(Received for publication, November 30, 1995; and in revised form, January 8, 1996)
From the
Enhancer-dependent transcription in enteric bacteria depends
upon an activator protein that binds DNA far upstream from the promoter
and an alternative factor (
) that binds with
the core RNA polymerase at the promoter. In the photosynthetic
bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins
(RcNtrB and RcNtrC) are putative members of a two-component system that
is novel because the enhancer-binding RcNtrC protein activates
transcription of
-independent promoters. To
reconstitute this putative two-component system in vitro, the
RcNtrB protein was overexpressed in Escherichia coli and
purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB
autophosphorylates in vitro to the same steady state level and
with the same stability as the Salmonella typhimurium NtrB
(StNtrB) protein but at a lower initial rate. MBP-RcNtrB
P
phosphorylates the S.typhimurium NtrC (StNtrC) and RcNtrC
proteins in vitro. The enteric NtrC protein is also
phosphorylated in vivo by RcNtrB because plasmids that encode
either RcNtrB or MBP-RcNtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and
autophosphatase activity of phosphorylated RcNtrC (RcNtrC
P) are
comparable to the StNtrC protein. However, the RcNtrC protein appears
to be a specific RcNtrB
P phosphatase since RcNtrC is not
phosphorylated by small molecular weight phosphate compounds or by the
StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC
P
binds the upstream tandem binding sites of the glnB promoter
4-fold better than the unphosphorylated RcNtrC protein, presumably due
to oligomerization of RcNtrC
P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to
other NtrC-like systems, where specific RcNtrB phosphotransfer to the
RcNtrC protein results in increased oligomerization at the enhancer but
with subsequent activation of a
-independent
promoter.
The NtrB and NtrC proteins of enteric bacteria form a
two-component signal transduction system that has been extensively
characterized genetically and biochemically (see (1) for
review). Under conditions of nitrogen limitation, the NtrB sensor
kinase autophosphorylates on a specific histidine residue (2, 3, 4, 5) and transfers the
phosphate to the NtrC response regulator protein on a specific
aspartate residue(2, 4, 6) . Phosphorylated
NtrC (NtrCP) (
)is a transcriptional activator of genes
involved in nitrogen metabolism such as glnA (glutamine
synthetase). NtrC
P has enhanced DNA binding activity(7) ,
presumably due to increased oligomerization on the DNA
template(8, 9) , and an ATPase
activity(10, 11) , which may also be due to the
oligomerization of the NtrC phosphoprotein(12, 13) .
These properties of oligomerization and ATPase activity are essential
for transcriptional activation in vitro(14) and in vivo(15, 16, 17) .
Members of
this class of proteins share certain properties. (a) They bind
to DNA at tandem sites far upstream (>100 bp) of the promoters that
they activate (see (18) for review); (b) they contain
an ATP binding motif, and possess ATPase activity(11) ; and (c) they require a specific factor, called
, that binds with the core RNA polymerase at highly
conserved promoters (see (19, 20, 21) for
reviews). The NtrC protein binds to sites over 100 bp upstream of the glnA promoter(22, 23) , and DNA looping
occurs between the NtrC protein bound at the enhancer and the
/RNA polymerase holoenzyme (which forms a stable
closed complex) bound at the promoter (24, 25, 26, 27) . Interaction
between the activated NtrC protein and the
/RNA
polymerase holoenzyme, in combination with the ATPase activity of the
NtrC protein results in a dramatic stimulation of the expression of the glnA gene(12, 14) .
The NtrC protein from
the photosynthetic bacterium Rhodobacter capsulatus (RcNtrC)
is a novel enhancer-binding protein that does not require the
factor to activate transcription of the R.
capsulatus nifA1, nifA2, and glnB genes(28, 29, 30, 31, 32) .
The promoters of these genes have been defined by lacZ translational fusions and primer extension analysis; they are
expressed in strains lacking
and have no sequence
homology to
promoters. The proteins encoded by the nifA1 and nifA2 genes are themselves transcriptional
activators that induce nitrogen fixation (nif) gene
expression, using the
RNA polymerase under
conditions of nitrogen and oxygen limitation(29, 33) .
The glnB gene is part of a glnBA operon; the GlnB
protein putatively acts to repress R. capsulatus NtrB (RcNtrB)
function under conditions of nitrogen excess (34, and see 28 and 35 for
reviews). The RcNtrC protein also binds to sites on the DNA greater
than 100 bp upstream from the promoters that it activates. The RcNtrC
binding sites have been characterized by extensive deletion analysis of
the nifA1 and nifA2 promoters(29, 31) . In vitro, DNase I
footprinting directly demonstrates that RcNtrC binds to tandem sites of
dyad symmetry at the nifA1, nifA2, and glnB upstream regions(31, 32) . In addition, the
RcNtrC protein has an ATP binding motif, which by homology with other
ATP-binding proteins is predicted to bind and hydrolyze
ATP(36) ; mutations in this motif prevent transcriptional
activation by the RcNtrC protein in vivo(31) .
The
RcNtrB and RcNtrC proteins are putative members of a two-component
system based on sequence homology to the enteric NtrB/NtrC proteins,
especially in the regions that are highly conserved in other
two-component systems(37) . Genetic evidence demonstrates that
the R. capsulatus ntrB and ntrC genes are members of
an operon, and both genes are essential for transcriptional activation
of nif genes in vivo(38, 39) . The
present study demonstrates that the RcNtrB and RcNtrC proteins comprise
a two-component regulatory system, that RcNtrBP is a specific
substrate for RcNtrC, and that the phosphorylated RcNtrC protein has
increased DNA binding activity in vitro at RcNtrC tandem
upstream binding sites. The R. capsulatus proteins are
compared to their counterparts in enteric bacteria.
Phosphorylation of NtrC proteins by acetyl phosphate was carried out
with a method modified from a procedure graciously supplied by Dr.
Tracy Nixon (Pennsylvania State University, University Park, PA).
Radiolabeled acetyl phosphate was prepared by the incubation of acetate
kinase (2 units; Sigma) with 60 mM potassium acetate and 5
µl of [-
P]ATP (6000 µCi; Amersham)
in 25 mM Tris-HCl and 10 mM DTT at 24 °C. After
15 min the reactions were added to an equal volume of either RcNtrC or
StNtrC proteins (500 nM). After either 10 or 40 min at 24
°C, the reactions were stopped and loaded onto a 10%
SDS-polyacrylamide gel for analysis of radiolabeled proteins.
Figure 1: Purification of the MBP-RcNtrB and RcNtrC proteins. The 10% SDS-polyacrylamide gel was loaded as follows: lane 1, molecular size markers (Bio-Rad); lane 2, sonicated cell extracts of uninduced cells that contain the pMBPRcB plasmid; lane 3, sonicated cell extracts of overproduced MBP-RcNtrB; lane 4, ultracentrifugation supernatant; lane 5, flow-through of amylose affinity column; lane 6, column wash; lane 7, elution of the MBP-RcNtrB protein from the amylose column with 10 mM maltose; lane 8, gel filtration fraction of the purified RcNtrC protein with size standards are shown at left. Details of protein purification are described under ``Experimental Procedures.''
To determine if the R. capsulatus NtrB
protein is a histidine kinase capable of autophosphorylation,
MBP-RcNtrB was incubated with [-
P]ATP at 37
°C. MBP-RcNtrB became labeled, indicating that it
autophosphorylates (Fig. 2, lane 3). To compare the
autokinase activity of RcNtrB to the enteric NtrB protein directly,
purified S. typhimurium NtrB protein (StNtrB) was also
incubated with [
-
P]ATP (Fig. 2, lane 1). Time-course experiments demonstrated that the StNtrB
protein became labeled more rapidly than the MBP-RcNtrB protein, but at
maximal phosphorylation both proteins incorporated an equivalent level
of label, indicating that both proteins were phosphorylated to the same
degree (Fig. 3). MBP-RcNtrB
P was separated from ATP and
determined to be stable for over 2 h at 37 °C, which is comparable
to the enteric NtrB
P (4) and to other histidine
autokinases (see (43) for review).
Figure 2:
Autophosphorylation of the MBP-RcNtrB
protein and phosphate transfer to the StNtrC and RcNtrC proteins in
vitro. Phosphorylation reactions are described in detail under
``Experimental Procedures.'' All reactions contained 100
µM cold ATP and 0.2 µM [-
P]ATP and were allowed to proceed
for 15 min unless otherwise indicated. Reactions were carried out at 37
°C and 1 mM DTT, except those that involved RcNtrC, which
were performed at 24 °C with 10 mM DTT unless otherwise
indicated. Protein concentrations are as follows unless otherwise
indicated: StNtrB (200 nM), StNtrC (500 nM),
MBP-RcNtrB (200 nM), and RcNtrC (500 nM). The gel was
loaded as follows: lane 1, StNtrB alone; lane 2,
StNtrB and StNtrC; lane 3, MBP-RcNtrB alone; lane 4,
MBP-RcNtrB and StNtrC; lane 5, RcNtrC alone; lane 6,
StNtrB and RcNtrC; lane 7, MBP-RcNtrB (500 nM) and
RcNtrC; lane 8, MBP-RcNtrB (500 nM) and RcNtrC
incubated for 1 h; lane 9, MBP-RcNtrB (500 nM) and
RcNtrC in 1 mM DTT; lane 10, StNtrB (600 nM)
and StNtrC. Autoradiogram of the SDS-polyacrylamide gel was exposed for
5 h. Size standards are shown at left and radiolabeled
proteins at right.
Figure 3:
Autophosphorylation of the MBP-RcNtrB and
StNtrB proteins. Comparison of the autophosphorylation of StNtrB (open squares) and MBP-RcNtrB (diamonds) proteins
(200 nM) incubated in the presence of
[-
P]ATP. The x axis represents
time in minutes, and the y axis represents the level of
phosphorylated NtrB proteins in cpm.
Two-component sensor
proteins autophosphorylate on a specific histidine residue in the
conserved C terminus of the
protein(6, 44, 45) . The RcNtrB protein
contains a histidine at position 214 that is completely conserved with
the enteric NtrB proteins and other sensor proteins. To demonstrate
that the RcNtrB protein is a histidine kinase, purified
MBP-RcNtrBP was blotted directly onto nitrocellulose, washed in 50
mM Tris-HCl, and exposed to neutral, acidic, or basic
conditions prior to radiodetection. MBP-RcNtrB
P was sensitive to
acidic conditions (a 13-fold loss of signal was observed in the
presence of 1 N HCl) and stable in the presence of basic
conditions (no loss of signal was observed in the presence of 0.5 N NaOH; data not shown). These results are consistent with the
properties of histidinyl-phosphate residues(46, 47) ,
indicating that phosphorylation of the MBP-RcNtrB protein probably
occurs on a histidine residue.
Figure 4:
RcNtrB mediated activation of a nifL-lacZ fusion by NtrC in E. coli. Colonies that
were transformed with the nifL-lacZ fusion plasmid and pETRcB (A), pET21B (B), pMBPRcB (C),
pmal-C2 (D), or pETRcC (E) were picked onto
LB plates that contained drug (ampicillin and tetracycline), 0.5 mM IPTG to induce gene expression, and 50 µg/µl
5-bromo-4-chloro-3-indol--D-galactopyranoside to observe
the level of expression of the nifL-lacZ gene.
Previous experiments have shown that the
enteric NtrC protein can be phosphorylated in vitro by a
variety of sensor kinases, including the CheA protein(49) . To
determine if MBP-RcNtrBP could phosphorylate the enteric NtrC
protein, MBP-RcNtrB
P was incubated with the Salmonella
typhimurium NtrC protein (StNtrC) in vitro. Label
disappeared from both major forms of the MBP-RcNtrB protein and
appeared in the StNtrC, indicating that the MBP-RcNtrB
P is a
substrate for the StNtrC protein (Fig. 2, lane 4). To
directly compare the phosphotransfer properties of the RcNtrB to the
enteric NtrB protein, both MBP-RcNtrB
P and StNtrB
P (at 200
nM dimers) were incubated with the StNtrC protein, and the
transfer of phosphate was measured. Both MBP-RcNtrB
P and
StNtrB
P labeled StNtrC to a comparable level (Fig. 2,
compare lanes 2 and 4). The autophosphatase activity
of StNtrC
P was determined to be the same irrespective of
phosphorylation by the MBP-RcNtrB or StNtrB proteins (see below). Thus,
the StNtrC protein probably interacts with a highly conserved
functional domain of both NtrB proteins (supported by amino acid
sequence homology in the region of the conserved histidine).
Figure 5:
Phosphorylation of the RcNtrC protein. A, the optimal concentration of MBP-RcNtrB protein required to
phosphorylate RcNtrC protein (at 1 µM) was determined. The y axis refers to the concentration of labeled RcNtrCP (in
cpm) as determined by scintillation counts of polyacrylamide gel slices
that contained the radiolabeled proteins. The x axis refers to
the concentration of MBP-RcNtrB (in nM dimers), and reactions
were performed at 24 °C for 15 min. B, comparison of the
phosphorylation of StNtrC
P and RcNtrC
P by the
MBP-RcNtrB
P protein. The MBP-RcNtrB protein was phosphorylated by
incubation with [
-
P]ATP and then mixed with
either StNtrC or the RcNtrC protein (at 500 nM). The x axis represents time (in minutes) and the y axis
represents the concentration of RcNtrC
P (diamonds) or the
amount of StNtrC
P (open squares) in cpm. The reactions
are described under ``Experimental
Procedures.''
The phosphotransfer reaction and autophosphatase activity of
RcNtrC were studied under the optimal conditions described above.
RcNtrCP was detectable within the first 30 s of incubation with
MBP-RcNtrB
P and reached a steady state maximal level after 15 min (Fig. 2, lane 7) that was stable for at least 1 h (Fig. 2, lane 8). RcNtrC alone did not label in the
presence of [
-
P]ATP under any conditions (Fig. 2, lane 5). To directly compare the
phosphorylation of the RcNtrC and StNtrC proteins, we used the
MBP-RcNtrB
P protein to phosphorylate both proteins. The RcNtrC and
StNtrC proteins were phosphorylated at approximately the same rate by
the MBP-RcNtrB protein; the maximal level of RcNtrC
P was
comparable to the StNtrC
P (Fig. 5B). Previous work
demonstrated that the enteric NtrC
P has an autophosphatase
activity that recycles the protein to its unphosphorylated form.
(Phosphate is also removed from RcNtrC
P by a mechanism called
regulated dephosphorylation, which requires both the NtrB and GlnB
proteins ((3) ).) To determine if the RcNtrC
P has an
autophosphatase activity, RcNtrC
P was formed and its decay was
measured and compared to the decay for the StNtrC
P protein.
RcNtrC
P had an autophosphatase activity with a half-life of
2-4 min (Fig. 6) similar to the 3-5 min observed for
StNtrC
P (Fig. 6, and see (3) ).
Figure 6:
The autophosphatase activity of the RcNtrC
protein. Graph of the disappearance of phosphate from the RcNtrCP
and StNtrC
P proteins. The y axis refers to the log of the
labeled phosphoprotein (in cpm), and the x axis refers to time
(in minutes). Open squares denote StNtrC
P, and diamonds RcNtrC
P. Autophosphatase assays are described in
the text.
In order to
address the specificity of the RcNtrC protein for phosphate donors (or
kinase proteins), we tested the ability of RcNtrC to be phosphorylated
by a variety of substrates. Small molecular weight high energy
phosphate compounds (acetyl phosphate, carbamyl phosphate, and
phosphoramidate) have been shown to phosphorylate the enteric NtrC
protein (50) and other members of the response regulatory
protein family in vitro(51) . Radiolabeled acetyl
phosphate was prepared and incubated with both the StNtrC and RcNtrC
proteins for 10 and 40 min; StNtrCP was detected after 10 min and
increased in concentration during the following 40 min; however, no
label was incorporated into the RcNtrC protein after 40 min (data not
shown). Unlabeled acetyl phosphate (Sigma), carbamyl phosphate (Sigma),
and either ammonium or potassium phosphoramidate (each at 20
mM) did not inhibit the phosphorylation of RcNtrC by the
MBP-RcNtrB
P (data not shown), whereas these compounds can compete
with the enteric NtrB
P for enteric NtrC
phosphorylation(52) . Additionally, DNase I footprinting (see
below) of the RcNtrC protein was not enhanced by any of these small
molecular weight compounds. The enteric NtrB protein can phosphorylate
other response regulatory proteins, including the CheY protein in
vitro(49) . To determine if the RcNtrC protein could be
phosphorylated by the enteric NtrB protein in vitro,
phosphorylated StNtrB protein was prepared and incubated in the
presence of the RcNtrC protein. No loss of label was observed from the
StNtrB
P, and no label was incorporated into the RcNtrC protein (Fig. 2, lane 6). In this respect it is interesting
that R. capsulatus ntrB mutants that are not polar on R.
capsulatus ntrC are still Nif
(38) ,
suggesting that cross-talk with other kinases or RcNtrC phosphorylation
by small molecular weight compounds does not occur in vivo.
This is in contrast with the enteric NtrC protein (53) and
consistent with a hypothesis that the phosphorylation domain of RcNtrC
may form a structure that makes it less accessible than the domain of
the StNtrC protein for other substrate kinases.
Phosphorylation of
RcNtrC protein is predicted to occur on an aspartate residue within the
conserved N terminus; RcNtrC has an aspartate at residue 53 that is
highly conserved between other members of the NtrC class of
enhancer-binding proteins(54) . Using the same methods
described for RcNtrBP, the RcNtrC
P was determined to be
sensitive to base (a 3-fold loss of signal was observed in 0.5 N NaOH) and stable in acid (less than 10% loss of signal was
observed in 1 N HCl; data not shown), indicative of a serine,
threonine, or aspartate residue(4, 55, 56) .
Figure 7: The RcNtrC protein is a dimer. A, the graph represents the elution profile of the purified RcNtrC protein off of a Sephacryl S200-HR (gel filtration) column calibrated by size standards. The x axis represents the fraction number off of the column, and the y axis represents the concentration of purified RcNtrC protein (in µg/µl). Arrows denote the elution of size standards, except for the arrow at approximately 110 kDa that indicates the peak elution fraction of the RcNtrC protein. B, Western analysis of a semi-native 10% SDS-polyacrylamide gel (see ``Experimental Procedures'') that was transfered to nitrocellulose and probed with antibodies raised against the RcNtrC protein as detected by phosphorescence of protein A peroxidase (ECL kit). The gel was loaded with 20 nM (lane 1), 100 nM (lane 2), or 500 nM (lane 3) of the purified RcNtrC protein. Lane 4 shows a Western analysis of extracts of 10 µg of wild type R. capsulatus (SB1003) probed with antibodies against the RcNtrC protein. Size standards are indicated at left. Western blots were performed as described under ``Experimental Procedures.''
The
enteric NtrC protein binds in vitro and in vivo to
specific sites on DNA far upstream of
promoters(22, 23, 59) . Phosphorylation of
the enteric NtrC protein stimulates enhancer binding in vitro by 4-20-fold by oligomerization at tandem binding
sites(7, 8, 15) . We wanted to analyze this
property of the RcNtrC protein. DNase I footprinting was performed with
the phosphorylated RcNtrC protein to determine if, like the enteric
NtrC, RcNtrCP has enhanced DNA binding activity compared to
unphosphorylated RcNtrC. RcNtrC was phosphorylated by the
MBP-RcNtrB
P under optimal conditions (various concentrations of
RcNtrC were incubated for 15-45 min at 24 °C with 1
µM MBP-RcNtrB protein in the presence of 10 mM DTT) and allowed to bind to an end-labeled DNA probe containing
the glnB promoter region. DNase I digests were performed as
described under ``Experimental Procedures,'' and the regions
that were protected from digestion were compared to the regions
protected by the unphosphorylated RcNtrC protein. Complete protection
was observed at the upstream binding sites of the glnB promoter at 160 nM unphosphorylated RcNtrC (Fig. 8A) but at 40 nM with phosphorylated
RcNtrC (Fig. 8C). The binding of unphosphorylated
RcNtrC was unaffected by the presence of ATP (Fig. 8, compare A and B). This increase in binding is also clearly
indicated by the hypersensitive site induced by RcNtrC binding at the
tandem sites (Fig. 8, see arrowheads). We conclude that
phosphorylation of RcNtrC increases DNA binding by approximately
4-fold, similar to the enteric NtrC system. Similar to the enteric
NtrC
P, RcNtrC may show enhanced DNA binding activity due to
increased oligomerization at the enhancer(12) .
Figure 8: Enhanced DNase I footprinting protection by the phosphorylated RcNtrC protein. Figures shows protection of the upper strand of the glnB promoter region from DNase I digestion by RcNtrC. A, RcNtrC alone; B, RcNtrC and 1 mM ATP; C, RcNtrC, 1 mM ATP and 1 µM MBP-RcNtrB. The concentration of probe was 0.1 nM for each reaction. RcNtrC concentrations (nM) are shown above each reaction. Shaded bars mark the strong tandem binding sites of RcNtrC (see text); numbers refer to the distance from the transcriptional start; large and small arrowheads mark areas of increased DNase I sensitivity. Brackets indicate the regions of protection described previously(32) .
The R. capsulatus and enteric NtrB/NtrC system were directly compared. The RcNtrB protein autophosphorylates to the same level and stability as the StNtrB protein, although at a lower initial rate. Both proteins are capable of phosphate transfer to the StNtrC protein, but only the RcNtrB protein phosphorylates the RcNtrC protein, indicating that the RcNtrC protein is a more specific phosphatase than the enteric NtrC protein. This specificity is supported by other genetic and biochemical evidence, including the inability of RcNtrC to use small molecular weight phosphate compounds in vitro and probably in vivo. Conditions for the phosphorylation of RcNtrC protein were optimized, facilitating future biochemical studies on this novel transcriptional activation system.