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J Biol Chem, Vol. 273, Issue 48, 31788-31794, November 27, 1998
From the Department of Bacteriology, University of Wisconsin,
Madison, Wisconsin 53706-1567
The cobB gene of
Salmonella 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 within
cobB 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 and cobB 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 intermediate
N1-(5-phospho- 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 (Me2Bza). This step is catalyzed by the CobT enzyme, which
converts Me2Bza to its 5'-mononucleotide (also known as
As predicted by the biochemistry of the CobT reaction, cobalamin
biosynthesis in mutants lacking CobT can be restored by providing In this paper we report the identification of the cobB gene
whose product is required to compensate for the lack of
NaMN:Me2Bza PRTase activity in cobT mutants.
cobB has been defined genetically by mutation analysis and
physically by its nucleotide sequence (GenBankTM 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 was monitored at 650 nm
with a Spectronic 20D spectrophotometer (Milton Roy Co., Rochester,
NY). Five-ml cultures were grown with shaking (~200 rpm) in 18 × 150-mm PyrexTM tubes. Doubling times were calculated graphically
from semi-log plots of A650 as a function of
time (hours).
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 in
Salmonella typhimurium LT2*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-D-ribosyl)-5,6-dimethylbenzimidazole
(also known as -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 and YTQNID 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).
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-ribazole-5'-phosphate) by transferring the phosphoribosyl group
from NaMN to Me2Bza (Fig. 1)
(2, 4).
View larger version (13K):
[in a new window]
Fig. 1.
Nucleotide loop assembly pathway.
AdoCbl, adenosylcobalamin; AdoCbi,
adenosylcobinamide; AdoCbi-P, adenosylcobinamide phosphate;
AdoCbi-GDP, adenosylcobinamide-GDP. CobU, AdoCbi
kinase, AdoCbi-GDP guanylyltransferase; CobT, nicotinate
mononucleotide (NaMN):5,6-dimethylbenzimidazole
(Me2Bza) phosphoribosyltransferase;
CobC, -ribazole-5'-phosphate phosphatase;
CobS, cobalamin synthase CobB, alternative
NaMN:Me2Bza phos-
phororibosyltransferase.
-ribazole-5'-phosphate in the medium (4). What was unexpected, however, was the finding that all previously reported
Me2Bza 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:Me2Bza
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 Me2Bza than CobT to explain the requirement
for additional Me2Bza (2).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Strains and plasmids
Genetic Techniques
Transductions-- All genetic crosses were performed with the high transducing mutant phage P22 HT105 int-201 (8, 9) as described elsewhere (7). Crosses that selected for kanamycin-resistant (Kmr) or chloramphenicol-resistant (Cmr) transductants were first preincubated nonselectively for 2 h at 30 °C without shaking (10).
Point Mutations--
Point mutations in cobB were
isolated by localized chemical mutagenesis using hydroxylamine as
described elsewhere (7, 11). A P22 phage lysate grown on strain JE2761
was mutagenized for 51 h which achieved an ~2 log reduction of
the lysate titer from 2.5 × 1011 to 2.6 × 109 (pfu/ml). Mutagenized lysate was used as donor to
transduce strain JE1857 (cobT109::MudJ) to
chloramphenicol resistance on NB medium containing Cm (20 µg/ml) and
EGTA (10 mM). Transductants were replica-printed onto E
minimal medium (12) containing glucose and either dicyanocobinamide
((CN)2Cbi, 20 nM) and 5,6-dimethylbenzimidazole (Me2Bza, 300 µM) or cyano-cobalamin (CNCbl),
20 nM. cobB mutants were identified by their
failure to grow on medium containing (CN)2Cbi and
Me2Bza and by their growth on medium containing CNCbl, i.e. a nucleotide loop assembly (Nla
)phenotype.
The propionate (Prp) phenotype of cobB mutants was assessed
as described elsewhere (13, 14).
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 (~80,000) 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 µg/ml) and CaCl2 (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 (~50,000) 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 µg/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 Kmr Tcr and
nucleotide loop assembly 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 (Tcs) and kanamycin-resistant (Kmr) clones that retained the inability to grow on minimal medium containing (CN)2Cbi and Me2Bza 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 Tcs 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 µl 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+ zhb-1624::Tn10d(Tc)) as donor and strain JE2445 (cobB1176::Tn10d(Tc) as recipient, selecting for Kmr transductants, patching 500 transductants on NB, Km, EGTA, and replica-printing 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)2Cbi 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+ his+) 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 (Apr) transductants were selected on NB medium containing Ap (30 µg/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 wild-type allele of cobB was recovered from a sized pool of Sau3A fragments (~9 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. Cmr transductants were replica-printed to E minimal medium supplemented with glucose, (CN)2Cbi, Me2Bza, 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 KitTM (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+ were identified by their ability to grow on minimal medium supplemented with (CN)2Cbi and Me2Bza.
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Plasmid pCOBB2-- An ~5.5-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 pCOBB3-- A derivative of plasmid pCOBB2 carrying the cobB1176::Tn10d(Tc) element was constructed by transduction. Phage P22 grown on strain JE2445 (cobB1176::Tn10d(Tc)) was used to transduce strain JE2600 (DEL1184cobB/pCOBB2 cobB+) to Tc resistance. Insertion of the cobB1176::Tn10d(Tc) element into cobB on the plasmid prevented strain JE2600 from growing on minimal medium containing (CN)2Cbi and Me2Bza. The new plasmid carrying the cobB1176::Tn10d(Tc) allele is referred to as plasmid pCOBB3.
Plasmid pCOBB4-- An ~1.8-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 ~1.3-kb fragment was cloned into plasmid pSU19 cut with the same enzymes.
Plasmid pCOBB6--
The NdeI
NruI
fragment of plasmid pCOBB5 (~1 kb) was cloned into the overexpression
vector pT7-7 cut with the same enzymes.
Overexpression of cobB+-- Plasmid pCOBB6 (cobB+) was moved by electroporation into strain JE2587 which carried plasmid pGP1-2 (T7 rpo+). The resulting strain, JE4349, was grown at 30 °C in LB broth containing 40 µg/ml kanamycin and 30 µg/ml ampicillin to a cell density of ~2 × 108 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 × 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 5'-TCAACAGCTCGCATGCATAT-3', and primer 2 was
5'-TGAGCAGCAGTCGCAGCGAG-3'. Plasmid pCOBB4 DNA was isolated from
E. coli strain DH5F' using Promega's Wizard Mini-Prep
KitTM (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 nucleotide sequence of
cobB is available from GenBankTM under the
accession number U89687.
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 × 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 × 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)
[2-14C]Me2Bza (40 nmol, specific
activity = 31.5 µCi/µmol) glycine/NaOH buffer, pH 10, crude
cell-free extract (~63 µg of protein) in a final volume of 20 µl.
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 × g for 5 min in
a Marathon 13K/M microcentrifuge. Products and reagents in the mixture
were resolved by TLC on silica using a CHCl3:MeOH (3:2)
solvent system. The product of the reaction, -ribazole-5'-phosphate,
was retained at the origin and was clearly separated from radiolabeled
Me2Bza (RF = 0.86) (data not shown).
Detection and quantitation of radiolabeled -ribazole-5'-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-5'-phosphate, Cbl, or methionine.
An overnight culture of strain JE2607 grown in nutrient broth was used
to test if CobB could synthesize -ribazole-5'-phosphate from NaMN
and Me2Bza 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)2Cbi
(20 nM). A 15-µl sample of the reaction mixture was
applied directly onto the overlay, and the plate was incubated
overnight at 37 °C. A 5-µl sample of a 0.015 mM CNCbl
stock solution (i.e. 75 pmol) was applied elsewhere on the
plate as positive control; a 5-µl sample of a 60 mM stock
solution of Me2Bza (i.e. 300 nmol) was applied
onto the overlay as negative control.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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+ cobT),
strain JE2501 (cobB cobT) was
unable to synthesize Cbl from (CN)2Cbi and
Me2Bza, 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+cob+), the resulting
strain JE2445 was still able to synthesize Cbl from
(CN)2Cbi in the absence of exogenous Me2Bza.
This result suggested that cobB function may only be
required for AdoCbl biosynthesis under some physiological conditions.
We noted, however, a <2-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).
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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 and Prp
phenotypes, strongly suggesting that the lack of CobB, and not of a
gene 3' 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+ gene)
into JE2501 (and its recombination-deficient derivative strain JE2607)
was sufficient to allow the cell to synthesize Cbl from
(CN)2Cbi in the presence of exogenous Me2Bza
(Table II) and to use propionate as the sole source of carbon and
energy (data not shown).
Genetic Mapping-- cobBwas located to the 27-centisome region of the chromosome using Benson and Goldman's (18) mapping kit. Two-factor crosses demonstrated that cobB was co-transducible 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 3' to the potABCD whose location relative to pepT and phoQ is known in Escherichia coli (32) and S. typhimurium (33).
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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 
10 and 35 regions,
for cobB were identified 217 and 240 bp 5' to
cobB, respectively; these sequences were separated by 17 bp.
Whereas the 35 sequence is close to the consensus sequence TTGACA (4 out of 6 match), the putative 10 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.
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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 5' to cobB may also be different in S. typhimurium.
Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole
PRTaseActivity 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 ~30% 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:Me2Bza 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-5'-phosphate (Fig. 5A, Rxn.
product). In contrast, assay mixtures containing cell-free extract
of control strains that did not carry the overexpression plasmid pCOBB6
failed to synthesize -ribazole-5'-phosphate as judged by the lack of
growth of the indicator strain JE2607
(cobT cobB) (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.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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CobB, a Putative New NaMN:Me2Bza 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:Me2Bza 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 operon3 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 GAGISAESGIRTFR 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
(37-42). 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 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.
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ACKNOWLEDGEMENTS |
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We thank J. Trzebiatowski for helpful discussions and C. G. Miller and E. Groisman for strains.
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FOOTNOTES |
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* 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. The 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 GenBankTM/EMBL Data Bank with accession number(s) U89687.
To whom correspondence should be addressed: Dept. of Bacteriology,
University of Wisconsin, 1550 Linden Dr., Madison, WI 53706-1567. Tel.:
608-262-7379; Fax: 608-262-9865; E-mail:
jcescala{at}facstaff.wisc.edu.
The abbreviations used are:
Cbl, cobalamin; NB, nutrient broth; MudJ, MudI1734; Tn10d(Tc), Tn10DEL16DEL17; PRTase, phosphoribosyltransferase; Me2Bza, 5,6-dimethylbenzimidazole; -ribazole-5'-phosphate, synthesizes
N1-(5-phospho--D-ribosyl)-5,6-dimethylbenzimidazole; -ribazole, N1-(-D-ribosyl)-5,6-dimethylbenzimidazole; (CN)2Cbi, dicyanocobinamide; AdoCbl, adenosylcobalamin; CNCbl, cyanocobalamin; Prp, propionate; PRPP, phosphoribosylpyrophosphate; Tc, tetracycline; Tcr, tetracycline-resistant; Cm, chloramphenicol; Cmr, chloramphenicol-resistant; Km, kanamycin; Kmr, kanamycin-resistant; pfu, plaque-forming unit; kb, kilobase pair.
2 A. W. Tsang and J. C. Escalante-Semerena, unpublished results.
3 Tsang, A. W., Horswill, A. R., and Escalante-Semerena, J. C. (1998) J. Bacteriol. in press.
4 A. R. Horswill and J. C. Escalante-Semerena, submitted for publication.
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Abstract
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