From the Department of Biochemistry and Center for Microbial Pathogenesis, State University of New York at Buffalo, Buffalo, New York 14214
Received for publication, March 5, 2001
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ABSTRACT |
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Bradyrhizobium japonicum transports
oligopeptides and the heme precursor Bacteria utilize small peptides as nutrients, chemoattractants,
and quorum sensing signals, and their metabolism is a target for
antibiotics (1-3). Escherichia coli and Salmonella
typhimurium contain distinct dipeptide
(Dpp)1 and oligopeptide
permease (Opp) systems with some overlap in substrate
specificity. The two permease systems are structurally homologous, each
one containing five proteins, including a periplasmic peptide-binding
protein (4, 5). The Opp system binds peptides two to five peptides in
length, with the highest affinity for tripeptides (6, 7). For both
permease systems, the amino acid side chain appears not to be important
for specificity, and therefore these systems transport peptides
independent of sequence.
Our interest in oligopeptide transport in the bacterium
Bradyrhizobium japonicum is founded on studies of heme
biosynthesis, where it has been demonstrated that the heme precursor
The phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS)
couples the transfer of a high energy phosphoryl group from PEP to a
sugar with the concomitant transport into the cell (13-15). Paralogues
of the PTS enzymes EI, Hpr and IIA, called EINtr, Npr, and
IIANtr, respectively, have been identified in several
organisms (16-21), and evidence for a parallel phosphoryl transfer
chain has been presented (22). Phenotypes of mutants in the parallel
PTS system (herein called the PTSNtr system), gene
organization and the structure of EINtr, suggest a role for
this system coordinating nitrogen and carbon metabolism. However, the
physiological functions of the PTSNtr system is unclear.
Although the conventional PTS system transports sugars, no solute
transport activity has been linked to the PTSNtr proteins.
Herein, we demonstrate a role for EINtr, encoded by the
ptsP gene, in oligopepide transport in B. japonicum. Furthermore, a novel function for the amino acid
biosynthesis enzyme aspartokinase was also implicated, and evidence for
interaction between EINtr and aspartokinase is presented.
Chemicals and Reagents--
All chemicals were reagent grade and
were purchased from Sigma Chemical Co., St. Louis, MO, Fisher
Scientific, Fair Lawn, NJ, or from J. T. Baker Inc., Phillipsburg,
NJ. Granulated agar and yeast extract were obtained from Difco
Laboratoies, Detroit, MI. Bacterial Strains, Plasmids, Media, and Growth--
B.
japonicum strain I110proC is a proC mutant derivative
of strain I110 and is a proline auxotroph (23). All B. japonicum strains were grown at 29 °C in GSY media or minimal
media as described previously (24). Tetracycline (75 µg/ml) was added
to the media for growth of strains bearing the broad host range plasmid
pVK102 (25) and its derivatives. Strains I20, Q5, I110I20, and I110Q5 were grown in the presence of kanamycin (50 µg/ml) and streptomycin (50 µg/ml). Strains I110proC, I20, and Q5 were grown in the presence of spectinomycin (50 µg/ml) and streptomycin. Strains I110proC, I20,
and Q5 required the addition of proline (500 µg/ml) for growth. E. coli strain DH5 Tn5 Mutagenesis and Construction of B. japonicum Mutant Strains
I110I20 and I110Q5--
A random transposon Tn5 mutagenesis of
B. japonicum strain I110proC was carried out as described
previously (26). Approximately 3500 mutant colonies were streaked onto
media containing either proline or prolyl-glycyl-glycine (50 µg/ml)
to screen for mutants that are unable to use the latter as a proline
source. Two mutants displaying this phenotype were identified and
designated strains I20 and Q5. Southern blot analysis revealed that Tn5
from each mutant were contained on EcoRI fragments ~12 kb
in length, which were then isolated as described previously (26). These
fragments, cloned into pBR322, were used to generate strains for the
corresponding mutants in parent strain I110 by homologous
recombination as described previously (26). The nucleotide sequence of
both strands of the HpaI-XhoI region bearing the
B. japonicum lysC and ptsP genes was determined.
ALA Uptake Activity by Cells--
B. japonicum cells
grown to mid-log phase in 100 ml of minimal media were washed and
assayed for [14C] ALA (150 µM; 2.67 Ci/mol)
as described previously (8). Strains I110, I110I20, and I10Q5 were used
in the studies for monitoring uptake in wild type, ptsP, and
lysC strains, respectively.
In Trans Complementation of Mutants--
Plasmids pVK102,
pVK-lysC, and pVK-ptsP were mobilized by
conjugation into B. japonicum strains I110proC,
I20, and Q5, and strains harboring plasmids were selected by
maintaining the strains on GSY agar containing tetracycline (75 µg/ml), streptomycin (100 µg/ml), spectinomycin (100 µg/ml), and
proline (500 µg/ml). Plasmid pVK102, and its derivatives, are broad
host range vectors and can be maintained in low copy in B. japonicum. Strains I110proC, I20, and Q5, containing
plasmids pVK102, pVK-lysC, or pVK-ptsP, were
tested for growth by streaking cells on GSY agar containing the
appropriate antibiotics and either no proline source, proline (500 µg/ml), prolyl-glycine (50 µg/ml), or prolyl-glycyl-glycine (50 µg/ml).
Isolation of RNA and mRNA Analysis--
Total RNA was
prepared and analyzed as described previously (27, 28). Analysis of RNA
levels of specific genes was carried out by a quantitative RNase
protection assay. Plasmid pAspK(RV) is a derivative of
pBluescriptSK+ that carries a 347-bp EcoRV
fragment containing a portion of the lysC gene. Plasmid
pPtsP(XhoI) is a derivative of pBluescriptSK+
that carries a 402-bp XhoI fragment containing a portion of
the ptsP gene. Each plasmid was used as a template to
synthesize antisense RNA as described previously (28, 29).
Expression and Purification of c-myc/HisX6 Fusion
Proteins--
AspK and EINtr were overexpressed as
c-myc/HisX6 fusion proteins from the pTrcHis2 expression
vector (Invitrogen). PCR reactions were performed using
5'-GGCCATGGCCCGCCTCGTGATG-3' and 5'-CCTAAGCTTGATCGAGGCCGTAGAGCG-3' as the forward and reverse primers to amplify the lysC
coding region and introduce NcoI and HindIII
sites, respectively. The pTrcHis2B-ptsP construct was
generated by introducing a Csp45I site at the 5'-end and a
HindIII site at the 3'-end of the ptsP open
reading frame by PCR using the primers
5'-GGTTCGAA-GCGCGTCGGGAGGTCC-3' and
5'-GCCAAGCTTAAGGCCAGGCCTTCGGC-3', respectively. One liter of LB,
containing 200 µg of ampicillin per ml of media, was inoculated with
10 ml of an overnight culture of E. coli XL-1 Blue cells containing either pTrcHis2B-ptsP or
pTrcHis2C-lysC, grown and induced as described previously
(28). The cleared lysates were applied to columns each
containing 1 ml of nickel-nitrilotriacetic acid settled resin charged
with 400 mM NiSO4 according to the manufacturer's instructions (Novagen). The resin was washed with 15 volumes of PO4 binding buffer followed by 25 volumes of
PO4 wash buffer (50 mM potassium phosphate,
10% glycerol, 300 mM NaCl, and 30 mM
imidazole, pH 8.0). Proteins retained by the resin were eluted with 3 volumes of PO4 elute buffer (50 mM potassium
phosphate, 10% glycerol, 300 mM NaCl, and 250 mM imidazole, pH 8.0) and dialyzed overnight at 4 °C
against 1 liter of 50 mM potassium phosphate, 10%
glycerol, 300 mM NaCl, and 1 mM
Expression of GST Fusion Proteins--
AspK and
EINtr were overexpressed as GST fusion proteins from the
pGEX-6P-2 expression vector (Amersham Pharmacia Biotech). The pGEX-6P2-lysC construct was generated by introducing a
BamHI site at the 5'-end and a SmaI site at the
3'-end of the lysC open reading frame by PCR using the
primers 5'-CTCGGATCCATGAGCCGCCTCGTGAT-3' and
5'-GAGCCCGGGCTAAGCCTGATCGAGGC-3', respectively. The
pGEX-6P2-ptsP construct was generated by introducing a
BamHI site at the 5'-end and a SmaI site at the
3'-end of the ptsP open reading frame by PCR using the
primers 5'-CTCGGATCCATGCGGAGCGCGTCGGG-3' and
5'-GAGCCCGGGCCCGCTACAAGGCCA-3', respectively. Expression was
induced by adding 1 mM
isopropyl- GST Pull-down Assay--
Crude lysates of E. coli
BL21(DE3)pLysS containing the overexpressed proteins GST, GST-AspK, or
GST-EINtr were prepared as described. 1 ml of each cleared
lysate preparation was added to a separate Eppendorf tube containing 50 µl of prewashed glutathione-Sepharose beads (Amersham Pharmacia
Biotech) and rotated on an Orbitron for 30 min at 4 °C. Beads
were washed five times with PBS + 0.1% Triton X-100 by centrifugation
at 2000 × g for 5 min at 4 °C followed by
aspiration of supernatant. Beads were resuspended in 250 µl of PBS + 0.1% Triton X-100, and the amount of protein bound to the beads was
determined using the Bradford protein assay (Bio-Rad). The results of
this assay were normalized against a control containing unbound
glutathione-Sepharose beads. 5 µg of purified B. japonicum
EINtr-c-myc/Hisx6 fusion protein was added to a
volume of beads that contained 20 µg of GST or GST-AspK, and 5 µg
of purified B. japonicum AspK-c-myc/Hisx6 fusion
protein was added to a volume of beads that contained 20 µg of GST or
GST-EINtr, in a total volume of 0.5 ml. Reactions were
rotated on an Orbitron for 60 min at 4 °C. Beads were washed five
times with PBS plus 0.1% Triton X-100 by centrifugation at 2000 × g for 5 min at 4 °C followed by aspiration removing
any trace of supernatant. Beads were resuspended in 25 µl of 2×
SDS-sample buffer, boiled for 5 min, and resolved by SDS-PAGE. After
transferring to Immobilon-P (0.45 µm polyvinylidene difluoride,
Millipore), proteins were detected with
anti-c-myc/peroxidase conjugate (Roche Molecular Biochemicals) by chemiluminescence (Renaissance, PerkinElmer Life Sciences).
Assay for Aspartokinase Activity--
Aspartokinase activity was
determined by measuring asparthydroxamate produced when aspartate is
incubated with enzyme in the presence of ATP and hydroxylamine (30).
The assay mixture contained 10.4 mM Mg-ATP, 94 mM Tris, HCl buffer (pH 8.0), 1.6 mM
MgSO4, 10 mM PEP-dependent Phosphorylation of
EINtr--
B. japonicum
EINtr-c-myc/HisX6 fusion was overexpressed and
purified as described above. [32P]PEP was synthesized in
a 0.1-ml reaction mixture containing 0.1 M triethylamine
(pH 7.6), 3 mM MgCl2, 15 mM KCl, 1 mM pyruvate, 0.1 mM phosphoenolpyruvate
(cyclohexammonium salt), 10 µM [ ATP-dependent Phosphorylation of
GST-EINtr--
GST-EINtr was used as the
substrate for phosphorylation in the presence of cell extracts so that
the protein could be separated from the reaction mixture after
completion with glutathione-Sepharose beads. In a final volume of 30 µl, 12 µg of purified GST-EINtr (3.7 µM)
or 21 µg of GST (27 µM), 4.5 µg of aspartokinase (2.7 µM), and 18 µg of cell extracts prepared from
lysC strain I110Q5 were added. The final concentrations of
other components, where added, were as follows: 95 mM Tris,
pH 8, 1.6 mM MgSO4, 10 mM Isolation of Oligopeptide Uptake Mutants of B. japonicum--
B. japonicum strain I110proC is disrupted in
the proline biosynthesis gene proC and requires an exogenous
source of proline for growth (23). We established that strain I110proC
could use the proline-containing peptides Pro-Gly or
Pro-Gly-Gly, to satisfy its auxotrophy as discerned by growth in
liquid or solid media supplemented with 50 µg/ml of either
compound (Table I). Thus, the
strategy for obtaining oligopeptide transport mutants was to screen for
mutants of strain I110proC that could not use prolyl-glycyl-glycine (Pro-Gly-Gly) as a proline source. Strain I110proC was mutagenized with
Tn5, and kanamycin-resistant colonies were screened for those that
could no longer use Pro-Gly-Gly as a proline source, but still grew on
proline. Two mutants, strains I20 and Q5, exhibited this phenotype on
plates and in liquid cultures and were also unable to use Pro-Gly as a
proline source (Table I). Both mutant strains retained the ability to
grow on proline as well as strain I110proC.
Mutations in Strains I20 and Q5 Affect ALA
Uptake--
Radiolabeled tripeptides were not commercially available,
but [14C]ALA was available to carry out uptake
experiments. The mutations in strains I20 and Q5 were reconstructed in
a wild type background, and ALA uptake activity was measured. The
mutants were constructed by isolation of the Tn5-containing
EcoRI fragment from strains I20 and Q5 followed by
introduction into the genome of strain I110 by homologous recombination
to generate strains I110I20 and I110Q5 (see "Materials and
Methods"). The uptake of [14C]ALA by the mutant and
wild type strains was assessed using cells cultured in minimal media.
Both mutants had severely reduced ALA uptake activities compared with
the parent strain I110 (Fig. 1). These
data show that the mutations in strain I20 or Q5, which affect their
ability to use proline-containing peptides, also severely inhibit ALA
uptake activity. The defect in uptake of ALA, a dipeptide analogue,
indicates that the inability of Pro-Gly or Pro-Gly-Gly to satisfy the
proline auxotrophy in strains I20 and Q5 is the result of a defect in
transport rather than another step of oligopeptide metabolism.
Identification and Characterization of the lysC Gene and Its
Product Aspartokinase--
Initial subcloning and analysis of
Tn5-containing genomic fragments from strains I20 and Q5 revealed that
the transposon from each strain was inserted into one of two different
regions of the same EcoRI fragment. Consequently, the
EcoRI fragment isolated from strain I20 contained the wild
type copy of the DNA mutated in strain Q5, and vice versa. Therefore,
from these EcoRI fragments, the wild type genes were cloned
and sequenced, and DNA fragments containing one or the other gene were
constructed (see "Materials and Methods"). These two open reading
frames have the same orientation, and are separated by a 288-bp
intergenic region (Fig.
2A).
The upstream gene corresponding to that mutated in strain Q5 is 1257 bp
in length and encodes a 418-amino acid polypeptide with extensive
similarity to aspartokinase from numerous organisms, with the greatest
identity (44.6%) to that from Corynebacterium glutamicum
(32) (Fig. 3). Aspartokinase, encoded by
the lysC gene, catalyzes the ATP-dependent
phosphorylation of aspartate yielding aspartyl
Aspartyl Identification and Characterization of the ptsP Gene and Its
Product Enzyme INtr--
The downstream open reading frame
corresponding to the gene mutated in strain I20 is 2268 bp in length
and encodes a 755-amino acid polypeptide that is homologous to an
unusual protein called Enzyme INtr (EINtr),
which has been identified in several organisms. B. japonicum EINtr shows greatest identity (36.4%) to EINtr
from Azotobacter vinelandii (21) (Fig.
4). EINtr is a paralogue of
Enzyme I (EI) of the PTS system except that it contains an additional
domain at the N terminus that is homologous to the N-terminal sensory
domain of NifA from A. vinelandii (36). NifA is a regulatory
protein of the
We overexpressed the ptsP gene in E. coli, and
found that the recombinant EINtr can be phosphorylated by
PEP, but not by ATP (Fig. 2C), similar to what is observed
for other EI proteins, including the EINtr from E. coli (22). The current work indicates that like EI, EINtr is involved in solute transport into cells. However,
EINtr is involved in transport of oligopeptides, which is
not a known PTS substrate. Furthermore, the ptsP strain, as
well as the lysC mutant, grew as well as the parent strain
on glycerol, succinate, or glucose as sole carbon sources. Therefore,
it is unlikely that the phenotypes of those mutants is an indirect
consequence of a defect in the ability to metabolize a carbon source.
Data base searches reveal that the gene organization of lysC
and ptsP in B. japonicum is not found in other
bacteria where either gene, along with flanking sequence, has been identified.
Evidence that lysC and ptsP Are Expressed as Separate
Transcriptional Units and Are Both Required for Oligopeptide
Transport--
The genetic organization of lysC and
ptsP led us to ask whether the phenotype of the mutants was
due to disruption of the respective gene, or whether it was due to a
polar effect of the Tn5 on a downstream gene. To evaluate the necessity
for each gene in the utilization of oligopeptides, we tested for
complementation of lysC strain Q5 and ptsP strain
I20 in trans using wild type copies of lysC or
ptsP, respectively, harbored in the broad host range vector
pVK102. The results show that each mutant strain can be complemented
in trans for growth on Pro-Gly or Pro-Gly-Gly by a wild type
copy of the respective gene (Table I) and indicates that the phenotypes
exhibited by strains Q5 and I20 are due to the loss-of-function of
lysC and ptsP, respectively.
Expression of lysC and ptsP was examined at the
RNA level to determine the effect of the Tn5 on the transcription of
both genes in the mutant strains. RNase protection analyses of total RNA isolated from strains Q5 and I20 and the parent strain
revealed that lysC and ptsP mRNA accumulated
in the parent strain, confirming that they are expressed genes (Fig.
5). lysC mRNA was not
detected in the lysC mutant, but that strain did accumulate
normal levels of ptsP transcript. Therefore, the Tn5
inserted in lysC did not have a polar affect on the
downstream ptsP gene, which is consistent with the
complementation data. Finally, lysC mRNA was found in the ptsP mutant; the levels were somewhat higher than found
in the parent strain, for which we offer no explanation. These results support the conclusion that lysC and ptsP each
have an effect on oligopeptide and ALA uptake in B. japonicum, and disruption of either gene results in a loss of
those activities.
Overexpression of ptsP in Trans Compensates for the lysC
Mutation--
lysC and ptsP are each involved in
the same cellular process, but their relationship to each other does
not appear to be at the level of gene activation. Nevertheless, we
found that ptsP complemented the lysC mutant
in trans for growth on oligopeptides, suggesting that
ptsP can compensate for the lysC mutation when expressed on a low copy plasmid (Table I). This is an interesting result given that transcription of the endogenous ptsP gene
is normal in lysC strain Q5. However, lysC did
not complement ptsP strain I20 when expressed in
trans, indicating aspartokinase is insufficient to promote
oligopeptide utilization in the absence of EINtr. The data
indicate that aspartokinase normally affects EINtr activity
in some manner that can be compensated for by expression of
ptsP from a multicopy plasmid. This idea is addressed
further as described below.
Evidence for Direct Interaction between Aspartokinase and Enzyme
INtr--
Complementation analysis suggests that
aspartokinase affects EINtr activity. Therefore, we
examined whether the two proteins interact with each other by
"pull-down" experiments using purified, recombinant proteins. In
the first experiment, we tested for the ability of a GST-aspartokinase
fusion, immobilized on glutathione-Sepharose, to interact with an
myc-tagged EINtr protein. The results show that
the myc-tagged EINtr was pulled down by the
GST-aspartokinase fusion protein but not when GST alone was used as
bait. The reverse experiment was carried out using a
GST-EINtr fusion and a myc-tagged aspartokinase
protein, and the results show an interaction between these two proteins
as well (Fig. 6). The findings suggest
that the mechanism by which aspartokinase affects EINtr
activity involves direct protein-protein interaction.
Recombinant EINtr Is Phosphorylated by ATP in the
Presence of B. japonicum Cell Extract, Which Is Negatively Affected in
the Presence of Aspartokinase--
Aspartokinase catalyzes the
transfer of phosphate from ATP to aspartate, whereas EINtr
is autophosphorylated by PEP. Initial experiments were carried out to
determine whether PEP and ATP were interchangeable in the respective
reactions, either alone or in combination with the other protein. Using
purified recombinant proteins, we did not find evidence supporting
phosphoryl transfer between aspartokinase and EINtr, nor
did one protein affect the activity of the other in vitro. It seemed plausible that a cellular factor was required for a functional interaction that was not present in these preliminary experiments. Therefore, we carried out a series of experiments where
B. japonicum cell extract was included in the reactions. Purified GST-EINtr fusion protein was used so that it could
be separated from the other components after the reaction was completed
using glutathione-Sepharose beads, and subsequently analyzed by
autoradiography of SDS-PAGE gels. GST-EINtr was not
phosphorylated when incubated by [ The intriguing primary structure of Enzyme INtr
suggested a role in transport, but previous studies had not
demonstrated a physiological role for it or for the other
PTSNtr. In this study, we show that EINtr is
involved in oligopeptide transport, and therefore it, and perhaps the
PTSNtr system as a whole, has a role in transport of a
non-sugar solute. The N-terminal domain of EINtr is similar
to the N-terminal sensory domain of NifA, and therefore it is probably
significant that this protein is involved in transport of a
nitrogen-containing compound. The present study identified a novel role
for aspartokinase, the lysC gene product, in oligopeptide transport. The B. japonicum lysC strain is not an amino acid
auxotroph, and thus its role in transport differs from its amino acid
biosynthetic function. Collectively, the data indicate that
aspartokinase interacts directly with EINtr to control its activity.
EINtr (GST-EINtr) was phosphorylated by ATP in
the presence of cell extract in vitro, indicating a cellular
factor that acts as an EINtr kinase or that allows
EINtr to phosphorylate itself.
Roseman's group identified two ATP-dependent EI kinase
activities in E. coli (39, 40), and therefore the input
signal to EI and EINtr is not limited to PEP.
ATP-dependent phosphorylation of EINtr was
severely inhibited in the presence of aspartokinase, which provides a
plausible regulatory function that the complementation data suggested.
These findings, along with the fact that both aspartokinase and
EINtr are required for oligopeptide uptake, strongly
suggests that EINtr positively affects transport activity
in the unphosphorylated state. Thus, it is likely that the
lysC mutant was complemented in trans by the
ptsP gene from a multicopy plasmid, because the overexpressed protein was primarily in the under phosphorylated state,
thereby obviating the need for aspartokinase. An activity for
unphosphorylated EINtr to promote oligopeptide transport
contrasts sharply with conventional EI enzymes, which transfer a
phosphoryl group to the next protein in the cascade, ultimately
resulting in phosphorylation and concomitant uptake of a sugar. In that
case, the high energy phosphate drives the transport process. However,
oligopeptide transport complexes bind ATP (4), and therefore
EINtr need not directly couple transport with energy. The
ability of EINtr to function differently depending on the
phosphorylation status may allow it to serve as a branch point in
different signal transduction systems. Recent studies involving a
ptsN mutant of Pseudomonas putida indicate that
the PTSNtr protein IIANtr is a general
regulator in that organism (17). Bacterial proteome and transcriptosome
analyses should help reveal the extent of control that these PTS
paralogues exert on cellular activities.
-aminolevulinic acid (ALA) by a
common mechanism. Two Tn5-induced mutants disrupted in the
lysC and ptsP genes were identified based on
the inability to use prolyl-glycyl-glycine as a proline source and were
defective in [14C]ALA uptake activity. lysC
and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in
trans complementation analysis showed that lysC and
ptsP are transcribed separately, and that both genes are
involved in oligopeptide transport. Aspartokinase, encoded by
lysC, catalyzes the phosphorylation of aspartate for
synthesis of three amino acids, but the lysC strain is not
an amino acid auxotroph. The ptsP gene encodes Enzyme INtr (EINtr), a paralogue of Enzyme I of the
phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In
vitro pull-down experiments indicated that purified recombinant
aspartokinase and EINtr interact directly with each other.
Expression of ptsP in trans from a multicopy
plasmid complemented the lysC mutant, suggesting that
aspartokinase normally affects Enzyme INtr in a manner that
can be compensated for by increasing the copy number of the
ptsP gene. ATP was not a phosphoryl donor to purified EINtr, but it was phosphorylated by ATP in the presence of
cell extracts. This phosphorylation was inhibited in the presence of
aspartokinase. The findings demonstrate a role for a PTS protein in the
transport of a non-sugar solute and suggest an unusual regulatory
function for aspartokinase in regulating the phosphorylation state of
EINtr.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-aminolevulinic acid (ALA) is taken up by a system that also
transports oligopeptides (8). ALA is structurally similar to
glycyl-glycine. It is taken up by the Dpp system in E. coli
(9) and S. typhimurium (10), but ALA is taken up in a
dpp mutant if the opp system is activated (8).
B. japonicum lives as a free-living soil bacterium or as an
endosymbiont of soybeans within root nodules. In symbiosis, B. japonicum may utilize ALA synthesized by the plant host for heme
formation (11, 12).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Aminolevulinic acid was purchased from
Porphyrin Products, Logan, UT. [14C]ALA (47.6 mCi/mmol),
[
-32P]ATP (3000 Ci/mmol), and
[
-32P]dCTP (3000 Ci/mmol) were purchased from
PerkinElmer Life Sciences, Boston, MA. [
-32P]UTP (800 Ci/mmol) was purchased from ICN Biomedicals, Irving, CA. Peptides were
obtained from Bachem, Torrance, CA.
was used for propagation of plasmids
and was grown at 37 °C on Luria-Bertani (LB) medium with appropriate
antibiotics. E. coli strains HB101(pDS4101) and
HB101(pRK2013) were grown in media containing ampicillin (200 µg/ml)
and kanamycin (50 µg/ml), respectively, for tri-parental matings.
Plasmid pSUP1011 is a pACYC184 derivative carrying the transposon Tn5
(26).
-mercaptoethanol, pH 8.0. A precipitate formed in the eluted
AspK-c-myc/HisX6 fraction after dialysis and was removed by
centrifugation. Concentration of proteins was determined using the
Bradford protein assay (Bio-Rad).
-D-thiogalactopyranoside to the media, and
cultures were grown overnight at 15 °C. Cells were lysed and cleared
as described above, and protein was purified with prewashed
glutathione-Sepharose beads (Amersham Pharmacia Biotech) according to
the manufacturer's instructions.
-mercaptoethanol, 10 mM L-aspartate, 800 mM
NH2OH, 800 mM KCl, and purified B. japonicum AspK-c-myc/Hisx6 fusion protein in a total
volume of 0.5 ml. After incubation at room temperature for 15 min, the
reaction was stopped by the addition of 0.5 ml of a 1.7% solution of
FeCl3 in 1 N HCl. After centrifugation, the
optical density of the asparthydroxamate-iron complex was measured at
540 nm using a Beckman DU spectrophotometer. Enzyme activity is
expressed as the optical density units × 1000. A blank reaction
mixture that contained all components except for enzyme served as a
control. Each reaction was performed in triplicate in the presence or
absence of Mg-ATP.
-32P]ATP
(2 × 104 Ci/mol), and 4 units of pyruvate kinase
(Sigma Chemical Co.) (31). The mixture was incubated for 90 min at
30 °C. Because this is an exchange reaction, the concentrations of
pyruvate and PEP do not change; therefore, the specific activity of the
[32P]PEP can be calculated from the known specific
activity of the [
-32P]ATP and the initial ATP and PEP
concentrations to be ~2 × 103 Ci/mol. The reaction
mixture was used as a [32P]PEP source without further
purification. The EINtr phosphorylation assay contained, in
a 20-µl volume, 94 mM Tris, HCl buffer (pH 8.0), 1.6 mM MgSO4, 10 mM
-mercaptoethanol, 4 µg of purified B. japonicum
EINtr-c-myc/HisX6 fusion protein, and either 125 µM [32P]PEP (220 Ci/mol) or 10 mM [
-32P]ATP (50 Ci/mol). The reactions
were incubated for 30 min at 37 °C. Reactions were stopped by adding
an equal volume of 2× SDS-sample buffer and incubated at room
temperature before resolving on a 7.5% SDS-PAGE. Gels were stained to
visualize protein standards, and labeled proteins were detected by autoradiography.
-mercaptoethanol, 0.1 mM [
-32P]ATP (9 µCi/mM). Reactions were carried out for 10 min at 37 °C.
Afterward, glutathione-agarose beads were added, centrifuged, washed,
and then analyzed as autoradiograms of SDS-PAGE gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Complementation of B. japonicum strains 120 and Q5 for growth on
proline-containing peptides with ptsP or lysC expressed in trans
1 media, 50 µg of prolyl-glycine ml
1
media, or 50 µg of prolyl-glycyl-glycine ml
1 media. +,
growth;
, no growth. The complementing genes were harbored in pVK102.
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Fig. 1.
ALA uptake activity in B. japonicum strains I110, I110I20, and I110Q5. Cells were
grown to mid log phase in minimal media. Cells were washed and
resuspended in 50 mM phosphate buffer, pH 7.4. At time = 0, 150 µM [14C]ALA was added to the
buffer, and aliquots of cells were removed at 5-min intervals and
washed. Incorporation of [14C]ALA was measured by liquid
scintillation. Each time point is an average of duplicate samples. The
uptake data are shown for parent strain I110 (closed
squares) and for mutant strains I110Q5 (open circles)
and I110I20 (closed circles).
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Fig. 2.
Identification of lysC and
ptsP, and characterization of their products
aspartokinase and Enzyme INtr. A,
organization of lysC and ptsP in the B. japonicum genome. The triangles denote the site of the
Tn5 insertions in strains Q5 and I20. B, aspartokinase
activity of B. japonicum lysC gene product. Purified,
recombinant aspartokinase was assayed for activity as described in the
text. Absorbance of the product was measured at 540 nm, and activity is
represented by the A540 × 1000. Reactions were carried out in the presence (closed squares)
or absence (closed triangles) of ATP. Each data point is an
average of three reactions. Standard deviations were calculated and
represented by error bars. C,
PEP-dependent phosphorylation of B. japonicum
EINtr. 4 µg of purified, recombinant EINtr
was incubated with [32P]PEP or [ -32P]ATP
and analyzed by SDS-PAGE and autoradiography.
-phosphate. The
putative lysC gene was analyzed further by overexpression of
the gene in E. coli (Fig. 2B). The purified
recombinant had aspartokinase activity as determined by as the
production asparthydroxamate from aspartate, ATP, and hydroxylamine
(30). Thus, a bona fide lysC gene was identified.
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Fig. 3.
Amino acid sequence comparison of
aspartokinase from B. japonicum and C. glutamicum. Solid lines denote amino acid
identity, and dotted lines represent similar residues
-phosphate formed by aspartokinase is the first
intermediate in the lysine, threonine, and methionine biosynthetic pathways (33). Although only one gene was detected using the lysC open reading frame as a probe against B. japonicum wild type DNA in a Southern blot (data not shown), the
lysC mutant strain I110Q5 is not an amino acid auxotroph.
Thus, it is likely that B. japonicum contains an additional
aspartokinase gene. Indeed, E. coli contains three
aspartokinase isozymes, all of which must be mutated to obtain an amino
acid auxotrophic phenotype (34, 35). We note, however, that the
phenotypes of the B. japonicum lysC mutants indicate that a
putative second aspartokinase gene cannot compensate for the mutated
gene described in this study. Finally, addition of lysine, threonine,
or methionine, or combinations of them, to growth media did not
complement lysC strain Q5 for oligopeptide-dependent growth in the absence of proline. We
suggest that lysC has a role other than, or in addition to,
amino acid biosynthesis in B. japonicum.
54-dependent family of
transcriptional activators responsible for the activation of genes
related to nitrogen fixation in a wide variety of diazotrophs (37),
including B. japonicum (38).
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Fig. 4.
Amino acid sequence comparison of
EINtr from B. japonicum and
Azotobacter vinelandii. Solid lines
denote amino acid identity, and dotted lines represent
similar residues.
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Fig. 5.
RNase protection analysis of ptsP
and lysC expression in B. japonicum
strains I110proC, I20, and Q5. Cells were grown in a yeast
extract-based media. 8 µg of total RNA was analyzed per
reaction.
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Fig. 6.
Protein-protein interactions between B. japonicum EINtr and AspK. A,
purified, recombinant EINtr (His-myc-tagged) was incubated
with either GST or GST-AspK, and protein bound to glutathione-Sepharose
beads was analyzed by Western blots using anti-myc antibodies.
B, purified, recombinant aspartokinase (His-myc-tagged) was
incubated with either GST or GST- EINtr, and protein bound
to glutathione-Sepharose beads was analyzed by Western blots using
anti-myc antibodies.
-32P]ATP alone, but
it was strongly radiolabeled when cell extracts from the
lysC strain I110Q5 were included in the reaction (Fig. 7). Extracts from the lysC
strain were used so that the only aspartokinase present was that added
as purified protein. GST alone was not phosphorylated under those
conditions. These observations indicate a factor in B. japonicum extracts that allow GST-EINtr to be
phosphorylated by ATP. However, when recombinant aspartokinase was
included in the reaction, GST-EINtr was substantially
underphosphorylated (Fig. 7). Addition of aspartate or dialysis of cell
extracts did not affect the phosphorylation of GST-EINtr,
nor did it affect the inhibition by aspartokinase (data not shown).
Thus, the underphosphorylated GST-EINtr in the presence of
aspartokinase could not be explained by consumption of ATP from the
enzyme activity of aspartokinase. We suggest that aspartokinase
controls EINtr function by regulating the phosphorylation
state of EINtr. The complementaton data (Table I),
indicating that aspartokinase exerts an affect on EINtr,
are consistent with the in vitro phosphorylation
experiments.
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Fig. 7.
ATP-dependent phosphorylation of
EINtr in the presence of cell extracts and inhibition by
aspartokinase. GST-EINtr or GST was incubated with
[ -32P]ATP either alone or with one of the components
labeled in the figure. When the reaction was complete,
glutathione-agarose beads were added, centrifuged, washed, and proteins
that bound were analyzed by autoradiography of SDS-PAGE gels.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-0077628 and United States Department of Agriculture Grant 99-35305-8062 (to M. R. O.).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) AF323675.
To whom correspondence should be addressed: Dept. of Biochemistry,
140 Farber Hall, State University of New York at Buffalo, Buffalo, NY
14214. Tel.: 716-829-3200; Fax: 716-829-2725; E-mail: mrobrian@buffalo.edu.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101982200
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ABBREVIATIONS |
---|
The abbreviations used are:
Dpp, dipeptide
permease;
Opp, oligopeptide permease;
ALA, -aminolevulinic acid;
EINtr, Enzyme INtr;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
PEP, phosphoenolpyruvate;
PTS, phosphoenolpyruvate:sugar phosphotransferase
system;
kb, kilobase(s);
bp, base pair(s);
PCR, polymerase chain
reaction;
PAGE, polyacrylamide gel electrophoresis.
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