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J Bacteriol, April 1998, p. 1814-1821, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification and Function of the pdxY Gene, Which
Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of
Pyridoxal 5'-Phosphate Biosynthesis in Escherichia
coli K-12
Yong
Yang,
Ho-Ching Tiffany
Tsui,
Tsz-Kwong
Man, and
Malcolm E.
Winkler*
Department of Microbiology and Molecular
Genetics, University of Texas
Houston Medical School, Houston,
Texas 77030-1501
Received 9 December 1997/Accepted 4 February 1998
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ABSTRACT |
pdxK encodes a pyridoxine (PN)/pyridoxal
(PL)/pyridoxamine (PM) kinase thought to function in the salvage
pathway of pyridoxal 5'-phosphate (PLP) coenzyme biosynthesis. The
observation that pdxK null mutants still contain PL kinase
activity led to the hypothesis that Escherichia coli K-12
contains at least one other B6-vitamer kinase. Here we
support this hypothesis by identifying the pdxY gene
(formally, open reading frame f287b) at 36.92 min, which
encodes a novel PL kinase. PdxY was first identified by its homology to
PdxK in searches of the complete E. coli genome. Minimal
clones of pdxY+ overexpressed PL kinase
specific activity about 10-fold. We inserted an omega cassette into
pdxY and crossed the resulting
pdxY::
Kanr mutation into the
bacterial chromosome of a pdxB mutant, in which de novo PLP
biosynthesis is blocked. We then determined the growth characteristics
and PL and PN kinase specific activities in extracts of
pdxK and pdxY single and double
mutants. Significantly, the requirement of the pdxB pdxK
pdxY triple mutant for PLP was not satisfied by PL and PN, and
the triple mutant had negligible PL and PN kinase specific activities.
Our combined results suggest that the PL kinase PdxY and the PN/PL/PM
kinase PdxK are the only physiologically important B6
vitamer kinases in E. coli and that their function is
confined to the PLP salvage pathway. Last, we show that
pdxY is located downstream from pdxH (encoding
PNP/PMP oxidase) and essential tyrS (encoding
aminoacyl-tRNATyr synthetase) in a multifunctional operon.
pdxY is completely cotranscribed with tyrS, but
about 92% of tyrS transcripts terminate at a
putative Rho-factor-dependent attenuator located in the
tyrS-pdxY intercistronic region.
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INTRODUCTION |
Pyridoxal 5'-phosphate (PLP) is the
active form of vitamin B6 and acts as an essential,
ubiquitous coenzyme in many aspects of amino acid and cellular
metabolism (3, 7, 10). PLP is synthesized de novo in
Escherichia coli by a pathway that is thought to condense
4-phosphohydroxy-L-threonine (4PHT) and
D-1-deoxyxylulose to form pyridoxine 5'-phosphate (PNP)
(9, 12, 18-21, 25, 41, 45-47). PNP is then oxidized by the
PdxH oxidase to form PLP, the active coenzyme (Fig.
1) (4, 24, 26, 27, 38, 48). In
addition, PLP can be synthesized by a salvage pathway that utilizes
pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) taken up
from the growth medium (Fig. 1) (20, 44). In the salvage
pathway, PL, PN, and PM are first phosphorylated by kinases to form
PLP, PNP, and pyridoxamine 5'-phosphate (PMP), respectively (Fig. 1).
PNP and PMP are oxidized by the PdxH oxidase, which functions in both
the salvage and de novo pathways (20, 26, 29, 48). Similar
salvage pathways are present in mammalian cells, which lack a de novo
PLP biosynthetic pathway (5, 6, 17). In mammalian cells, PLP
homeostasis is further maintained by the offsetting activities of PL
kinases and a PLP-specific phosphatase (13-15). A
cytoplasmic PLP phosphatase activity has been detected in E. coli K-12, but it has not yet been determined whether this
phosphatase is specific for PLP (43).
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FIG. 1.
De novo and salvage pathways for PLP biosynthesis in
E. coli K-12. The de novo pathway illustrates that the
intermediate 4PHT is synthesized from erythrose 4-phosphate (E4P) by a
series of steps, one of which is catalyzed by the PdxB dehydrogenase
(9, 25, 32). 4PHT can be produced in pdxB mutants
from GA or 4HT by alternative pathways that normally do not contribute
to de novo PLP biosynthesis (12, 20, 46). PNP, which is the
first B6 vitamer synthesized by the de novo biosynthetic
pathway, is formed by the condensation of 4PHT and
D-1-deoxyxylulose (DX) (18, 20, 21, 46). PNP
formation from the de novo pathway does not require the
activities of PL and PN kinases, which phosphorylate PL, PN, and PM
taken up from the environment. The PNP/PMP oxidase PdxH functions in
both the de novo and salvage pathways. As shown here, PdxY is a PL
kinase in vivo, whereas PdxK is a PN kinase that can also phosphorylate
PL and PM. See the text for additional details.
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We recently reported the identification of the pdxK gene,
which encodes a PN kinase (44). Previously, a PN kinase with
additional PL and PM kinase activities was purified from E. coli, and it is likely that pdxK encodes this PN/PL/PM
kinase (39). This was the first identification of a gene
encoding a PN/PL/PM kinase in any organism and led to the rapid
identification of a gene encoding a PL kinase in humans
(17). A reverse genetics approach was used in the protozoan
Trypanosoma brucei to identify a gene encoding a PL kinase,
which showed significant homology to E. coli PdxK
(34).
We showed previously that a pdxK null mutant lacks PN kinase
activity but still contains PL kinase activity that is detectable in
bacteria in which de novo PLP biosynthesis is blocked (44). This finding led to the hypothesis that E. coli K-12
contains at least one other PL kinase that converts PL to PLP. Here we confirm this hypothesis by identifying the pdxY gene, which
encodes a novel PL kinase whose function is confined to the
B6 vitamer salvage pathway. We show further that
pdxY is located in a multifunctional operon that contains
the gene for the PdxH PNP/PMP oxidase, which functions in both the de
novo and salvage pathways of PLP synthesis (Fig. 1), and the essential
tyrS gene, which encodes aminoacyl-tRNATyr
synthetase.
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MATERIALS AND METHODS |
Materials.
Restriction endonucleases, T4 DNA polymerase, T4
DNA ligase, T7 RNA polymerase, SP6 RNA polymerase, RQ1 DNase, and
Wizard Miniprep DNA Purification Systems were purchased from Promega Corp. (Madison, Wis.). Some restriction endonucleases, Vent DNA (exo+) polymerase, and 10× PCR buffer were
purchased from New England Biolabs, Inc. (Beverly, Mass.). PN, PL, PM
(98% pure), PLP, glycolaldehyde (GA), antibiotics, hydroxylamine, and
zinc chloride were purchased from Sigma Chemical Co. (St. Louis, Mo.).
RNase T2 and custom DNA oligomers were purchased from Gibco-BRL, Inc.
(Gaithersburg, Md.). 4-Hydroxy-L-threonine (4HT) was a
generous gift from Ian Spenser (McMaster University, Hamiton, Ontario,
Canada). Bacto-Agar was obtained from Difco Laboratories (Detroit,
Mich.). [3H]PN substrate was synthesized by reduction of
PL with sodium [3H]borohydride as described previously
(44). [
-32P]CTP (10 mCi/ml; >4,000
Ci/mmol) used to radiolabel RNA probes was purchased from Amersham
Corp. (Arlington Heights, Ill.).
Bacterial strains, plasmids, and media.
Bacterial strains
and plasmids used in this study are listed in Table
1. Strains isogenic to NU426 were
constructed by generalized transduction with P1vir
bacteriophage (28, 35). Cloning and genetic manipulations
were performed by standard methods (31).
Vogel-Bonner minimal salts (1 × E) plus 0.4% (wt/vol) glucose
medium (MMG) was supplemented with 1 µM PN, 1 µM PL, 1 µM PM,
1 mM GA, or 6 µM 4HT where indicated. Solid medium contained 1.5%
(wt/vol) Bacto Agar. Luria-Bertani (LB) medium (10 g of NaCl per
liter)
was prepared from capsules purchased from Bio 101, Inc.
(Vista,
Calif.). Antibiotics were added to MMG and LB at the following
concentrations where indicated: kanamycin, 12.5 and 50 µg per
ml,
respectively; chloramphenicol, 20 and 25 µg per ml, respectively;
tetracycline, 10 µg/ml; and ampicillin, 50 to 100 µg per ml.
Cloning of pdxY.
Putative genes encoding pyridoxal
kinases were identified by Blast searches of the complete E. coli genome for homologs of pdxK (see Results and
Discussion). A hypothetical reading frame (f287b) of 287 amino acids was found to encode PL kinase and was designated
pdxY. It was amplified on 1.1- and 1.6-kb fragments from
E. coli genomic DNA by using standard PCR with Vent
(exo+) DNA polymerase. The primers used to
amplify pdxY on the 1.1- and 1.6-kb fragments were S1
(5'-AGAAGCTTGTCTGTTTGGTCGTTTTA-3' in tyrS)/S2
(5'-AACTGAATTCGGAAGGGTTAGAGCAC-3' in gst) and L1
(5'-TGCAAGCTTCCCGTGGTCAGGCA-3' in tyrS)/L2
(5'-CCTGAATTCCTGCTGGATGACGGTA-3' in gst),
respectively. The S1 and L1 or S2 and L2 primers contain
HindIII or EcoRI sites, respectively. The PCR
fragments were ligated into the HindIII and
EcoRI sites of vector pUC19 to form plasmids pTX608 and
pTX618 (Table 1; Fig. 2). The
orientation of the pdxY reading frame was the same as
that of the lacZ segment in the pUC19 vector, and
isopropyl-
-D-thiogalactopyranoside (IPTG) induced
pdxY expression (see Results and Discussion).
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FIG. 2.
Structure and transcription of the region surrounding
the pdxY gene at 36.9 min in the chromosome of E. coli K-12. The figure is drawn to scale. The orientations of the
reading frames of pdxH (encoding PNP/PMP oxidase),
tyrS (encoding tyrosine aminoacyl-tRNA synthetase), and
gst (encoding glutathione S-transferase) and the
location of the pdxY::Kanr insertion
constructed herein are indicated by arrows. Locations of the
PpdxH and PtyrS promoters
are from reference 24, and the Rho-factor-dependent
AtntyrS attenuator and
TerpdxY and Tergst
terminators were localized as described in the text. Horizontal lines
represent the pdxY+ inserts, generated by
high-fidelity PCR, used to construct plasmids pTX618 and pTX608
(minimal pdxY+ clone) and the inserts in the
indicated plasmids used to synthesize RNA probes 1 to 5 and 1o to 5o
for mapping of in vivo transcripts by RNase T2 protection assays (see
the text and Materials and Methods).
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Culture growth, preparation of S150 crude extracts, and enzyme
assays.
Five milliliters of starter cultures containing LB medium,
the supplements indicated, and appropriate antibiotics were grown overnight at 37°C with vigorous shaking. One to two milliliters of
the overnight cultures was inoculated into 200 ml of fresh LB
containing the indicated supplements. Antibiotics were omitted from
final cultures except for maintaining plasmids, in which case 50 to 100 µg of ampicillin per ml was added. The cultures were grown with
shaking at 37°C to a turbidity of 70 Klett (660 nm) units
(
6.1 × 108 cells per ml) and were harvested by
centrifugation at 4,420 × g for 15 min at 4°C. Cells
were resuspended in 20 ml of cold 20 mM KPO4 buffer (pH
7.2) and were collected by centrifugation. Following a second round of
washing, pellets were resuspended in 2 ml of cold 40 mM
KPO4 buffer (pH 7.2) containing 1 mM dithiothreitol. Cells
were disrupted by passage through a French pressure cell (20,000 lb/in2), and suspensions were centrifuged at 150,000 × g for 60 min. The S150 supernatants were assayed for PL
and PN kinase activities.
PL kinase activity in S150 crude extracts was measured by a
fluorometric assay as described in reference
36.
Briefly, reaction
mixtures (3 ml) contained 40 mM KPO
4 (pH
7.2), 0.1 mM PL, 1 mM
ATP, 1 mM dithiothreitol, 0.5 mM zinc chloride,
and about 2 mg
of S150 crude extract. Reactions were started by
addition of the
S150 crude extract, and reaction mixtures were
incubated at 37°C
for 60 min. Hydroxylamine was then added to a final
concentration
of 1 mM, and fluorescence intensity (excitation
wavelength, 380
nm; emission wavelength, 450 nm) was determined 90 s later to
maximize the signal-to-background ratio of hydroxylamine
adducts
of PLP over PL. The combined fluorescence intensities of
control
reaction mixtures lacking either crude extract or PL were
subtracted
for each sample. Protein concentrations were determined by
using
a Bradford Protein Assay Kit with bovine serum albumin as the
standard (Bio-Rad, Inc., Torrance, Calif.). The linearity of the
PL
kinase assay was confirmed for incubations of at least 60 min
and for
reaction mixtures containing 1 to 4.5 mg of S150 crude
extract (data
not shown).
PN kinase activity in S150 crude extracts was determined by conversion
of [
3H]PN to [
3H]PNP as described before
(
44), except that the specific activity
of the
[
3H]PN substrate was decreased to 3.8 mCi/mmol.
Construction of a pdxY::Kanr
mutant.
pTX618 was digested with EcoRV, which cuts in
the middle of the pdxY reading frame (Fig. 2). An
Kanr cassette was isolated from a BamHI and
ScaI digestion of plasmid pHP45Kanr
(30), and the BamHI ends of the cassette were
made blunt by filling them in with T4 DNA polymerase. A ligation
mixture containing the digested pTX618 and Kanr cassette
was used to transform strain DH5, and transformants were selected on
LB medium containing ampicillin and kanamycin. Restriction analysis of
plasmid pTX623 purified from one transformant confirmed the location of
the Kanr cassette in the middle of the pdxY
gene.
The
pdxY::
Kan
r insertion mutation was
crossed into the bacterial chromosome by transforming strain JC7623
with pTX623 that
was linearized by digestion with
EcoRI
(
2,
40). One transformant,
designated TX4017 (Table
1), that
grew on LB medium containing
kanamycin contained no plasmids and was
sensitive to ampicillin.
The location of the
pdxY::
Kan
r insertion at the expected
location in the chromosome (36.92 min)
(Fig.
2) was confirmed by TX2768
(
pdxH::
Cm
r) × P1
vir(TX4017)
crosses which showed that the Kan
r marker was 100% linked
to the Cm
r marker in
pdxH. The
pdxY::
Kan
r cassette insertion mutation
was moved from TX4017 into strains
NU426, TX3689, TX4015, and TX4016 by
P1
vir transduction to form
strains TX4021, TX4023, TX4022,
and TX4024, respectively (Table
1).
RNase T2 protection assay.
Total RNA was purified from 15-ml
cultures grown in LB medium at 37°C to a turbidity of 50 Klett (660 nm) units. RNase T2 protection assays were performed as described
before (37). RNA probes 1 to 5 and complementary probes 1o
to 5o (Fig. 2) were synthesized in vitro by using the following phage
RNA polymerases (RNAP) and linearized plasmid templates: for probe 1, T7 RNAP and pTX303 cut with HindIII; for probe 1o, SP6
RNAP and pTX303 cut with EcoRI; for probe 2, SP6 RNAP and
pTX628 cut with EcoRI; for probe 2o, T7 RNAP and pTX628 cut
with HindIII; for probe 3, SP6 RNAP and pTX632 cut with
EcoRI; for probe 3o, T7 RNAP and pTX632 cut with
HindIII; for probe 4, T7 RNAP and pTX630 cut with HindIII; for probe 4o, SP6 RNAP and pTX630 cut with
EcoRI; for probe 5, T7 RNAP and pTX629 cut with
HindIII; and for probe 5o, SP6 RNAP and pTX629 cut with
EcoRI. Protected regions of probes were analyzed by
electrophoresis on gels containing 7 M urea and 6% polyacrylamide
(37). No self-protection of any probes was detected for
control hybridizations that contained tRNA instead of mRNA.
Radioactivity in bands on dried gels was measured directly by using an
Instant Imager (Packard Instrument Co., Meriden, Conn.). Sizes of
protected fragments were estimated from standard curves of mobility
versus size for RNA standards of known lengths, as described before
(37).
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RESULTS AND DISCUSSION |
Identification of pdxY encoding PL kinase.
We ran
Blast searches of the recently reported E. coli genome
to identify homologs of pdxK that might encode a missing PL kinase. These searches turned up three candidates: f287b (at
36.92 min; encoding a product 30% identical and 42% similar to PdxK over its whole length); yeiI (at 48.49 min; encoding a
product 19% identical and 29% similar to PdxK over its whole length), and yeiC (at 48.63 min; encoding a product 20% identical
and 35% similar to PdxK over its whole length). We amplified each of
these reading frames by PCR under conditions that minimize errors and cloned them downstream of the Plac promoter in
the high-copy-number vector pUC19. We induced expression of these
reading frames by addition of IPTG to cultures of cells containing the
clones, and we determined PL kinase specific activity in cell crude
extracts of several different clones of each construct (Table
2) (Materials and Methods). Minimal
clones, such as pTX608 (Table 1; Fig. 2), containing the
f287b reading frame in a 1.1-kb fragment and clones containing f287b in a slightly larger, 1.6-kb fragment, such
as pTX618 (Table 1; Fig. 2), increased PL kinase specific activity 8- to 11-fold (Table 2, JM109 strains). This result suggested that
f287b encoded a PL kinase, and we renamed the reading frame pdxY. Clones containing the yeiI and
yeiC reading frames did not have increased PL kinase
specific activity (data not shown) and were not studied further.
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TABLE 2.
PL and PN kinase specific activities in strains
overexpressing the PdxY and PdxK proteins and in pdxK,
pdxY, pdxY pdxK, and pdxH
mutantsa
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pdxY functions as a PL kinase in vivo.
We inserted
an omega cassette into the pdxY reading frame and crossed
the pdxY::Kanr insertion mutation
into the E. coli K-12 chromosome (Materials and
Methods). We then moved the pdxY::Kanr
mutation into the isogenic strains listed in Tables 2 and
3. pdxY,
pdxK, and pdxY pdxK mutants were not auxotrophs
(TX3689, TX4021, and TX4023 [Table 3]). This result shows that
the de novo pathway of PLP biosynthesis functions in the absence of the PN/PL/PM kinase PdxK and the PL kinase PdxY and is consistent with the
model in which these kinases function solely in the salvage pathway
(Fig. 1) (see below). In this model, the phosphate ester group of PNP,
which is the first B6 vitamer synthesized de novo, is
provided by the intermediate 4PHT (46).
We next tested the growth requirements of
pdxY and
pdxK mutants in strains containing a
pdxB
mutation, which blocks the de
novo PLP biosynthetic pathway upstream of
4PHT (Fig.
1).
pdxB mutants can synthesize 4PHT only
when supplemented with GA or
4HT by an alternative
pathway involving ThrB homoserine kinase
(
12,
46). The
pdxB pdxK pdxY+ mutant grew when supplemented
with 1 µM PL, but not when supplemented
with 1 µM PN, as shown
previously (TX4016 [Table
3]) (
44).
Given that the
B
6 vitamers are present in
E. coli in
relatively
small amounts and need to be added as supplements (
8,
9),
this result confirms the conclusion that the PdxK gene
product
is the major PN kinase in
E. coli K-12.
Addition of 100 µM PN
allowed growth of the
pdxB pdxK
pdxY+ mutant; however, this result cannot be
interpreted, because our
high-performance liquid chromatographic
analyses demonstrated
that commercial PN contains a contaminant that
could be PL at
very low levels (data not shown). Likewise, growth tests
with
PM were inconclusive, because commercial PM is contaminated with
as much as 2% (wt/wt) PL. The
pdxB pdxK+ pdxY
mutant grew when supplemented with PN or PL (TX4022 [Table
3]),
consistent with the previous conclusion that the purified
PdxK enzyme
possesses PL, as well as PN, kinase activity (
39).
Most
significantly, the PLP requirement of the
pdxB pdxK pdxY triple mutant was not satisfied by PN, PL, or PM, and the triple
mutant grew only when supplemented with GA or 4HT (TX4024 [Table
3]). This result shows that PdxK and PdxY are the only physiologically
significant PL, PN, and PM kinases in
E. coli.
Moreover, growth
of the
pdxB pdxK pdxY triple mutant on GA
and 4HT, but not on
PL, PN, or PM, strongly supports the model in which
B
6 vitamer
kinases participate only in the salvage pathway
of PLP biosynthesis
(Fig.
1).
PL and PN kinase assays of crude extracts (Table
2) gave results
consistent with the conclusions from the growth experiments
(Table
3).
The
pdxB pdxK+ pdxY+ mutant had the
same PL and PN kinase specific activities as the
pdxB+ parent strain (NU426, NU402, and TX4015
[Table
2]). The
pdxB pdxK pdxY+ double mutant
lacked significant PN kinase activity but contained
unchanged PL kinase
specific activity (NU426 and TX3634 [Table
2]). Compared to the
pdxB pdxK+ pdxY+ parent, the PL
kinase specific activity was reduced about threefold
in the
pdxB
pdxK+ pdxY double mutant, which still had full PN
kinase activity (TX4015
and TX4022 [Table
2]). Finally, the
pdxB pdxK pdxY triple mutant
lacked PN kinase activity and
contained reduced PL kinase activity
compared to the
pdxB
pdxK+ pdxY double mutant (TX4015, TX4022, and TX4024
[Table
2]). The
apparent residual PL and PN kinase activities in the
pdxB pdxK pdxY triple mutant were not physiologically
significant, because
the triple mutant failed to grow when supplemented
with PN, PL,
or PM (Table
3 and data not shown). These residual
activities
may simply reflect background in the enzyme assays
containing
crude extracts. In particular, the PL kinase assay has a
high
background, because hydroxylamine forms fluorescent oxime adducts
at a slower rate with the PL substrate than with the PLP product
(
36). A residual background could result if the formation of
PL-oxime was slightly greater in reaction mixtures containing
crude
extract than in control mixtures lacking extract. Alternatively,
other
carbohydrate kinases, which are evolutionarily related to
PdxK and PdxY
(
39), may use PL and PN at low levels in in vitro
enzyme
assays. Consistent with this notion, bacteria containing
suppressors of
the
pdxB pdxK pdxY triple mutant readily appear
on MMG
plates supplemented with PN, PL, or PM at 37°C (
43).
We further tested the conclusion that PdxK functions as both a PL
kinase and a PN kinase by overexpressing the PdxK protein.
The PN and
PL kinase specific activities in crude extracts increased
80- and 3-fold, respectively, in the strain overexpressing PdxK
from
plasmid pTX485 compared to the
pdxB pdxK pdxY+
strain lacking the plasmid (TX3634 and TX3636 [Table
2]). We
also
tested whether the PdxY kinase possessed a low-level PN kinase.
Overexpression of PdxY did increase PN kinase about 10-fold over
the
background level in the
pdxB pdxK pdxY+ strain
(TX3634 and TX4037 [Table
2]) and allowed growth on MMG
containing 2 µM PN (data not shown). However, the PdxY PN kinase
activity was not
physiologically significant under the growth
conditions tested, because
a
pdxB pdxK pdxY+ mutant failed to grow when
supplemented with PN (Table
3). Thus,
the combined amino acid
homology, growth, and enzyme assay data
support the conclusions
that
pdxK encodes a PN kinase with moderate
PL kinase
activity and
pdxY encodes a PL kinase with a low level
of PN
kinase activity that can be detected when PdxY is overexpressed.
Comparisons of PdxY and PdxK with other B6-vitamer
kinases.
Previously, we made the observation that the PdxK
kinase was a member of a superfamily of carbohydrate kinases that
includes phophofructokinases and ribokinases (44). This
finding was unexpected because of the different structures of the
carbohydrates and the substituted pyridine ring of the B6
vitamers. Figure 3 presents an updated
alignment that includes the E. coli PN/PL/PM kinase PdxK and the E. coli PL kinase PdxY, PL kinases from
humans and T. brucei (17, 34), and proteins from
Haemophilus influenzae, Caenorhabditis elegans,
Rattus norvegicus, Saccharomyces cerevisiae, and
Salmonella typhimurium that likely are PL or PN kinases.
This alignment indicates conserved motifs that may be involved in
substrate binding or catalysis, including signature motifs found in the PfkB superfamily of carbohydrate kinases (regions 1 and 4) (42, 44); degenerate P-loop motifs (regions 1 and 4), which may be involved in ATP binding (34); candidate tyrosine residues
(marked 2), one of which cannot be modified following PL binding
(33); candidate aspartic and glutamic acid residues (marked
3), which may act as general bases in phosphate transfer
(34); and a region (marked 4) that is affinity labeled by
the bisubstrate analog adenosine tetraphosphate in the PL kinase
isolated from sheep brain (11). A degenerate Walker B motif
located in the T. brucei PdxK kinase (near region 3 in the
third panel of the alignment) was speculated to play a role in
Mg2+ binding (34) but is not well conserved in
the different B6-vitamer kinases.
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FIG. 3.
Amino acid alignments of the E. coli
PN/PL/PM kinase PdxK and the E. coli PL kinase PdxY
with homologs from other organisms. Kinase activities have been
demonstrated directly only for E. coli PdxK and PdxY
(see the text) (44), human PKH (human homolog of pyridoxal
kinase) (17), and T. brucei PdxK (34);
the other sequences are putative homologs identified by Blast searches.
The sequences have the following database accession numbers:
E. coli PdxK (PdxK_ecoli), GenBank U53700; S. typhimurium Yfei (yfei_salty), SW P40192; E. coli
PdxY (PdxY_ecoli), DDBJ D90807 cds10; H. influenzae Yfei
(yfei_haein), SW P44690; S. cerevisiae Yn8fp (yn8f_yeast),
SW P53727; S. cerevisiae Yec9p (yec9_yeast), SW P39988;
T. brucei PdxK (PdxK_tbruc), GenBank U96712; C. elegans PdxK (PdxK_celeg), GenBank AF003142; R. norvegicus Plk (Plk_rnorv), GenBank AF020346; and human PKH
(PKH_human), U89606. Conserved motifs that may be involved in substrate
binding or catalysis are overlined and discussed in the text. Solid
background, identical amino acids; shaded background, similar amino
acids.
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Dendrogams, phylograms, and cladograms of these sequences
compiled by different evolution programs in the University of
Wisconsin
Genetics Computer Group package all suggest that the
eukaryotic
and the
H. influenzae proteins are more closely
related to PdxY
than to PdxK, whereas the
S. typhimurium protein, which is encoded
from an analogous region of
the chromosome, is more closely related
to PdxK than to PdxY.
Presumably,
S. typhimurium contains an unidentified
homolog
of PdxY. The existence of PdxK and PdxY in
E. coli
raises
the possibility that PN- and PL-specific kinases are present in
other eubacteria and in eukaryotes. This possibility has implications
for current efforts to exploit B
6 vitamer kinases in the
uptake
of selectively toxic analogs for the treatment of certain
parasitic
diseases (
34). For example, it may be possible to
exploit one
of the two B
6 vitamer kinases present in some
organisms to enhance
the selectivity of drug uptake. Finally, it was
recently communicated
to us that the
E. coli
PN/PL/PM kinase PdxK also possesses a hydroxymethylpyrimidine
kinase activity and therefore may function in thiamine (vitamin
B
1) biosynthesis (
22). It remains to be
determined whether other
PL and putative B
6 kinases (Fig.
3) have these dual functions
that possibly allow cross talk between the
vitamin B
6 and B
1 pathways.
Structure and expression of pdxY.
pdxY is located
at 36.9 min in the E. coli chromosome in the same
orientation immediately downstream of pdxH (encoding PNP/PMP oxidase [Fig. 1]) and tyrS (encoding
tRNATyr-aminoacyl synthetase) and in the opposite
orientation to gst (encoding glutathione
S-transferase) (Fig. 2). Previously, we showed that
pdxH and tyrS are transcribed from separate
promoters in vivo, but about 20% of tyrS transcripts
are present as pdxH-tyrS cotranscripts (24).
No Rho-factor-independent terminators are obvious in the
sequences of the tyrS-pdxY or pdxY-gst junctions (Fig. 2), and previously we did not detect transcription termination between pdxH and tyrS (24). Therefore,
we mapped the pdxY transcript by RNase T2 protection assays
(see Table 1 and Fig. 2 for probes) in order to determine the
transcription relationship of these genes and to learn whether
pdxH and pdxY are somehow cotranscribed.
Consistent with earlier results, hybridization to probe 1, corresponding to the
pdxH and
tyrS noncoding
strand, showed two
bands of 297 and 560 nucleotides (nt) (Fig.
4, lane 4), representing
transcription
from the P
tyrS and P
pdxH
promoters,
respectively (Fig.
2). Hybridization to probes 2 and 3, corresponding
to the
tyrS and
pdxY noncoding
strands, each gave a large, faint,
protected band and a much more
intense, smaller, protected band
followed by degradation products (Fig.
4, lanes 7 and 10). The
large, faint, protected bands are smaller than
the undigested
probes, which contain linker regions (Fig.
4, lanes 5 and 8),
and represent contiguous
tyrS-pdxY
cotranscripts. Because probes
2 and 3 end at the same place in
pdxY (Fig.
2), the intense, shorter
1,050- and 530-nt bands
observed with probes 2 and 3, respectively,
must correspond to
terminated
tyrS transcripts. The sizes of the
bands place
the termination point in the
tyrS-pdxY intercistronic
region
about 20 nt downstream from the translation termination
codon of
tyrS (Fig.
2). No bands were detected in hybridizations
with
probes 1o, 2o, and 3o, corresponding to the
pdxH,
tyrS, and
pdxY coding strand, indicating that
there is no antisense transcription
of
tyrS and
pdxY in vivo and no DNA contamination in our total-RNA
preparations (data not shown).
View larger version (86K):
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|
FIG. 4.
RNase T2 protection assays of transcripts from the
pdxH-tyrS-pdxY-gst region of the E. coli
K-12 chromosome. Total RNA was purified from strain NU426 growing
exponentially in LB medium at 37°C, and RNase T2 protection assays
were performed as described in Materials and Methods. The positions of
RNA probes 1 to 5 used for this assay are indicated in Fig. 2. Probes 1 to 5 correspond to the pdxH, tyrS, and
pdxY noncoding strands and hybridized with pdxH,
tyrS, and pdxY transcripts. Probes 4o and 5o are
complementary to probes 4 and 5, respectively, and hybridized to
gst transcripts. See the text for additional details. a,
unhybridized RNA probes; b, RNA probes hybridized with 50 µg of tRNA
(self-hybridization control); c, RNA probes hybridized with 50 µg of
total cellular RNA; s, RNA size standards (see Materials and Methods)
(37); arrowheads, protected RNA species described in Results
and Discussion.
|
|
Using probe 1, 2, or 3, we did not detect any protected band that would
indicate the presence of an independent
pdxY promoter.
Therefore,
tyrS and
pdxY seemed to be
cotranscribed, and the terminator
between
tyrS and
pdxY (Atn
tyrS; Fig.
2)
functioned as
an attenuator to decrease
pdxY expression
relative to that of
tyrS. To test further the transcription
linkage between
tyrS and
pdxY, we tested whether
a polar omega-cassette insertion mutation
in
pdxH decreased
expression of the PdxY gene product. Previously
we showed that blockage
of transcription from P
pdxH by
a
pdxH::Mu
dI-8 insertion reduced the
tyrS transcript amount by
about 20% (
24). We
found that a
pdxH::
Cm
r insertion also
reduced PL kinase activity by about 25% (NU816
and TX2768 [Table
2]). This result is consistent with the interpretation
that all
pdxY transcription originates at the
P
pdxH and
P
tyrS promoters
and that there is a low level of coupling
between the transcription of
pdxH and that of
pdxY. Finally, quantitation
of
the radioactivity in gel bands indicated that about 92% of
tyrS transcripts terminate at the
Atn
tyrS terminator
and only about 8% read
through into
pdxY. Since there is no Rho-factor-independent
terminator structure in the
tyrS-pdxY intercistronic region,
Atn
tyrS is likely a Rho-factor-dependent
transcription terminator.
The
tyrS-pdxY cotranscript is terminated at a terminator
(Ter
pdxY; Fig.
2) located in the
pdxY-gst intercistronic
region. Hybridization to probe 4, corresponding to the
pdxY noncoding
strand, gave 202- and
1,120-nt protected bands (Fig.
4, lane 21),
which represent terminated
tyrS transcripts at Atn
tyrS and
tyrS-pdxY cotranscripts at Ter
pdxY,
respectively.
Hybridization to probe 5, corresponding to the
pdxY noncoding
strand, gave a faint 148-nt protected band
(Fig.
4, lane 24) consistent
with the location of
Ter
pdxY, which is about 35 nt downstream
from
the
pdxY translation stop codon (Fig.
2). Last, we located
termination of the oppositely transcribed
gst transcript at
Ter
gst in the
pdxY-gst intercistronic
region about 4 nt downstream from
the
gst translation stop
codon (Fig.
2). Hybridization to probes
4o and 5o, corresponding to the
gst noncoding strand, gave 450-nt
protected fragments
representing termination at Ter
gst (Fig.
4,
lanes 14 and 17). We also detected some (
12%) readthrough
of the
Ter
gst terminator (Fig.
4, lane 14, series of
bands above the 450-nt protected fragment, and lane 17, upper
band),
which would produce an antisense
pdxY transcript. However,
as determined by the length of the read-through transcripts, this
antisense transcription did not extend past the last quarter of
the
pdxY reading frame. This is consistent with our observation
that no antisense
pdxY transcript was detected with probes
1o,
2o, and 3o (see above). Thus, it is unlikely that an antisense
pdxY transcript extends to the
pdxY ribosome
binding site. As
is the case for Atn
tyrS, no
Rho-factor-independent structures
are obvious for
Ter
pdxY and Ter
gst, and
these
terminators may be Rho factor dependent.
Detailed molecular genetic analyses have shown that genes
encoding aminoacyl-tRNA synthetases are regulated by a
variety of
mechanisms in
E. coli, including
transcription (Ala-tRNA synthetase)
and translation (Thr-tRNA
synthetase) autoregulation and transcription
attenuation
(Phe-tRNA synthetase) (reviewed in reference
16).
Currently, the regulation of
tyrS in
E. coli is largely unknown.
Precedents from genes
encoding other aminoacyl-tRNA synthetases
in
E. coli
suggest that
tyrS may be regulated positively by growth
rate
and by tyrosine limitation (
16,
24). The cotranscription
of
pdxY and
tyrS demonstrated here may provide a
point of genetic
integration that coordinates incorporation of amino
acids into
proteins with PLP coenzyme supply. Ongoing studies
are aimed at
elucidating the regulation and expression of the
pdxH-tyrS-pdxY multifunctional operon and the roles of the
PL kinase PdxY and
the PL/PM/PN kinase PdxK in maintaining PLP
homeostasis.
| |
ACKNOWLEDGMENTS |
We thank the colleagues cited in Table 1 for bacteria and
bacteriophage stocks, Ian Spenser for generously providing 4HT and
sharing information about PLP biosynthesis, and T. Begley, D. Cane, and
D. Downs for helpful discussions about the role of kinases in PLP and
thiamine biosynthesis.
This work was supported by Public Health Service grant GM37561 from the
National Institute of General Medical Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, University of TexasHouston
Medical School, 6431 Fannin; JFB 1.765, Houston, TX 77030-1501. Phone: (713) 500-5461. Fax: (713) 500-5499. E-mail:
mwinkler{at}utmmg.med.uth.tmc.edu.
| |
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Abstract
Introduction
