORF4 (Fig. 3). The inverted sequencesare 12 bp apart and have the potential for forming a stem-loop structure in the intergenic region between cobW andcobN.
Genetic analysis of the 13.1-kb fragment. In order tolocalize the cob genes among the nine identified ORFs, the13.1-kb fragment was mutagenized. Thirteen different Tn5insertions were selected within the insert of pXL189, whichcontains ORF5 to ORF9 (Fig. 4). pXL189 complementsAgrobacterium tumefaciens Cob mutants G632 and G633(12). All the TnS-inserted plasmids except the one carryinginsertion 58 (the only one mapped within ORF5) were stillable to complement G632 and G633. This indicated thatORF5 is a cob gene, and it was named cobO.A Spr resistance cassette from pHP45Qk was precisely
inserted into the chromosome of SC510 Rif within ORF6 toORF9, resulting in insertions fQ2, Qfl, fQ3 and Q4, respec-tively (Fig. 4). The four strains carrying these insertions allexhibited a Cob' phenotype, indicating that these fourORFs were not cob genes.The 9.1-kb EcoRI fragment from pXL156 was subcloned
into pRK290 to generate pXL1560. This plasmid was muta-genized with transposon TnS spr, resulting in 27 insertions(Fig. 4) which were introduced through marker exchangemutagenesis into the SC510 Rif' chromosome. Eighteeninsertions of the 27 exhibited a Cob- phenotype (Fig. 4). Allthe mutants for which the insertion was within ORF1 (i.e.,insertions 19, 32, 24, 27, 37, 39, 26, 11, and 14; Fig. 4) had aCob- phenotype and were complemented by plasmidpXL617, whose insert carried only ORF1. We concludedthat ORF1 is a cob gene, and we named it cobQ. PlasmidpXL221, carrying only part of cobQ, did not complementthese mutants, indicating that the ClaI site is internal to the
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urogen III I
ID methylations at C-2 and C-7
precorrln-2
| methylation at C-20
precorrin-3
methylations at C- 17. C-I Iand C-I
elimination of C-20
reduction of the macrocycle
methylations at C- IS and C-S
decarboxylation
methyl migration fromC-1l toC-12
cob alt insertionFcobrinic acid8 mediating reaction
m a,c amidations
0 adenosylation
b.d. e. and g amidations
addition ofR- I -amino-2-propanol
cobinam Ide
Iphosphoryiationaddition of GMP
GDP-cobinam ide
IE incorporation of DBIR(P)
n zyme DBIRP+nicotinate
CobAC
CobI A
CobF'
CobG A
CobH A
CobJa A
CobK A
CobL' A
CobM' A? BZ- CobT
CobS BCobN C
CobB C
CCobO
CobQC
CCobCCobD C
CobP C
DCobV
E transglycosylation
DBI + NaMN
DCobU
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Ikb
LGROUP A
HE H B BX V|B H B Ix V SSIS II I I 1 11
H S X11 T I
F G H I J K L M
| Xl;E~~~E 15x |
X ES XHB BHHl IX S B HS E X XBl ElB SX B X H EI sGROUP B I I I I I 1 1 I I 1 I I II I
-
I II1
ORF2 S T-4.8 kb
ORF 1
H X
S ES X B SXS S B E B SX H H E SH H B B E X X
GROUP CQ p W N O QB 02£ E A B C D
18.4kbORF4 ORF6
GROUP DS S X B E S
U ORF83.9 kb
ORF7 V
FIG. 2. Restriction map of the four P. denitrificans complementation groups. Below each group, the cob genes are shown by theircorresponding letters. A hatched line represents the sequenced fragments, and their lengths are indicated on the right. A line over the hatchedline indicates that the coding strand goes 5'--3' from left to right. The ORFs of unknown functions are also represented. B, BamHI; E, EcoRI;H, HindIII; S, SstI; X, XhoI.
cob gene; this is in agreement with the sequence results.Plasmid pXL622, containing ORF2 only (Fig. 4), comple-mented two A. tumefaciens Cob mutants, G642 and G2043(12). For this reason, ORF2 is a cob gene named cobP.Three A. tumefaciens Cob mutants (G622, G623, and
G630) are complemented by plasmid pXL156. No otherplasmid so far described that carries a small subclonedfragment from pXL156, such as pXL189 (carrying cobO as
the only cob gene), was found to complement these mutants(12). pXL1908, which contains the ORF3 3' end, ORF4,cobO, and ORF6, complemented these mutants (Fig. 4).Since ORF6 is not a cob gene and since cobO cannot beresponsible for the complementation, we concluded thatORF4 is a cob gene, and we named it cobN. Six TnS Sprinsertions (i.e., insertions 30, 22, 40, 35, 10, and 17) mappedwithin cobN resulted in a Cob- phenotype when present in
FIG. 1. Pathway for coenzyme B12 synthesis from urogen III and reactions catalyzed by the P. denitrificans Cob proteins. Structures ofurogen III (A), cobyrinic acid (B), and coenzyme B12 (C) are shown. Reactions occurring between the indicated intermediates are presentedon the right and represent what is currently known (4, 5, 23, 34, 40, 44, 46). Between precorrin-3 and cobyrinic acid, the order of the stepsis not known except for the methylations; however, the structure of precorrin-6x (44, 46) indicates that reduction of the macrocycle,methylations at C-5 and C-15, decarboxylation at C-12, and methyl migration from C-il to C-12 occur last. The position of the cobaltinsertion-mediating reactions is discussed in the text (** indicates that cobalt insertion takes place on hydrogenobyrinic acid a,c-diamide. Theadenosylation reaction is shown to occur on cobyrinic acid a,c-diamide (18); therefore, all the following intermediates are adenosylated.Identified enzymes: 1, S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase; 2, S-adenosyl-L-methionine:precorrin-2 methyl-transferase; 3, cobyrinic a,c-diamide synthase; 4, cob(I)alamin adenosyltransferase; 5, cobyric acid synthase; 6, cobinamide kinase-cobinamide phosphate guanylyltransferase; 7, cobalamin (5'-phosphate) synthase, which uses either 5,6-dimethylbenzimidazole-ribose(DBIR) or 5,6-dimethylbenzimidazole-ribose-5'-phosphate (DBIRP) as a substrate (11); 8, nicotinate-nucleotide:dimethylbenzimidazolephosphoribosyltransferase. DBI, 5,6-dimethylbenzimidazole; NaMN, P-nicotinic acid mononucleotide; R, 5'-deoxy-5'-adenosyl; X and Y,axial cobalt ligands (e.g., H20); *, proposed methyltransferases intervening after synthesis of precorrin-3. Superscript letters after Cobproteins refer to the complementation group (A to D) to which the corresponding structural gene belongs (12).
8.7 kb
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TABLE 1. Bacterial strains, phage, and plasmids used
Bacterial strain, Markers and Relevant prope.ies Referencephage, or plasmid replicons or source
E. coliTG1C600C600 Rifr113-3CbllLE392MC1060MC1060::Tn5 Spr
P. denitrificansSC510 Rifr
A. tumefaciensC58-C9 Rif' NalrG622G623G630G632G633
A(lac-pro) thi supE hsdDS/F' proAB lacI"ZAMJ5thi thr leuB lacY supE tonARif' isolate (100 SLg/ml) of C600metE cannot convert cobinamide into cobalaminF- hsdR514 supE44 supF58 lacYI galK2 galT22 metBi trpRSS X-A(lacIOPZYA)X74 galU galK strA2 hsdRTnS Spr chromosomal insertion in MC1060Rif' isolate (100 ,ug/ml) of SC510
Rif Nalr isolate (100 and 20 ,u.g/ml, respectively) of C58-C9 (26)Cob-, derived from C58-C9 Rift, complemented by pXL156Cob-, derived from C58-C9 RifT, complemented by pXL156Cob-, derived from C58-C9 Rif, complemented by pXL156Cob-, derived from C58-C9 Rif, complemented by pXL227Cob-, derived from C58-C9 Rif, complemented by pXL227
241This laboratory1217141316
121212121212
Xb221 rex::Tn5 c1857 Oam29 Pam8O
Ampr Spr ColElKmr RSF1010Kmr ColElTpr R751Tetr RK2Tetr RK2Ampr ColElKmr RSF1010Kmr RSF1010
Kmr RSF1010
Kmr RSF1010
Kmr RSF1010
Kmr RSF1010Kmr RSF1010
Ampr ColEl
Kmr RSF1010Kmr RSF1010
Kmr RSF1010Kmr RSF1010Kmr RSF1010
Kmr RSF1010
Kmr RSF1010
Kmr RSF1010
Contains Spr cassetteCarries Mob locus of RSF1010Carries tra genes of RK2Carries tra genes of R751Carries Mob locus of RK2Carries Mob locus of RK2Multicloning siteCarries Mob locus of RSF101014.2-kb P. denitrificans Sau3AI fragment cloned into BamHI site ofpXL59
13.2-kb P. denitrificans Sau3AI fragment cloned into BamHI site ofpXL59
P. denitrificans Sau3AI-ClaI fragment cloned into BamHI-ClaI site ofpXL59; cobO, ORF6 to ORF8
5.2-kb BamHI from pXL156 cloned into BamHI site of pKT230;cobO, ORF6
1.6-kb EcoRI-ClaI from pXL156 cloned into pXL592.5-kb EcoRI-SstI fragment from pXL157 cloned into pKT230; cobO,ORF6 to ORF7
4.3-kb EcoRI-Clal fragment from pXL157 cloned into pUC13; cobO,ORF6 to ORF9
Multicloning site3.1-kb BamHI fragment from pXL156 cloned into pKT230; cobP,cobW
2.3-kb SstI-BamHI fragment from pXL156 cloned into pKT230; cobQ1.7-kb BamHI-PstI fragment from pXL156 cloned into pXL59; cobP9.1-kb EcoRI fragment from pXL156 cloned into pRK290; cobP,cobQ, cobW
6.5-kb XhoI-BamHI fragment from pXL156 cloned into pXL435;cobN, cobO, ORF6
8.4-kb BamHI fragment from pXL156 cloned into pXL435; cobP,cobQ, cobW, cobN, cobO, ORF6
6.5-kb XhoI-BamHI fragment from pXL156 cloned into pXL435;cobN, cobO, ORF6
the chromosome of SC510 Rif. These mutants were notcomplemented by pXL189, pXL208, or pXL1938 but werecomplemented by plasmid pXL1908, confirming that theirCob- phenotype is due to a lack of cobN expression. ThepXL1938 and pXL1908 plasmids have the same insert clonedin the opposite orientation on the pXL435 vector. Thedifference in complementation between the two plasmidsimplies that in pXL1908, but not in pXL1938, the amplifica-tion of the cobN gene might be driven by the Kmr gene
promoter, suggesting that the cobN promoter is located
upstream of the XhoI site bordering the insert of pXL1908and pXL1938.Tn5 Spr insertions 23, 13, and 12 are positioned within
ORF3 (Fig. 4). When these insertions were introduced intothe SC510 Rif' chromosome, a Cob- phenotype was ob-served. These mutant strains were complemented by plas-mid pXL593 harboring cobP and ORF3 but were not com-plemented by pXL622 carrying only cobP. Thus, ORF3 is acob gene named cobW. Plasmid pXL1908, which includescobN, cobO, ORF6, and only the 3' end of cobW, partially
17Phage X467PlasmidspHP45QkpKT230pRK2013pR751pRK290pRK415pUC13pXL59pXL156
pXL157
pXL189
pXL208
pXL221pXL227
pXL332
pXL435pXL593
pXL617pXL622pXL1560
pXL1908
pXL1909
pXL1938
39322302132511212
12
12
This study
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complemented the mutants having insertions within cobW (apartial complementation represents cobalamin synthesis ofbetween 0.5 and 0.05% of the SC510 Rif' level of cobalaminsynthesis). It is proposed either than expression of the cobW3' end yields a partially active cobW gene product or thatcobN amplification partially complements a cobW mutation.cobQ is the structural gene encoding cobyric acid synthase.
Cobyric acid synthase, which catalyzes amidations at posi-tions b, d, e, and g on adenosylcobyrinic a,c-diamide (Fig.1), with glutamine as the amide group donor has beenpurified from SC510 RifT(pXL617) (7). Plasmid pXL617,which carries cobQ, amplifies this activity in SC510 Rif.This enzyme is a homodimer for which the estimated molec-ular weight per subunit is 57,000, which is close to that of theCobQ protein (51,982). The 16 NH2-terminal residues of thisenzyme (7) are identical to the NH2-terminal sequence of theCobQ protein (Fig. 3) except that the amino-terminal methi-onine has been removed. Therefore, cobQ is the structuralgene for cobyric acid synthase.CobQ and CobB (16) together catalyze the six glutamine-
dependent amidations of the cobalamin pathway (Fig. 1); thetwo are homodimeric enzymes with similar molecularweights, i.e., 45,600 and 52,000, respectively. They show19.6% identity over nearly all their length (data not shown).No other proteins in the data bases exhibited this degree ofsimilarity over such a length. Thus, the identity betweenCobQ and CobB is significant, and the two proteins might bestructurally similar.
Identification of structural gene encoding bifunctional cobi-namide kinase-cobinamide phosphate guanylyltransferase.The purification of a single P. denitrificans enzyme thatcatalyzes both the ATP-dependent phosphorylation of ade-nosylcobinamide and the addition of GMP to adenosylcobi-namide phosphate (Fig. 1) has been described previously (8).These activities are amplified in strain SC510 Rif harboringpXL622, which carries cobP. The enzyme is a homodimerwith an Mr of 20,000 per monomer, which is close to that ofCobP (19,442). The amino-terminal sequence of the first 10residues of the monomer (8) is identical to that of the CobPprotein (Fig. 3) except that the amino-terminal methioninehas been removed. This proves that cobP is the structuralgene of the bifunctional cobinamide kinase-cobinamidephosphate guanylyltransferase.
Biochemical studies of CobP have given evidence that ithas two separate nucleotide binding sites, one for ATP andone for GTP (8). The sequence GX4GKT/SX6I/V, which iscommon to a number of ATP-binding proteins and is in-volved in phosphate binding (47), was found at Gly-13 (Fig.3), but no match with the other phosphate-binding sequence,
R/KX3GX3K-hydrophobic-hydrophobic-hydrophobic-D,was detected (47). The consensus sequence DX2G, which iscommon to a conserved region among GTP-binding proteinsand has been shown to participate in phosphate binding, was
found to start at Asp-70 (49) (Fig. 3). In contrast, no
sequence specific for guanine selectivity or guanine bindingwas found (49). Therefore, there should be other sequences
involved in the binding of the two nucleotides.CobO is cob(I)alamin adenosyltransferase. The P. denitri-
ficans cob(I)alamin adenosyltransferase, which catalyzes theATP-dependent adenosylation of Cobalt(I) corrinoids (Fig.1), has been purified from strain SC510 Rif(pXL227) (18).Plasmid pXL227, carrying cobO, led to an increase ofcob(I)alamin adenosyltransferase activity in strain SC510Rif'. The purified enzyme has a molecular weight of 28,000per monomer, which is close to the molecular weight of thecobO-encoded polypeptide (24,321). The NH2-terminal se-
quence of the purified cob(I)alamin adenosyltransferase,NH2-SDETXVGGEAPAKK ... (18), was found to matchthe amino-terminal sequence of the CobO protein (Fig. 1)except that (i) the undetermined residue is a threonine; (ii)instead of a lysine at position 13 there is a glutamic acidresidue, probably because of an error in the NH2-terminalsequencing; and (iii) the amino-terminal methionine has beenremoved. Therefore, cobO is the structural gene for cob(I)alamin adenosyltransferase. It was found that a 28,000-Mrprotein was overproduced when plasmid pXL227 waspresent in SC510 Rif (see Fig. 6); microsequencing of thisprotein indicated that it was CobO.The CobO protein was found to have 43% identity with the
E. coli BtuR protein over most of its length (35) (Fig. 5).BtuR has a molecular weight of 21,979, which is close to thatof CobO, and has been proposed to be involved in convert-ing reduced cobalamin into adenosylcobalamin (35). Thehomology we found supports the proposed BtuR function.The conserved regions include the suggested nucleotide-binding domain of BtuR except that in the aspartic acid-containing domain, Lys-113 rather than Arg-114 is the basicresidue (Fig. 5).
Visualization of CobW protein and comparison with pro-teins in the data base. Strain SC510 Rif' harboring plasmidpXL593, which carries cobP and cobW, overexpressed aprotein of Mr 43,000, which is close to the Mr of CobW(38,121), as analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (Fig. 6). The amino-terminal se-quence of this protein corresponded exactly to the amino-terminal sequence of the CobW protein (Fig. 3) except thatthe amino-terminal methionine has been removed. Thisindicated that the overexpressed protein was CobW.CobW showed 26.3% homology with the NAD(H)-binding
domain of the bovine nicotinamide nucleotide transhydroge-nase from residues 28 to 195 (50) (Fig. 5). This may besignificant, since no protein in the GenBank data baseexhibited this degree of identity over such a length, indicat-ing that CobW might have an NAD(H)-binding site. If this isthe case, CobW mnight be involved in an NAD(H)-dependentoxidoreduction step in the pathway, which should be thereduction step leading to Cobalt(I) corrinoids, the substratesfor adenosylation (27). However, an ATP-binding consensussequence of the ATP-binding 1cx3 fold (47) was present inthe CobW sequence (Fig. 3), indicating that CobW might beresponsible for a reaction different from the one proposed.
Visualization of CobN protein and biochemical studies ofCob mutants complemented by cobN. SC510 Rif' harboringplasmid pXL1909, which amplifies cobP, cobW, cobN, andcobO as cob genes, overexpressed three proteins in SC510Rif' (Fig. 6). Two of them comigrated with the 43- and28-kDa proteins overexpressed in the presence of plasmidspXL593 and pXL227, respectively, and correspond to CobWand CobO (see sections on cobO and cobW above). The thirdprotein has an estimated Mr of 140,000, which is close to thatof CobN protein (138,055), confirming that cobN encodes ahigh-molecular-weight protein.The expression of CobN protein is visualized with plasmid
pXL1909 but not with plasmids pXL1908 and pXL1938 (datanot shown), in which cobN is downstream from an 889-bpfragment encompassing the 109-bp intergenic region be-tween cobN and cobW plus 780 bp of the 3' end of cobW.Thus, CobN and CobW expression should be dependent onthe same promoter.The intracellular contents of corrinoids and metal-free
corrinoids in A. tumefaciens Cob mutants G622, G623, andG630, which are complemented by cobN, were studied in
VOL. 173, 1991
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gagct cgaaggggc t tccgccccgatcgct ggcgt tagccgacgt tcgacgt gcggat gacgccgagcgggccgaagggcgcgt cgacgac gaggt tgcgt acgcgcgac tggc tggacg 120
gaacct tcgagt tccaggcgat ct goacgaaat tgggct tgct gaoaaat at acagcat ggacat gaacct t gagaggccggaggcct at cc tcccggggcgt gt tgct atgccgct gat at 240
aggt gt gcgc tgcaaaaatt gaat gccaaact cgccacgccat gt cgcat t ct ggct at cggccgcgocat t t t cgacaagcct t gcgaaagcgcgaaacaatgcgt gaaagggct t tg9 360
tcaattgcggcgaaatcgtgtcgaaacagacctttgccgctgcccgtttcagtgttaccgatggccgc RTG RCR CGC RGG RTC RTG TTG CRG GGR RCC GGC TCG GAT 467n T R R n L s0 G T G S O
--> ORFI (cobU)GTC GGR RRR TCG GTR TTG GTG GCG GGG CtC TGC CGG CTT GCC GCC RRT CRG GGC CTG RRG GTC CGG CCG TTC RRG CCG CRG RRC RTG TCG 557U G K S U L U R G L C R L A R N Q G L K U R P F K P Q N n s
RRC RRC GCC GCC GTT TCC GRC GRC GGC GGC GRG RTC GGC CGC GCG CRR TGG CTG CRG GCG CTG GCC GCG CGC GTG CCG TCG TCG GTG CRC 647N N R R U S D O G G E G R R Q U L Q R L R R R U P S S U H
RTG RRC CCG GTG CTC CTG RRG CCG CRG TCG GRC GTG GGC RGC CRG RTC GTC GTT CRG GGC RRG GTC GCC GGG CRG GCC RGG GGG CGG GRR 737nt N P U L L K P Q S D U G S Q0 U U Q G K U R G 0 R R G R E
TAT CRG GCG CTC RRG CCC RRG CTG CTG GGC GCt GTC RTG GRG RGT TTC GRR CRR RTR TCG GCC GGT GCC GRT CTC GTG GTG GTC GRR GGC 827Y Q R L K P K L L G R U n E S F E Q S R G R D L U U U E G
GCC GGC TCG CCG GCC GRR RTC RRC CTC RGG CCC GGC GRC RTC GCC RRT RTG GGC TTT GCG RCR CGG GCC RRT GTG CCG GTC GTG CTG GTC 917R G S P R E I N L R P G D I R N n G F R T R R N U P U U L U
GGC GRC RTC GRC CGC GGG GGG GTG RTC GCC TCG CTG GTC GGC RCG CRT GCG RTC CTG CCC GRG GRR GRC CGG CGC RTG GTG RCC GGC TRT 1007G D I O R G G U I R S L U G T H R L P E E D R R n U T G Y
CTC RTC RRC RRG TTC CGC GGC GRC GTC RCG CTG TTC GRC GRC GGC RTT GCT GCC GtC RRC CGC TRC RCC GGC TGG CCC TGC TTC GGC GTC 1097L N K F R G O U T L F D D G R R U N R Y T G U P C F G U
GTG CCG TGG CtG RRG GCG GCG GCR CGC CTO CCG GCG GRR GRT TCC GTC GTG CTG GRG RRG CTG RCG CGC GGC GRG GGG CGG GCG CTG RRG 1187U P U L K R R R R L P A E D S U U L E K L T R G E G R R L K
GTT GCC OTC CCG GTR CTG TCG CGC RTC GCC RRT TTC GRC GRC CTC GRT CCG CTC GCC GCC GRR CCG GRG RTT GAT CTC GTC TTC GTG CGG 1277U R U P U L S R I R N F D D L D P L R R E P E I D L U F U R
CCT GGC RGT CCC RTT CCG GTC GRC GCT GGC CTC GTC GTC RTT CCC GGG TCG RRR TCG RCC RTC GGC GRC CTC RTC GRT TTC CGT GCG CRR 1367P G S P I P U D R G L U U I P G S K S T G D L I D F R R Q
GGG TGG GRC CGT GRC CTC GRR CGT CRT GTG CGC CGG GGC GGC CGG GTC RTC GGC RTC TGC GGC GGC TRC CRG RTG CTC GGC CGG CGC GTC 1457G U D R D L E R H U R R G G R U G I C G G Y O n L G R R U
RCC GAT CCG CTC GGC RTC GRG GGC GGC GRR CGT GCG GTC GRG GGC CTC GGG CTG CTC GRG GTC GRG RCC GRG RTG GCG CCG GRR RRG RCG 1 547T D P L G I E G G E R A U E G L G L L E U E T E n R P E K T
GTG CGC RRC RGC CGC GCC TGG TCG CTG GRG CRT GRT GTG GTG CTC GAR GGC TRC GRR RTC CRT CTT GGC RRG RCG CRR GGt GCG GRC TGT 1637U R N S R R U S L E H D U U L E G Y E H L G K T Q G R D C
GGC CGG CCG TCG GTG CGC RTC GRC RRT CGC GCC GRC GGC GCC CTT TCG GCC GRT GGC CGC GTG RTG GGC RCC TRC CTG CRT GGG CTC TTC 1727G R P S U R I D N R A D G A L S R D G R U n G T Y L H G L F
RCC RGC GRC GCC TRT CGC GGC GCG CTG CTC RRG RGT TTC GGC RTC GRR GGC GGC GCC RRC RRC TRC CGC CRR TCG GTC GRT GCG GCG CTC 1817T S D R Y R G R L L K S F G I E G G R N N Y R O S U D R A L
GRC GAT GTC GCG RRC GRR CTG GRG GCT GTG CTC GRT CGT CGC TGG CTG GRC GRG TTG CTC RGG CRC TRG ggacgcggcaacggtcagccagcaggt 1913D D U R N E L E R U L D R R U L D E L L R H
ccggt acgtcgggcccaacaggagcaacgagct tat ccgacggaact acgct gcgacat cgt gctcct cgct tgcggct tcccagactt cccgcgcggcat ccaggt tcat cagggcaat 2033
ccccaggccgacgat caggt ccggccaggccgact gcccacgat aggct gt cgccagacccgcggcgat gatggccacat tggcgaaggcat cgt tgcgggccgagagaaat gct gcccg 2153
cgt gagcgt gccgct cgt gt gacggt aggcgacgagcagat aggcgcagaaoaggt tgaccaccagcgccccoagt ccggt cogggacagggcaaagggct ct ggcgggaccggat ccat 2273
gaac t tcgcccaggccgtccaaaggaaggccagcgccggt accagt agaat gaacgccat cgccat gccgacccgcgcgcgggt tcgcgccgt ccaggccagagcaaagaaaat cagcat 2393
gt tgacggoggcgt ct tcgaggaagtcgacgct gt cggccat gagggacaccgagccgat cgaaagcgcgacaaggagt tcgaccccgaaot agccaaggtt caacagggagacgat gag 2513
gacgocgcggcgcaggt cggt at ccactcgaaaggtt ccct t tctggcgagatt cgccct cggcact ttt tttggcgagat tcgccctcggcact t tggcocoggtgt tagcagcagt tt 2633
gc t at ccat agcactaggt t t cgacat cggt t ccgt t cacact gccgt cgt gcct gacgccegacaaat cgt cgcgt ggcgcaactcggccggggaggcgt cgcat gcgt cgat tgac tt 2753
t gggct gcccgct tccstaatcat caggt gt tggat ggt tccccssttgt cgt ggcgatct gggggaat aat tgggaat gt gacggat ggacccaaat cgggcatccstt atcgcagccgocc 2673
ccgcgact gt agaacggtcagggttcgccatcgggat tggt gccgggctgtcggccggtt gcat gggcaatcggggcaggt cggggatcaagccggoaaaagccactggcgtggcatcgtg 2993
at cagccgggt ttggacgcct ct tctt ctacgaat cgtccgcct ttcacgat gt ccct caagcgcccat gcgt cggagacgacgcgcaaaggt tcgct gt ggcoccggaaagacgccgg 3113
gaaggt gaggcgggccgct cgggccct gacat cggaccstt gccgt t taagggcgaggcgat gt tcggcccgt gacgccgt gagccaggagacct gccat ccggcot gggcat tccgccc 3233
gaggggact ttt gt ct ccacgccatcacggaggt tgt tttggct cgcagat gt tttcaagaacgcgcccgt ggcgcgt ccgatggct ttt gccaccgacggctgat ttgggaatgt tga 3353
ggcagccacg RTG RGC RGT CTC RGC GCC GGG CCC GTG CTG GTC CTT GGC GGC GCC CGT TCC GGC RRG TCC RGC TTT TCC GRG RGG CTC OTC 3t44It S S L S R G P U L U L fL G R R S G K S S F S E R L Q
--> ORf2 (cobP)GAR GCG TCC GGC TTC RCC RTG CRT TRT GTC GCC RCG GGC CGC GCC TOG GRC GRC GRR RTG CGC GRG CGC RTC GRC CRT CRC CGG RCG CGC 3534E R S G F T n H Y U A T G R R U D D E n R E R D H H R T R
CGC GGC GRG GGC TGG ACG RCG CRT GRG GRG CCG CTC GRT CTC GTC GGC RTC CTC RGR CGC RTC GRT GRT CCC RGC CRT GTG GTC CTG RTC 3624R G E G U T T H E E P L JI L U fi L R R D D P S H U U L
GRC TGC CTG RCG CTR TGG GTC RCC RRT CTC RTG CTG GRR GRG CGC GRC RTG RCG GCG GRG TTC GCC GCC CTT GTT GCG TRT CTG CCC GRG 371 tD C L T L U U T N L n L E E R D n T R E F R A L U R Y L P E
GCG CGG GCG CGC CTC GTC TTT GTT Tcc RRT GRG GTC GGC CTC GGC RTC GTG CCC GRG RRC CGC RTG GCC CGC GRG TTT CGC GRC CRT GCC 300tR R R R L U F U S N E U G L G U P E N R n R R E F R D H A
FIG. 3. Nucleotide sequence of the 13,144-bp SstI-SstI-SstI-B91II-SstI-Bg1II fragment from complementation group C (12). The beginningof each ORF is indicated along with the predicted arnino acid sequence of the gene. The DNA strand presented goes 5'--3' from SstI to Bglllsites. All the ORFs except ORF7 and ORF9 are on this strand. For the two remaining ORFs, the amino acid sequences of the putative encodedproteins were deduced from the other strand, although it is not represented. The positions of the putative ribosome-binding sites for ORF1to ORF9 are indicated above the sequence by horizontal lines. Noncoding DNA is represented in lowercase letters. Sequences sulch asinverted or direct repeats that are mentioned in the text are underlined. In addition to the 13,144-bp sequence, a 26-bp sequence (alre'adypiiblished [16]) at the Bglll boundary has been added to show the proposed initiation codon of ORF9 and the corresponding putativerihnosome-hinding s.ite. IUnderlined amnino acids. are the- same. as, thos.e fouind in k-nown ATP- or GTP-hinding consensus squen ces.ri-
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VOL. 173, 1991 P. DENITRIFICANS cobNOPQW GENES 6081
GGC CGG CTT CRC CRG RTC GTT GCG GRG RRR TCC GCT GRR GTT TRC TTT GTC GCG GCC GGT TTG CCG CTG RRR RTG RRG GGT TGR tcc RTG 3694G R L H Q U R E K S R E U V F U R R G L P L KR K G * n
--- > ORF3 (.so&U)RCC RCT GCG RGR GCC RRC CRG GGC RRG RTC CCG GCG RCC GTC RTC RCC GGC TTC CTC GGC GCC GGC RRG RCG RCG RTG RTC CGC RRC CTG 3981T T R R R N O G K P R T U T A F L G R G K T T 6 R N L
CTG CRG RRC GCC GOC GGC RRG CGC RTC GGC CTG RTC RTC RRC GRG TTC GGC GRT CTT GGC GTC GRC GGC GRT GTC TTG RRG GGC TGC GGT 1071L Q N R D G K R I G L I N E F G D L G U 0 GD U L K G C G
GCC GRG GCC TGC RCC GRG GRC GRC RTC RTC GRG CTC RCC RRT GGC TGC RTC TGC TGC RCC GTG GCT GRC GRT TTC RTC CCG RCC RTG RCG 4161R E R C T D D E L T N G C C C T U R D D F P T M T
RRG CTG CTC GRG CGT GRR RRC CGT CCT GRC CRC RTC RTC RTC GRR RCC TCG GGC CTT GCC CTG CCG CRG CCG CTG RTC GCC GCT TTC RRC 1254K L L E R E N R P D H E T S G L R L P 0 P L R R F H
TGG CCG GRT RTC CGC RGC GRR GTG RCC GTC GRT GGC GTC GTC RCC GTG GTC GRC RGC GCC GCC GTT GCC GCT GGC CGC TTT GCC GRC GRC 1341U P D R SE U T U D G U U T U U O S R R U R R G R F R D D
CRC GRC RRG GTC GRT GCG CTG CGC GTC GRG GRC GRC RRT CTC GRT CRC GRR RGC CCG RTC GRG GRG CTG TTC GRG GRT CRR CTG RCG GCT 1431H D K U D R L R U E D HN L D H E S P E E L F E D L T R
GCC GRT CTC RTC GTT CTC RRC RRG RCC GRT CTG RTC GRT GCC TCC GGC CTC RRG GCC GTG CGC GRC GRG GTG TCT TCG CGC RCC RGC CGC 4524RA L N K T D L D R S G L K R U RD CE U S S R T S R
RRG CCC RCG RTG RTC GRG GCG RRR RRC GGC GRR GTC GCC GCT GCC RTC CTG CTT GGC CTC GGT GTC GGC RCG GRA RGC GRT RTC GCC RRC 1611K P T 6 I E R K N G E U R R R I L L G L G U G T E S 0 R H
CGC ROG TCG CRT CRC GRG RTG GRG CRC GRG GCR GGT GRG GRG CRC GRT CRC GRC GRG TTC GRC RGC TTC GTC GTC GRG-CTC GGT TCG RTC 1704R K S H H EC E H E R 6 E E H OH D E F O S F U U E L G S
GCC GRT CCO GCC GCC TTC RTC GRT CGC CTG RRG GGC GTR RTC GCG GRG CRC GRC GTT CTG CGC CTC RRG GGT TTT GCR GRC GTG CCC GGC 4791A O P R R F 0 R L K G U R E H D U L R L K G F R D U P G
RRG CCG RTG CGC CTC CTG RTC CRG GCG GTC GGC GCC CGC RTC GRC CRR TRT TRC GRC CGC GCC TGG GGC GCT GGC GRO RRG CGC GGT RCG 1868K P n R L L Q R U G RA I10 Q Y D R R U G RA E K R G T
CGC CTC GTC GTC RTC GGC CTG CRC GRC RTG GRC GRG GCG GCG GTO CGC GCC GCG RTC RCC GCG CTC GTG TRG atcgttctttgaatgaaatgatc 1979R L U U G L H D n D E R R U R R R T R L U
._ ._.
taacgcattgaoatgatgcagttccggatggagaacgcttttagcgttttcgttcggaattgccccoacggacaagacga RTG CRT CTG CTT CTC GCC CRG RRR GGR RCG 5089R H L L L R Q K G T
---> ORFI (o.obNi)RTC GCC GRC GGC RRC GRG GCG RTC GRC CTT GGG CRR RCG CCG GCC GRT RTC CTT TTC CTR TCG GCC GCC GRC RCC GRG CTC TCC TCG RTC 5179
R D G N E R D L0 1 T P R D L F L S R R D T E L S S
GCC GCG GCT CRC GGC CGR CGC GRC GGR GGC TTG RGC CTG CGC RTC GCC RGC CTG RTG RGC CTG RTG CRC CCG RTG TCG GTC GRC RCT TRC 5269R R R H G R R D G G L S L R R S L Ss L R H P' nS U D T Y
GTC GRG CGC RCG GCG CGT CRC GCC RRG CTG RTC GTC GTC CGG CCG CTC GGT GGC GCC RGC TRT TTC CGT TRT CTG CTG GRR GCC CTG CRT 5359U E R T R RA R K L U U R P L G G R S Y F R Y L L E R L H
GCO GCT GCC GTC RCC CRT CGT TTC GRG RTC GCG GTT CTG CCG GGT GRC GOC RRG CCG GRT CCG GGG CTG GRG CCT TTC TCC RCC GTC GCR 5449R R A U H R F E I A U L P G 0 D K P O P G L E P F S OU R
GCC GRC GRC CGC CRG CGC CTT TGG GCT TRC TTC RCC ORR GGC GGC TCG GRC RRT GCC GGG CTG TTT CTC GRC TRT GCG GCC GCR CTG GTC 5539R D A 0 R L U R Y F T E G O S D R G L F L D V R R L U
RCR GGT GCG GRG RRG CCG CRG CCG GCR RRG CCC CTG TTG RRG GCC GGC RTC TGG TGG CCG GGT GCT GGT GTG RTC GGC GTC RGC GRR TGG 5629T GO E K P O P R C P L L K R 0 U U P G R G U G U S E U
CRG TCC CTT GTT CRG GGR CGG RTG GTR GCG RGG GRG GGR TTC GRR CCC CCG RCG GTC GGG RTC TGC TTT TRC CGC GCG CTC GTG CRG RGT 5719O S L U Q G R n U R R E G F E P P T U G I C F Y R R L U O S
GGC GRG RCR CGG CCT GTG GRG GCG CTG RTC GRT GCG CTG GRG GCT GRR GGT GTG CGG GCR CTG CCG GTG TTT GTC TCR RGC CTC RRG GRT 5809G E T R P U E R L I D R L E R E G U R R L P U F U S S L K D
GCC GTT TCC GTC GGC RCG CTG CRG GCG OTT TTT TCC GRG GCC GCR CCC GRC GTG GTG RTG RRC GCC RCT GGC TTT GCG GTC TCG TCG CCC 5699R U S U G0 L O R I F S E R R P D U U O N R OG F R U S S P
GGT GCC GRC CGT CRG CCG RCG GTG CTG GRR TCG RCC GGT GCG CCG GTG CTG CRG GTG RTT TTC TCC GGC TCG TCG CGG GCG CRR TGG GRR 5989G R AR Q P T U L E S T G R P U L U I F S G S S AR 0 U E
RCG TCG CCG CRG GGG CTG RTG GCG CGC GRC CTC GCC RTG RRC GTG GCR CTC CCC GRR GTC GRT GGC CGC RTC CTT GCG CGC GCC GTC TCC 6079T S P 0 G L n R R D L R n N U R L P E U AG R L R R R U S
TTC RRG GCG GCG TCG RTC TRT GRC GCC RRG GTG GRG GCC RRT RTC GTC GGC CRT GRG CCG CTC GRR GGC CGG GTG CGC TTT GCC GCT GRT 6169F K R R S I YV R K U E R N U G H E P L E G R U R F A R D
CTT GCC GTC RRC TGG GCG RRC GTG CGC CGG GCR GRG CCC GCC GRG CGC CGT RTT GCC RTC GTC RTG GCC RRC TRT CCG RRC CGC GRC GGT 6259L R U N UR N R R R E P R E R R I RA u0 R N Y P N R D G
CGC CTC GGC RRC GGT GTC GGG CTC GRC RCG CCG GCC GGT RCC GTC GRG GTG CTT RGC GCC RTG GCG CGG GAR GGC TRT GCG GTC GGT GRG 6349R L G N G U0 L 0 T P R G T U E U L S R n R R E G Y R U CE
GTT CCC 0CC GRT GGC GRC GCG CTG RTC CGC TTT CTG RTG GCC GGG CCG RCC RRT GCG GCG RGC CRT GRC CGT GRR RTC CGC GRG CGT RTT 6139U P R O G D R L I R F L n R G P T N R SH D0 R E R E R
TCG CTG RRC GRT TRC RRR RCG TTC TTC GRT TCG CTT CCG RRR CRG RTR RRG GRT GRR GTT GCC GGT CGC TGG GGC GTG CCG GRG GCC GRT 6529S L N D Y K T F F D S L P K K D E U R G R U G U P E R D
CCC TTT TTC CTC GRT GGC GCC TTC GCG CTG CCG CTC GCC CGC TTC GGC GRG GTG RTC GTC GGC RTC CRR CCG GCG CGC GGC TRC RRC RTC 6619P F F L D G R F R L P L R R F G E U I U G O P R R G Y N
GRT CCG RRG GRR RGC TRC CRT TCC CCG GRC CTC GTG CCG CCG CRT GGC TRT CTC GCC TTC TRC GCC TTC CTG CGC CRG CRG TTC GGR GCG 6709D P K E S Y H S P O L O P P H G Y L R F Y R F L R 0Q 0 F G R
CRG GCG RTC GTC CRC RTG GGC RRG CRC GGC RRT CTC GRR TGO CTG CCG GGC RRG GCG CTG GCG CTG TCG GRR RCC TGC TRT CCC GRR GCG 6799O R I U H n G K OG H L E U L P G K R L R L S E T C Y P E R
FIG. 3-Continued.
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RTC TTC GGG CCG CTG CCG CRC RTC TRT CCC TTC RTC GTC RRC GRT CCG GGC GRR GGT RCG CRG GCC RRG CGC CGC RCC RGC GCC GTC RTC 6889I f G P L P H I V P F U N 0 P G E 0 T Q R K R R T S R U I
RTC GRC CRC CTG RCC CCG CCC TTG RCG CGC GCC GRR TCC TRC GGC CCG CTC RRG GRT CTG GRR GCG CTC GTC GRC GRR TRT TRC GRC GCC 6979ID H L T P P L T R R E S V G P L K D L E R L U D E V Y 0 R
GCC GGC GGT GRT CCG CGC CGC CTC RGG CTG CTC RGC CGC CRG RTC CTC GRT CTC GTO CGC GRC RTC GGC CTC GRC RGC GRC GCR GGC RTC 7069R G G 0 P R R L R L L S R 0 I L 0 L U R D G L D S D R G
GRC RGG GGC GRC RGC GRC GRC RRG GCG CTG GAR RRG CTC GRC GCC TRT CTC TGC GRC CTC RAG GRR RTG CRG RTC CGC GRC GGC CTG CRC 7159o R G D S O D K R L E K L D R V L C D L K E n Q I R 0 G L H
RTC TTC GGC GTT GCG CCG GRR GGG CGG TTG TTG RCG GRC CTC RCC GTR GCG CTG GCG CGC GTG CCC CGR GGT CTC GGC ORG GGC GGC GRC 7249I F 0 U R P EC R L L T D L T U A L R R U P R G L G E G O D
CRG RGC CTG CRG CGG GCG RTC GCR GCG GRT GCG GGG CTO CGT G0G TTT GCT RTT CCC RCC TCG GCG GGG GGC RRC CCC GCR CGC GRC GCC 7339Q S L Q R R I R R D R G L R G F R I P T S R G G N P R R D R
CRR CCC TTC GRC CCO CTC GRC TOC GTC RTO TCC GRC RCC TGG RCR GGC CCO RRR CCG TCC RTC CTC GCT GRC CTC TCG GRC GCC CCC TGO 7429Q P F O P L D C U A S D T U T O P K P S L R D L S 0 R P U
CGC RCC GEE GGC ORT RCG GTC GRO CGC RTC GRG TTG CTT GCC GCR RRT CTC GTO TCG GGT GRR CTG GCT TGC CCO GRC CRC TGG GCC RRC 7519R t R G 0 T U E R I E L L R R 1 L U S G E L R C P O N U R N
RCC CGC GEE GTG ETC GGC OAR ATC GRR RCG CGC CTG RRG CCG TCG RTT TCR RRC TCG GGT GCC GCC GRO RTC RCC GGC TTC CTC RCC GGT 7609T R R U L G E I E T R L K P S I S N S G R R E n T O F L T G
CTC RGC GGC CGC TTC GTC GCC CCC GGT CCR TCG GGC GCG CCG RCG CGC GOC CGG CCO GRT GTG TTO CCO RCG 000 CGC RAT TTC TRC TCG 7699L S G R F U R P G P S G R P T R G R P D U L P T G R H F V S
GTC GRC RGC CGC GE GTGCCEO RCG CCG GCG GCT TRC GRG CTT GGC RRG RRR TCG GCC GRG CTT CTG RTC CGC CGC TRC CTG CRG GRC CRT 7789U O S R R U P T P R R V E L G K0 S R E L L R R V L 0 D H
GGC GRR TGG CCG TCC TCC TTT GGC CTG RCC GCC TGG GGC RCG GCG RRC RTG CGC RCC GGC GGC GRC GRC RTC GCC CRG GCC CTG GCG CTG 7879G E U P S S F 0 L T R U G T R N n R T O GO D I R Q A L A L
RTC GGC GCC RRG CCC RCC TGG GRC RTG GTC TCT CGC CGG GTG RTG GGC TRC GAO ATE GTGCEO ETC GCR GTC CTC GGC COC CCR CGC GTC 79691 G R C P T U 0 n U S R R U A G V E I U P L R U L G R P R U
GRC GTG RCC TTG CGC RTT TCC GGC TTC TTC CGC GRT GCC TTC CCG GRC CRG RTC GCG CTC TTC GRC RRG GCG RTC CGC GCC GTC GCG CTG 8059O U T L R I S G F F R 0 R F P 0 Q R L F 0 K R I R R U R L
GRG GRR GRC GRT GCC GRC RRC RTG RTC GCC GCR CGC RTG CGG GCG GRR RGC CGG CGG CTG GRG GCC GRR GGC GTG GRR GCC GCC GRG GCC 6149E E D D R 0D n I R R R n R R E S R R L E R E G U E R R E R
GCG CGT CGC GCC TCE TRC CGC GTC TTT GGC GCR RRG CCC GGT GCC TRT GGC GEE GCC CTG CRG GCG CTG RTC GRC GRG RRO GGC TGG GRR 8239R R R R S Y R U F G R C P G V G R R L R L I CE G U E
RCC RRR GCR GRT CTC GCC GRG GCC TRT CTT RCC TGG GGC GCC TRT GCC TRT GGC GCC GGC GRG GRG GGC RRG GCC GRG CGC GRT CTT TTC 8329T C RA L R E R V L T U GV R G E E G K R E R L F
GRG GRG CGC CTG CGC RCG RTR GRG GCG GTG GTG CRG RRC CRG GRC RRC CGC GRG CRC GRT CTG CTC GRC RGC GRC GRC TRC TRC CRG TTC 8419CE R L R T I E R U U OH 0 D N R E H D L L O S 0 0 Y Y O F
GRR GGC GGC RTG RGC GCT GCC GEC GRR CRG CTC GGC GGT CRC CGT CCG GCG RTC TRC CRC RRC GRC CRT TCC CGT CCG GRR RRG CCT OTG 8509E G G A S R R R E O L G G H R P R I Y H OD H S R P E K P U
RTC CGG TCG CTC ORR GRR GRG RTC GGC CGC GTG GTC CGG GCC CGC GTC GTC RRT CCC RRG TGG RTC GRT GGC GTC RTG CGC CRC GGR TRC 8599I R S L E E E G R U U R R R U U N P C U I D G U n R H G Y
RRG GGC GCC TTC GRG RTC GCT GCC RCG GTC GRC TRC RtG TTC GCC TTT GCC GCG RCC RCG GGT GCG GTG CGC GRC CRT CRT TTC GRG GCC 8689C G R F E I R R T U D Y n F R F R R T T G R U R D N H F E R
GCT TRT CRG GCG TTC RTT GTC GRC GRG CGC GTG GCT GRC TTC RTG CGC GRC RRG RRC CCG GCC GCC TTT GCC GRG CTT GCC GRR CGC CTG 6779R YV R F I U D E R U R O F H R HK I P R R F R E L R E R L
CTT GRR GCR RTC GRC CGC RRT CTC TGG RCG CCG CGC TCG RRT TCG GCG CGG TTT GRR CTT GCC GGC RTC GGC RCG GCR GCR RCC CGG CTT 0869L E R I D H L U T P R S N S R R F E L A G I O T R R T R L
CGT GCC GGC RRT GRR TRG agcggttccgggctggcggttatccgtccggaattgcttggaaacaaagacctggttccgtttcgctgctcagtgaagtgcgaaoaggaaccgaa 8962R R G 11 E s
gcgggacgagggcgtctgeccatcccgaacttgagaactgagggagtgatc RTG RGC GRC GRG RCG RCR GTR GGC GG GRR GCC CCG GCC GRG RRG GRC GRT 9064n S O E T T U G G E R P R E KC O
ORF5 (&ahf)GCC CGC CRC GCC RTG RAG RTG GCG RRG RAG RRG GCR GCR CGC GRR RRG RTC RTG GCG RCG RRG RCC GRC GRG RRG GGT CTG RTC RTC GTC 9174R R N R CK n R K K K R R R E K I A R T K T O E K G L I U
RRC RCC GGC RRR GGC RRG GGC RRG TCG RCC GCC GGC TTC GGC RTG RTC TTC CGC CRT RTC GCC CRC GGC RTO CCC TGC GCC GTC GTG CRG 9264N T G0 G K G K S T R G F G n I F R N R H GN P C R U U Q
TTC RTC RRG GGT GCG RTG GCR RCC GGC GRG CGC GRG TTG RTC GRO RRG CRT TTC GGC GRT GTC TGC CRG TTC TRC RCG CTC GGC GRG GGC 9354F I G AR n R T G E R E L E K H F G D U C O F Y T L G E G
ITC RCC TGG GRR RCG CRG GRT CGC GCC CGC GRT GTT GCG RTG GCT GRR RRG GCC TGG GRG RRG GCG RRG GRR CTG RTC CGT GRC GRG CGC 9141F T U E T OO R R R D U R n R E K R U E K R 1 E L I R D E R
RRC TCG RTG GTG CTG CTC GRC GRG RTC RRC RTT GCT CTG CGC TRC GRC TRC RTC GRC GTC GEE GRR GTG GTG CGC TTC CTG RRG GAR GRR 9534N S n U L L D E I M I R L R Y O Y I D U R E U U R F L C E E
RRG CCG CRC RTG RCG CRT GTO GTG CTC RCC GGC CGC RRC GCG RRR GRR GRE CTG ATE GRR GTC GCC GRT CTC GTC RCT GRG RTG GRG CTG 9624C P H n T H U U L T G RO R K E O L I E U RA L U T E R E L
RTC RRG CRT CCG TTC CGT TCC GGC RTC RRG GCG CRG ERG GGC GTG GRG TTC TGR RTG RGC CRG RGC TGG CRG TTC TGG GCG CTG CTT TCG 97131 K H P F R S G I K R 0 0 G U E F n S o S N o F U R L L S
--> ORF6GCC GCC TTC GCT GCG CTC RCG GCG GTG TTT GCC RRG GTC GGG GTT GCG CRG RTC RRC TCC GRC TTC GCR RCG CTG RTC CGC RCC GTC GTC 9603R R F R R L T R U F R K U G U RA M S O F R T L I R T U U
RTC CTC TGC GTG RTC GCC GCC RTC GTG GCG GCG RCR GGG CRG TGG CRG RRG CCR TCG GRR RTC CCG GGC CGC RCC TGG CTG TTC CTG GCG 9093I L C U I R R I U R R T G NUO K P S E I P G R T U L F L R
FIG. 3-Continued.
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VOL. 173, 1991 P. DENITRIFICANS cobNOPQW GENES 6083
CTG TCR GGG CTT GCG RCT GGC GCT TCC TGG CTT GCC TRT TTC CGC GCG CTG ARG CTC GGC GAC GCC GCC CGC GTG GCG CCG CTC GRC RRG 9903L S O L R T G R S U L R Y F R R L K L G AA R R U R P L D K
CTC TCG RTC GTC RTG GTC GCG RTC TTC GGC GTG CTC TTC CTC GGT GRR RRG CTC RRC CTG RTG RRC TGG CTC GGC GTC GCC TTC RTT GCC 10073L s uV u R I F G U L F L G E K L N L n H U L G U R F I R
GCC GGG GCG CTG TTG CTG GCG GTG TTT TGR gcgcgcctgctctggtgcctgttcactgaatgctcgcctcoatcoatccgtaatcccgacacotgcagtggttgtgaclO182R R L L L R U F *
ogcgggaggacggcot gcagot tgoaggcaat tggagcgogcgcc t tcc t gatccgt cgggccacgt cgcgcaogt tcggcaogacgc tggaagcgt cgcagcct gogggt gogc cc tgct 10301
TCR GRC CCR CCG GCG GRC RCG CCT GCR RTR GGC RCC GTR GGC GTC GCC GRR GRC CTT GGC GRG GTG GGT TTC CTC CRT GCG GRT CTG GTR 10391* U U R R U R R C V R G V R 0 G F U K R L N T E E n R I VY
GGR RRT CGR GRT CCR GGC GGR GRG CGC CRG CGC CRC CGR GRT GRC GTT GGG CRC CGC CRT CRC CGT GCC GRT CRG CGC GGT CRC CRT GCC 10401S I S I U R S L R L R U S I U N P U R n u T G I L R T u 1 G
GRC RTR GRT CGG GTT GCG CGR GRR GGC RTA GRG GCC TGR GGT CRC RRG CGG CGC GTC CTG CTT TTC RGG GRT GCC GRT CTT CCR OGR RTG 10571U V P N R S F R Y L O S T U L P R O Q K E P I I 1K U S H
RCG CRT CGC CCR TTG CGR CRG CRT CGT CRG CCC GCC GCC GRG CGT CRT CRG CGC CAG GCC GRC GGC GTG RRG GRT 6GG CGT GTC GAG CGC 10661R n R U O S L n T L G G 6 L T n L R L G U R N L I P T O L R
CGG GRT CCG GCC GRG GGC RGC RTC GRC GGR GGC CGG GRG CRT GGC GRC CGC CRG CRG GTG GRT CRC CRG CGC TGC GAC GRT CRG GCG GRR 10751P I R G L R R D U S R P L A R U R L L H I U L R VU I L R F
RRG CCT GCC CGC RRR CCC TTC CGC RTC GTC GCC RTR GGT TRG CRC GRC CGG CGR GCG GCC GGR TTG CRC GCG GCG GRG GRT CGC CRG CGC 10041L R G R F G E R DO G Y T L U U P S R G S O U R R L I R L R
GRG CGT GGR CRR TCC CRC GRC GRG CRT CRG GRT GOT GGG RRG GGT GGT GGR CRT ggoaaocctctggogcgogctgacaagocaggagcgcacgacgggtaglO942L T S L 6 U U L n L I T P L T T S n
ORF7 <---gcggccct ot gagcgtctacccggcgaagcatt ct gatcaccttgcaatctctogtaooctaogaggt tcaagcgt cggacctgt ccgactttcgtcgt ggttaccggotacttotttgccaal 1062
gcgttggaggctgtcotcgtcgcccccgccgtgtcggaaggtcggcaaaottcgtctct tgacggctgctccttccgtcgagcgattgcotoggcaggaggccgcacccatgt tagaccgl 1102
tcgacaggctaaataocgggt gaoccttgoagoat octetcagogctgcggt tggt gt cgcatcggtctt gctgt tettgt catcaggt gt ggcggggcaggcgcoaaccgt gaagagcggl 1302
ggc gt cacgagctcoagaaacgacgaccocccagaaggcgaaaccgaoooc toooocgacgcgcaagcaaagggct gcggat goagccaaggccaoggcgct cgccgoogcgcgccgt cc 1 1422
ocggatttgcaagacgcgggagagcgootgcagctatggcgcaggtccggtcggogagcagtgctcgtgctggtcgoaat ccggtgcgcctgatcttggcatoactgtcaggcgt tgacc 15142
gec cgcgaccsttcgcgcgggcaggcaagcgt gcgt cgct cgaagcgacgcct gacgcgataogaaatcaocgg gtcgc ct ggt tcgttctgaaogc t tgggot tgggt t t ggt gRT G GRR 1166 1n E
---) ORFSGCC GGC GTT GRR CGC RRR RTR RTG RTC GRT CTC GRG RRC RGC GCG CTC CRG TTT GCR RCC CGR GCR CRC GGC GRR CRG RRG CGT RRG TRT 11751R G U E R K I A I D L E N S R L Q F R T R R H G E OK R K Y
GRC GGT CGG CCC TRT RTC GTT CRT CCG RTT GCG GTG GCG GRG RTT GTT CGR RGC GTG CCC CRT RCG CCC GRR RTG RTC GCC GCR GCG CTG 11641O G R P V I U N P I R U R E I U R S U P H T P E n I R R R L
CTT CRC GRT RCG GTC GRR GRT RCC GRC GCG RCG CTG CTG GRG RTC RRG GRR GCG TTC GGC CCC RRG GTC GCR RCR CTG GTT GCG TGG CTC 11931L 0O T U E 0 T O R T L L E I K E R F G P K U R T L U R U L
RCC ONE RTR TCC RCT CCG TTC CRC GGC RRC CGR CRG GTG CGC RRG GRR CTG GRT CGC CRG CRC CTC GCR TCG GCG CCC GCC GCG GCG RRR 12021T O 1 S T P F N G I R O U R K E L O R Q H L R S R P R R R K
RCC GTC RRG CTC GEE ONE CTG ATC GRC RRT GCG RTR GCG RTC RRR GCC GGC GRT CCG RAT TTC TGG RRR GTG TTC GGC GCC GRG RTG RRR 12111T U K L RA L I ON R I R I OR G D P N F U K U F G R E n K
CGC TTG CTG GRG GTC TTG GGC GRC GGC GRC GRG RCC CTT CTC GCR RRG GCC CGT GCR TTR GCG CCG GRR TGR gagtgccgccgtttatcggcoag 12206R L L E U L G O G O E T L L RA R R R L R P E
cat gtet gt gccat gt cgocccggtcaaccggteotceoagatgeagcoaggac9ot gcat t tgcggtt t tgcccgccggt gtgg9cscOcagocgcct cocaggctgcgcggt tgcggcc 12326
gttaggacagcgcagaatttgccgaccgcgccgcgccTCR RTG CCC CRG CCR GRT CCG CRA GGG RTG CGT CGG RTC TGC GRG CRG CCG GRT CGC GRG CGC12426' H G L U I R L P H T P R LL R I R L R
GRT CER GRC GRT GRC GRG CRG CGG CTT GRT GRT CTT GGC GCC CTT GGC CRT GGC RTR GCG CGR GCC GRC CTO GGC GCC GRG GRR CTG GCC 12516I S U I U L L P K 1 K RN K R n R Y R S 0 U O R G L F OQG
GRO GCC CRT CRR CRG GCC GRC CTT CCR GRG RRC GGC GCC GRR GRR GAO GRA ORC GCC GRR GGC GCC GRC GTT GGR GCC RRR GTT GRG GRR 12606L G n L L G U K U L U R G F F L F U O F RN U N S G F N L F
CTT CGT GTG CGC CGT CGC CTT CRR CRC GCC GRR GCC GGC GRG GGT RRC GRR GCC GRG CRT GRA GRA CGR GCC GGT GCC GGG GCC GRA GRC 12696K T H R T R K L U G F O R L T U F G L A F F S G T G P G F U
GCC GTC RTR RRR GCC GRT TRG CGG CRC CRG TGT CRG CGT GRR GNE GRR 606 GOT GRC GCG GCT GTO CTG GTC GRC GTC GCC CRT GTT CGG 12706G VY F G I L P U L T L T F U F P T U R S N 0 0 UV G n N P
CTT CRG GCC GRR RTR NRG CGC RRT GGC GRT CRG CRG RRR GGG CRG GRT CGC CTT CRG CRC GTC GCC G6G RRC GRT GGT TGC GRG CRG GGC 12076K L G F Y L R I R I L L F P L I N K L U O G P U I T R L L R
GCC GRG CRC GGC GCC GGC GGC CGR CRT CRG CGC CRT CGG CRG CTG CTC TTT CRG GTT CAC GTG GCC GCG CCG GGC RTR GGR CRG CGT GGC 12966G L U RN G R S n L R n P L 0 E K L A U N G R R R Y S L T R
CGR GEE GG CGEE GRR CRR TCC CTG CRG CTT GTT GGT GCC GRG CGT CTG CRR GGG CGG GRT GCC CGC RAT GRG CRT GGC CGG RRT GGT GRT 13056S G S G F L G O L K N T G L T O L P P G R L n R P I T I
.RgillCRT GCC RCC GCC GCC GGC GRT CGR RTC GRT GRR GCC TGC GRT GRR GGC GGC GRC GRR CRG GRR GGC GRG CRG GTG GRR GGC GRG RTC TTG 13116n G G 0 0 R I S D I F ON IF N V F L F R LL N F R L DO
CRC ggcggggactcgagaggagag 13170n
ORF9 '---
FIG. 3-Continued.
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TABLE 2. ORFs of 13.1-kb sequenced fragment fromcomplementation group C
Position of': Mol wt ofORF First Last encoded
codon codon polypeptideb
1 429 1881 51,9822 3364 3883 19,4423 3892 4951 38,1214 5060 8882 138,0555 9034 9673 24,0276 9679 10098 14,9907 10895 10304 21,0578 11656 12178 19,1839 13149 12366 24,321
a Positions refer to the first bases of the codons on the sequence presentedin Fig. 3. The position of the ORF9 initiation codon is in the 5.4-kb sequencepreviously determined to be just 5 bp distant from the BglII site.
b Deduced from amino acid sequences.
order to determine the position in the pathway at which theCobN protein might act. The levels of hydrogenobyrinic acida,c-diamide accumulation in G622, G623, and G630 were211, 894, and 1,014%, respectively, with 100% correspond-ing to the level observed in the parent strain (C58-C9 Rif'NalD). Moreover, in G623 and G630, more hydrogenobyrinicacid accumulated than in the parent strain. This showed that
I kb
these mutants are blocked in a step after the building of thedescobaltocorrin nucleus. No corrinoids (except traces ofvitamin B12) were observed, indicating that the production ofcorrinoids is blocked at or before the cobalt insertion steps inthese mutants. These data argue for an involvement of theCobN protein in cobalt insertion-mediating reactions. Thedata base revealed no significant identity with the CobNprotein or between CobN and the Saccharomyces cerevisiaeferrochelatase (33), which might present some structuralsimilarities with the cobaltochelatase of the cobalamin path-way, since both enzymes chelate divalent cations into tet-rapyrrole-derived compounds.
DISCUSSIONIn the present study, five new P. denitrificans cob genes
have been identified on a 13.1-kb fragment from complemen-tation group C. Three of these cob genes are structural genesencoding proteins of known activities in P. denitrificans:CobO is the cob(I)alamin adenosyltransferase, CobQ cata-lyzes four of six amidations leading to cobyric acid, andCobP is a bifunctional enzyme transforming cobinamide intoGDP-cobinamide (Fig. 1). CobW might be involved in cobaltreduction leading to cobalt(I) corrinoids. The accumulationof intermediates in A. tumefaciens mutants complementedby cobN suggests that the CobN protein is involved, likeCobS and maybe CobT (13), in mediating cobalt insertionreactions or eventually in cobalt metabolism devoted to
73
6364 67Sau 581 60)l 62 |65 661 C]
pXL189 LIau III37
27 4124, i
25 19 26 141is 23 13 40
329 2 19 28
]1 2 2iI35 10 17
ES5 B S S B Bg E
ORF -
cobO
pXL617 SI B
pXL593 B r
ORF 2 ORF 3
cobP cobWORF4
cobN
02 0 1 03 04
1 1 IB S
ORF 5 ORF 6
cobOORF 8
cN r) 0
pXL593 - - -
pXL 1938 X r* *-
pXL 1908 _-x-X I
pXL208
lE pXL1560
pXL 189pXL 189 - - -
'ci
FIG. 4. Genetic analysis of the 13.1-kb fragment from complementation group C. Above the EcoRI-Bglll fragment are mapped the 27 Tn5Spr and the 4 Spr_fk (ill to Q14) chromosomal insertions. A plus or minus sign under the insertion site indicates the Cob phenotype. In addition,the insert of plasmid pXL189 is presented with 13 mapped TnS insertions; the plus or minus sign reveals whether or not the correspondingplasmid complements A. tumefaciens Cob mutants G632 and G633. The position of each ORF is indicated by an arrow showing the directionof transcription. The inserts of plasmids studied for complementation of mutants are presented. Signs above each insert indicate that theplasmid does (+) or does not (-) complement the Cob::TnS Spr mutants aligned with the signs. ., partial complementation (described in thetext). The complementation (+) or lack of complementation (-) ofA. tumefaciens Cob mutants G622, G623 and G630 by the plasmids studiedis also presented. B, BamHI; Bg, BglII; Cl, ClaI; E, EcoRI; S, SstI; Sau, Sau3AI.
Bg
pXL622 B I P
pXLl909 B I
pXL221 E I C1
pXL622 - - -
pXL 1 909 * +.
pXL1938- * *
pXL1908 + * *
pXL208 - - -
J. BACTERIOL.
BT8 *
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30 40 50 60 70 80 1 2 3 4DAVa4O%KqAEFIKIEMIWElKaf5qDG~~VV'FIGMM
10 20 30 40 50 60 7090 100 110 120 130 140 150
EERNLLEPH-G-V- W I EL Y80 90 100 110 120 130
160 170 180 190 200 210
L.EEVWUAINE-BPHQB2 ITII lBHtDIIEIAtUVSELIpKHAFDAG'IAIGIDY150 160 170 180 190
30 40 50 60 70 80 90B I8NI.iUfDGRIGIINEEU3LGVDGDN3CGAEACrEDDIIELT-CIOCr-VAoDFIPTM=E
VA_SG SMIF7IAALQKIU-LEVDIEE5 KG,LM-;i FiAEmK[EA190 200 210 220 230 240 250100 110 120 130 140 150 160
RENRPDBIIIErS;LALPOLLkaEWD1 IIW AD MDPR
QQDPM-ITAIPGEWIL-FN-KREMMKE9EGSV-VVDIAAE WEr-TKPELYVE,I260 270 280 290 300 310170 180 190
DHESPIEELEDQLT-AADLIV1kflLIDA
THIG-YTDIPSRWrMTLYSNNITKLKY320 330 340
FIG. 5. Alignments of amino acid sequences by the program ofKanehisa (31). (A) P. denitrificans CobO (upper sequence) and E.coli BtuR (lower sequence) proteins from positions 17 to 214 and 3to 196, respectively. Residues proposed to be important in ATPbinding are boxed. (B) P. denitrificans CobW (upper sequence) andbovine mitochondrial nicotinamide transhydrogenase (lower se-quence) from positions 28 to 195 and 186 to 347, respectively.Similarities are indicated as follows: =, same amino acid; -, aminoacid belonging to the same group (15). Horizontal bars representgaps in the alignments.
cobalt insertion. The step at which cobalt is introduced intothe cobalamin pathway is still unknown. The first possibilityis that cobalt insertion occurs at the stage at which hydro-genobyrinic acid a,c-diamide (the cobalt-free analog ofcobyrinic acid a,c-diamide) is produced, since (i) hydro-genobyrinic acid is a much better substrate for cobyrinica,c-diamide synthase than monocyanocobyrinic acid or dia-quacobyrinic acid (19), (ii) cobyrinic a,c-diamide synthase-deficient mutants accumulate hydrogenobyrinic acid (16,19), and (iii) cobN- and cobS-complemented mutants alsoaccumulate hydrogenobyrinic acid a,c-diamide. The secondpossibility is that cobalt is incorporated between precorrin-3and cobyrinic acid.The five cob genes studied are clustered and have the
same polarity. The intergenic region between cobQ andcobP is 1,481 bp, suggesting that these genes are not part ofthe same operon. cobW and cobN expression depends on thesame promoter; cobP, cobW, cobN, and cobO might be partof the same operon, since the intergenic regions betweencobP and cobW and between cobN and cobO are 3 and 150bp, respectively.
This study and other reports on the genetics of cobalaminsynthesis in P. denitrificans (11, 13, 15, 16) represent thecomplete analysis of four genomic clusters involved incoenzyme B12 synthesis in P. denitrificans. A total of 22 cobgenes as well as eight ORFs that encode polypeptides ofunknown function (Fig. 2) have been identified and se-quenced. These cob genes should represent most, if not all,of the P. denitrificans cob genes. However, at least one cobgene has been found at another locus (16) and is implicatedin the biosynthesis of (R)-1-amino-2-propanol (Fig. 1). Thisindicates that other cob genes might not be cloned. Noregulatory genes have been identified on the basis of homol-
CobN96
66 -4
45CobW
21 --
CobO
FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of total bacterial extracts stained with Coomassie blue. The cellextracts were prepared as described elsewhere (20). Lanes: 1,SC510 Rifr(pXL435); 2, SC510 Rif(pXL1909); 3, SC510 Rift'(pXL593); and 4, SC510 Rif(pXL227). The molecular weights of themarkers are indicated in thousands. The migration positions ofoverexpressed proteins are shown.
ogy with any known family of regulatory proteins. In addi-tion, the studied genes do not include the structural genes for5,6-dimethylbenzimidazole synthesis (12).Most of the cob genes are clustered in complementation
groups A and C, and the other two groups contain only fourcob genes (Fig. 2). The genetic organization of these genes inrelation to the part of the pathway on which their productsact is discussed below. All the mutants complemented bygenes from complementation group A (cobF to cobM) havebeen shown to accumulate no corrinoids (15) and reducedlevels of metal-free corrinoids compared with levels in theparent strain (6). These data indicate that all the geneproducts of cluster A are involved in building the corrinmacrocycle from precorrin-2 (Fig. 1). The first gene of thepathway, cobA, belongs to complementation group C. Then,most of the genes from this complementation group code forenzymes that are involved in the end part of the pathway, upto GDP-cobinamide (Fig. 1). Apart from CobE and CobW,for which the biochemical functions are unknown, the pro-teins CobB, CobQ, CobO, and CobP act in the same part ofthe pathway, since they are, respectively, cobyrinic acida,c-diamide synthase, cobyric acid synthase, cob(I)alaminadenosyltransferase, and cobinamide kinase-cobinamidephosphate guanylyltransferase (Fig. 1). The cobC and cobDgene products are supposed to be involved in the conversionof cobyric acid into cobinamide (16) (Fig. 1). The remainingprotein encoded by this cluster, CobN, should participate incobalt incorporation steps along with CobS and maybe CobT(13). These reactions should occur, as discussed above,either at the stage of hydrogenobyrinic acid a,c-diamideproduction or before cobyrinic acid production. If the firsthypothesis is true, all the cob genes from this cluster apartfrom cobA, cobE, and cobW code for proteins that are
involved in the reactions between hydrogenobyrinic acid andGDP-cobinamide (Fig. 1). If the second possibility is con-
firmed, then only CobB, CobC, CobD, CobO, CobP, andCobQ proteins either catalyze or take part in sequentialreactions along the pathway.The two cob genes from complementation group D (cobU
A
VOL. 173, 1991
20
._. .-. aw-.
"Ol,W -,.-il
qmpno I
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and cobV) encode proteins that catalyze sequential reactionsat the end of the pathway. The product of the reactioncatalyzed by CobU (nicotinate-nucleotide:dimethylbenzimi-dazole phosphoribosyltransferase) is the substrate of coba-lamin (5'-phosphate) synthase (i.e., CobV protein) (11) (Fig.1).From these data, a nice parallel between the arrangement
of the cob genes and the part of pathway on which the geneproducts act can be drawn (Fig. 1); it is not known whetherthis correlation results in common regulation of the expres-sion of genes belonging to the same cluster. However, it isstriking that in Salmonella typhimurium, cob genes are alsoorganized into three classes related to three parts of thepathway (28, 29). In Bacillus megaterium, a genetic cluster-ing of close biochemical functions has also been reported(10, 48).Comparison of the proteins encoded by ORF6 to ORF9
with proteins in data bases has not allowed us to find theactivities of the ORF6- to ORF9-encoded proteins or thecellular functions in which they are involved. However, it isstriking that three of these four proteins (encoded by ORF6,ORF8, and ORF9) have the characteristics of membraneproteins as analyzed by the program of Hopp and Woods(25). The clustering of these ORFs between cob genes (Fig.2) favors the possibility that some if not all of the encodedproteins form a transport system necessary for coenzymeB12 synthesis, such as a cobalt transport system. In addition,mutants in any of these four genes grow on M9 medium,indicating that they do not code for functions essential forgrowth in such conditions. The cobalamin phenotype thathas been studied is the synthesis of adenosylcobalamin (12).Therefore, we cannot exclude the possibility that thesegenes, or some ofthem, are involved in other reactions in thecorrinoid pathway such as the conversion of coenzyme B12into methyl cobalamin or the synthesis of 5,6-dimethylben-zimidazole.
ACKNOWLEDGMENTS
We thank P. E. Bost, J.-C. Brunie, J. Lunel, and J.-F. Mayaux fortheir support during this work. T. Ciora, D. Lagneaux, and I. Glayseare especially acknowledged for help in the nucleotide sequencing,and the CITI2 (Centre de Traitement Interuniversitaire d'Informa-tique A Orientation Biomddicale, Paris, France) is acknowledged fornucleic and protein sequence analysis programs. We thank D.Faucher and F. Cuine for the amino-terminal sequencing of pro-teins. F. DeBruijn, H. Krish, and D. Trollinger are acknowledgedfor their gifts of strains, plasmids, and phages.
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