Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation.

Abstract

A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pkn1, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coli under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-beta-lactamase produced in E. coli was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated beta-lactamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous beta-lactamase or related enzymes in response to an external signal yet to be identified.

Full text

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10.1101/gad.9.8.972Access the most recent version at doi:
1995 9: 972-983Genes Dev.

H Udo, J Munoz-Dorado, M Inouye, et al.
secretion of beta-lactamase by phosphorylation.
transmembrane protein serine/threonine kinase that blocks the
Myxococcus xanthus, a gram-negative bacterium, contains a


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Myxococcus xanthus, a Gram-negative
bacterium, contains a transmembrane
protein serine/threon!ne kinase
that blocks the secrenon of [ -lactamase
by phosphorylation
Hiroshi Udo, Jose Munoz-Dorado, ~ Masayori Inouye, and Sumiko Inouye 2
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 USA
A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein
serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the
protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279
residues show 37% identity to catalytic domain of Pknl, another protein serine/threonine kinase expressed
during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25%
identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In
the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of
18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coil under a T7 promoter was
phosphorylated at both serine and threonine residues. TEM-13-1actamase produced in E. coil was found to
serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent
molecular mass from 29 to 44 kD. The phosphorylated 13-1actamase was unable to be secreted into the
periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with
pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the
207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no
effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present
results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity
of endogenous 13-1actamase or related enzymes in response to an external signal yet to be identified.
[Key Words: Protein serine/threonine kinase; myxobacteria; transmembrane protein kinase; ~-lactamase]
Received January 25, 1995; revised version accepted March 9, 1995.
Protein kinase cascades play major roles in intracellular
signal transduction regulating various cellular functions
in both prokaryotes and eukaryotes. In prokaryotes, pro-
tein histidine kinases are known to be the key enzymes
for the cascades, and it has been believed for a long time
that protein serine/threonine kinases do not exist in
prokaryotes. These kinases, together with protein tyro-
sine kinases, are essential in signal transduction in eu-
karyotes. However, recently, the existence of such eu-
karyotic-like protein serine/threonine kinases in Myxo-
coccus xanthus, a Gram-negative bacterium, has been
demonstrated (Munoz-Dorado et al. 1991; Zhang et al.
1992). M. xanthus living in the soil shows rather spec-
tacular morphogenesis, including multicellular fruiting
body formation upon nutrient starvation (for review, see
tPresent address: University of Granada, Department of Microbiology,
Faculty of Sciences, Granada 18071 Spain.
2Corresponding author.
Shimkets 1990). Within the fruiting body, a significant
population of rod-shaped vegetative cells differentiate
into spherical heat-resistant spores. It is believed that M.
xanthus responds to a large number of signals in its nat-
ural habitat to regulate its cell-cell communication,
gliding motility, aggregation, fruiting body formation,
and differentiation to myxospores. The first prokaryotic
protein serine/threonine kinase, Pknl, has been charac-
terized extensively and shown to be required for normal
development of M. xanthus (Munoz-Dorado et al. 1991).
In this study we describe another protein serine/thre-
onine kinase from M. xanthus, designated Pkn2. In con-
trast to Pknl, Pkn2 was found to be a transmembrane
protein kinase, which was able to effectively phosphory-
late threonine residues of TEM-13-1actamase to block its
secretion across the membrane. We propose that Pkn2
senses an external signal yet to be identified to regulate
the activity of J3-1actamase and/or related enzymes by
phosphorylation. This is the first demonstration that a
972 GENES & DEVELOPMENT 9:972-983 ?9 1995 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/95 $5.00
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Transmembrane Ser/Thr kinase from M. xanthus
prokaryotic organism contains a transmembrane protein
serine/threonine kinase. The significance of the role of
Pkn2 in M. xanthus is discussed.
Results
Identification and cloning of the pkn2 gene
From the polymerase chain reaction (PCR) performed on
the M. xanthus chromosomal DNA, using degenerate
oligonucleotides designed according to the consensus se-
quences of catalytic subdomains VI and VIII of eukary-
otic protein kinases as primers, two different DNA se-
quences were obtained, PCRPK1 and PCRPK2 (Munoz-
Dorado et al. 1991). The sequence of PCRPK2 is shown
in Figure 1A. PCRPK2 has a length of 173 bp and con-
tains the consensus sequence DFG characteristic of sub-
domain VII in the same reading frame as subdomains VI
and VIII of eukaryotic protein kinases (Hanks et al.
1988). This indicates that M. xanthus contains another
eukaryotic-like protein kinase in addition to Pknl (Mu-
noz-Dorado et al. 1991). PCRPK2 seems to be quite dif-
ferent from PCRPK1 (Munoz-Dorado et al. 1991). It has
one extra codon over PCRPK1, and more significantly,
the identity of the deduced amino acid sequences be-
tween the two PCR products is only 26%, excluding the
sequences of the primers used in the reaction.
Using PCRPK2 as a probe, we attempted to isolate the
entire gene corresponding to the PCRPK2 sequence.
Southern blot hybridization analysis of M. xanthus chro-
mosomal DNA digestions revealed that only one DNA
fragment for each restriction enzyme digestion hybrid-
ized with the PCRPK2 probe (Fig. 1B). Positive clones
were obtained from an M. xanthus genomic DNA library
constructed in phage X with use of the same probe. Of
1632 phages screened, 2 were detected as positive and
the DNA from one of them was purified as described in
Materials and methods. After digestion of the phage
DNA with SalI, a 4.8-kb fragment was purified and
cloned into pUC at the SalI site. The plasmid thus ob-
tained was designated pJMPK2S. Because this fragment
did not contain the initiation codon of the open reading
frame (ORF}, shown below, another DNA fragment was
cloned from the same phage X DNA. A 2.5-kb XhoI frag-
ment was cloned into the pUX vector (Apelian and In-
ouye 1990) at the XhoI site, resulting in plasmid
pJMPK2X. Using both pJMPK2S and pJMPK2X, a new
construct designated pJMPK2HB was constructed, which
contains the 7.3-kb XhoI(a~-SalI(b) fragment encompass-
ing the entire ORF. The physical map of the region con-
taining the pkn2 gene is shown in Figure 2.
pkn2 encodes a putative protein serine/ threonine
kinase
The entire sequence of the 2.5-kb XhoI fragment of
pJMPK2X and most of the sequence of the 4.8-kb SalI
fragment of pJMPK2S was determined by using the strat-
egy described in Material and methods. The DNA se-
quence obtained, starting from the XhoI{a) site (Fig. 2) is
shown in Figure 3. The relationship between the two
fragments and the position of the pkn2 gene are shown in
Figure 2. The ORF was in the same reading frame as that
of PCRPK2 and contained the consensus sequences of
the 11 subdomains (Fig. 3) defined for the catalytic do-
main of eukaryotic protein serine/threonine kinases
(Hanks et al. 1988). There are no ATG codons upstream
of the consensus sequence of subdomain I starting from
base 421. Instead, there are two GTG codons (positions
341-343 and 392-394). However, the codons used be-
tween the two GTG codons show a low G + C usage at
the third position (10 of 16; 62.5%) in contrast to a high
G + C usage of the codons used after the second GTG
(92.5%), a typical codon usage in M. xanthus (Inouye et
al. 1989). Thus, the ORF for pkn2 is tentatively assigned
from the second GTG (base 392-394) to the TGA termi-
nation codon (base 2882-2884). A putative ribosome-
binding site, AAGATG, can be identified 8 bases up-
stream of the initiation codon. The ORF should encode a
protein (Pkn2) of 830 amino acid residues, with a calcu-
lated molecular weight of 87,594.
When the deduced amino acid sequence of Pkn2 was
examined for sequence similarities with other proteins,
it was found to contain the consensus catalytic domain
of eukaryotic protein serine/threonine kinases from
Figure 1. Identification of the pkn2 gene. (A}
The sequence of PCRPK2. The sequences of the
primers used for PCR are marked by long arrows,
and the highly conserved sequence in the subdo-
main VII of eukaryotic protein kinases is under-
lined. IB) Southern blot hybridization of the M.
xanthus chromosomal DNA with the PCRPK2
probe. The digestions used were BamHI, (lane I);
BamHI and EcoRI, (lane 2); BamHI and HindlII,
(lane 3); BarnHI and PstI, (lane 4); SalI (lane 5);
SalI amd XhoI (lane 6); and XhoI (lane 7). The
molecular mass standards are expressed in kilo-
bases.
GENES & DEVELOPMENT 973
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Udo et al.
~:i!i:i~i:i:i:!:!:i:!:i:i:i:i:i:i:~:i:i:i:i:i:i:i:i:i:!:!:i:i:i:i:i:i:i:i:i~
/
I
I
/
/
r
pkn2 \
N
I
\
\ lkb
\
\
1 Kinase
Domain
(5-274)
A/S/T/G/P
Rich Region
(275-373)
Hydrophobic
Domain
(606-623)
830
Figure 2. Restriction map of the pkn2 locus and a domain
structure of Pkn2. Numbers 1 and 830 represent the first and the
last amino acid in the ORF, respectively.
amino acid residue 20-274 (see Fig. 3) and the highest
identity to the catalytic domain of Pknl (37%), a protein
serine/threonine kinase of M. xanthus (Munoz-Dorado
et al. 1991). It also shows 27% identity with rat Ca2+/
calmodulin-dependent protein kinase (Lin et al. 1987)
and 25% identity with Bos taurus rhodopsin kinase
(Lorenz et al. 1991). After the catalytic domain, there is
an unusual sequence of 99 amino acid residues (from
residue 275 to 373) containing 20 Ser, 10 Thr, 23 Ala, 17
Gly, 10 Pro, and 0 charged residues (double-underlined in
Fig. 3). These amino acid residues account for 81% of the
sequence. This Ala/Ser/Thr/Gly/Pro-rich region is fol-
lowed by a sequence of 467 amino acid residues (from
residue 374 to 830), in the middle of which there is a
hydrophobic stretch of 18 amino acid residues (from res-
idue 606 to 623; boxed in Fig. 3J. This hydrophobic se-
quence is flanked by Gln-Arg-Arg-Arg-Glu (total net
charge is + 2) at the amino-terminal side and by Ser-Gln-
Arg-Asn-Asp (total net charge is 0) at the carboxy-termi-
hal side within a sequence of 5 residues. This is a typical
feature of a transmembrane domain (Gennity and Inouye
1991; Pugsley 1993; Gafvelin and von Heijne 1994; Kim
et al. 1994) and predicts that Pkn2 is a transmembrane
protein having the kinase domain in the cytoplasm and
the carboxy-terminal domain downstream of the trans-
membrane domain outside the cytoplasmic membrane.
Within the putative extracellular domain, the signature
sequence PKDRLRAHY, known for mitochondrial en-
ergy transfer proteins (Klingenberg 1990; Nelson et al.
1993), was found from residue 722 to 730 (underlined in
Fig. 31.
Pkn2 is a transmembrane protein
The amino acid sequence deduced from the DNA se-
quence of pkn2 showed the existence of a stretch of 18
hydrophobic amino acid residues (residues 606-623) pre-
ceded by positively charged residues in the middle of the
regulatory domain, suggesting that this domain func-
tions as an internal signal sequence (Kim et al. 1994). To
demonstrate that this region can act as an internal signal
sequence, fusion proteins, consisting of a fragment of
Pkn2 and the E. coli alkaline phosphatase (PhoA), were
constructed before and after the hydrophobic stretch as
shown in Figure 4.
Two fusion proteins were designated Pkn2-PhoA-1
and Pkn2-PhoA-2 (see Fig. 4). Their genes were con-
structed in pUC9 in such a way that their expression was
under the control of the lacZ promoter. In the presence
of 1 mM IPTG, Pkn2-PhoA-1 showed very low PhoA
activity (58 units) similar to the activity of the phoA-
host strain (16 units; see Fig. 4). However, when the hy-
drophobic stretch was added to the fusion protein, the
phoA activity of the resulting protein, Pkn2-PhoA-2 in-
creased dramatically to 5067 units, which was similar to
that of a phoA + control strain (4014 units; see Fig. 4).
Because PhoA is known to be active only when it is
translocated across the membrane (Manoil and Beckwith
1985), the present results demonstrate that the hydro-
phobic domain corresponding to residues 606 to 623
functions as an internal signal sequence to translocate
the PhoA domain fused downstream of the hydrophobic
domain. Because the intemal signal sequence appears to
have no cleavage site at the carboxy-terminal end, the
hydrophobic domain is likely to serve as a transmem-
brane domain. Therefore, the present results indicate
that Pkn2 is a transmembrane protein consisting of the
207-residue carboxy-terminal domain in the periplasmic
space and the 605-residue amino-terminal domain in the
cytoplasm, where the kinase domain exists.
Expression of pkn2 in E. coli
To characterize Pkn2, the pkn2 gene was expressed in E.
coli using a T7 RNA polymerase system. The pkn2 gene
was cloned under a T7 promoter in plasmid pET1 la as
described in Material and methods. In this construction,
the GTG initiation codon for pkn2 was substituted with
an ATG codon. The plasmid thus obtained was desig-
nated pETll/pkn2. After transformation of E. coli
BL21(DE3) with this plasmid, the expression of the pkn2
gene was induced with IPTG, and the proteins were la-
beled with Tran3SS-label in the presence of rifampicin.
As shown in Figure 5A, four new bands were induced in
the presence of IPTG. As described later, band a was
identified as Pkn2. Its apparent molecular mass (110 kD)
was higher than its expected molecular mass (88 kD).
This abnormal migration in a SDS-polyacrylamide gel is
considered to be the result of phosphorylation of the
product as observed for Pknl (Munoz-Dorado et al.
1991). The apparent molecular masses of bands b, c, and
d were 44, 31, and 29 kD, respectively, and all of them
were found to be derived from the bla gene in the pET1 ta
vector used as described later.
Next, we investigated phosphorylation of Pkn2 using
the method applied for Pknl (Munoz-Dorado et al. 1991).
E. coli cells harboring pET11/pkn2 were labeled with
inorganic 32p after induction with IPTG as described in
974 GENES & DEVELOPMENT
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Ttansmembtane Ser/Thr kinase from M. xanthus
CTCGAGGAAGCCGAAGTGCAGCTTCTTGCGCTGGTAGCGCGTGGCGCCGGCGCTGACGTTGAAGGGGAAGCT
GAGATGCGCAGTCCCGGCAGGACC TCCAGCGCC~GCC
CCGTGGCGGGCTTGGGAGAATCGCTTTCGGTAGCCATCGCGGGAGCAGCKT, ACGTTAGCACGGGCT CCAAGCCCT C GAA TTTCT GGCAAAC CCCAGCTTG
TGGGGCAAGCAACGAGGCGTGATGGCTCCCTGAATCCAGGGTGAAGGAAC~TGC
GACGTCC GGAATCTGGAGT GCC, AAGTCG 100
200
300
400
CACGAACGC CC TGCGGTCCAGCAGGCGCAATTC ~GGCGGC GCGCGCATCCAGCG
GCGCTC-GGCCGAAGTCGGACAAGATGCGCC CC~ GT~T ~C
v L A 3
CCTGACTCCCTTGTCCTCGACGGTCC-CTT CCGGGTT CTCCGTCCGCT GGGC TCC~
P D S L V L D G R F
GGAATGGGTGAGGTCTACCTTGGC GAACAGGT CTCCCTGGGCC
G M G E V y L G
I
500
37 R V L R P L G S G E Q V S L G R
GCAAC, GTGGCCATCAAGGTCCTGCACCATGACCTC CACGC C CAGGCCGGCATGGCGGAGCGCTTCAAGCGCGAGGCGC~
K V A I K V L H H D L H A
II
CCCGGCCGTGGTGCGCATCGTCGACTTCGGCGAATCCGGCGACCACGCCTGCC TGGTCATGGAGT TCGTCGAAGGTGAGAGCCTGTACGACGTGCT CACG
P A V V R I V D F G E S G D H A C
IV
CCGGGCCCCATGCCCCCGGGCCGCGCGCTGC CCCT GCTCCAGCAGTTGGCGGAGGGC CTGGC CGCCATCCACGACAAGG~T
P G P M P P G R A L P L L Q Q L A
CTCCTCT C C ~ C G T ~
L L S A
600
70
Q A G M A E R F K R E A R V E S
III
700
103 L V M E
V
F V E G E S L Y D V L T
~TC~CC
I
~ ~CCT~
R D
VI
~
A A
800
137
E G L A A I H D K G I H L K
AGCCGGAGAAC GTCTTCATCTCCAAGAGC GC CCGGGGCGAGCAGGC CCGGCTC CT GGACT TC GGCATCGCCC GG TTGGTCGAGCCAGACGC C
P E N V F I S K S A R G E Q A
G
S
900
170 R L L D F G I A R L V E P D S
VII
CGTCAGCCAGATT GGCGTGGTGCTGGGCACGCCGGAGTACCTGTCGCCGGAC-CAGGC C GT GGGCGCGAAGGT GGACACGCGCAGCGACC TGTACT CGTTC I000
V S Q I G V V L G T P E Y L S P E Q
VIII
GGCGT GCTGACCTACCGCGTGCTGTCCGGGCGGCTCCCCT TCGACGGGCCC CTGCCGC GAAACTTCC TGTC GCAGCAC GCCTCC GCCGCGCCGCTGCCCC 1100
G V L T Y R V L S G R L P F D G P L
IX
TGGACCGCGCGGC CCCCACGCTGTCGCGC TATGTGGGCCTGCT GTC GCTGGTGATGCGCC T GCT GGAGAAGGACGC~G~GTC
D R A A P T L S R Y V G L L S L V
A V G A K V D T R S D L Y S F 2O3
P R N F L S Q H A S A A P
X
L P L 237
CC ~GA~
P Q
GC~
A
1200
270 M R L L E K D A S K R
XI
S H
CGAACTGGCCGAC GCGCTGGCCGCGGCGCACTCCGCCCTCTCCGC CTTCACGCCCGGC CT GGGCAC CC CTGCC TACGT C CCC, CAC.CC CGGGAGTGGCGCC 1300
E L A D A L A A A H S A L S A F T P G L G T P A Y V P Q P G S G A 303
ACGCCCTCCTCGGC-CACGTCGGTGTTCGGCACCGGGAGTGCCTCGGGGAGCAGTTC GGGCC C CACGGGCACGGCGGC CT TCGCGGGAGT CGCT C CGGCGC 1400
T P S S G T S V F G T G S A S G S S S G P T G T A A F A G V A P A P 337
CGCAGGCGAGCAGT GGTACCGCCGCGTTCGGC GTGGCCTCCAGCAGCC~TCGGC CTCGGGC GCCCTGC CGC~GGC CTC CCCC.CA~C ~ A C ~CGTC
Q A S S G T A A F G V A S S S G
1500
370 S A S G A L P A A S P H T G T A S
CTTCGGACTGAAGTCCTCCGGCGGCGTGGCCGCAGTGACGGC4~C-GCAACGC GTCGGTGGTGAAGCC GCAGAACCTCAC GGTGATGCT CACCGACAT CCAG 1600
F G L K S S G G V A A V T G G N A S V V K P Q N L T V M L T D I Q 403
GGCTT CACCGAGCGCACCAGCCGGCAGAC GCACGAAGAGAAC GC GCGGATGCT GGAGAC GCA C GACAAC, CTGCT GATC.C CCCTGGT GAAGGAGCAC GACG 1700
G F T E R T S R Q T H E E N A R M L
GGCGACTGGTCCAGAAGCGTGGGGACGCGTTGCTGGTGGT GTT CCGCTCCCC CACC GCGGGC GTGCTGT GTGGCATGGC CATGCAGGAC C GGCTGT GGCG 1800
R L V Q K R G D A L L V V F R S P
CCACAACCAGACGGTGCCGGAAGTGGACC GCCTCAACGT C-CC, CGT C TC-CC TGCACGCGGGCGAGGTGCT C GC CAC GCC GGACTC CGTGCT C.GGCGAGCCC 1900
H N Q T V P E V D R L N V R V C L H
E T H D K L L M P L V K E H D G 437
T A G V L C G M A M Q D R L W R 4~0
A G E V L A T P D S V L G E P 503
ATGGAGGTCATCGAGGCCGTGGAGCATGTCGCCAGCGCGGGT GAGGT CACCTTCACTGA.%GC GGTGAACC TGGCGCGCAACC~~
M E V I E A V E H V A S A G E
AGCCCTGCGGCGC CATCACCCTCCCCGGC CGCAACGAGCAACTC CAGCTCTACC GCTGCCAGC GC GC CGC GGAGGGC CC ~C CTT T~AGACC ~TTC ~
P C G A I T L P G R N E Q L Q L
CTCGCAAGGCAGCCGGC~CGCGCTCGCGCCCCTGCTGGCGAAGCTCCAGGC
S Q G S R G N A L A P L L A K L Q
CGCGAGGCGGCGCTGGTCGCCGGCGCGGTGGTGCTCCTGGGCGCTGGCGCCGCGTGGCT GAGCCAGCGCAAC~CG~CCC~
R E]A A L V A G A V V L L G A G A
TGGAGGATGGGAAGCTGAACGAAGCCCTGGCGCTGATGGACGCCGC CACCGACGAGGAGAAGGAACT C, CCCTCGCT GAGGC ~
E D G K L N E A L A L M D A A T
CCATGCCAAGGGGCACCACATCAGCGAACGGACTGCC-CTCAGCCACCTGAAGGAGGAGGAGC T C ~ C G T ~ C G C T ~ T CCT C~C~CT
H A K G H H I S E R T A L S H L K
GAGGACTACGGCAAGGAGCCTCTCACCGTCCTGGGGAAT GCCCTGGCCCGGTTC CC GAAGGAC CGCTT GC GC GCCCACTACGAGGACCT GGCCGAGGAGG 2600
E D Y G K E P L T V L G N A L A R F
CGTACT CCCTC-CGCCAGTGGGGCGCGCTGCGCTACCTGGAGTTTGT GAAGGCCC, C GGACGGGGTGAACCTGGTTCC.CC-CCTATAGCGAC, C.CC.CTGAACTC 2700
Y S L R Q W G A L R Y L E F V K A
C CCGGACTGCGACATCCGCACCCAGGCCGCCAACCGTCTC GCGGGC C TGGGTGACGCGGATGCCATCC C C GC GATGGAGCGCGTCACCTC GCTCCC CAAG 2800
P D C D I R T Q A A N R L A G L G D
GCGAAGGGCCTCC TC~GCAAGGACTGCGGCCACGAAGCAGCGGC GACC GCCATCAAGT CACTCAA~GAAGAGCGACTGACTCAGTCATCCCGCCG
A K G L L G S K D C G H E A A A T A
GGCGTGCCCCTTCTTCTTGCCGCGC CCGC GCCCGCGCC GGTCATCGTCGTCCCAGTCATGTC GGCGGGC CTT CACCTTGTCATC CACCCGCAGGAGGCTG 3000
CC-CGAGTACGCATCGAAGTCCAGGTGCAGGTGGC CCTTCGCCCTCCGCGTGGAGGCATGGAACTTCACC CC, C CACACGT CCCGC CCCTTGCGGTCC GCGT 3100
CCTT CAGGCGGCACTCATAGCCACGCGAGCGACATTGGTTGAAGCCCAGGTTCACCGCTTCC CGGTACGTCATCGCCAC CGGACGATGC GGAGGAGGCGG 3200
TGGATGCGGAGAAGGTGAGCGGGTGTGCAGGACACAACCCGAAGCGAGCATCAGACAC-CCAAT GACAA~
CGCC, GCACATCTT CAAAGCCACGCAGGGTATGAGCCAGGCACTGGAAAGTTACAGCCCTTCAGGCGACGCGAACGCCGC
GGCCCGCGAGCCCTTCGCGCCGGGGCGGATGTAGTCATTGACCACGGGCCCGCCCTGCTC
CGCGCGACGCCCT TGCCGCCCCGGTG~T GAAGGTGACGGTGCCAT GCGCATC CACGGCCTCCACCACGCCAATGTGTGTCATGCCGT~C~
CATCGCGGTTGCGGTCATACGT CTCACGGAAGAAGACGAGGTCTCC C.GGAC GCGGCGGCTGGTGGTGGACGGA~C~TC~CC~C
GCGGAGAGGCGTTCTCTCCCGCGCGGAAGCGTGCGCCACCAGGT CGAT GCCGG
~CC ~CG
T
2000
537
2100
570
2200
603
2300
637
2400
670
2500
703
V T F T E A V N L A R N R A E A A E
Y R C Q R A A E G P P F G D R F A
CGTGAAGC TGC C CACCGGACTGGGGGA~T~T
V K L P T
~ G T ~ G ~
R Q
CTTC~T
A F A
G T ~ C ~
R V A
A G L G E L L R R
~
A W L1S Q R N D A G T R L L
D E E K E L P S L R R A A N
~G
A E E E L E D V E P L I L D G L
P K D R L R A H Y E D L A E E A 737
A D G V N L V R A Y S E A L N S 770
A D A I P A M E R V T S L P K
380
2900
830
I K S L K Q K S D
~GTC~AT~CC~T ~C~T~G 3300
3400
3500
3600
3700
G A A ~ T ~ C C ~ T A
CAACTGGTC, CCTCGACGGGAAGGACAGGTTCAACCGGCTT
~TG
Figure 3. DNA sequence of 3753 bases
encompassing pkn2 (GenBank/EMBL ac-
cessiort no. M94857) and the deduced
amino acid sequence of Pkn2. Roman nu-
merals represent conserved subdomains in
the catalytic domain of kinases. The Ala/
Ser/Thr/Gly/Pro-rich region is doubly un-
derlined. The sequence homologous to mi-
tochondrial energy transfer protein is un-
derlined. The hydrophobic stretch is
boxed,
Materials and methods. After a 3-hr induction, inclusion
bodies were isolated and analyzed by SDS-PAGE. As
shown in Figure 5B, of four bands (a, b, c, and dl only
bands a and b were labeled with 3~p (lane 2). To identify
amino acid residues phosphorylated in these bands, they
were extracted from the gel and hydrolyzed in 6 N HC1.
The hydrolysates were separated by two-dimensional
thin layer chromatography. The results are shown in Fig-
ure 5, C and D. Pkn2 (band a) was phosphorylated at both
serine and threonine residues (Fig. 5C), whereas the band
b protein was mainly phosphorylated at threonine resi-
dues (Fig. 5D).
Characterization of bands b, c, and d
In addition to band a, the induction of bands b, c, and d
was puzzling. To determine whether these bands were
derived from the pkn2 gene, we examined the precursor-
product relationship between these bands. For this pur-
pose, the cells harboring pETll/pkn2 were pulse-la-
beled for 5 min with TranaSS-label after induction with
IPTG for 1 hr and chased for 7.5, 15, 30, and 60 min. As
shown in Figure 6, Pkn2 (indicated with arrow a) was
detected with a molecular mass of 106 kD during the
5-min pulse (lane 1 }. When chased, the position of Pkn2
gradually shifted to higher molecular mass, which was
probably caused by continuous phosphorylation of Pkn2.
In addition to band a, bands b, c, and d of 44, 31, and 29
kD, respectively were pulse-labeled with Tran3ss-label.
The intensity of band c decreased with a concomitant
mcrease of the intensity of band b during the chase pe-
riod. These results suggest that band c at 31 kD was
chased into band b at 44 kD, probably because of the
post-translational modification of band c. Band b is un-
likely to be derived from Pkn2 {band a}, because the in-
tensity of band a remained constant throughout the
chase period tested (Fig. 6).
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Udo et al.
Figure 4. Construction of Pkn2-PhoA hybrids
and their PhoA activity in E. coli CCl18
(phoA-). The enzyme assay was performed ac-
cording to the method described previously
(Brickman and Beckwith 1975).
Deletion analysis of pkn2 in E. coli
To confirm further that band a is the pkn2 product, var-
ious deletion mutations of the pkn2 gene were con-
structed, as described in Materials and methods, using
the unique restriction enzyme sites in the gene [SacII
(1221-1226), KpnI (1415-1420), MluI (1548-1553), NcoI
(1899-1904), and XhoI (2462-2467; see Fig. 3)] as shown
in Figure 7A. The deletion constructs were expressed
under the control of the T7 promoter in pET1 la. The
mutation designated AKn was constructed by introduc-
ing an NdeI site at Met-398 (see Fig. 3) followed by clon-
ing the resulting 3.5-kb NdeI-BamHI fragment into
pET1 la at its NdeI and BamHI sites. Therefore, the re-
suiting plasmid, designated pAKn, encodes a carboxy-ter-
minal fragment of Pkn2 consisting of 433 amino acid
residues, which lacks the entire kinase domain.
The cells harboring pET11/pkn2 and the deletion con-
structs were labeled with TrangSS-label for 30 min with
or without IPTG induction in the presence of rifampicin.
The products were then analyzed by SDS-PAGE as
shown in Figure 7B. In the case of pETll/pkn2, Pkn2
Figure 5. Expression of Pkn2 and identification of phosphorylated amino acid residues. {A) Expression of pkn2 under the T7 promoter
in E. coli BL21 (DE3). Cells harboring pET/pkn2 were incubated in the presence (lane 1 ) or the absence (lane 2) of 1 mM IPTG, and then
labeled with Tran3SS-label in the presence of rifampicin (150 ~tg/ml) as described in Materials and methods. Total cellular proteins
were subjected to SDS-PAGE. Proteins were blotted on an immobilon-P membrane, which was then exposed to X-ray film. Letters with
an arrow indicate the positions of Pkn2 (band a) and other products from pET11/pkn2 (bands b,c, and d) (see text). Bars with numbers
at left indicate the positions of molecular mass markers {kD). (B) Phosphorylation of the products from pET11/pkn2. Cells harboring
pET11/pkn2 were labeled with either Tran3SS-label (lane I) or ortho-32p (lane 2) after 2-hr induction with IPTG in the presence of
rifampicin as described in Materials and methods. (Lane 1) SDS-gel analysis of total cellular proteins labeled with TranaSS-label; (lane
21 SDS-gel analysis of inclusion bodies labeled with ortho-32p. Inclusion bodies were isolated by a low centrifugation after sonification
as described in Materials and methods. SDS-PAGE was carried out as in A. (C,D) Phosphoamino acid analysis of Pkn2 and band b in
lane 2 (in B) (described in Materials and methods). The positions of the nonradioactive phosphoamino acid standards liP-Set) phos-
phoserine; (P-Thr) phosphothreonine; (P-Tyr) phosphotyrosine] identified by ninhydrin and the position of inorganic phosphate (Pi) are
shown by broken lines. The first and second dimensions used for the separation of the phosphoamino acids are indicated by numbers
1 and 2, respectively. The origin is indicated by a dot at the intersection of arrows 1 and 2.
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Transmembrane Ser/Thr kinase from M. xanthus
Figure 6. Pulse-chase experiment of E. coli BL21 (DE3) har-
boring pET11/pkn2. Cells harboring pET11/pkn2 were labeled
with Tran3SS-label for 5 min in the presence of 1 mM IPTG (lane
1) and chased in the presence of nonradioactive methionine for
various times indicated at the top of the gel (lanes 2,3,4, and 5;
chase for 7.5, 15, 30, and 60 min, respectively). Letters with an
arrow indicate positions of Pkn2 (bands a) and other products
from pET11/pkn2 (bands b, c,d). Bars with numbers at left in-
dicate the positions of molecular mass markers {kD).
{band a) as well as bands b, c, and d were detected (lane 2)
as shown previously in Figure 5A. However, in all of the
deletion mutations (lanes 3-14}, band a disappeared com-
pletely and a new band appeared as indicated by a dot for
each deletion mutation. The apparent molecular masses
of these bands were estimated to be 105 kD for AX-B
(lane 4), 79 for AN-B (lane 6), 56 for AM-B (lane 8), 41 for
AK-B (lane 10), 32 for AS-B (lane 12), and 50 for aKn {lane
14). The observed reduction of molecular masses for all
of the construct is consistent with the deletion muta-
tions depicted in Figure 7A, demonstrating that band a is
the product of pkn2.
The total amounts of the 13-1actamase products {bands
b+ c+ dl are different depending on the constructs {Fig.
7B, see lanes 1-14). It is not known at present whether
this is attributable to the readthrough from the T7 pro-
moter or the effect of Pkn2 products on the ~-lactamase
production.
Band b is the product of the bla gene
In the deletion analyses of pkn2 shown in Figure 7B, one
can observe that in contrast to band a, band b did not
disappear in &X-B and AN-B mutations (lanes 4 and 6,
respectively). When the 3'-end region of pkn2 was de-
leted beyond the NcoI site, the production of band b be-
came almost undetectable {Fig. 7B, lanes 8, 10, and 12).
In the case of AM-B, a faint band appeared slightly below
the band b position, suggesting that this faint band may
have something to do with band b. Note that the band at
41 kD in AK-B {lane 10) is the truncated product of Pkn2
but not the band b product as discussed later. Interest-
ingly, band c became a major product, and no band b was
produced in AKn (lane 14).
It should be noted that band b was still produced
in &N-B {lane 61 and that the plasmid for 2xN-B contained
the 1.5-kb fragment from the M. xanthus chromo-
somal DNA that encompasses only the amino-terminal
fragment of Pkn2 from residue 1 to 502. Therefore, band
b was not likely to be derived from the M. xanthus
DNA fragment. We thus speculated that the bla gene for
ampicillin resistance encoding TEM-B-lactamase in
the pET1 la vector is responsible for the production of
band b.
As shown in Figure 8A, the bla gene locates down-
stream of the pkn2 gene in the same orientation in a
circular map of pET 11/pkn2. To determine that band b is
really a product of the bla gene, the bla gene in pET11/
pkn2 was replaced with the kan r gene for kanamycin
resistance from the Tn5 transposon as described in Ma-
terials and methods, and the resultant plasmid was des-
ignated pETllkm/pkn2. The cells harboring pETll/
pkn2 and pETllkm/pkn2 were labeled with Tran3SS -
label for 30 rain in the presence of rifampicin with or
without the addition of IPTG. The labeled products were
then analyzed by SDS-PAGE. As shown in Figure 8B,
band a was still clearly detected in the presence of IPTG
in cells harboring pET1 lkm/pkn2 (lane 4) as well as in
cells harboring pET11/pkn2 (lane 2). However, band b,
together with bands c and d, completely disappeared in
the cells harboring pET1 lkm/pkn2 in which the f~-lac-
tamase gene was replaced with the kan r resistant gene
(compare lane 4 with lane 2).
To confirm further that band b was derived from the
bla gene, the same samples used in Figure 7B were ana-
lyzed by Western blot using anti-13-1actamase antibody.
As shown in Figure 7C, strong bands corresponding to
band b were detected not only in the cells harboring
pETll/pkn2 (lane 2) but also in the cells harboring
pAX-B (lane 4), and pAN-B (lane 6). A faint band slightly
below the band b position was also detected in cells har-
boring pAM-B (lane 8), which also coincides well with
the faintly labeled band at the same position in Figure
7B, lane 8.
In contrast to band b, band d was detected in all lanes
in Figure 7C, indicating that it is 13-1actamase, the prod-
uct of the bla gene. The apparent molecular mass of band
d (29 kD) also agrees well with the molecular mass of
13-1actamase. It is not known at present why band d ap-
pears as a doublet form. The intensities of band d were
also very similar in all lanes regardless of the absence or
the presence of IPTG, indicating that 13-1actamase at
band d was synthesized from its own promoter. On the
other hand, the production of bands b and c was observed
only in the presence of IPTG and, thus, likely to be under
the control of the T7 promoter of pET1 la. On the basis of
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Figure 7. Analysis of the band b product
using various pkn2 deletion mutants. (A)
Schematic diagram of pkn2 and its deletion
mutants. The method for constructing the
deletion mutations is described in the text.
(B) Expression of pkn2 and its deletion mu-
tants. E. coli BL21 (DE3) harboring a plas-
mid containing individual pkn2 mutants
was labeled with Tran3SS-label for 2 hr in
the presence of rifampicin. (- and +)
Without and with IPTG, respectively.
{Lanes 1,2) Total cell extract from cells har-
boring pET11/pkn2; {lanes 3,4) from pAX-
B; (lanes 5,6) from pAN-B; (lanes 7,8) from
pAM-B; Ilanes 9,10) from pAK-B; {lanes
11,12 from pAS-B; (lanes 13,14) from pAKn.
The products corresponding to individual
deletion mutations are marked with dots.
(C) Western blot analysis using antiq3-1ac-
tamase antiserum. The same sets of sam-
ples as used in B were transferred on PVDF
membrane to detect cross-reactive materi-
als. Lanes 1 to 14 were the same as shown
in B. Letters with an arrow and bars with a
number are the same as in Fig. 6.
the apparent molecular mass of band c (31 kD), this band
is considered to be the secretory precursor of ~-lacta-
mase, which still contains the uncleaved signal peptide
of 23 amino acid residues.
The present results indicate that TEM-~-lactamase
serves as a substrate for Pkn2, which can be phosphory-
lated very effectively at multiple threonine residues (Fig.
5D), resulting in the shift of the apparent molecular mass
from 31 (band c) to 44 kD {band b). It is interesting to
note that the Ala/Ser/Thr/Gly/Pro-rich domain of Pkn2
(see Fig. 7A) is required for kinase activity, because AK-B
and AS-B were unable to phosphorylate ~3-1actamase (Fig.
7C, lanes 10 and 12, respectively).
Figure 8. Circular map of pET11/pkn2 (A) and SDS-PAGE pat-
terns of cells harboring pET11/pkn2 and pET1 lkm/pkn2 (B).
(A) Locations of pkn2, bla, and lacI genes are represented by
boxes, and their orientations are indicated by arrows in boxes.
(B) E. coli BL21 (DE3I harboring pETll/pkn2 and pET1 lkm/
pkn2 were labeled with Tran3SS-label for 2 hr with (+) or with-
out ( - ) IPTG. (Lanes 1,2) Cells harboring pET11/pkn2 without
( - ) and with ( + ) IPTG, respectively; (lanes 3,4) cells harboring
pET1 lkm/pkn2 without (-) and with (+) IPTG, respectively.
Assignments are the same as in Fig. 6.
Phosphorylation of ~-lactamase and the Pkn2
regulatory domain
Next, we examined whether Pkn2 phosphorylates 13-1ac-
tamase produced by a separate plasmid. For this purpose,
E. coli cells were transformed with both pET1 lkm/pkn2
and pAKn. The cells transformed with either plasmid or
both were labeled with Tran35S-label in the presence of
rifampicin. As shown in Figure 9A, Pkn2 became the
major product in the cells harboring pETllkm/pkn2
when IPTG was added {band a in lane 2). In the cells
harboring pAKn, three major bands appeared in the pres-
ence of IPTG Ibands c, d, and f in lane 4)--band d for
~3-1actamase, band c for pro-13-1actamase, and band f for
the truncated product of Pkn2. The same pattern was
observed in Figure 7B, lane 14.
When cells harboring both plasmids were analyzed,
two extra broad bands (bands b and e in lane 6) were
detected in addition to bands a, c, d, and f. When the
same sample used in Figure 9A was analyzed by Western
blot using anti-13-1actamase antibody, not only bands c
and d, but also band b, were detected as shown in Figure
9B, lane 6, indicating that Pkn2 produced from a separate
plasmid could phosphorylate [3-1actamase precursor.
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Transmembrane Ser/Thr kinase from M. xanthus
lactamase and f~-lactamase, respectively. It should be
noted that bands c and d could not be detected with
anti-Pkn2 serum (lanes 4,101. These results demonstrate
that phosphorylated 13-1actamase forms a complex with
Pkn2.
Figure 9. Phosphorylation of 13-1actamase by pkn2 expressed
by a separate plasmid. (A) Protein patterns of E. coli BL21 (DE3)
harboring pETllkm/pkn2 (lanes 1,2}, AKn (lanes 3,4}, and
pETllkm/pkn2 plus AKn (lanes 5,6). Cells were labeled with
Tran3SS-label for 2 hr and analyzed by SDS-PAGE as described
in Materials and methods. (e and f, with arrow) The positions of
phosphorylated hKn and nonphosphorylated hKn polypeptide,
respectively. (B) Western blot analysis of [3-1actamase phospho-
rylated by Pkn2. The same samples used in A were analyzed
by Westem blot with anti-[3-1actamase serum. {Lanes 1-6)
The same as lanes 1-6 in A. The assignments are the same as in
Fig. 6.
Cellular localization of phosphoryla ted [3-1actamase
To examine the cellular localization of phosphorylated
~-lactamase, cells harboring pETll/pkn2 were incu-
bated with IPTG for 60 min, and cellular fractionation
was carried out by the method of Neu and Heppel (1965).
As shown in Figure 11, phosphorylated f3-1actamase, to-
gether with the precursor form of b-lactamase (band c),
was detected in both the membrane and the cytoplasmic
fractions (lanes 4 and 5, respectively), but not in the
periplasmic fraction (lane 3). In contrast, the mature
form of ~3-1actamase was found primarily in the periplas-
mic fraction (lane 3). The periplasmic localization of ma-
ture ~-lactamase is particularly evident in the control
experiment with p2AKn (lane 8). Note that no band b
was produced in the control experiment (lanes 6-10).
These results indicate that the phosphorylation of 13-1ac-
tamase blocks its secretion across the membrane result-
ing in the accumulation of phosphorylated 13-1actamase
in the cytoplasm as well as in the membrane fraction.
Expression of pkn2 in M. xanthus
M. xanthus strain pkn2/Z was constructed, which con-
tained intact pkn2 as well as pkn2-1acZ fusion. The
pkn2/Z strain was plated on CYE agar plate for vegeta-
tive growth as well as on CF agar plate for fruiting body
formation. X-gal (5-bromo-4-chloro-3-indolylq3-D-galac-
Most importantly, Pkn2 was also able to phosphorylate
the carboxy-terminal fragment of Pkn2 resulting in the
formation of band e when both AKn and pkn2 were co-
expressed.
Association of Pkn2 with phosphorylated [3-1actamase
Because eukaryotic protein kinases are known to be
associated with their substrates, we then examined
whether Pkn2 forms a complex with ~3-1actamase.
BL21(DE3) cells harboring pETll/pkn2 were labeled
with TrangSS-label with or without rifampicin, as de-
scribed in Materials and methods. Cell lysates were im-
munoprecipitated with anti-Pkn2 or anti-~-lactamase se-
rum. As shown in Figure 10, lanes 4 and 10, phosphory-
lated 13-1actamase (band b) was coprecipitated with Pkn2
(band a) when anti-Pkn2 serum was used. Similarly,
when the cell lysate was treated with anti-~-lactamase
serum, Pkn2 (band a) was coprecipitated (lanes 6,12).
Note that bands c and d in lanes 6 and 12 are pro-~-
Figure 10. Coimmunoprecipitation of Pkn2 and 13-1actamase.
E. coli BL21 (DE31 harboring pETll/pkn2 was labeled with
Tran35S-label for 2 hr in the presence (lanes 1--6) or the absence
(lanes 7-12) of rffampicin. Immunoprecipitation was performed
with anti-Pkn2 {lanes 3,4,9,10) or antiq3-1actamase (lanes
5,6,11,12), as described in Materials and methods. (Lanes
1,2,7,8) Total cell extracts without(-}and with ( + ) IPTG. Let-
ters with an arrow and bars with a number are the same as in
Fig. 6.
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Udo et al.
the chromosome was disrupted by inserting the kanr
gene from Tn5 into the unique SalI site that lies within
the coding region for subdomain IV of the Pkn2 catalytic
domain forming strain Apkn2. Strain apkn2 was found
to grow normally as the wild-type strain in CYE me-
dium, indicating that pkn2 is not essential for vegetative
growth. Strain hpkn2 was also able to form well-defined
fruiting bodies on CF agar plates. The fruiting body mor-
phology of strain Apkn2 was somewhat different from
that of the wild-type strain; strain Apkn2 produced con-
tinuous mounds that appeared loosely packed, in con-
trast to the well-separated round mounds of the wild-
type strain (not shown).
We also quantitated the number of myxospores pro-
duced during development. The Apkn2 spore production
was -30%-50% of the level of the wild-type strain. The
Apkn2 myxospores were viable at the same level as the
wild-type spores (not shown).
Figure 11. Cellular localization of phosphorylated f3-1acta-
mase. E. coli BL21 (DE3) harboring pETll/pkn2 or pETll/
pkn2AKn were labeled with Tran3SS-label for 2 hr and fraction-
ated into the periplasmic membrane and cytoplasmic fractions,
as described in Materials and methods. After SDS-PAGE, the
proteins were transferred to PVDF membrane and detected us-
ing anti-13-1actamase serum. (Lanes 1,2,6,7) Total cell lysate;
(lanes 3,8) periplasmic fraction; (lanes 4,9) membrane fraction;
(lanes 5,10) cytoplasmic fractions. Equal portions of the
periplasmic, membrane, and cytoplasmic fractions were applied
to the gel. The assignments are the same as in Fig. 6.
topyranoside) was added to the agar plates to detect [3-
galactosidase expression. Blue colonies became visible
after -48 hr incubation at 30~
whereas no blue color was developed on the CF plates.
These results indicate that pkn2 is expressed only during
the vegetative growth but not during development.
A more quantitative analysis of the expression of pkn2
was obtained by assaying [3-galactosidase activity in the
pkn2/Z strain during both vegetative growth and the de-
velopmental cycle. The specific t3-galactosidase activi-
ties were between 3 and 5 U/rag of protein/rain during
the exponential growth and sharply dropped to < 1 dur-
ing the stationary phase. [3-Galactosidase activity on CF
agar plates decreases rapidly and stayed at a very low
level of <2 U/mg of protein/min. These results are con-
sistent with the plating experiment described above, in-
dicating that pkn2 is mainly expressed during the late
exponential growth phase.
on the CYE plates,
Effects of a disruption mutation of pkn2
As described in Materials and methods, the pkn2 gene on
Discussion
In this study we demonstrated that M. xanthus contains
a transmembrane protein serine/threonine kinase, des-
ignated Pkn2. We have described previously that M. xan-
thus contains a cytoplasmic protein serine/threonine ki-
nase, Pknl (Munoz-Dorado et al. 1991). Pkn2, consisting
of 830 amino acid residues, contains the 11 subdomains
characteristic of the eukaryotic protein kinases at the
amino-terminal region. Immediately after the last con-
served subdomain XI, there is a 99-residue region (from
residue 275 to 3731 enriched with serine (20 residues),
threonine (10), alanine (23), glycine (17), and proline (10).
These residues account for 81% of this region. This do-
main may be the site for phosphorylation, having a total
35 serine plus threonine residues in this region, and the
phosphorylation of this domain may be involved in the
regulation of Pkn2 function.
From the PhoA fusion analysis, the 207-residue car-
boxy-terminal domain immediately after the 18-residue
hydrophobic domain was found to be translocated across
the membrane, which is likely to serve as a receptor to
sense an external signal yet to be identified. Most signif-
icantly, it was found that Pkn2 is a protein kinase that is
able to phosphorylate threonine residues of TEM-[3-1ac-
tamase specifically. Our data indicate that only nascent
[3-1actamase was phosphorylated, which prevented the
translocation of [3-1actamase across the membrane.
TEM-13-1actamase contains a total of 20 threonine resi-
dues, of which 5 residues are found in known phospho-
rylation site motifs (R/KXXT and R/KXT; Hunter 1991).
Consistent with this fact, five radioactive tryptic pep-
tides were obtained when phosphorylated [3-1actamase
was digested with trypsin. These results indicate that
Pkn2 phosphorylates [3-1actamase at multiple sites to
block its secretion. At present, it is not known whether
phosphorylated [3-1actamase still contains the signal pep-
tide.
M. xanthus is known to be highly resistant to ampi-
cillin, and a [3-1actamase, which is cross-reactive with
anti-TEM-[3-1actamase serum has been identified in this
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Transmembrane Ser/Thr kinase from M. xanthus
organism (K. O'Connor and D. Zusman, pers. comm.). It
is tempting to speculate that Pkn2 may regulate the ac-
tivity of 13-1actamase and/or other enzymes related to
[3-1acatamase such as penicillin-binding proteins by
phosphorylating them under certain conditions. In this
fashion, cell viability, cell shapes, or morphology may be
regulated under certain circumstances.
In eukaryotes, most protein serine/threonine kinases
are soluble localizing in the cytoplasm. However, a few
protein serine/threonine kinases, such as transforming
growth factor (TGF)q3 receptor (Massaqu6 et al. 1994)
and IRE1 (Cox et al. 1993), are known to be transmem-
brane protein kinases. Pkn2 is the first transmembrane
protein serine/threonine kinase ever found in prokary-
otes. In prokaryotes, a large number of transmembrane
protein histidine kinases are known, which function as
sensors for various external signals (Stock et al. 1989). It
remains to be elucidated how M. xanthus differentially
utilizes two types of transmembrane kinases in terms of
their functions. It is also an intriguing question how M.
xanthus acquired the serine/threonine kinase gene dur-
ing the course of evolution.
Materials and methods
Materials
[~-s2P]dCTP, [,/-3~p]ATP, [et-3SS]dATP, and 32p were purchased
from Amersham, and TranSSS-label containing [3SS]methionine
and [3SS]cysteine from ICN. DNA sequencing was performed
with a Sequenase kit obtained from U.S. Biochemical. Taq poly-
merase for PCR was purchased from Perkin-Elmer Cetus, re-
striction enzymes from New England Biolabs, and T4 DNA li-
gase from Bethesda Research Laboratories.
Bacterial strains and growth conditions
M. xanthus DZF1 was grown at 30~ in CYE medium (Campos
and Geisselsoder 1978), and 40 ~g/ml of kanamycin was added
when necessary.
M. xanthus fruiting bodies were obtained on CF agar plates
(Hagen et al. 1978), and the samples were harvested and treated
as reported previously (Munoz-Dorado et al. 1991).
E. coli JM83 IVieira and Messing 1982) and E. coli CL83
(Lerner and Inouye 1990) were used as recipient strains for trans-
formation and Pl infection. Cells were grown in LB medium
(Miller 1972) supplemented with 50 I~g/ml of ampicillin, 25
I~g/ml of kanamycin, and/or 10 I~g/ml of chloramphenicol
when necessary. E. coli BL21(DE3) was used for the T7 RNA
polymerase expression system (Studier et al. 19901. This strain
was grown in M9 medium containing 19 amino acids, excluding
methionine, when cells were labeled with TrangSS-label or in a
low phosphate medium when the cells were labeled with 32p
(Munoz-Dorado et al. 1991). E. coli strains were grown at 37~
E. coli CC118 was used for the alkaline phosphatase assay (Man-
oil and Beckwith 1985).
Plasmids and phages
pUC9 (Vieira and Messing 1982) and pUX (Dhundale et al. 1988)
were used for cloning, subcloning, and sequencing, pET1 la was
used for the expression of M. xanthus genes in E. coli by the T7
RNA polymerase system (Studier et al. 1990). pP1EK was de-
rived from pUC19 and contained a 5.4-kb EcoRI-KpnI fragment
encoding Pl-specific incompatibility (Hsu et al. 1989). pKM005
was derived from plNIII-A3 and contained the lacZ gene with-
out a promoter from E. coli IMasui et al. 1983}. pUC7Kan5 and
pUC9Kn(Pst-} were derivatives of pUC7 and pUC9, respec-
tively, constructed by S. Inouye (unpubl.); they contained the
kanramycin resistance gene from TnS. Phage PlchlOOCm
(Rosner 19721 was used to transduce cloned DNA from E. coli to
M. xanthus.
DNA manipulation and sequencing
M. xanthus chromosomal DNA was prepared according to the
method of Avery and Kaiser (1983) and phage ~ DNA according
to the method of Maniatis et al. t1982). Southern blot analysis
was performed by the method described by Southern (Southern
1975). Phage plaques were blotted to nitrocellulose filters by
contact, and M. xanthus and E. coli colonies were grown on
Whatman 3MM papers; the filters were treated as described
previously lInouye and Inouye 1987). The DNA probes were
labeled by nick translation, and hybridization was carried out at
42~ in 50% formamide as reported {Maniatis et al. 1982). DNA
sequencing was determined by the dideoxynucleotide chain ter-
mination method (Sanger et al. 1977} using double-stranded
plasmid DNA and the universal primers and the synthetic oli-
gonucleotides designed from newly obtained DNA sequences as
primers.
Construction of pkn2 and its deletion mutants under a
T7 promoter
To clone the pkn2 gene under the control of the T7 promoter in
pET1 la, an NdeI site was first created at the initiation codon of
the pkn2 gene by PCR, using the following two primers; 5'-
AAGATAAGCTTCATATGCTGGCCCCTGACTCCCTT-3';
which annealed at position 393--412 (Fig. 3) and contained NdeI
and HindIII sites, and 5'-GCAGGCGTGGTCGCCGGATT-3';
which annealed at position 633-652, immediately downstream
of the SalI site (619-624). The fragment amplified was digested
with HindIII and SalI and ligated to pJMPK2S with HindIII di-
gestion and partial digestion with SalI. The plasmid thus ob-
tained (pJMPK2HB) was then digested with NdeI and BamHI,
and the resulting 5-kb fragment was ligated to pET1 la digested
with the same enzymes. The plasmid thus obtained was desig-
nated pETll/pkn2. In the case of aKn, the primers used
were 5'-AACAAGCTTCATATGCTCACCGACATCCAG-3'
(position 1583-1600), which contained HindIII and NdeI sites,
and 5'-ACATGCTCCACGGCCTCG-3' (position 1912-1929),
which annealed downstream of the NcoI site at position 1899-
1904 (see Fig. 3). The fragment amplified was digested with
HindIII and NcoI and ligated to pJMPK2HB digested with Hin-
dIII and partially with NcoI. The plasmid obtained was then
digested with NdeI and BamHI, and the resulting 4-kb fragment
was ligated to pET1 la linearlized with the same enzymes. This
plasmid was designated pAKn.
The carboxy-terminal deletion mutations of Pkn2 (pAX-B,
pAN-B, pAM-B, pAK-B, and pAS-B) were constructed by digest-
ing pET11/pkn2 at the BamHI site and another unique restric-
tion enzyme site described in the text and Figure 8, followed by
ligation after filling in at the cleaved sites with the Klenow
fragment of DNA polymerase I.
Expression of pkn2 and its deletion mutants in E. coli
Using E. coli BL21 (DE3) harboring pET11/pkn2 or its deletion
constructs, the expression of pkn2 or its mutated genes was
induced by addition of 1 mM IPTG for 1 hr. Rifampicin (150
lag/ml} was then added, and the culture was incubated further
for 15 min. After treatment the cells were labeled with Tran3SS -
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Udo et al.
label for a period specified in each experiment. Pulse-chase ex-
periments were performed by labeling for 5 rain without adding
rifampicin and chasing in the presence of 200 ~tg/ml of nonra-
dioactive methionine at various times.
Western blot analysis
After SDS-PAGE, the samples were transferred onto a PVDF
membrane using a Sartblot semidry transfer apparatus as rec-
ommended by the manufacturer {Sartorious, Gottingen, Ger-
many). [3-Lactamase was detected by anti-13-1actamase serum
subjecting anti-rabbit IgG conjugated to alkaline phosphate
(Bio-Rad) with chromogenic substrates.
Immunoprecipitation of Pkn2 and its mutants,
and f3-1actamase
Ten-milliliter cultures of E. coli BL21(DE3) harboring various
plasmids were labeled with Tran35S-label as described previ-
ously. The cells were harvested, washed with 50 mM Tris-HCl
(pH 8.0), and then suspended in 50 txl sonication buffer [50 mM
Tfis-HC1 (pH 8.0), 1 mM PMSF, 1 mM EDTA, and 2 mM [3-mer-
captoethanol]. After sonication, cell debris was removed by low
centfifugation and 500 ixl of TENN [50 mM Tris-HCl (pH 8.0),
140 mM NaCI, 5 mM EDTA, 0.5% NP-40] was added. After the
addition of 5 Ixl of anti-13-1actamase serum, the mixture was
incubated on ice for 1 hr, and then 5 ~1 of Staphylococcus au-
reus ghosts purchased from Sigma was added. After 15 min at
room temperature, precipitates were recovered by centrifuga-
tion (10,000g for 1 rain), washed three times with TENN, sus-
pended in SDS-PAGE sample buffer [80 mM Tris-HCl {pH 6.8),
2% SDS, 0.2 M 13-mercaptoethanol, 10% glycerol], and analyzed
on 15% SDS-PAGE after boiling. The gel was dried and autora-
diographed.
Cellular fractionation
Isolation of periplasmic, membrane, and cytoplasmic fractions
was carried out according to the method by Neu and Heppel
{1965). Briefly, E. coli cells grown at Klett 100 in 10 ml of M9
medium with or without induction of IPTG were harvested by
centrifugation at 5000g for 10 min at 4~ and the cell pellet was
washed with 10 mM Tris-HC1 (pH 8.0). The washed cells were
resuspended into 1 ml of 20% sucrose in 30 mM Tris-HCl (pH
8.0) containing 1 mM EDTA, and the suspension was incubated
for 10 rain at room temperature. The cell suspension was then
spun at 13,000g for 10 rain at 4~
of ice-cold water to the cell pellet, the cell suspension was in-
cubated for 10 min at 4~ The cell suspension was centrifuged
at 5000g for 10 rain at 4~ and the supernatant was used as the
periplasmic fraction. The cell pellet was then disrupted by son-
ication in 0.5 ml of 50 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 1 mM
phenylrnethane sulfonyl fluoride (PMSF), and 2 mM [3-mercap-
toethanol. The cell debris and unbroken cells were removed by
centrifugation at 5000g for 10 min at 4~ and the supernatant
was fractionated into the membrane and cytoplasmic fractions
by ultracentrifugation at 100,000g for 30 rain at 4~
and after the addition of 1 ml
Construction of pkn2-phoA fusion genes and PhoA assay
The DNA fragments, with or without the region encompassing
a putative transmembrane domain, were amplified by PCR us-
ing pJMPK2HB as a template and the following oligonucleotides
as primers. P1 (5'-CTAAGCTTGGGCAACGCGCTCGCG3-')
corresponds to the region from amino acid 576 to 580 in Figure
3. This primer contains a HindIII site at the 5' end that enables
in-frame fusion of the pkn2 fragments with the a-fragment of
lacZ in pUC9. P2 (5'-CTCTGCAGCCTCGCGCCTCCGCTG-
3') and P3 (5'-CTCTGCAGCGGCGGCGTCCATCAG-3')cor-
respond to the region from residue 601 to 605 and from residue
648 to 652 in Figure 3, respectively. They have a PstI site at the
3' end to enable the in-frame fusion of the pkn2 fragments with
the phoA gene in pCH2 (Hoffman and Wright 1985). PCR-am-
plified fragments were digested with HindIII and PstI and cloned
in the HindIII and PstI sites of pucg. The resulting plasmids
were digested with PstI, and the 3-kb PstI fragment containing
the phoA gene from pCH2 was ligated into the PstI site, fol-
lowed by determination of the correct orientation of the phoA
gene. Using the resulting plasmids, ppkn2-phoA-1 and -2, the
expression of the phoA gene was under the control of the lac
promoter-operator. Before PhoA assay, cells were incubated in
M9 medium in the presence of 1 mM IPTG for 2 hr. PhoA ac-
tivity was measured as described previously (Brickman and
Beckwith 1975).
Phosphoamino acid analysis
E. coli BL21 (DE3) haboring pETll/pkn2 was labeled in vivo
with ortho-3~P as described previously (Munoz-Dorado et al.
1991). Phosphoamino acid analysis was carried out as described
previously (Kamps and Sefton 1989).
Construction of the pkn2/Z strain
To fuse the pkn2 gene to lacZ, a BamHI site was created in the
pkn2 gene in-frame with the BamHI site of lacZ in pKM005 by
PCR. The reaction was performed with the oligonucleotides
5'-GGCCGCGGGATCCAGGGGCAGCGG-3', which anneals
at position 1090-1113 (Fig. 3} and contains a BamHI site;
and the oligonucleotide 5'-AATTTCACACAGGAAACAGC-
TATG-3', which anneals to pUC9 near the EcoRI site of the
polylinker region. After 25 cycles, the 1.1-kb PCR product ob-
tained was purified, digested with EcoRI and BamHI, and ligated
to pUC9 digested with the same enzymes. This plasmid was
designated pJMPK2EB, and its sequence was confirmed by par-
tial sequencing. The 1.1-kb fragment obtained after digestion of
pJMPK2EB with EcoRI and BamHI, the 6.2-kb BamHI-SalI frag-
ment of pKM005, and the 1.45-kb SalI-EcoRI fragment of
pUC9Km(Pst--) were then ligated to plasmid pP1EK digested
with SalI. The resulting plasmid was then transferred from E.
coli to M. xanthus by P1 transduction as reported previously
(Shimkets et al. 1983). M. xanthus colonies containing the fused
gene were selected with kanamycin, and one of them was char-
acterized further for the expression of pkn2. This strain was
designated pkn2/ Z.
Construction of a Apkn2 strain in M. xanthus
The pkn2 gene was disrupted by insertion of kanr of Tn5 (ob-
tained by the SalI digestion of pUC7Kan5) into the unique SalI
site of pJMPK2X (see Fig. 2). The plasmid thus obtained was
designated pJMPK2XKan. From this plasmid the disrupted gene
was obtained by digestion with XhoI, and the 3.8-kb fragment
was ligated to pP1EK digested with SalI. This plasmid was
then transferred from E. coli to M. xanthus by P1 transduction
(Shimkets et al. 1983). The kanamycin-resistant colonies were
screened for double crossover events using pUC9 as a probe. The
replacement of the wild-type gene by the disrupted gene was
confirmed by Southern blot analysis using the 4.8-kb SalI frag-
ment containing pkn2 as a probe. This strain was designated
Apkn2.
Acknowledgments
We acknowledge Dr. W.A. Hanlon for his critical reading of this
manuscript. This work was supported by a grant from the Na-
982 GENES & DEVELOPMENT
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Transmembrane Ser/Thr kinase from M. xanthus
tional Institutes of Health--National Institute of General Med-
ical Sciences (GM44012).
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 USC section
1734 solely to indicate this fact.
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