(Received for publication, February 24, 1997, and in revised form, April 30, 1997)
From the Department of Microbiology and Immunology,
University of Tennessee and the Veterans Administration Medical Center,
Memphis, Tennessee 38163 and the § Department of
Microbiology and Immunology, Bowman Gray School of Medicine at Wake
Forest University, Winston-Salem, North Carolina 27157-1064
The exopolysaccharide alginate is an important
virulence factor in chronic lung infections caused by the bacterium
Pseudomonas aeruginosa. Two positive activators for
alginate synthesis, algB and algR, are members
of a superfamily of response regulators of the two-component regulatory
system. AlgB belongs to the NtrC subfamily of response regulators and
is required for high-level production of alginate. In this study, an
open reading frame encoding a polypeptide of 66 kDa, designated
kinB, was identified immediately downstream of
algB. The sequence of KinB is homologous to the histidine
protein kinase members of two-component regulatory systems. Western
blot analysis of a P. aeruginosa strain carrying a
kinB-lacZ protein fusion and studies of
kinB-phoA fusions indicate that KinB localizes
to the inner membrane and has a NH2-terminal periplasmic domain. A KinB derivative containing the COOH terminus of KinB was
generated and purified. In the presence of [-32P]ATP,
the purified COOH-terminal KinB protein was observed to undergo
progressive autophosphorylation in vitro. Moreover, the phosphoryl label of KinB could be rapidly transferred to purified AlgB.
Substitutions of the residues conserved among histidine protein kinases
abolished KinB autophosphorylation. These results provide evidence that
kinB encodes the AlgB cognate histidine protein kinase.
Chronic pulmonary infection with the bacterium Pseudomonas
aeruginosa is a major factor in the poor prognosis and high
mortality rate of patients with cystic fibrosis
(CF)1 (1). Most P. aeruginosa
strains isolated from the CF respiratory tract overproduce an
exopolysaccharide called alginate, which gives the colonies a mucoid
morphology (2). This highly viscous polysaccharide plays a role in the
pathogenesis of P. aeruginosa by imparting antiphagocytic
properties (3) and an adherence mechanism (4). Most of the genes
involved in alginate biosynthesis are in a tightly regulated operon at
34 min on the 75-min chromosome (5). High expression of the alginate
biosynthetic genes requires the activation of an alternative sigma
factor (22) encoded by algT (algU)
at about 68 min on the chromosome (for review, see Ref. 6). In
addition, a cascade of several positive regulators are also required
for high expression of alginate genes (7, 8). Two of these, AlgB and
AlgR (AlgR1), belong to the superfamily of response regulators of
prokaryotic two-component regulatory systems (9, 10).
Two-component regulation is a mechanism for signal transduction to control cellular adaptations in response to environmental or physiological changes (for review, see Ref. 11). Observed in many bacterial species (12, 13), as well as in yeasts (14) and plants (15), two-component systems generally include a histidine protein kinase and a cognate regulator protein. In general, the histidine protein kinase senses a specific environmental stimulus and undergoes autophosphorylation at a histidine residue present in a highly conserved carboxyl-terminal domain of the protein. This phosphate group is subsequently transferred to an aspartate residue in the amino terminus of the response regulator, resulting in a change in the activity of the response regulator that leads to an adaptive response (11, 12). Response regulators can also catalyze kinase-independent phosphorylation and dephosphorylation by low-molecular weight phosphorylated compounds (e.g. acetyl phosphate, carbamyl phosphate, etc.), which may serve to integrate environmental control with the physiological status of the cell (16).
Alginate overproduction by P. aeruginosa is generally seen in strains causing pulmonary infection of CF patients. Specific signals present in the environment of the CF lung (e.g. dehydration, high osmolarity, limiting nutrients, antibiotics) may play a role in stimulating alginate production (for review, see Ref. 17). However, the role or requirement for any particular in vivo signal in the expression of alginate genes has not been well established. The discovery of two-component response regulators (i.e. AlgB and AlgR) suggests that environmental signals may play a role in the regulation of alginate production. Moreover, inhibitors of the two-component regulatory pathway inhibit the expression of alginate biosynthetic genes (18). However, proteins in P. aeruginosa with sensor kinase activity that can phosphorylate AlgB or AlgR have not been demonstrated. A gene adjacent to algR was recently identified that encodes a protein (FimS, AlgZ) with homology to an atypical two-component sensor (19, 20), but whether it functions as a kinase of AlgR is unknown. In this study we identified a gene called kinB, located immediately downstream of algB, that encodes a protein with high similarity to typical histidine protein kinases of two component systems. Our data indicate that KinB is an inner membrane protein with histidine protein kinase activity that is capable of promoting autophosphorylation and rapid transfer of the phosphate to AlgB.
The P. aeruginosa strains utilized in this study were FRD1, an
alginate-overproducing (Alg+) CF isolate and its derivative
FRD444 (Alg,
algB::Tn501), which contains a mercury
resistance (Hgr) transposon marker in algB (21).
Escherichia coli strains HB101 and JM109 were used in
routine cloning manipulations (22); BL21(DE3) was used to express
His6-tagged KinB; XL-2 Blue was used to overexpress AlgB. L
broth (10.0 g of tryptone (Difco), 5.0 g of yeast extract (Difco),
5.0 g of NaCl/liter, pH 7.5) was used for the routine culture of
P. aeruginosa and E. coli. A 1:1 mixture of
Pseudomonas isolation agar (Difco) and L agar was used to
select for P. aeruginosa following triparental matings.
Selective antibiotics used for P. aeruginosa were
carbenicillin at 300 µg/ml and tetracycline at 100 µg/ml; selective
antibiotics used for E. coli were ampicillin at 100 µg/ml,
kanamycin at 35 µg/ml, and tetracycline at 15 µg/ml. HgCl2 was used at 18 µg/ml both for P. aeruginosa and E. coli.
Cloned DNA
fragments utilized in this study are shown in Fig. 1. Most routine
genetic manipulations were performed as described elsewhere (22).
Plasmid DNA was isolated from E. coli using Qiagen columns
and procedures (Qiagen Corp.). Genomic DNA of P. aeruginosa
was prepared using a protocol previously described (21). Restriction
endonucleases were purchased from Boehringer Mannheim and New England
Biolabs. To isolate DNA that included sequences located downstream of
algB, chromosomal DNA from FRD444 (algB::Tn501) was digested with
BamHI (where Tn501 is not cut by
BamHI), ligated into cosmid vector pEMR2 (23), packaged
in vitro into particles (Gigapack II cloning kit,
Stratagene), and transduced into HB101. One representative clone
(pDJWA10, Fig. 1) that conferred Hgr contained
approximately 15-kb DNA upstream and 10-kb DNA downstream of
algB. Plasmid pDJW130 (Fig. 1) had a 0.8-kb
XhoI-EcoRI fragment from pJG12 (24) cloned into
vector pKS(
) that was used as a hybridization probe; it was
digoxigenin-labeled by the polymerase chain reaction using T3 and T7
primers. This probe was used to identify a 4-kb
ClaI-HindIII fragment from pDJWA10 which
contained a portion of algB and the entire kinB
gene (below), which was then cloned into pUC19 to form pSM67 (Fig.
1).
DNA Sequencing and Analysis
To prepare the DNA downstream
of algB for sequence analysis, the 4-kb
ClaI-HindIII fragment of pSM67 was digested with
PstI or partially digested with Sau3AI, and the
resulting fragments were subcloned into the PstI or the
BamHI site of M13mp19 (New England Biolabs), respectively.
Single-stranded DNA templates were prepared from these M13mp19 clones
using a sample preparation protocol (Applied Biosystems). DNA
sequencing reactions were performed with a Taq Dyedeoxy
terminator cycler sequencing kit (Applied Biosystems) using a
Perkin-Elmer DNA thermal cycler and run on an Applied Biosystems 373A
DNA sequencer. DNA fragments were sequenced on both strands, and the
sequence data obtained were aligned using SeqMan software (DNASTAR) on
an Apple Macintosh computer. To verify alignment of the sequence
contigs, six additional sequences were obtained by manual sequencing of
pSM67 (Fig. 1) using T7 DNA polymerase version 2, the 7-deaza-dGTP
sequencing kit (Life Sciences), and synthesized oligonucleotide
primers: p50 (5 CGGCTGTCCTTCTCCAGGTC 3
), p51 (5
CCACTACACCTCCACCGATC
3
), p52 (5
CAAGCGCACGGTATCACC 3
), p53 (5
GCATATCGACGCTGAGCATG 3
),
p54 (5
CGGTGGTGCTGGCCTGG 3
), and p55 (5
CGCCATTGTCTTCCACCGC 3
).
Homology searches and alignments were performed with the Basic Local
Alignment Search Tool (BLAST) Network Service at the National Center
for Biotechnology Information, National Institutes of Health (25).
To
construct a kinB-lacZ protein fusion, a 2.6-kb
EcoRI fragment containing algB-kinB was cloned
into pMLB1034 (26), resulting in pSM78; this was followed by the
introduction of a mob site on a EcoRI fragment
(27) to form pSM82 (Fig. 1). An algB-lacZ protein
fusion containing the amino-terminal 379 amino acids of AlgB was
constructed by cloning a 3.4-kb SmaI-DraI
fragment of pMLB1034 containing lacZ into the
EcoRV site of algB in pJG221 (21) to form pSM33;
this was followed by the introduction of a mob site on a
HindIII fragment (27) to form pSM35 (Fig. 1). Plasmids were
later moved into P. aeruginosa FRD1 by triparental mating as
described previously (24), which resulted in their integration into the
chromosome by homologous recombination. Expression of LacZ fusion
proteins in P. aeruginosa was evident by their
-galactosidase activity as detected by the formation of blue colonies on L agar plates containing
5-bromo-4-chloro-3-indolyl-
-D-galactoside at 75 µg/ml.
For immunoblot analyses of LacZ protein fusions, overnight cultures of
the P. aeruginosa strains carrying lacZ fusions
(FRD1::pSM82 and FRD1::pSM35) were diluted 1:50 in
100 ml of L broth with antibiotics and agitated at 37 °C to
A600 0.7. Cells were resuspended in 10 ml of A
buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM dithiothreitol), passed twice through a French press (15,000 p.s.i.), and centrifuged at 10,000 × g for 10 min at 4 °C to remove unbroken cells. The
supernatant obtained was used as the whole cell extract. A sample was
centrifuged at 200,000 × g for 60 min at 4 °C, and
the supernatant was regarded as the fraction enriched for cytoplasmic
proteins; the pellet was resuspended in 1.0 ml of A buffer and regarded
as the fraction enriched for membrane proteins. After determining the
protein concentration by the Bradford method (28), samples were diluted in sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris
hydrochloride, pH 6.8, 2% SDS, 10% glycerol, 0.1 mg of bromphenol
blue/ml, 5% 2-mercaptoethanol), and 30 µg of each fraction were
subjected to electrophoresis on a SDS-8% polyacrylamide gel. Proteins
were electrotransferred to nitrocellulose membrane, and LacZ fusion proteins were detected with rabbit anti-
-galactosidase polyclonal antibody (5 Prime
3 Prime, Inc., Boulder, CO; at a 1:5,000
dilution) as the primary antibody, and goat anti-rabbit horseradish
peroxidase conjugate (Sigma; 1:30,000 dilution) was used as the
secondary antibody. Protein bands were visualized with chemiluminescent Western blot detection reagents ECL (Amersham Corp.) and visualized on
film (Kodak X-Omat AR) exposed for 2 min.
Constructions containing kinB-phoA translational fusions were based on the plasmid pSM111 (Fig. 1). Polymerase chain reaction amplification was used to generate DNA fragments starting at the MluI site in algB to sites in kinB terminating at codons for Asp-148 or Ile-211, at which the primers generated BamHI sites. These MluI-BamHI fragments were each joined to a 2.6-kb BamHI-XbaI fragment containing phoA from pPHO7 (29), and cloned into pSM111, replacing the existing MluI-XbaI fragment, to form pSM126 and pSM127, respectively. A KinB-PhoA fusion with a junction at residue F379 was constructed by using a linker to join a MluI-EcoRI restriction fragment containing "algB-kinB" to a 2.6-kb SmaI-XbaI fragment containing phoA from pPHO7; this was cloned into pSM111, replacing the existing MluI-XbaI fragment, to form pSM128 (Fig. 1). Protein fusions containing PhoA (alkaline phosphatase) were verified by Western blot analysis using rabbit anti-alkaline phosphatase (Sigma). Colonies containing PhoA fusions with alkaline phosphatase activity (i.e. localized to the periplasm) were screened for blue color on L agar containing 5-bromo-4-chloro-3-indolyl phosphate at 40 µg/ml.
Purification of the COOH Terminus of KinB (C-KinB)To
construct plasmids that overexpressed a His6-tagged
carboxyl-terminal (Gly-198 to Val-595) fragment of KinB (HC-KinB), a
1.6-kb AscI fragment of pSM67 (Fig. 1) was cloned into
pNEB193 (New England Biolabs) to form pSM93; this was subsequently
digested with SacI and HindIII, and the 1.6-kb
fragment containing kinB was cloned in pET28.b (Novagen),
resulting in pSM95 (Fig. 1). E. coli BL21(DE3) harboring
pSM95 was agitated overnight at 37 °C in 100 ml of L broth with
kanamycin. The cells were harvested by centrifugation and resuspended
in 100 ml of L broth with kanamycin and 1 mM isopropyl
-D-thiogalactopyranoside for induction of the
tac promoter (Ptac). After incubation at 30 °C
with aeration for 2 h, the cells were harvested and incubated in
20 ml of 20 mM Tris-HCl, pH 8.0, containing lysozyme (100 µg/ml) for 30 min at 4 °C. Following sonication, the lysate was
centrifuged at 12,000 × g for 15 min, and the
supernatant was filtered (0.45-µm disc filter, Millipore). HC-KinB in
the cell extract was purified on a 2.5-ml His-Bind nickel column
(Novagen) according to manufacturer's protocol. As estimated by
SDS-PAGE and Coomassie Blue staining, HC-KinB was over 95% pure. The
His6 tag was removed from HC-KinB by digestion with 25 units/ml thrombin (Novagen) for 2 h at 22 °C to form C-KinB,
which was subjected to an amino-terminal sequence analysis
(Biotechnology Center, St. Jude Children's Research Hospital, Memphis,
TN).
To obtain C-KinB
proteins with substitutions in conserved residues, the
SacI-HindIII fragment in pSM95 (Fig. 1) was
cloned into pAlter1 (Promega) and mutagenized using the altered sites mutagenesis system (Promega) according to the procedure suggested by
the manufacturer. The mutagenic primers used were: E257Q (5 TGCTTTCCGGCCAGCGGCGCCTG 3
), H385K (5
CTGCGCGCCTCCAAGGAACTGCGCACG 3
),
H385Q (5
CGCGCCTCCCAGGAACTGCG CACG 3
), N504Q (5
CAACCTGCTGGAACAGGCCCTGCGCCATA 3
), D532E (5
CGGTGGAAGAAAATGGCGAAG 3
),
D532N (5
CGCGGTGGAAAACAATGGCGA 3
), and G560A (5
GGCGGCGCCGCTCTCGGCCTG 3
). After confirming the mutations by DNA
sequence analysis, the SacI-HindIII fragment of
each kinB allele was cloned into pET28.b, and mutant C-KinB proteins were expressed and purified as described above.
E. coli XL21 Blue harboring
pDJW52 (Fig. 1) expresses algB under the control of
Ptac as described previously (9). Cells from 400 ml of
overnight culture of XL21 Blue(pDJW52) were resuspended and agitated in
400 ml of fresh L broth with ampicillin and 1 mM isopropyl
-D-thiogalactopyranoside at 30 °C for 3 h. Cells were harvested and then incubated for 30 min at 4 °C in 20 ml of 20 mM Tris-HCl, pH 8.0, containing lysozyme (100 µg/ml).
Following sonication, the lysate was centrifuged at 12,000 × g for 10 min, and the supernatant was centrifuged at
200,000 × g for 60 min. Proteins in the clear
supernatant were precipitated with 35% ammonium sulfate (J. T. Baker
Inc.), and the precipitant was resuspended and dialyzed against 15 mM BisTris propane, pH 7.0, 20 mM NaCl. A
sample (20 ml containing 8.4 mg of protein) was loaded onto an AP-2
column (Waters) packed with Protein-PAK DEAE 40HR anion exchange matrix
(Waters), and a linear 20-160 mM NaCl gradient in 15 mM BisTris propane, pH 7.0, was used to elute proteins from the column. AlgB eluted at 130 mM NaCl and was estimated by
SDS-PAGE and Coomassie Blue staining to be >90% pure.
Autophosphorylation of
C-KinB was performed at 22 °C in P buffer (50 mM
Tris-HCl, pH 7.5, 50 mM KCl, 5 mM
MgCl2). C-KinB was diluted to a final concentration of 2.5 µM and distributed in 9-µl aliquots for each reaction.
Each reaction was started by adding [-32P]ATP (30 Ci/mmol, Amersham) to a final concentration of 33.3 µM
and was stopped by the addition of 3 µl of 5 × SDS sample
buffer. Unincorporated label was removed by passage through a 1-ml
Sephadex G-25 (Pharmacia Biotech Inc.) column, and samples were
electrophoresed on a SDS-10% polyacrylamide gel and examined by
autoradiography. To examine the time course of C-KinB
autophosphorylation, phosphorylation reactions were stopped by adding
10 µl of 200 mM sodium acetate, pH 4.0, and immediately
spotting the mixture onto a phosphocellulose membrane (Beckman)
pre-equilibrated with 25 mM sodium acetate, pH 4.0. The
membranes were washed three times for 10 min each in 800 ml of buffer
containing 25 mM sodium acetate, pH 4.0, and the
radioactivity on the dried membranes was measured (TRI-CARB 2000 liquid
scintillation analyzer). In studies demonstrating the transfer of
phosphoryl label from C-KinB to AlgB, 13 pmol of KinB was
phosphorylated for 60 min at 22 °C in a 10-µl mixture under the
conditions described above. AlgB (40 pmol) was added to the mixture,
and the reaction was terminated after 90 s by adding 3 µl of
5 × SDS sample buffer. The samples were passed through a 1-ml
Sephadex G-25 column to remove unincorporated label and analyzed by
SDS-10% PAGE, followed by autoradiography.
The nucleotide sequence data and inferred amino acid sequence reported here for kinB have been deposited in the GenBankTM data base under accession number U97063.
We examined whether a gene
(kinB) encoding a sensor kinase was closely linked to a
known gene (algB) encoding a response regulator that
controls the alginate biosynthetic operon in P. aeruginosa. Several studies on bacterial two-component regulatory systems have
shown that genes encoding a response regulator and its cognate histidine protein kinase are often linked (12). A 25-kb
BamHI fragment containing the DNA flanking algB
(pDJWA10, Fig. 1) was obtained from genomic DNA of
P. aeruginosa FRD444, a strain with an
algB::Tn501 allele (21) that provided a
selectable marker (mercury resistance) for the DNA in this region. A
4-kb ClaI-HindIII fragment was then subcloned
from the region immediately downstream of algB (pSM67, Fig.
1). This was subjected to a sequence analysis, and the putative
kinB ORF of 1,788 bp was observed in the same direction of
transcription as algB (Fig. 2). The
kinB ORF had a translation initiation codon that overlapped
with the algB termination codon, suggesting that expression
of algB and kinB may be translationally coupled.
The kinB ORF predicted a polypeptide of 595 amino acids with
a molecular weight of 66,078. Two hydrophobic domains at the amino
terminus of KinB were observed (underlined in Fig. 2). An
11-base pair inverted repeat sequence was located 75 bp downstream of
the kinB ORF that may serve as a factor-independent
terminator (shown as hatched lines in Fig. 2).
The kinB Gene Encodes a Protein with Homology to Histidine Protein Kinases
A homology search showed that the KinB sequence was
similar to a number of histidine protein kinases in two-component
regulatory systems. Fig. 3 depicts an alignment of KinB
sequences with that of PhoR, a similar sized histidine protein kinase
in Bacillus subtilis (30). Overall, KinB shows 31% identity
and 59% similarity with PhoR. The most conserved sequences were in
four regions that are characteristic of histidine protein kinases
(marked with hatched boxes in Fig. 3). The H box is proposed
to be the phosphorylation domain and may also be involved in the
dimerization of the kinase monomers; the N, D/F, and G boxes are
proposed to form a nucleotide binding surface in the tertiary structure
within the active site (31). The residues in these boxes that are
believed to be critical (marked with triangles in Fig. 3)
were all conserved in KinB and PhoR. In addition, both KinB and PhoR
contained two hydrophobic domains that were similarly positioned in
their amino termini (underlined and overlined in
Fig. 3). Both hydrophobic regions of KinB were sufficient in length to
form transmembrane domains, suggesting that KinB may be localized to
the inner membrane, as is PhoR.
Membrane Localization of KinB-LacZ in P. aeruginosa
A
KinB-LacZ fusion protein (encoded by pSM82, Fig. 1) was constructed to
test the expression of the kinB ORF in P. aeruginosa. The KinB-LacZ was predicted to be a 157.4-kDa peptide,
since the amino-terminal 379 amino acids of KinB (41.6-kDa) was fused
to a lacZ derivative expressing all but the first eight
amino acids of LacZ (115.8 kDa). As a control, an AlgB-LacZ fusion
protein of 151.4 kDa was constructed (encoded by pSM35, Fig. 1). The
plasmids containing the kinB-lacZ and algB-lacZ
fusion genes were in suicide vectors, and their mobilization to
P. aeruginosa FRD1 resulted in chromosomal integration at
the site of DNA homology (Fig. 4A). FRD1::pSM82 and FRD1::pSM35, harboring the
respective kinB-lacZ and algB-lacZ fusions in
single copy, both showed -galactosidase activity, indicating that
each ORF expressed a protein in P. aeruginosa. The
kinB-lacZ and algB-lacZ encoded fusion proteins
were also analyzed in a Western blot analysis of whole cell extracts,
using polyclonal antibody specific for LacZ (Fig. 4B, lanes
1 and 4); this showed that their electrophoretic
mobilities were consistent with the sizes predicted. The KinB-LacZ
fusion produced in FRD1::pSM82 contained a large
amino-terminal fragment of KinB that included both putative
transmembrane domains. To test whether the KinB-LacZ hybrid localized
to the membrane, whole cell extracts of FRD1::pSM82 were used
to obtain fractions enriched for either cytoplasmic or membrane
proteins. Extracts containing the AlgB-LacZ (FRD1::pSM35) were processed in parallel. Using anti-LacZ in the Western blot analysis, KinB-LacZ was detected in the membrane fraction, but not in
the cytoplasmic fraction (Fig. 4B, lane 2), suggesting that
KinB was indeed associated with the membrane. In contrast, the
AlgB-LacZ fusion protein, which does not contain a potential transmembrane domain (9), was detected in the cytoplasmic fraction (Fig. 4B, lane 6), but not in the membrane fraction.
Study of Membrane Topology with KinB-PhoA Fusions
The two
hydrophobic domains in the amino terminus (residues 13-39 and
170-190) of KinB, which may serve as transmembrane domains, were
evident in the hydrophilicity plot (Fig. 5A).
Thus, the region between the two putative transmembrane domains
(residues 40-169) of KinB was predicted to be in the periplasmic
space. To test this, KinB-PhoA fusions were constructed with junctions
at residue Asp-148, Ile-211 and Phe-379 (Fig. 5B). All three
KinB-PhoA fusions expressed proteins of the predicted size that were
readily detected in whole cell extracts of E. coli using a
Western blot analysis and antibody specific for PhoA (Fig.
5C). In that the phoA gene encodes the
periplasmic enzyme alkaline phosphatase, such protein fusions are
enzymatically active (PhoA+) only if translocated to the
periplasm (32). The KinB(D148)-PhoA fusion retained the first
transmembrane domain and was PhoA+ in E. coli
(Fig. 4B), suggesting that the amino terminus of KinB between the two transmembrane domains was periplasmic. In contrast, bacteria expressing KinB(I311)-PhoA and KinB(F379)-PhoA, where fusions
were downstream of the two transmembrane domains, were not
enzymatically active for PhoA (Fig. 5B). This suggests that the COOH terminus of KinB was localized to the cytoplasm. Thus, the
KinB amino terminus appears to be the periplasmic sensor domain and the
C terminus contains the cytoplasmic histidine kinase domain.
Autophosphorylation of KinB
The localization of KinB to the
membrane complicated the purification of native protein for studies of
its potential histidine protein kinase activity. However, all of the
conserved sequences for kinase activity were present in the cytoplasmic
carboxyl terminus. Thus, we tested the possibility that a
carboxyl-terminal fragment of KinB may be enzymatically active, as is
the case for several other membrane associated sensor kinases (33-36).
DNA encoding a carboxyl-terminal fragment of KinB from Gly-198 to the
end (Val-595) was cloned in frame into the His tag vector pET28.b to
form pSM95 (Fig. 1). This plasmid expressed a His6-tagged
fusion protein (HC-KinB), which was purified using a nickel sulfate
affinity column. To remove the His6 sequence, the purified
fusion protein was digested with thrombin, which recognizes a site
between the His6 tag and C-KinB sequence. However, an
amino-terminal sequence analysis of C-KinB revealed that thrombin
(which has arginine as its preferred site) also cleaved the KinB
protein between residues Arg-243 and Gln-244 to generate a 39.4-kDa
C-KinB polypeptide. Nevertheless, this C-KinB fragment still retained
all the sequences predicted to function as a histidine protein kinase
(see Fig. 3). To test this, C-KinB was incubated with
32P-labeled ATP, and then the samples were subjected to
SDS-PAGE and autoradiography. C-KinB (25 pmol) incubated with
[-32P]ATP (33 µM) showed progressive
autolabeling over the 1-60-min period examined (Fig.
6A, lanes 1-6). Incubation with
[
-32P]ATP at 33 or 66 µM for 40 min
showed similar labeling of C-KinB (compare lanes 5 and
7), suggesting that ATP was not a limiting factor in these
reactions. Accordingly, incubation with 50 pmol of C-KinB for 40 min
did show increased labeling (Fig. 6A, compare lanes
5 and 8). As a control, C-KinB (25 pmol) was incubated
with [
-32P]ATP (15 µM) for 60 min (Fig.
6A, lane 9), and no labeling was observed; this ruled out
the possibility of nonspecific binding of ATP by C-KinB. The
autoradiogram showing autolabeling of C-KinB suggested that the level
of protein phosphorylation (i.e. the balance of
autophosphosphorylation and dephosphorylation) was not maximum by 60 min. Thus, a quantitative time course of C-KinB autophosphorylation was
performed using liquid scintillation (Fig. 6B). This showed
that the maximum level of phosphorylated C-KinB under these conditions
did not reach a plateau until approximately 5 h of incubation. One
possible reason for this overall slow reaction was a high rate of
C-KinB dephosphorylation. However, this appeared not to be the case
because the phosphoryl label on C-KinB was stable after incubation with
a chase of cold ATP (333 µM) for 30 min (Fig.
6C).
C-KinB Mutants Altered at Conserved Sequences Are Affected in Autophosphorylation
We tested whether autophosphorylation
activity required sequences in KinB that are homologous to those of
other sensor kinases. Critical residues in histidine protein kinases
that were conserved in C-KinB (described above, see Fig. 3) were
altered by site-directed mutagenesis of kinB. Mutant alleles
of kinB were generated that expressed the following mutant
HC-KinB proteins: H385K and H385Q, in which His-385 in the H box
(i.e. the predicted site of phosphorylation) was changed to
Lys and Gln, respectively; N504Q, where Asn-504 in the N box was
mutated to Gln; D532N and D532E, in which Asp-532 of the D/F box was
changed to Asn or Gln, respectively; and G560A where Gly-560 in the G
box was substituted for Ala. Mutant derivatives of HC-KinB were
purified in the same manner as wild-type HC-KinB and estimated to be
>95% pure by SDS-PAGE. The His6 tags on these proteins
were also removed by thrombin digestion. Equivalent amounts of
wild-type and mutant C-KinB derivatives, after treatment with thrombin,
were examined by SDS-PAGE for relative stability of the proteins (Fig.
7A). Only C-KinB D532E (Fig. 7A, lane
7) showed any evidence of degradation beyond removal of the
His6-Arg-243 peptide (despite 27 other Arg residues, the
preferred site of thrombin cleavage). When each protein (2.5 µM) was incubated with [-32P]ATP (33 µM), the wild-type C-KinB sequence showed strong
autophosphorylation activity (Fig. 7B, lane 1). However,
labeling of the mutant proteins was undetectable except for the C-KinB
D532N derivative in which a trace amount of phosphorylated protein was
detected (Fig. 7B, lane 5). The C-KinB E257Q protein had a
substitution at a nonconserved residue, and it showed
autophosphorylation that was comparable with that of wild-type (Fig.
7B, lanes 8).
Phosphotransfer from C-KinB to AlgB
To determine whether
AlgB-KinB may function as a two-component regulatory pair, the ability
of phosphorylated C-KinB to donate a phosphate group to AlgB was
examined. AlgB was overexpressed in E. coli and purified
(>90%) using standard chromatographic procedures (Fig.
8A, lane 4). Purified AlgB alone was not
autophosphorylated when it was incubated with
[-32P]ATP as determined by SDS-PAGE and
autoradiography (Fig. 8B, lane 1). As shown above, purified
C-KinB (1.3 µM) incubated with [
-32P]ATP
for 60 min showed autophosphorylation (Fig. 8B, lane 2). However, when AlgB (40 pmol) was incubated for 90 s with
autophosphorylated C-KinB (K*), AlgB became radiolabeled, and complete
dephosphorylation of C-KinB was also observed (Fig. 8B, lane
4). Other studies of response regulators (e.g. CheY)
indicate that Mg2+ is required for phosphorylation (37).
This also appears to be the case with AlgB, since no AlgB
phosphorylation was observed when the protein was preincubated with
EDTA to chelate divalent cations (Fig. 8B, lane 3). In other
experiments, maximum phosphotransfer from 32P-C-KinB to
AlgB was observed after only 20-40 s of incubation (data not shown).
Taken together, the above results show that KinB in P. aeruginosa is a member of the sensor kinase superfamily with
histidine kinase activity that can rapidly phosphorylate its cognate
response regulator, AlgB.
The genes involved in alginate biosynthesis are under complex control by a cascade of regulators (6, 8). Two positive regulators of alginate production, AlgB and AlgR, affect transcriptional activation of the alginate biosynthetic operon at algD, and both have sequence similarity to the family of response regulators of two-component systems (9, 10). This suggested that the production of alginate by P. aeruginosa is influenced by environmental factors, some of which may be found in the unique environment of the CF lung (17). Prior to the recent description of FimS and its association with AlgR (19), no putative cognate sensor for AlgR had been recognized. However, FimS (also known as AlgZ) does not possess sequence similarity to typical histidine protein kinases (19, 20). The goal of this study was to identify KinB, a cognate sensor for AlgB, followed by tests for their potential interaction via phosphorylation. In that genes encoding histidine protein kinases are often closely linked to genes for their cognate response regulators (12), we examined the DNA immediately downstream of algB, and as a result kinB was discovered. KinB had a predicted molecular mass of 66 kDa and showed sequence similarity to many histidine protein kinases of two-component regulatory systems. KinB had all four conserved "boxes" characteristic of histidine protein kinases. Like many of them, KinB also had two hydrophobic domains at the amino terminus that are of sufficient length and hydrophobicity to span the inner membrane. These observations led to an analysis of a KinB-LacZ fusion protein in P. aeruginosa that suggested that KinB was indeed a membrane protein. An analysis of KinB-PhoA fusions supported the predicted membrane topology of KinB that the region between the two hydrophobic domains was in the periplasm. The COOH terminus of KinB, which contained amino acid residues conserved with other sensor kinases, was apparently localized to the cytoplasm. During appropriate in vivo conditions, the amino-terminal domain may act as an environmental sensor of some unknown factor(s) and transduce that information to the cytoplasmic domain to affect its kinase activity. It is difficult to speculate at this time just what environmental signal(s) might activate KinB, as its periplasmic domain has no significant similarity with any other known protein.
Most sensor kinases studied are capable of undergoing
autophosphorylation at a conserved histidine residue in the H domain of
the protein (38). Purified C-KinB was shown in this study to undergo
progressive autophosphorylation when incubated with [-32P]ATP. Interestingly, the level of
autophosphorylated protein did not reach its maximum until about 5 h at room temperature in the presence of excess
[
-32P]ATP. This rate is quite slow when compared with
the autophosphorylation of other sensor proteins under similar
conditions. These sensors include derivatives of ArcB (35) and EnvZ
(39) that were deleted of their amino-terminal transmembrane domains,
and they have been shown to reach maximum autophosphorylation within
minutes. Since the phosphorylated form of C-KinB appeared quite stable,
a high intrinsic phosphatase activity is not likely, and an explanation for the atypically slow autophosphorylation of C-KinB is not currently available. However, it is possible that the deletion of the amino terminus affected its autophosphorylation activity, even though C-KinB
contained the entire kinase domain. The oligomeric state of many sensor
kinases is important for their autophosphorylation activity (40-42).
The periplasmic domain of some kinases facilitates dimerization when it
is bound by environmental stimulatory ligands (43, 44). The rapid
autophosphorylation seen in amino-truncated ArcB and EnvZ may be due to
strong protein-protein interactions that remain between the monomers,
which is suggested by the observed aggregation and precipitation of
truncated ArcB and EnvZ with the membrane fraction when overexpressed
in E. coli (35, 39). In contrast, when C-KinB was
overexpressed, it remained soluble. It is currently not clear whether
the native form of KinB forms a dimer or whether dimerization affects
KinB autophosphorylation activity. Another explanation for the observed
kinetics of C-KinB phosphorylation also relates to the soluble nature
of C-KinB. When "tethered" to a membrane, as is the case for native
KinB, the effective concentration of KinB may be higher than that
observed with the soluble C-KinB used in these studies. In addition,
the reaction condition for the C-KinB autophosphorylation assay used here may not be optimal for this protein, although similar conditions were used in the phosphorylation of truncated ArcB and EnvZ (35, 39).
Since the sequence of KinB showed high homology with other sensor kinases, substitutions of the conserved residues were made to verify that KinB is a new member of this conserved superfamily of histidine protein kinases. When the predicted histidine phosphorylation site in KinB (His-385 in the H box) was changed to either a lysine or a glutamine, autophosphorylation of C-KinB was completely lost. Moreover, mutations affecting other conserved boxes all had deleterious effects on the kinase activity, suggesting that KinB is a typical histidine protein kinase. Interestingly, while no phosphorylated protein was detected when Asp-532 in the D/F box was substituted for a glutamate, changing the same residue to an asparagine permitted some residual C-KinB autophosphorylation.
The ability of phospho-C-KinB to phosphorylate the purified response regulator AlgB was also demonstrated. When AlgB was incubated with the phosphorylated C-KinB at a molar ratio of 3 to 1, the phosphoryl group was rapidly transferred to AlgB and completed by 40 s. This rate is similar to that observed between other sensor-regulator pairs (37, 45). Also, similar to the phosphorylation of other response regulators (37, 45), AlgB phosphorylation was inhibited by EDTA, suggesting the requirement of Mg2+ in the phosphorylation reaction. Magnesium has been shown to bind at an aspartate-rich acid pocket within the active site of the response regulator phosphorylation domain. Binding of Mg2+ causes conformation changes in the response regulator, and this likely facilitates the phosphotransfer reaction between histidine protein kinases and response regulators (46-49). Previous studies with the alginate response regulator AlgR demonstrated that AlgR was capable of being phosphorylated by the well characterized histidine protein kinase CheA and by small phospho-donor molecules (50). Despite numerous attempts, AlgB could not be phosphorylated by CheA (data not shown). This suggests that phosphorylation of AlgB by C-KinB has a relatively high specificity. The possibility of AlgR phosphorylation by C-KinB, as well as the involvement of small phospho-donor molecules in AlgB phosphorylation, are currently being examined.
At least three other sensor kinase-regulator pairs have been reported in P. aeruginosa, but this is the first case that in vitro phosphorylation of the sensor and the regulator has been demonstrated in this organism. Besides AlgB-KinB, there are two other typical two-component regulatory systems: PilS-PilR are involved in the regulation of expression of type IV fimbriae (49), and PfeS-PfeR control the expression of the ferric enterobactin receptor, PfeA (51). The genes for the histidine protein kinase and the response regulator in each of these two systems are also next to each other (49, 51). The organization of pfeR-pfeS is strikingly similar to that of algB-kinB, in that the start codon for pfeS also overlaps the stop codon for pfeR (51). The three kinases, PilS, PfeS, and KinB, all have conserved residues characteristic of histidine protein kinases, but little homology beyond that. It appears likely that KinB responds to signals different from that of PilS and PfeS. Recently, another sensor-kinase pair, FimS-AlgR, has been suggested to belong to a new family of transmitter-receiver response regulators (19, 20). However, in that the predicted FimS (AlgZ) sequence lacks a conserved H box, it has been postulated that FimS may not undergo autophosphorylation, although it may still be able to transfer a phosphate group to AlgR (19). It will be of interest to determine to what extent the roles of algB-kinB system and fimS-algR system overlap in control of the virulence factors in this opportunistic pathogen.
We thank Kimberly Prince of the Molecular Resources Center of the University of Tennessee, Memphis for excellent assistance in collecting sequencing data. We also acknowledge the Molecular Resources Center of the University of Tennessee, Memphis for providing oligonucleotides and the Biotechnology Center in St. Jude Children's Research Hospital for amino-terminal sequencing analysis. The gift of CheA from Dr. Phil Matsamura, University of Illinois, Chicago is appreciated.