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JOURNAL OF BACTERIOLOGY,
0021-9193/98/$04.0010Dec. 1998, p. 6764–6768 Vol. 180, No. 24
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
A Novel Serine/Threonine Protein Kinase Homologue of
Pseudomonas aeruginosa Is Specifically Inducible within
the Host Infection Site and Is Required for Full
Virulence in Neutropenic Mice
JINGYI WANG,
1
CAIHE LI,
1
HONGJIANG YANG,
1
ARCADY MUSHEGIAN,
2
AND SHOUGUANG JIN
1
*
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences,
Little Rock, Arkansas 72205,
1
and Axys Pharmaceuticals, Inc.,
La Jolla, California 92037
2
Received 21 July 1998/Accepted 8 October 1998
A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during
infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type
serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice
compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli
or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the
toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial
cells.
We have previously reported a method for the isolation of
genes induced upon infection of neutropenic mice, using
Pseudomonas aeruginosa PAK. After five rounds of selections,
22 different genetic loci were identified through characteriza-
tion of 45 randomly picked isolates (10). To identify a locus
that is the most highly inducible in vivo, two additional rounds
of selection were conducted with mice as described earlier
(10). A total of 48 colonies were picked and analyzed by South-
ern hybridization followed by DNA sequencing, as described
previously (10). Fusion sites in 29 of them were identical to the
np6 locus and the remaining 19 were identical to the np1 locus
from our initial selection study (10). We focused our attention
on the np1 locus since it appears to encode a previously un-
characterized gene product. The strains and plasmids used in
this study are listed in Table 1.
The np1 locus is highly and specifically inducible in host
tissue. To confirm the in vivo inducibility of the np1 locus, we
compared the in vivo (neutropenic mice) and in vitro (minimal
TABLE 1. Strains and plasmids used
Strain or
plasmid Genotype or description Source or reference
E. coli DH5aendA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 D(lacZYA-argF)U169 l-f80 dlacZDM15;
recipient for recombinant plasmids Bethesda Research Laboratories
P. aeruginosa
PAK Wild type David Bradley
PAK-AR2 PAK strain with purEK gene deleted 10
PKN-A PAK strain with ppkA gene mutated by Vinsertion This study
Plasmids
pTZ18R E. coli cloning vector U.S. Biochemicals
pGEX5x-1 GST fusion vector Pharmacia Biotech
pUC19VpUC19 plasmid carrying a 2-kb Vfragment 8
pVK-np1 Cosmid clone containing the ppkA gene in a 15-kb insert This study
pNP1 np1::purEK fusion rescued from the chromosome of NP1 This study
pPKN-Sa4-kb SalI fragment containing 59two-thirds of ppkA gene in pTZ18R, ppkA in the same
direction as lacZa9
This study
pSJ9711 2-kb Vfragment inserted into the ppkA gene in pPKN-SaThis study
pHJY9 In-frame fusion of the ppkA gene behind gst of pGEX5x-1 This study
pHJY10 D-to-N change at position 129 of PpkA encoded in pHJY9 This study
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Mail Slot 511, 4301 W. Markham, Little Rock,
AR 72205. Phone: (501) 296-1396. Fax: (501) 686-5359. E-mail:
JINSHOUGUANG@EXCHANGE.UAMS.EDU.
6764
medium A [MinA]) (1a) replication rates between the original
isolate, NP1, and the parent purEK deletion strain, PAK-AR2.
Since purines are limited under either the in vivo or in vitro
assay conditions, the growth rate of the NP1 strain should be
proportional to the strength of the np1 promoter, which con-
trols purEK gene expression. Equal numbers of the two bacte-
rial strains were mixed and injected intraperitoneally into six
neutropenic mice (2 310
5
cells per mouse). Bacterial cells
were recovered from livers of the mice 24 and 48 h after
inoculation. The numbers of each bacterium were determined
under conditions that allowed the growth of both strains (L
agar) or NP1 only (L agar containing 150 ml of carbenicillin per
ml). Assuming that the two bacterial strains were cleared
equivalently by the host defense system, the ratios of the two
bacterial strains in the animal tissue reflect their relative rep-
lication rates in vivo, which is indicative of the np1 promoter
strength. As an in vitro control, the same bacterial mixture
was inoculated into 100 ml of MinA medium, with a final
concentration of 10
5
/ml, and incubated at 37°C. As shown in
Fig. 1, by 24 and 48 h of infection (in vivo), the NP1 strain
had overgrown PAK-AR2 by 84- and .6310
5
-fold, respec-
tively, whereas in vitro the approximate 1:1 ratios were main-
tained at all times. These data clearly indicate that the np1
locus is specifically and highly expressed in the in vivo envi-
ronment.
The np1 locus encodes a putative serine/threonine protein
kinase. A cosmid clone containing the np1 locus was identified
from a cosmid clone bank of the PAK chromosomal DNA (5)
by colony hybridization, using a partial np1 gene fragment in
pNP1 as a probe. DNA fragments surrounding the original
purEK fusion site were subcloned and sequenced. An open
reading frame (ORF) was identified with the predicted direc-
tion of transcription of the np1 gene, encoding a 1,032-amino-
acid protein, which bears no obvious signal sequence in its N
terminus.
The N-terminal third of the protein is similar to serine/
threonine protein kinases found in bacteria and eukaryotes.
The greatest similarity observed was to the putative kinases
from Myxococcus xanthus (7), Mycobacterium leprae (2), Myco-
bacterium tuberculosis (1), and Streptomyces coelicolor (9). Mul-
tiple sequence alignment of the putative bacterial kinases with
their better-studied eukaryotic counterparts revealed pro-
nounced conservation of at least 10 of the known 12 motifs that
define the Ser/Thr protein kinase superfamily in eukaryotes (3,
4), including an ATP-binding glycine loop in subdomain I, an
invariant lysine residue involved in interaction with a- and b-
phosphates in subdomain II, a “kinase loop” motif with an
invariant catalytic aspartate in subdomain Vib (amino acid
129), and a threonine residue in motif VIII that is frequently
autophosphorylated (Fig. 2). The C-terminal two-thirds of the
np1 ORF, rich in proline, shares no similarity to any known
sequences. Prediction of globular and nonglobular regions,
using local sequence complexity measures (11), detects a non-
globular, elongated protein segment in the area spanning
amino acids 280 to 660 and a globular structure in the remain-
ing C-terminal portion of the protein.
The purEK gene fusion in the original isolate, NP1, occurred
at amino acid 561. Further analysis of the other isolate, NP6,
indicated that it had a purEK fusion to the same gene, but the
fusion site resided in the N-terminal end, at amino acid posi-
tion 198. These results indicate that we had actually isolated a
single locus that is the most highly inducible in vivo, having the
purEK gene fused at two different sites. This locus is designated
ppkA (Pseudomonas protein kinase).
The ppkA locus is required for full bacterial virulence in
neutropenic mice. To investigate the role of the ppkA gene in
bacterial virulence, a ppkA insertional null mutant was gener-
ated. An Vfragment (8), coding for resistance to spectinomy-
cin and streptomycin, was inserted into the 59structural ppkA
gene on pPKN-Sa, resulting in pSJ9711. The mutant ppkA
gene was then introduced into the chromosome of the wild-
type PAK strain by electroporation (6). The ppkA null mutant
strain, designated PKN-A, was confirmed by Southern hybrid-
ization of the chromosomal DNA (Fig. 3).
The PKN-A strain did not show any traits distinguishable
from those of wild-type PAK when grown on either rich or
minimal medium; however, a clear difference in virulence in
neutropenic mice was observed. Tests of virulence in neutro-
penic mice were conducted as described earlier (10), and the
number of animal deaths was observed at 6-h intervals for a
total of 72 h. As shown in Fig. 4, about 10-fold-more PKN-A
cells were needed to cause a similar lethal effect in neutropenic
mice compared to the wild-type PAK strain. Furthermore, the
ppkA mutant caused on average 8 to 12 h of delay in the times
of the animal deaths compared to the wild type.
To see whether the delay in the times of animal deaths
caused by PKN-A is a direct result of the ppkA mutation or of
FIG. 1. Comparison of replication rates between NP1 and PAK-AR2 under
in vivo and in vitro growth conditions. Numbers of each bacterial strain recov-
ered from livers of neutropenic mice (A) or from MinA medium (B) after 24 and
48 h are shown. The in vivo data represent an average from three mice at each
time point. The in vitro data represent an average of three independent mixed-
culture tests.
VOL. 180, 1998 NOTES 6765
a polar effect on downstream genes, the pPKN-Saplasmid,
containing a 39-end-truncated version of the ppkA gene, was
electroporated into PKN-A cells and a single crossover
through the left arm (59to the “V” insertion site of the ppkA
gene in PKN-A) was selected for, as depicted in Fig. 3. The
resulting strain, PKN-AC, has a single-copy, stably maintained
ppkA gene (Fig. 3 and data not shown). As shown by the
virulence test results in Fig. 4, PKN-AC caused an animal
death rate similar to that caused by the wild-type PAK strain.
These results indicated that the N-terminal two-thirds of the
PpkA protein is sufficient to complement the PKN-A mutant
and that the ppkA gene is solely responsible for the reduced
bacterial virulence of the mutant strain PKN-A. Furthermore,
searching the database of the unfinished contigs from the
Pseudomonas genome projects (accessible at http://www.ncbi
.nlm.nih.gov/BLAST/pseudoabl.html) revealed that, in addi-
tion to PpkA itself, there are at least three other PpkA-related
sequences, which may account for the moderate reduction of
virulence of PKN-A.
The kinase domain of PpkA affects growth of E. coli and P.
aeruginosa. To confirm the size of the PpkA ORF as well as to
study the biochemical properties of PpkA, we attempted to
overproduce the PpkA protein in E. coli. The first ATG codon
of PpkA was fused in frame behind the glutathione S-trans-
ferase (GST) gene in pGEX5x-1, resulting in pHJY9. This
construct was sequenced to confirm the in-frame gene fusion.
E. coli harboring the fusion construct, pHJY9, grows slowly
and forms small colonies on media even in the absence of
isopropyl-b-D-thiogalactopyranoside (IPTG), compared to E.
coli harboring the fusion vector only. In the presence of IPTG
FIG. 2. Alignment of PpkA with related bacterial and eukaryotic Ser/Thr-like protein kinases. Unique identifiers in SWISSPROT or PDB databases are shown.
Distances between the ungapped blocks of the highest similarity and the protein termini are indicated by numbers. Invariant residues are shown in boldface. Highly
conserved bulky hydrophobic residues (I, L, M, V, F, Y, and W) and small-side-chain residues (A, G, and S) are also highlighted. Functionally important residues in
motifs I, II, VIb, and VIII (see the text) are underlined. In the motif line, the conserved motifs in Ser/Thr kinases, as defined in Hanks and Hunter (3, 4), are indicated.
In the secondary structure line, a-helices and b-strands in the known three-dimensional structure of twitchin (1KOA) are indicated.
6766 NOTES J. BACTERIOL.
(.0.1 mM), E. coli containing pHJY9 hardly grows on minimal
or rich medium and prolonged incubation in liquid medium
leads to bacterial lysis, indicating that the PpkA portion of the
fusion protein is toxic to E. coli.
We next asked if the kinase activity of PpkA plays any role
in toxicity. Since the conserved aspartic acid residue (amino
acid 129), residing within the catalytic loop of the kinase do-
main, is required for the catalytic activity of the enzyme in all
characterized Ser/Thr protein kinases (3, 4), it was mutated to
an asparagine by site-directed mutagenesis. The mutant gene,
ppkA(D129N), was then fused behind the gst gene as in pHJY9,
resulting in pHJY10. In contrast to E. coli harboring pHJY9, E.
coli harboring pHJY10 grows normally on media in the pres-
ence or absence of IPTG. Furthermore, a 150-kDa GST-
PpkA(D129N) fusion protein was highly and specifically pro-
duced in the presence of IPTG (Fig. 5), demonstrating that the
toxic effect of PpkA is due to its kinase domain. Excluding the
25-kDa GST portion, the size of the PpkA protein in the above
fusion construct is in good agreement with the molecular mass
predicted from the DNA sequence. By using antibody against
FIG. 3. Southern hybridization of chromosomal DNA from strains PAK, PKN-A, and PKN-AC. (Top) Restriction map of the region containing the ppkA gene in
PKN-A and PKN-AC. B, BamHI; P, PstI; R, EcoRI. (Bottom) Chromosomal DNA from strains PAK (lanes 1, 3, and 5), PKN-A (lanes 2, 4, and 6), and PKN-AC (lane
7) were digested with EcoRI (lanes 1 and 2), BamHI (lanes 3 and 4), or PstI (lanes 5, 6 and 7). A 4-kb SalI fragment containing the 59ppkA gene was used as a probe.
VOL. 180, 1998 NOTES 6767
GST, an IPTG induction-specific 150-kDa GST-PpkA fusion
protein was also detected in E. coli containing pHJY9 (data not
shown), including a series of smaller bands, mainly the visible
40-kDa protein band (Fig. 4, lanes 3 and 4), representing
breakdown products.
Although the tac promoter that drives the expression of the
gst gene is not as strong a promoter in P. aeruginosa as it is in
E. coli, the same toxic effect of GST-PpkA on P. aeruginosa was
observed when pHJY9 was introduced into the chromosome of
PAK by single crossover (PAK3pHJY9), whereas pHJY10
had no toxic effect. Taken together, the above observations
clearly indicate that the kinase domain of PpkA has an enzy-
matic function within bacterial cells and high-level substrate
phosphorylation might have led to the inhibitory effect on
bacterial growth.
Nucleotide sequence accession number. The nucleotide se-
quence of ppkA has been submitted to the GenBank databases
under accession number AF035395.
We thank Marie Chow for many suggestions and stimulating discus-
sions, Linda Thompson for statistical analysis of the data, and Allen
Gies for running the automated DNA sequencer.
This work was supported by NIH grants R29AI39524 and
5P20RR11815.
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FIG. 4. Survival rates of neutropenic mice infected with strain PAK, PKN-A,
or PKN-AC. Bacteria were injected intraperitoneally at doses of 10
3
,10
4
, and 10
5
cells, and animal death was observed at 6-h intervals for a total of 72 h. The
numbers of dead animals by 72 h over the total numbers of animals tested are
shown next to the strains used.
FIG. 5. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel
of total bacterial cell extracts. E. coli DH5aharboring GST fusion vector
pGEX5x-1 (lanes 1 and 2), GST-PpkA fusion construct pHJY9 (lanes 3 and 4),
or GST-PpkA(D129N) fusion construct pHJY10 (lanes 5 and 6) was grown in the
presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of 0.1 mM IPTG. Cell
extracts from the same number of bacterial cells were loaded in each lane.
6768 NOTES J. BACTERIOL.
