CobB, a New Member of the SIR2 Family of Eucaryotic Regulatory Proteins, Is Required to Compensate for the Lack of Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase Activity in cobT Mutants during Cobalamin Biosynthesis inSalmonella typhimurium LT2*

The cobB gene ofSalmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of ∼28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element withincobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3′ of the potABCD operon, with the potABCD operon andcobB being divergently transcribed. cobB was overexpressed to ∼30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediateN 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole (also known as α-ribazole-5′-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997)J. Biol. Chem. 272, 17662–17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR andYTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996)J. Bacteriol. 178, 7016–7019).

The genetic analysis of the late steps of cobalamin (Cbl) 1 biosynthesis (also known as nucleotide loop assembly) in Salmonella typhimurium (1) has raised important questions about the biochemical capabilities of the enzymes involved (2,3). The specific step of the nucleotide loop assembly pathway that we address in this paper is the activation of the lower ligand base 5,6-dimethylbenzimidazole (Me 2 Bza). This step is catalyzed by the CobT enzyme, which converts Me 2 Bza to its 5Ј-mononucleotide (also known as ␣-ribazole-5Ј-phosphate) by transferring the phosphoribosyl group from NaMN to Me 2 Bza ( Fig. 1) (2,4).
As predicted by the biochemistry of the CobT reaction, cobalamin biosynthesis in mutants lacking CobT can be restored by providing ␣-ribazole-5Ј-phosphate in the medium (4). What was unexpected, however, was the finding that all previously reported Me 2 Bza auxotrophs (5) were alleles of cobT (2), including insertions that eliminated CobT completely from cell-free extracts (6). This phenotype was difficult to explain in light of the documented biochemical activity of CobT as the NaMN: Me 2 Bza phosphoribosyltransferase (PRTase) (EC 2.4.2.21) (4). This finding basically said that increasing the substrate for CobT would circumvent the lack of this enzyme. To help explain this paradox, we postulated the existence of an alternative enzyme that could perform the CobT reaction, with the qualification that such an enzyme would have less affinity for Me 2 Bza than CobT to explain the requirement for additional Me 2 Bza (2).
In this paper we report the identification of the cobB gene whose product is required to compensate for the lack of NaMN: Me 2 Bza PRTase activity in cobT mutants. cobB has been defined genetically by mutation analysis and physically by its nucleotide sequence (GenBank TM accession number U89687). Computer analysis of the primary amino acid sequence of CobB shows it to be a member of the SIR2 family of eucaryotic regulatory proteins.

Bacteria, Culture Media, and Growth Conditions
A list of strains, plasmids, and their genotypes are presented in Table  I. Culture media composition, concentrations of antibiotics, and nutritional supplements were as reported (2,5,7). Increase in cell density * This work was supported by National Institutes of Health Grant GM40313, U. S. Department of Agriculture Hatch Grant WIS3765 (to J. C. E.-S.), and by the College of Agricultural and Life Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) U89687.
Insertional Inactivation of cobB-Transposable elements inserted in cobB were isolated. One Tn10d(Tc) element inserted in cobB was isolated from a pool of strains (ϳ80,000) each of which was assumed to carry one Tn10d(Tc) element inserted randomly in the genome. P22 phage grown on this pool was used as donor to transduce strain JE1857 (cobT109::MudJ) to tetracycline (Tc) resistance on NB medium containing Tc (20 g/ml) and CaCl 2 (1 mM). Strain JE2445 carrying insertion cobB1176::Tn10d(Tc) was identified as described above for the point mutants.
A similar strategy was used to isolate MudJ (Casadaban's MudI1734 (15)) elements inserted in cobB. For this purpose a phage lysate grown on a pool of strains (ϳ50,000) each carrying a MudJ element in their genome was used as donor to transduce strain JE1856 (cobT111::Tn10d(Tc)) to kanamycin resistance on NB medium containing Km (50 g/ml) and EGTA (10 mM). Replica printing to E minimal medium with glucose and appropriate supplements (see above) was used to identify strains carrying cobB::MudJ insertions. Four independently isolated mutants displaying Km r Tc r and nucleotide loop assembly Ϫ phenotypes were obtained.
Deletions of cobB-Deletions of cobB were isolated in vivo by the method of Bochner et al. (16) as modified by Maloy and Nunn (17). A 0.3-ml sample from an overnight culture of strain JE2501 (cobT109::MudJ cobB1176::Tn10d(Tc)) grown in NB medium containing tetracycline was spread on freshly prepared Bochner plates and incubated 12-16 h at 42°C. Tc-sensitive (Tc s ) and kanamycin-resistant (Km r ) clones that retained the inability to grow on minimal medium containing (CN) 2 Cbi and Me 2 Bza were assumed to carry a deletion of cobB. The extent of the deletions was not determined.

Genetic Mapping of cobB
General Location-The general location of cobB in the chromosome was determined by using the Mud-P22 mapping kit of Benson and Goldman (18,19). Strain JE2445 was grown in NB medium containing Tc and plated on freshly prepared Bochner plates (16,17) to increase the frequency of isolation of Tc s strains. Phage P22 lysates were prepared from a collection of 72 strains each containing one MudP or MudQ insertion in a known site of the chromosome. Lysates were prepared after mitomycin C induction as reported (20) and delivered onto plates by means of a multiprong device. Each prong delivered approximately 10 l of lysate.
Two-and Three-factor Crosses-A more accurate location of cobB was obtained by 2-and 3-factor crosses with genetic markers within the region suggested by the Mud-P22 mapping experiments to contain cobB. Separate 2-factor crosses were performed using phage lysates grown on strain TN2757 (leuBCD485 pepT7::MudJ oxrB ϩ zhb-1624::Tn10d(Tc)) as donor and strain JE2445 (cobB1176::Tn10d(Tc) as recipient, selecting for Km r transductants, patching 500 transductants on NB, Km, EGTA, and replicaprinting onto NB, Km, Tc, EGTA, and NB, Km plates to assess the frequency of loss of the cobB1176::Tn10d(Tc) element. All strains used for 3-factor crosses carried mutations metE205 ara-9 and were derived from strain TR6583. E minimal medium containing glucose (11 mM) was used in all crosses (12). (CN) 2 Cbi and CNCbl were at concentrations described above.
Direction of cobB Transcription-The direction of transcription of cobB was determined to be clockwise by the method of Hughes and Roth (21) as described elsewhere (22). The titer of phage lysates (pfu/ml) grown on the appropriate strains was determined as described (7). The cobB1206::MudJ element was converted to cobB1206::MudA as reported (15) to increase the homology between insertions and increase the frequency of recombination between co-infecting Mud elements. cobT mutant JE1857 (cobT cobB ϩ his ϩ ) was co-infected with phage lysates grown on JE3231 (cobB1206::MudA) and JE1391 (hisF9951::MudA) or JE1392 (hisF9954::MudA) each at an approximate multiplicity of infection of 1. Ampicillin-resistant (Ap r ) transductants were selected on NB medium containing Ap (30 g/ml), incubated overnight at 37°C, and replica-printed to E minimal medium containing glucose (11 mM) as carbon/energy source and methionine (0.5 mM).

Recombinant DNA Techniques
Plasmid Isolation, Plasmid pCOBB1-A plasmid carrying the wildtype allele of cobB was recovered from a sized pool of Sau3A fragments (ϳ9 kb) obtained by partial digestion of the S. typhimurium. The Sau3A fragments were cloned into the BamHI site of the tetA gene of plasmid pBR328 (kindly provided by C. G. Miller, University of Illinois, Urbana). This plasmid is referred to hereafter as pCOBB1, and it is shown in Fig. 2. Phage P22 grown on the clone bank was used to transduce strain JE2607 (cobB1176::Tn10d(Tc) cobT109::MudJ recA1) to Cm resistance on NB, Cm, and EGTA medium. Cm r transductants were replicaprinted to E minimal medium supplemented with glucose, (CN) 2 Cbi, Me 2 Bza, Cm, and EGTA. Transductants able to grow on this medium were rid of phage (23). Plasmid DNA was isolated from strains carrying putative complementing plasmids using a Magic Mini-Prep Kit TM (Promega, Madison, WI). Plasmid DNA was electroporated into strain JE2607 to verify that complementation of the phenotype correlated with inheritance of the plasmid. Conditions for electroporation of plasmids into S. typhimurium LT2 were as reported (24). Plasmids carrying cobB ϩ were identified by their ability to grow on minimal medium supplemented with (CN) 2 Cbi and Me 2 Bza.
Plasmid pCOBB2-An ϳ5.5-kb EcoRI fragment of plasmid pCOBB1 was cloned into the multiple cloning site of plasmid pSU19 (25) digested with EcoRI. This plasmid complemented strain JE2607.
Plasmid pCOBB4 -An ϳ1.8-kb SalI fragment of plasmid pCOBB2 was cloned into the multiple cloning site of plasmid pSU19 cut with SalI. This plasmid complemented strain JE2607.
Plasmid pCOBB5-Plasmid pCOBB4 was digested with SalI and NdeI. The resulting ϳ1.3-kb fragment was cloned into plasmid pSU19 cut with the same enzymes. Plasmid pCOBB6 -The NdeI-NruI fragment of plasmid pCOBB5 (ϳ1 kb) was cloned into the overexpression vector pT7-7 cut with the same enzymes.
Overexpression of cobB ϩ -Plasmid pCOBB6 (cobB ϩ ) was moved by electroporation into strain JE2587 which carried plasmid pGP1-2 (T7 rpo ϩ ). The resulting strain, JE4349, was grown at 30°C in LB broth containing 40 g/ml kanamycin and 30 g/ml ampicillin to a cell density of ϳ2 ϫ 10 8 colony-forming units/ml. Cultures were then incubated for 2 h in a 42°C water bath with shaking, followed by overnight incubation at 37°C. One-liter cultures were incubated in an orbital shaker (LabLine Instruments, Melrose, IL) rotating at 220 rpm; cultures were cells were harvested by centrifugation (10,415 ϫ g, Sorvall RC-5B, GSA rotor; Dupont, Wilmington, DE) at 4°C for 10 min.
DNA Sequencing-Two oligonucleotide primers complementary to each end of the tetA gene were used to sequence the flanking regions of the cobB1176::Tn10d(Tc) element. Primer 1 was 5Ј-TCAACAGCTCG-CATGCATAT-3Ј, and primer 2 was 5Ј-TGAGCAGCAGTCGCAGCGAG-3Ј. Plasmid pCOBB4 DNA was isolated from E. coli strain DH5␣FЈ using Promega's Wizard Mini-Prep Kit TM (Promega, Madison, WI). Sanger's dideoxy sequencing method (26) was used to sequence using Sequenase kit version 2.0 (U. S. Biochemical Corp.) as per manufacturer's directions. Additional primers were designed as the sequence flanking the cobB1176::Tn10d(Tc) element was obtained. This strategy was used to obtain the entire sequence of the region containing cobB. The

Biochemical Techniques
Preparation of Cell-free Extracts-Two-liter cultures of strain JE4349 grown under overexpression conditions (see above) were harvested by centrifugation (10,415 ϫ g) at 4°C for 10 min with a Sorvall GSA rotor and RC-5B refrigerated centrifuge (DuPont). Cell-free extracts were obtained by sonication using a Sonic Dismembrator model 550 (Fisher). For this purpose, cells were resuspended in 50 mM Tris-Cl buffer, pH 7.5, and sonicated twice at 50% duty at a setting of 3 for 5 min. Cell-free extracts were allowed to cool for 2 min on ice after the first 5-min sonication period. Cell debris was discarded by centrifugation at 43,140 ϫ g at 4°C using a Sorvall SS34 rotor (DuPont).
Phosphoribosyltransferase (PRTase) Enzyme Activity Assay-In vitro experimental conditions described by Trzebiatowski and Escalante-Semerena (4) for assaying the activity of the CobT protein were used to test for PRTase activity in cell-free extracts enriched for CobB. Cell-free extracts containing high amounts of the CobB enzyme (Fig. 4) were obtained from strain JE4349, a strain carrying a deletion of cobT and the overexpression plasmid pCOBB6. The typical reaction mixture contained NaMN (20 nmol) [2-14 C]Me 2 Bza (40 nmol, specific activity ϭ 31.5 Ci/mol) glycine/NaOH buffer, pH 10, crude cell-free extract (ϳ63 g of protein) in a final volume of 20 l. The reaction mixture was incubated at 37°C for 15 min and terminated by heating to 100°C for 10 min. Denatured protein was pelleted at room temperature at 4,800 ϫ g for 5 min in a Marathon 13K/M microcentrifuge. Products and reagents in the mixture were resolved by TLC on silica using a CHCl 3 :MeOH (3:2) solvent system. The product of the reaction, ␣-ribazole-5Ј-phosphate, was retained at the origin and was clearly separated from radiolabeled Me 2 Bza (R F ϭ 0.86) (data not shown). Detection and quantitation of radiolabeled ␣-ribazole-5Ј-phosphate was performed with a Molecular Dynamics PhosphorImager model 4451 (Molecular Dynamics, Sunnyvale, CA).
Protein Determinations and Electrophoresis-Protein concentrations were determined by the methods of Kunitz (27) and Bradford (28). Protein mixtures were resolved by 12% SDS-PAGE using the Laemmli system (29) and visualized by staining with Coomassie Brilliant Blue (Sigma) (30).
Biological Activity Assays-In these bioassays, growth of the indicator strain JE2607 cobB1176::Tn10d(Tc) cobT109::MudJ) depended on the activity of the Cbl-dependent methionine synthase (MetH) enzyme. This enzyme catalyzes the methylation of homocysteine to methionine (31). Synthesis of Cbl in strain JE2607 was only restored when the medium was supplemented with ␣-ribazole-5Ј-phosphate, Cbl, or methionine.
An overnight culture of strain JE2607 grown in nutrient broth was used to test if CobB could synthesize ␣-ribazole-5Ј-phosphate from NaMN and Me 2 Bza in vitro. Cells were resuspended in 3 ml of 0.7% molten agar and overlaid on E minimal glucose medium (12) supplemented with glucose (11 mM) and (CN) 2 Cbi (20 nM). A 15-l sample of the reaction mixture was applied directly onto the overlay, and the plate was incubated overnight at 37°C. A 5-l sample of a 0.015 mM CNCbl stock solution (i.e. 75 pmol) was applied elsewhere on the plate as positive control; a 5-l sample of a 60 mM stock solution of Me 2 Bza (i.e. 300 nmol) was applied onto the overlay as negative control.

RESULTS
Isolation of cobB Mutants-A total of 30 cobB mutants were isolated. Of these, 10 carried independently isolated cobB deletions, 1 carried a cobB::Tn10d(Tc) insertion, 4 carried a cobB::MudJ insertion, and 15 carried independently isolated, hydroxylamine-generated point mutations.
Cobalamin Biosynthesis (Cob) Phenotype of cobB Mutants-Data presented in Table II illustrate the effect of cobB mutations on Cbl-dependent growth of cobT mutants of S. typhimurium. Unlike strain JE1857 (cobB ϩ cobT Ϫ ), strain JE2501 (cobB Ϫ cobT Ϫ ) was unable to synthesize Cbl from (CN) 2 Cbi and Me 2 Bza, hence it became a Cbl auxotroph. The fact that strains with this phenotype were isolated strongly supported our hypothesis for the existence of an alternative phosphoribosyltransferase enzyme that can compensate for the lack of CobT in the assembly of the nucleotide loop of cobalamin in cobT mutants (2,4). When the cobB1176::Tn10d(Tc) mutation was introduced into the Cbl-proficient strain TR6583 (cbi ϩ cob ϩ ), the resulting strain JE2445 was still able to synthesize Cbl from (CN) 2 Cbi in the absence of exogenous Me 2 Bza. This result suggested that cobB function may only be required for AdoCbl biosynthesis under some physiological conditions. We noted, however, a Ͻ2-fold but reproducible effect of a mutation in cobB on the doubling time of cultures of cobB mutants even when Cbl was provided in the medium (compare the doubling times of strains JE2445 and TR6583, Table II).
Propionate (Prp) Phenotype of cobB Mutants-We previously reported that insertions in cobB prevented S. typhimurium from using propionate as carbon/energy source (14). We assessed the Prp phenotype of 15 independently isolated cobB point mutants to investigate if this phenotype was due to polar effects of the insertions on a gene downstream of cobB. All point mutants displayed both Cob Ϫ and Prp Ϫ phenotypes, strongly suggesting that the lack of CobB, and not of a gene 3Ј to cobB, was responsible for both of the observed phenotypes. This idea proved to be correct since introduction of plasmid pCOBB5 (carried only the cobB ϩ gene) into JE2501 (and its recombination-deficient derivative strain JE2607) was sufficient to allow the cell to synthesize Cbl from (CN) 2 (Table II) and to use propionate as the sole source of carbon and energy (data not shown).
Genetic Mapping-cobB was located to the 27-centisome region of the chromosome using Benson and Goldman's (18) mapping kit. Two-factor crosses demonstrated that cobB was cotransducible with genetic markers in the 27-centisome region. phoQ was 25% co-transducible with cobB, whereas pepT was 82% co-transducible with cobB. Strain JE2699 (cobB pepT) was constructed for the purpose of determining the gene order of cobB relative to phoQ and pepT using 3-factor crosses. The rare class of recombinant strain (relative frequency of 0.22%, or 1 in 451 analyzed) (Table III) can be explained as the product of four recombination exchanges if the gene order was cobB pepT phoQ. These results were consistent with our sequencing data which placed cobB 3Ј to the potABCD whose location relative to pepT and phoQ is known in Escherichia coli (32) and S. typhimurium (33).
Analysis of cobB and Its Product-The nucleotide sequence of cobB and the predicted primary sequence of the protein is shown in Fig. 3. Also included in Fig. 3 is the location of the cobB1176::Tn10d(Tc) element. We have tentatively assigned the AGAG sequence located 11 bp away from the methionine codon as the ribosome-binding site (also known as Shine-Dalgarno). Other putative Shine-Dalgarno sequences are located 16 bp (GAGA) and 29 bp (GAGGA) away from the translation initiation codon. Putative promoter sequences, i.e. the Ϫ10 and Ϫ35 regions, for cobB were identified 217 and 240 bp 5Ј to cobB, respectively; these sequences were separated by 17 bp. Whereas the Ϫ35 sequence is close to the consensus sequence TTGACA (4 out of 6 match), the putative Ϫ10 sequence is notably away from the consensus sequence TATAAT. It should be emphasized that both the Shine-Dalgarno and promoter sequences are putative, and their role in cobB expression needs to be demonstrated.
We note that the analysis of the cobB sequence showed that this gene lacked its native promoter in plasmid pCOBB1 which was isolated from the gene library. Only 21 bp separated cobB from vector sequence. However, since this plasmid was isolated as capable of complementing the phenotypes of cobB mutants, we inferred that expression of the promoterless cobB was under the control of an unidentified promoter within the vector. No effort was made to identify such a promoter.
Computer comparisons of the cobB and CobB sequences with available gene and protein sequence data bases failed to identify any genes of known function. Interestingly, neither cobB nor CobB showed homology to cobT or CobT. The cobB homolog in E. coli, however, is annotated as a putative member of the SIR2 family of proteins (32). The residues that constitute the core domain of these proteins is underlined in Fig. 3. There are differences in the location of the cobB gene in S. typhimurium and E. coli. In E. coli the potABCD operon and the cobB homolog are separated by 1,424 bp, whereas in S. typhimurium the intervening sequence between these loci is only 81 bp, suggesting that the region 5Ј to cobB may also be different in S. typhimurium.
Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole PRTase Activity in Cell-free Extracts Enriched for CobB-To facilitate the biochemical analysis of the CobB function, the level of CobB in the cell was increased to ϳ30% of the total protein by placing cobB under the control of a phage T7 promoter and ribosome binding site (Fig. 4, lane C). Cell-free extracts obtained from the cobB overexpressing strain JE4349 were used to test if CobB had CobT-like NaMN:Me 2 Bza PRTase activity. Data presented in Fig. 5, A and B, show a correlation between the presence of CobB in the extract and the synthesis of ␣-ribazole-5Ј-phosphate (Fig.  5A, Rxn. product). In contrast, assay mixtures containing cellfree extract of control strains that did not carry the overexpression plasmid pCOBB6 failed to synthesize ␣-ribazole-5Ј-phosphate as judged by the lack of growth of the indicator strain JE2607 (cobT Ϫ cobB Ϫ ) (Fig. 5B, Rxn. product). As expected, reaction mixtures lacking a source of protein failed to stimulate growth of the indicator strain. Although these results suggest that CobB has PRTase activity, a demonstration of activity using homogeneous CobB protein is needed. DISCUSSION CobB, a Putative New NaMN:Me 2 Bza Phosphoribosyltransferase Enzyme-The gene encoding the alternative enzyme for CobT has been characterized both physically and genetically. From the data in hand, it appears that cobB is monocistronic and not part of an operon. One interpretation of the genetic and biochemical data presented in this paper would be that cobB encodes a new NaMN:Me 2 Bza phosphoribosyltransferase enzyme that is specific for NaMN, and as with CobT, phosphoribosylpyrophosphate (PRPP) does not substitute for NaMN in the reaction. 2 If CobB has this activity, it is not as efficient an enzyme as CobT. Whereas CobB can satisfy the cell requirement for cobalamin for the purpose of synthesizing methionine, CobB phosphoribosyltransferase activity is not sufficient when the demand for cobalamin is increased, e.g. during growth on ethanolamine or 1,2-propanediol as carbon and energy source. A different interpretation would suggest that CobB is not an enzyme, but it is somehow required for the synthesis of the alternative phosphoribosyltransferase enzyme. This possibility is discussed below.
CobB Is a Member of the SIR2 Family of Eucaryotic Regulatory Proteins-We have previously reported the requirement of CobB for the catabolism of propionate in S. typhimurium (14). Although no specific biochemical activity for CobB in this system has been identified so far, we know that a lack of cobB function prevents transcription of the prpBCDE operon 3 which encodes the enzymes responsible for the degradation of propionate in this bacterium (34). 4 This observation is interesting for the following reasons. The CobB protein appears to be a procaryotic member of the SIR2 family of eucaryotic transcriptional regulators. CobB contains the sequences GAGISAES-GIRTFR and YTQNID (Fig. 3) which are a good match to the consensus sequences GAGISTS(L/A)GIPDFR and YTQNID. These sequences are diagnostic of this family of regulatory proteins (36). It is interesting that in addition to these sequences, the 17 residues immediately preceding the YTQNID sequence comprise a region of high hydrophobicity. Of these 17 residues, 6 are leucines, 4 are alanines, 1 is isoleucine, 1 is valine, and 1 is phenylalanine. The large number of leucines and isoleucine in this region would be consistent with the observation that members of this family of proteins appear to have a leucine zipper in this region. The identification of CobB as a member of the SIR2 family of regulators would be consistent with the effect of CobB on the expression of the prpBCDE operon. In light of this finding, we are currently addressing the possibility that CobB may not have the alluded enzymatic activity, but instead it may be required for the expression of the alternative PRTase enzyme. Unequivocal assessment of the enzymatic activity of CobB requires homogeneous protein. This work is in progress. 2 CobB, an Enzyme, Regulator, or Both?-The presence of PRTase activity in regulatory proteins is not unprecedented. The PyrR protein of Bacillus subtilis is a member of a family of proteins that regulate transcription attenuation by binding to mRNA (37)(38)(39)(40)(41)(42). PyrR is also a PRPP-dependent uracil phosphoribosyltransferase, but it is not clear whether this activity is needed for the regulatory function of PyrR. In fact, the physiological significance of the uracil phosphoribosyltransferase activity of PyrR in B. subtilis is also unclear since at physiological pH this activity is very low (43). In addition, this bacterium contains an alternative enzyme encoded by the upp a Cm-resistant transductants were selected on NB medium containing Cm (20 g/ml) and EGTA (10 mM) after a 2-h preincubation of the bacteria/phage suspension at 30°C without shaking. The inferred map order was cobB pepT phoQ. b The abbreviations used are: R, resistant; S, sensitive. A, Rxn. Product, reaction mixture containing cell-free extract (CFE) of the cobB overexpressing strain JE4349, NaMN, Me 2 Bza, and buffer as described under "Experimental Procedures." B, Rxn. Product, reaction mixture containing cell-free extract of strain JE4350 (carrying plasmid pT7-7 without cobB ϩ ), NaMN, Me 2 Bza, and buffer. In both panels cobalamin (Cbl) and Me 2 Bza were used as positive and negative controls, respectively. gene (44). It has been suggested that PyrR evolved from an ancestral PRTase in which UMP and PRPP-binding sites were retained and an RNA-binding surface arose (45). Unlike PyrR, work presented herein shows that the putative PRTase activity of CobB is physiologically significant. CobB may be the first NaMN-dependent PRTase with regulatory function.