Download Eukaryotic-type protein kinases in Streptomyces

Microbiology (2003), 149, 1609–1621
Review
DOI 10.1099/mic.0.26275-0
Eukaryotic-type protein kinases in Streptomyces
coelicolor: variations on a common theme
Kateřina Petřı́čková and Miroslav Petřı́ček
Correspondence
Kateřina Petřı́čková
Laboratory of Physiology and Genetics of Actinomycetes, Institute of Microbiology ASCR,
Vı́deňská 1083, 14220 Prague, Czech Republic
[email protected]
The increasing number of genes encoding eukaryotic-type Ser/Thr protein kinases (ESTPKs) in
prokaryotes, identified mostly due to genome-sequencing projects, suggests that these enzymes
play an indispensable role in many bacterial species. Some prokaryotes, such as Streptomyces
coelicolor, carry numerous genes of this type. Though the regulatory pathways have been
intensively studied in the organism, experimental proof of the physiological function of ESTPKs is
scarce. This review presents a family portrait of the genes identified in the sequence of the
S. coelicolor A3(2) genome. Based on the available experimental data on ESTPKs in
streptomycetes and related bacteria, and on computer-assisted sequence analyses, possible roles
of these enzymes in the regulation of cellular processes in streptomycetes are suggested.
Overview
Ser/Thr/Tyr-specific protein phosphorylation represents
one of the fundamental regulatory mechanisms in eukaryotes. Though, generally, His/Asn phosphorylation plays a
more important role in prokaryotes, some groups of
bacteria were recently shown to employ both systems. The
first Ser/Thr protein kinase (ESTPK; stands for eukaryotictype Ser/Thr protein kinase) gene was isolated from
Myxococcus xanthus by Munoz-Dorado et al. (1991). Since
then, numerous genes have been identified in diverse
bacterial species thanks to genome sequencing projects and
other studies. Recent data show that even though ESTPKs
are not as widely and universally utilized in bacteria as in
eukaryotes, their more or less conserved homologues may be
traced across the prokaryotic world (Kennelly, 2002).
Certain bacteria (Mycobacterium, Streptomyces and some
cyanobacteria) harbour numerous representatives of these
enzymes. Their common feature seems to be a complex
life cycle, including morphological and physiological
differentiation, sophisticated cell communication, or
interactions with host cells.
The genome of Streptomyces coelicolor A3(2) (Bentley et al.,
2002) was screened for the presence of ESTPK genes in the
present study. This organism is an extensively studied model
of bacterial differentiation that to some extent resembles
moulds (mycelial growth, sporulation and secondary metabolite production). Though 34 putative ESTPK genes can be
revealed in the genome sequence and some of them have
already been reported, their roles remain quite unclear. Only
two examples, afsK and ramC, have been shown to be
involved in the antibiotic production regulatory cascade
(Matsumoto et al., 1994; Umeyama & Horinouchi, 2001;
Lee et al., 2002) or aerial hyphae formation (Hudson et al.,
0002-6275 G 2003 SGM
2002; O’Connor et al., 2002), respectively. The role of some
others in differentiation of S. coelicolor still remains speculative (Umeyama et al., 2002; Petrickova et al., 2000). The
functions of some ESTPKs have been investigated in other
streptomycete species: Pkg2 is required for aerial mycelium
formation in Streptomyces granaticolor (Nadvornik et al.,
1999) and StoPK-1 of Streptomyces toyocaensis NRRL 15009
influences the oxidative stress response in connection with
glucose metabolism (Neu et al., 2002).
The aim of this review is to compile all available ESTPKrelated experimental data in actinomycetes with the results
of computer-assisted analysis of the S. coelicolor genome and
also other bacterial genomes. Based on this, we suggest
possible roles for these enzymes in the regulation of cellular
processes in streptomycetes.
The S. coelicolor A3(2) genome carries 34
putative ESTPK genes
Based on the screening of the chromosome sequence data
(Bentley et al., 2002) using the standard BLAST program
(Altschul et al., 1990) with some already identified
streptomycete ESTPKs (AfsK, PkaA and PkaC) as queries,
we identified 37 suspected ESTPK genes with P(N) values
below 0?1. Their predicted amino acid sequences were first
analysed with reciprocal BLAST searches; these excluded the
SCK13.20 gene, which was assigned as adenylate cyclase.
Secondly, the remaining 36 were aligned with the eukaryotic
consensus (Hardie & Hanks, 1995). As a criterion, conservation of all catalytic subdomains in the range common
for the eukaryotic protein kinases, except subdomain I, was
applied (see Fig. 1 and the supplementary figure in the
online version of this paper at http://mic.sgmjournals.org).
According to recent studies, subdomain I, which serves as
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K. Petřı́čková and M. Petřı́ček
Fig. 1. Consensus amino acid sequence of the catalytic domains in S. coelicolor ESTPKs. The first line shows the Hardie &
Hanks’ eukaryotic consensus with essential amino acid residues (Hardie & Hanks, 1995). The second line represents the
consensus amino acid sequence of the S. coelicolor ESTPKs. Numbers in parentheses within the S. coelicolor consensus
show the length range of spacers between particular subdomains. The strength of the consensus (i.e. fraction of sequences
conformable with the consensus) is indicated graphically below. The Hardie & Hanks’ consensus sequences are given
according to the following code: uppercase letters, invariant residues; lowercase, nearly invariant residues; o, nonpolar
residues; *, polar residues; +, small residues with near neutral polarity. Catalytic subdomains are indicated with roman numerals.
the ATP-binding site (P-loop), may be substituted with
different ATP-binding motifs (Shi et al., 1998). After the
sequence inspections, two more candidates were excluded
from the study: SCC24.21, with well-conserved subdomains
I–VIB, but with a speculative following section; and
SCC24.15c, with a totally missing subdomain VIII. The
remaining 34 genes were numbered according to their order
on the chromosome (PK01–PK34), disregarding their
previous characterization (Table 1).
The arrangement of the ESTPK genes in the S. coelicolor
chromosome is not even: the central part of the chromosome, encoding the essential life functions, contains the
majority (Fig. 2). Some of the genes are juxtaposed, in either
the same (PK07–PK08, PK19–PK20) or the opposite (PK12–
PK13) direction. Five genes (PK22–PK26) lie ‘head to tail’ in
a group.
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The length of the putative ESTPKs varies from 380 to 1557
amino acid residues. The catalytic domain, of typical length
of 260–275 amino acid residues, is usually N-terminal. The
catalytic domain of the PK23 kinase is interrupted between
the VII and VIII subdomains by an Ala-rich insert (372
residues) of unknown function. The structure of PK30, the
longest kinase, is exceptional; it contains two ESTPK
catalytic domains in its N-terminal half and a putative
C-terminal basic helix–loop–helix motif, which usually
serves as a dimerization domain. A similar structure was
reported in eukaryotes (Jones et al., 1988), but there is no
evidence of a double ESTPK catalytic domain in a single
protein in prokaryotes. However, a somewhat comparable
multi-domain structure is common for some regulatory
proteins of cyanobacteria. They combine features of ESTPKs
and two-component system proteins in a single molecule
(Ohmori et al., 2001).
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Microbiology 149
http://mic.sgmjournals.org
Table 1. Summary of the properties of Streptomyces coelicolor A3(2) ESTPKs
Gene no.
Gene code
Name*
Location
(kbp)
Size
(aa)
TMD
Extra
domains
Nearby genes/
encoded proteinsd
SCL6.25c, SCO1468
SCL11.07, SCO1551
1568
1659
774
493
2
T
2
2
nusB, fmt, rpe, guaB2
cobA, cobT, cbiE, trpE
PK03
SCI11.13, SCO1724
1844
550
D
2
PK04
SC6E10.04, SCO2110
pkaF
2267
667
T
PASTA
PK05
PK06
PK07
PK08
PK09
SC1G2.06c, SCO2244
SC6D10.09, SCO2666
SCE59.32c, SCO2973
SCE50.02c, SCO2974
SCE41.11c, SCO3102
pkaB
pkaA
pkaE
2415
2900
3234
3236
3399
686
903
417
543
510
D
2
T
D
2
WD-40
2
2
2
2
PK10
PK11
PK12
SCE7.11, SCO3344
SC66T3.32c, SCO3621
SCGD3.21c, SCO3820
3700
4001
4200
720
783
522
D
T
T
PQQ
2
2
PK13
SCGD3.22, SCO3821
4201
556
D
PASTA
PK14
PK15
PK16
PK17
PK18
SCH69.18, SCO3848
SCH69.30, SCO3860
SCD10.09, SCO4377
SC6F11.21, SCO4423
SCD69.01, SCO4481
4234
4245
4790
4839
4900
673
576
580
799
632
T
T
T
2
D
PASTA
2
2
PQQ
Sugar-b.
ABC transporter, sigma factor,
exocellular hydrolases
Oxidoreductases, DNA repair
endonucleases
glnA
Sugar transport system
ftsX, ftsE, prfB, smpB
ftsX, ftsE, prfB, smpB
TRCF, cytochrome P450,
Rpf-like growth factor
ilvD, proC
recR, sigma factors, gabT, asd2, ask
ABC transporter, pyruvate dehydrogenase
complex, quinone oxidoreductase
ABC transporter, pyruvate dehydrogenase
complex, quinone oxidoreductase
ftsW, PBP, PP2C
DNA-binding proteins
mutT, FA synthase components
afsR, afsS, kbpA
ccdA, ccsA
PK19
PK20
SCD69.07, SCO4487
SCD69.08, SCO4488
4905
4907
592
626
D
T
LamGL
Sugar-b.
ubiDAX operon
ubiDAX operon
PK21
PK22
PK23
PK24
PK25
PK26
PK27
SCD35.14, SCO4507
SCD63.07, SCO4775
SCD63.08, SCO4776
SCD63.09, SCO4777
SCD63.10, SCO4778
SCD63.11, SCO4779
SC2A6.02c, SCO4817
4927
5189
5192
5195
5196
5198
5248
586
717
979
599
380
548
452
D
T
T
T
T
D
D
2
2
2
2
2
2
PG-binding
PK28
PK29
SC2A6.05c, SCO4820
SCK13.03, SCO4911
5251
5344
712
670
T
D
SLT
Solute-binding
pksC
afsL
afsK
pkaG
pkaH
pkaD
pkaI
pkaJ
FA synthesis, scoF2 cold shock, ftsK
Metabolic genes, sigma factor
Metabolic genes, sigma factor
Metabolic genes, sigma factor
Metabolic genes, sigma factor
Metabolic genes, sigma factor
gbsA, mdh, folD
gbsA, mdh, folD
Sigma factor, afsQ operon
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RNA metabolism
Cobalamine biosynthesis, amino acid
biosynthesis
Hydrolytic enzyme regulation
DNA repair
Metabolic regulation?
Sugar uptake and degradation
Translation, cell-division regulation
Translation, cell-division regulation
DNA repair, catabolism of glycans,
growth regulation
Amino acid biosynthesis
DNA recombination, nitrogen metabolism
Energy metabolism: pyruvate/succinate
dehydrogenase, electron transport
Energy metabolism: pyruvate/succinate
dehydrogenase, electron transport
Cell division
Transcription regulation?
DNA repair, FA metabolism
Differentiation, secondary metabolism
Receptor kinase (sugar signals),
energy metabolism
Energy metabolism
Receptor kinase (sugar signals),
energy metabolism
FA synthesis, cell division, cold shock
Nucleotide, sugar metabolism
Nucleotide, sugar metabolism
Nucleotide, sugar metabolism
Nucleotide, sugar metabolism
Nucleotide, sugar metabolism
Receptor kinase (peptidoglycan signals),
osmotic adaptation
Osmotic adaptation
Differentiation, secondary metabolism
Eukaryotic-type protein kinases in S. coelicolor
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PK01
PK02
Proposed roled
1612
2
2
KLC
2
D
2
2
T
930
565
745
538
7420
7631
8047
8097
SC5A7.31, SCO6681
SC7F9.13c, SCO6861
SC7A12.07, SCO7240
SC5H1.01, SCO7291
PK31
PK32
PK33
PK34
ramC
pknA
SC1F2.23, SCO6626
PK30
*Previously reported genes: afsK (Matsumoto et al., 1994; Ueda et al., 1996); pkaA and pkaB (Urabe & Ogawara, 1995); pkaD–pkaG (Ogawara et al., 1999); pkaH–pkaI (Bentley et al., 2002); afsL
(Umeyama et al., 2002); ramC (O’Connor et al., 2002); pknA (Petrickova et al., 2000).
DPrediction of transmembrane (TM) helices: T, TMHMM-predicted (http://www.cbs.dtu.dk/services/TMHMM-2.0/); D, additional, DAS-predicted (http://www.sbc.su.se/~miklos/DAS); 2, no
transmembrane helices predicted.
dFA, fatty acid; TRCF, transcription-repair coupling factor; PBP, penicillin-binding protein; PP2C, protein phosphatase, 2C-type; IS oxidoreductase, iron–sulphur (2Fe–2S) oxidoreductase. For
abbreviations of the extra protein domains see the Fig. 4 legend.
Differentiation, energy metabolism
?
Respiration, electron transport
Detoxification, degradation, stress response
Secondary metabolism
ATP/GTP-binding proteins,
abaA-like locus
ram cluster, IS oxidoreductase
Possible bacterial regulator
Respiratory genes
Stress genes, hydrolases
HLH
1557
7348
D
Nearby genes/
encoded proteinsd
Size
(aa)
Gene no.
Table 1. cont.
Gene code
Name*
Location
(kbp)
TMD
Extra
domains
Proposed roled
K. Petřı́čková and M. Petřı́ček
Fig. 2. Arrangement of the putative ESTPK genes along the
S. coelicolor A3(2) linear chromosome. The central part of the
chromosome, shown in black, has been reported to carry
mainly ‘house-keeping’ genes, whereas both arms, in grey,
encode mostly adaptive functions. The size scale (in Mbp) and
position of the oriC origin of replication are indicated. The
chromosome scheme is adapted from the work of Bentley
(2002).
The amino acid sequences of the catalytic domains suggest
in most of the proteins their Ser/Thr specificity (see the
supplementary figure in the online version of this paper at
http://mic.sgmjournals.org), though for some (AfsK, PK17
in the study) dual specificity, i.e. Ser/Thr together with Tyr,
has been reported (Matsumoto et al., 1994). Only two of the
S. coelicolor ESTPKs, PK30 (in its second ESTPK domain)
and PK31, resemble the eukaryotic Tyr-specific consensus
to some extent, mainly in subdomain VI. PK23 and the
first domain of PK30 deviate from both groups (see the
supplementary figure at http://mic.sgmjournals.org).
The catalytic domains of the S. coelicolor putative ESTPKs
were aligned together with those of Mycobacterium
tuberculosis, the most related bacterium whose entire
genome has been sequenced, and several representatives of
eukaryotic ESTPKs. The phylogenetic tree based on the
alignment is shown in Fig. 3. Generally, the presented
kinases tend to group in an origin-specific manner (shown in
different colours), though only some groups (in dashed
ellipses) are well defined by the bootstrap test (i.e. bootstrap
values >70 %). The tree revealed two putative pairs of
homologues from S. coelicolor and M. tuberculosis: PknB–
PK14 and PknL–PK04. Further inspection of their domain
architectures and relevant gene regions showed the striking
similarity of the first pair, PknB and PK14, which is
discussed later in the review.
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Microbiology 149
Eukaryotic-type protein kinases in S. coelicolor
Fig. 3. Dendrogram of kinase domains from the Streptomyces coelicolor and Mycobacterium tuberculosis ESTPKs and several
representatives of eukaryotic ESTPKs. The aligned sequences were processed using the PHYLIP 3.6a3 software package: the tree
file was calculated using a protein sequence parsimony method (Protpars). Groups supported with high bootstrap values (>70 %,
out of 100 replicates) are indicated by dashed ellipses. The tree contains the following sequences: (a) 34 ESTPKs from S. coelicolor
A3(2): PK01–PK34 (shown in green). In the case of PK30, both catalytic domains were included, assigned PK30-1 (N-terminal)
and PK30-2 (C-terminal). (b) Eleven ESTPKs from Mycobacterium tuberculosis (shown in blue): PknA (P71585), PknB (P71584),
PknD (O05871), PknE (P72001), PknF (P72003), PknG (P96256), PknH (Q11053), PknI (Q10964), PknJ (Q10697), PknK
(P95078) and PknL (O53510). (c) Representatives of major groups of eukaryotic ESTPKs (shown in purple): AKT8, kinase of
murine leukaemia virus (P31748); BARK, b-adrenergic receptor kinase 1 of Bos taurus (P21146); CAMKIIA, calcium/calmodulindependent protein kinase type II alpha chain of Rattus norvegicus (P11275); CAPKA, human cAMP-dependent protein kinase
(P17612); CDCIIHS, human cyclin-dependent kinase 1 (P06493); CKIIA, human casein kinase II alpha chain (P19138); ERK1,
MAP kinase 1 of Rattus norvegicus (P21708); GSK3A, glycogen synthase kinase-3 alpha of Rattus norvegicus (P18265); and
PKCA, human protein kinase C, alpha type (P17252).
The majority of the kinases contain additional
functional domains, mostly non-enzymic
In addition to the ESTPK catalytic domain, several other
domains can be predicted in the ESTPK sequences. Thus
27 contain possible membrane-spanning regions, suggesting their membrane localization. Several ESTPKs carry
binding domains for various substrates, such as the ricin B
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sugar-binding domain (PF00652), the bacterial peptidoglycanbinding domain 1 (PF01470), the bacterial extracellular
solute-binding domain 3 (PF00497) and the PASTA (PBP
and serine/threonine kinase associated) b-lactam binding
domain (PF03793). Several kinases encompass repetitive
domains, some of them with predicted protein-interactive
roles, e.g. WD-40 (PF00400) and PQQ (PF01011). PK28 is
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K. Petřı́čková and M. Petřı́ček
Fig. 4. Domain structure analysis of ESTPKs from S. coelicolor A3(2). The programs SMART at http://smart.emblheidelberg.de (Schultz et al., 1998), BLOCKS at http://www.blocks.fhcrc.org (Henikoff et al., 1999), InterProScan at http://
www.ebi.ac.uk/interpro/scan.html (Mulder et al., 2002) and SBASE at http://hydra.icgeb.trieste.it/~kristian/SBASE (Vlahovicek
et al., 2002) were used for domain predictions. Transmembrane helices predicted with TMHMM (version 2.0) software at http://
www.cbs.dtu.dk/services/TMHMM-2.0/ (Sonnhammer et al., 1998) are shown as black dots; additional, predicted by the DAS
prokaryotic protein topology server at http://www.sbc.su.se/~miklos/DAS (Cserzo et al., 1997), are shown as grey dots. The
following domains are presented (domain accession nos in the PROSITE, Pfam, ProDom or SMART databases are indicated in
parentheses): (a) ESTPK domains, black rectangles; (b) predicted transmembrane regions, black or grey dots; (c) enzymic
SLT transglycosylase domain, a yellow rectangle; (d) binding domains, green rectangles or pentagons in the case of repetitive
domains, including: ‘Sugar-b.’, ricin B sugar-binding domain (PF00652); ‘PG-b.’, bacterial peptidoglycan-binding domain 1
(PF01470); ‘EC solute-b.’, bacterial extracellular solute-binding domain 3 (PF00497); ‘PASTA’, PASTA b-lactam binding
domain (PF03793); ‘ATP-b.’, ATP/GTP-binding site, P-loop (PS00017); (e) repetitive domains with putative protein–protein
interaction roles (pentagons): ‘WD-40’, b-transducin repeat (PF00400); ‘PQQ’, bacterial PQQ repeat (PF01011); ‘KLC’,
kinesin light-chain repeat (PD148673); (f) repetitive domains of unknown function, white rectangles together with the
repetitive motifs; (g) other domains: ‘HLH’, dimerization domain of the eukaryotic basic helix–loop–helix type (PS00038);
‘LamGL’, LamG-like jellyroll fold domain (SM0560); (h) regions rich in a particular amino acid: proline (blue rectangles), and
alanine, ‘Ala-rich’; and (i) regions of unknown function shared by some ESTPKs: SR1, SR2 and SR3.
1614
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Microbiology 149
Eukaryotic-type protein kinases in S. coelicolor
the only enzyme that carries a recognizable additional
enzymic domain, the SLT transglycosylation domain
(PF01464), putatively involved in murein degradation. In
addition to previously characterized protein domains, we
have identified three types of protein regions, shared by
some of the ESTPKs, designated SR1, SR2 and SR3 (discussed later). A schematic representation of the domain
structures of the proteins is shown in Fig. 4.
ESTPK function predictions
ESTPK topology – putative receptor kinases
Two-thirds of the S. coelicolor ESTPKs are putative
membrane proteins resembling the structure of eukaryotic
receptor kinases. One of the membrane-associated ESTPKs,
RamC, was recently reported to be required for aerial
hyphae formation. Transcription of ramC is developmentally regulated and probably activated by the RamR response
regulator (O’Connor et al., 2002). RamC protein kinase
activity is weak in vitro. It has been speculated that as a
primary role it might phosphorylate different targets, e.g.
lipopolysaccharides (Hudson et al., 2002).
For the topology predictions, two algorithms were generally
used (TMHMM and prokaryote-specific DAS), the results of
which differ in some cases (for details see Fig. 4). A receptive
function of some ESTPKs is supported by the presence
of C-terminal ligand-binding domains, probably located
outside the cell. Three membrane kinases (PK04, PK13 and
PK14) carry a C-terminal PASTA domain, a small globular
fold composed of three b-sheets and an a-helix. The domain
is common to several penicillin-binding proteins and bacterial ESTPKs and is responsible for binding of b-lactam
antibiotics and their peptidoglycan analogues. It is probable
that it may act in sensing free peptidoglycan units as signals
for cell wall biosynthesis (Yeats et al., 2002). We can find
support for the theory in the case of PK14 and its close
homologue PknB of M. tuberculosis (Av-Gay et al., 1999):
both relevant genes are clustered with genes implicated in
cell wall biosynthesis and with other regulatory genes involved in protein Ser/Thr phosphorylation/dephosphorylation
(Fig. 5). The pknB gene is pertinently expressed in
M. tuberculosis cells in vitro as well as in animal host cells
(Av-Gay et al., 1999). A highly similar architecture of the
chromosomal regions together with the lack of a comparable
cluster in the genomes of other, less related, prokaryotes
(Eschericha coli, Helicobacter pylori and Bacillus subtilis)
suggest the presence of a specific, cell-division-associated
regulatory pathway common to the high-GC branch of the
Gram-positive bacteria.
The other two PASTA-containing ESTPKs, PK04 and PK13,
do not have close mycobacterial homologues. Considering
their gene neighbours, they might be involved in DNA repair
and energy metabolism regulation, respectively, in response
to peptidoglycan compounds.
The PK27 kinase may be involved in the transmission of
peptidoglycan signals too, since it carries a C-terminal
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peptidoglycan-binding domain I (PF01471). The domain
probably has a general peptidoglycan-binding function and
is found in a variety of enzymes implicated in bacterial cell
wall degradation. The PK27-encoding gene is clustered with
several genes encoding hypothetical secreted proteins and
with the PK28-encoding gene. PK28 is a multifunctional
protein kinase with a C-terminal SLT transglycosylase
(PF01464). The SLT domain degrades murein by cleaving
the 1,4-b-glycosidic bond between N-acetylmuramic acid
and N-acetylglucosamine. Moreover, nearby genes are
responsible for the biosynthesis of the osmoprotectant
glycine betaine (Boch et al., 1997). Thus in response to
changes of the peptidoglycan structure caused by osmotic
shock, PK27 may transmit a signal to the enzymes involved
in osmotic adaptation. As possible results of the pathway
activation, glycine betaine is synthesized and the cell wall is
modified by the SLT transglycosylase activity of the activated
PK28 (Fig. 6).
Two ESTPKs, PK18 and PK20, carry C-terminal, probably
extracellular, carbohydrate-binding domains of the ricin
type. Both kinases are also highly similar in their catalytic
domains and their genes are situated close to each other
in the chromosome (Fig. 7). The carbohydrate-binding
domain, originally found in the legume lectin ricin, is
present in many carbohydrate recognition proteins where it
binds simple sugars, such as galactose or lactose (Hazes,
1996). Both kinases may thus respond to sugar signals. The
third kinase-encoding gene (PK19) in the region may also be
associated with sugar signals; its additional LamGL domain
seems often to be linked with enzymes involved in carbohydrate metabolism. However, its exact function is not
clear. Products of other surrounding genes, putatively responsible for cytochrome and quinone biosynthesis, may
serve as possible targets of regulation by Ser/Thr phosphorylation. Moreover, expression of the PK18 gene is possibly
developmentally regulated in a bldA-dependent manner,
since it contains the TTA rare leucine codon (Leskiw et al.,
1991). The PK18, PK19 and PK20 kinases might be involved
in the control of the respiratory chain in response to simple
sugar availability in a developmental-stage-specific manner.
A bacterial extracellular solute-binding domain of the family
3 (PF00497) is present in PK29. In Gram-positive bacteria,
such domains are part of receptor proteins that trigger or
initiate translocation of the solute through the membrane by
interaction with a specific transport system. In some cases,
they also initiate sensory transduction pathways. The family
3 members seem to be specific for amino acids and opines
(Tam & Saier, 1993). In the case of PK29, the domain is most
similar (25 % identity) to that of the GluR0 receptor of the
glutamate-gated potassium channel of Synechocystis (Chen
et al., 1999). The surrounding genes encode enzymes
responsible for degradation of carbon compounds and
nucleotide interconversion. In addition, the afsQ1–Q2
two-component regulatory system genes, involved in
antibiotic production, and a possible s factor gene are
located nearby. Though no transport systems that could
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K. Petřı́čková and M. Petřı́ček
Fig. 5. Comparison of two closely related ESTPKs, PknB of Mycobacterium tuberculosis and PK14 of Streptomyces
coelicolor. (a) Alignment of PK14 and PknB amino acid sequences. Identical residues are highlighted; ESTPK and PASTA
domains are indicated. (b) Arrangement of the PK14 and PknB chromosomal regions. The gene encoding PK14 and the pknB
gene are shown by patterned arrows. The regions contain genes encoding the following proteins or RNAs: PP2C, Ser/Thr
protein phosphatase of the PP2C type; ftsW, probable FtsW/RodA/SpoVE family cell division protein; PBP, secreted
penicillin-binding protein; pknA, Ser/Thr protein kinase; trpG, probable glutamine amidotransferase (anthranilate synthase II);
ppiA, probable peptidyl-prolyl cis-trans isomerase; Leu, tRNA Leu – anticodon CAG; probable transmembrane proteins (small
grey arrows), unknown or similar to hypothetical proteins (small black arrows).
putatively be regulated by the PK29 in a glutamate-specific
manner are encoded in the region, implication of the kinase
in glutamate/other solute-mediated signal transduction in
S. coelicolor is a subject for further investigation.
There are more putative ESTPKs in S. coelicolor that seem
to be integrated in the membrane, but their predicted
extracellular parts do not share any homology with known
proteins. In some cases possible targets of their regulatory
actions may be suggested based on the composition of
regions around their genes (Table 1). As an example, PK07
(PkaB) and PK08 (PkaA), the genes of which lie in an
operon followed by genes involved in translation (prfB
encoding the chain release factor 2, and smpB encoding the
small protein B) and cell division (ftsX, ftsE), may be
involved in translation and cell division control in response
to environmental stimuli.
1616
ESTPKs as organizers of signal transmission
complexes
Within the set of S. coelicolor ESTPKs, four contain
repetitive domains, which are generally responsible for
protein–protein interactions and assembly of multi-protein
complexes: PK10 and PK17, bacterial PQQ repeat, PF01011;
PK05, b-transducin WD-40 repeat, PF00400; and PK33,
kinesin light-chain repeat, PD148673. All the repetitive
motifs share a typical core repeat length (about 40 amino
acid residues); in addition, PQQ and WD-40 form the same
b-propeller structure. PQQ occurs in enzymes with pyrroloquinoline quinone as a cofactor, in Ser/Thr kinases, and in
prokaryotic dehydrogenases (Oubrie et al., 1999). Though
the PQQ-containing protein kinases are present in both
prokaryotes and eukaryotes, their exact domain architecture varies according to the origin: in all prokaryotic
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Microbiology 149
Eukaryotic-type protein kinases in S. coelicolor
already been studied. In S. coelicolor, the best-characterized
kinase, AfsK (PK17 in the study), is necessary for antibiotic
production, whereas in Streptomyces griseus (AfsK-g), it is
conditionally needed for morphological differentiation
(Umeyama et al., 1999). AfsK activity is regulated by
the KbpA protein, which binds to the catalytic domain of
AfsK and inhibits its autophosphorylation (Umeyama &
Horinouchi, 2001). The active form of AfsK phosphorylates
the AfsR global regulator, which is needed for the biosynthesis of actinorhodin and undecylprodigiosin. Recent
work has shed more light on the mechanism of regulation.
The phosphorylated form of AfsR binds efficiently to a
promoter region of afsS and initiates its transcription (Lee
et al., 2002). The product of the gene then, directly or
indirectly, activates transcription of actII-ORF4, the
actinorhodin-pathway-specific transcriptional activator
(Umeyama et al., 2002).
Fig. 6. Tentative model of the roles of PK27 and PK28 in
osmoprotection. Osmotic changes alter the peptidoglycan (PG)
architecture of the cell wall, CW (1), which serves as a signal
received by the peptidoglycan-binding domain (PGb) of PK27
(2). The signal reception causes activation of the PK27 ESTPK
catalytic domain (K27). As a result, the genes responsible for
glycine betaine biosynthesis (gbs) are expressed (3), probably
via activation of specific transcription factors, and the osmoprotective glycine betaine is produced (4). Next, the active
PK27 activates (by phosphorylation?) the kinase domain of
PK28 (K28), which subsequently causes activation of the extracellular SLT domain (5). The SLT transglycosylase modifies the
peptidoglycan in a way that improves cell wall resistance to
osmotic pressure (6).
representatives the PQQ domain is situated in the
C-terminus of the kinase molecule, whereas in eukaryotes
it is always N-terminal (based on the SMART protein
architecture database).
Some of the PQQ-containing ESTPKs in actinomycetes have
In S. granaticolor, three ESTPKs containing a PQQ domain
have been characterized, but none of them seems to be
a close homologue of those found in S. coelicolor. Pkg2
is probably involved in aerial mycelium development
(Nadvornik et al., 1999). The genes encoding the next
two, Pkg3 and Pkg4, are organized in an operon. Their
physiological role is not clear (Vomastek et al., 1998).
Similarly to the PQQ repeats, the WD-40 repetitive domain
forms a propeller structure (Smith et al., 1999). The domain
is characteristic of the b-subunits of trimeric G-proteins and
many other eukaryotic regulatory proteins; its presence in
prokaryotic proteins is quite rare. According to the gene
region content, the only S. coelicolor gene encoding an
ESTPK containing a WD-40 domain (PK05) may be
involved in metabolic regulation.
The last of the protein-interactive domains, the KLC repeats
(present in PK33), were first identified in the light chain of
the eukaryotic kinesin motor protein. In the multimeric
kinesin molecule they are involved in the coupling of a cargo
to the heavy-chain subunits and in the modulation of its
Fig. 7. PK18–19–20 chromosomal region. Genes encoding PK18 and PK20 are shown by patterned arrows. The region
contains genes encoding the following products: ccdA, similar to cytochrome-c-type biogenesis protein; ccsA, possible
cytochrome c assembly protein; MCM, possible transferase, MCM family signature (PS00847); SH, possible secreted
hydrolase; ubiD, probable 3-octaprenyl-4-hydroxybenzoate carboxy-lyase; ubiA, probable 4-hydroxybenzoate octaprenyltransferase; ubiX, probable octaprenyl carboxylase; DPR, putative DNA polymerase related protein; AcT, putative
acetyltransferase; gltP, putative proton transport protein – glutamate transporter. Genes encoding transcription regulators
are shown by bold grey arrows, transmembrane proteins by small grey arrows, and unknown (similar to hypothetical) proteins
by small black arrows. Genes containing the TTA codon are indicated with asterisks.
http://mic.sgmjournals.org
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1617
K. Petřı́čková and M. Petřı́ček
ATPase activity (Gauger & Goldstein, 1993). It was also
shown that the protein-binding abilities of KLC are
regulated by phosphorylation (Ichimura et al., 2002).
Considering the gene organization around the gene
encoding the KLC-containing PK33, the kinase might be
involved in the regulation of respiration and electron
transport. Phosphorylation of the KLC repetitive domain by
the kinase domain may modulate its interactions with
protein ligands.
What the exact physiological functions of the proteininteractive domains in ESTPKs are remains to be elucidated.
Some hints were given in the case of the gene pkwA from
the thermophilic actinomycete Thermomonospora curvata.
The gene encodes the WD-40-containing ESTPK, which is
probably membrane-associated. It was shown that the WD40 domain of PkwA itself could be phosphorylated by an
unknown kinase activity in the T. curvata cell-free extracts
and also by the Pkg2 kinase of S. granaticolor. However, the
presence of the PkwA catalytic domain prevents phosphorylation in vitro (Joshi et al., 2000). How the phosphorylation
of the WD-40 domain affects kinase function has not been
investigated yet. Our latest experiments have revealed that
pkwA gene expression is developmentally regulated and is
strictly associated with the early exponential growth phase.
In young T. curvata mycelium, PkwA is predominantly
present in the form of high-molecular-mass protein complexes (unpublished). Among the genes near pkwA are those
putatively encoding subunits of DNA polymerase III and
DNA helicases. Both enzyme families are certainly involved
in replication and may thus be required in the fast-growing
cells in the early exponential phase.
Other domains that may give ESTPKs extra functions
Many ESTPKs contain regions that are particularly rich in
proline residues (over 30 % of all amino acid residues) (see
Fig. 4). Proline-rich domains are often discussed in
connection with their protein-binding abilities. Specific
proline-rich motifs interact with the eukaryotic WW, SH3
and other protein-interactive domains (Einbond & Sudol,
1996; Kay et al., 2000). They are frequently involved in signalling pathways of eukaryotes. It is possible that the prolinerich regions may also specify functions of the bacterial
ESTPKs by coupling them to their targets or regulators.
Some of the identified ESTPKs share regions that do not fit
to any protein pattern and motif in the databases. In the set
of the S. coelicolor ESTPKs we have identified three types,
SR1 (over 200 amino acid residues), SR2 (about 60 residues)
and SR3 (about 100 residues), seen in Fig. 4. Their functions
are clearly speculative. SR1 may serve as an extracellular
(signal?)-binding domain. Three of four SR1-encoding
ESTPK genes (PK22, PK23 and PK24) lie in a group at the
chromosome and might also be products of gene duplication. SR2, present in PK11 and PK21, closely follows the
ESTPK domain, so it may be involved in substrate binding
or kinase activity regulation.
1618
Implications of the content of the ESTPK gene
region
As was mentioned before, the character of surrounding
genes may provide clues to a predicted possible role of
an ESTPK. Considering their possible targets of regulation, they might control membrane transport systems (ABC
transporters and other permeases), biosynthetic pathways
(primary or secondary), energy metabolism, cell division,
stress response and differentiation. While conveying their
signals, they may interact with other ESTPKs, protein
phosphatases, members of two-component systems, transcription regulators and s factors (see also Table 1).
PK04 (PkaF) may be given as an example. Just downstream
of pkaF we found a gene similar to many DNA-repair
endonuclease genes. Its protein product follows the consensus of the AP endonuclease class, i.e. apurinic or apyrimidinic site-specific DNA lyases. Based on it we hypothesize
that the kinase might be involved in the DNA-repair or
stress response. Moreover, PkaF contains the extracellular
PASTA peptidoglycan-binding domain that may sense
stress-induced peptidoglycan changes of the cell wall as
was discussed before in the case of PK14. Association of
cell-wall-related genes with oxidative stress defence has
been discussed in other bacteria (Thibessard et al., 2002).
Recently, the PkaF gene homologue StoPK-1 was characterized in S. toyocaensis. Disruption of the gene causes
increased sensitivity to oxidative stress and the kinase
activity is needed for the wt phenotype. It was also shown
that PkaF of S. coelicolor could fully substitute StoPK-1 (Neu
et al., 2002). Taking the information together, it is conceivable that the oxidative-stress-protective role of PkaF/
StoPK-1 is mediated by the control of the AP endonuclease
and the signal transduction pathway affects DNA-repair
control.
For details on other ESTPK genes see Table 1.
Evolutionary insights into the presence of
ESTPKs in prokaryotes
In 1991, Munoz-Dorado and coworkers first reported a
eukaryotic-type Ser/Thr protein kinase, Pkn2, in bacteria
and broke the traditional classification of protein kinases as
eukaryotic Ser/Thr/Tyr-specific and prokaryotic His/Asnspecific (Munoz-Dorado et al., 1991). Since then, many
bacterial Pkn2-type kinases have been characterized, and
over a hundred others have been identified thanks to
genome-sequencing projects in many bacterial species,
including archaea. However, it is important to note that
these enzymes are not universally spread within the prokaryotes and their occurrence is limited to particular
bacterial groups. It seems that they are dispensable for the
accomplishment of the ‘basic’ prokaryotic way of life,
represented by E. coli, but they are required for cell
differentiation, a multicellular lifestyle, complex secondary
metabolism, circadian cycling, pathogenicity, etc. (Ogawara
et al., 1999). Perhaps, derived from a common ancestor, they
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Microbiology 149
Eukaryotic-type protein kinases in S. coelicolor
became involved in the prokaryotic regulatory pathways
only when the more complex lifestyle demanded them.
According to recently available data, two prokaryotic groups
exhibit high numbers of Pkn2-type ESTPK genes in their
genomes: cyanobacteria and actinomycetes. Other bacterial
species usually do not contain more than just two or three
such genes (e.g. Bacillus, Chlamydia, Salmonella, Yersinia,
etc.) or do not have them at all (Escherichia, Neisseria,
Borrelia, etc.). It should be taken into account that the
choice of organisms for genome-sequencing projects affects
the overall image of ESTPK distribution in prokaryotes.
Recently, new groups of putative ESTPK homologues,
ABC1, RIO1, piD261, AQ578 and others, were assigned in
bacterial genomes and eukaryotes (Leonard et al., 1998; Shi
et al., 1998; Ponting et al., 1999; Kennely, 2002). Though
they are evidently more distant from the Hardie & Hanks’
consensus sequence, they seem to share all the structural
features with eukaryotic protein kinases. In the case of
piD261 of Saccharomyces cerevisiae, the protein kinase
activity was clearly shown (Stoccheto et al., 1997); other
cases remain hypothetical. Recently, RIO1 group members
were shown to act as lipopolysaccharide kinases in Gramnegative bacteria (Krupa & Srinivasan, 2002). Thus it is
quite probable that some of these kinases predominantly
phosphorylate non-protein targets. Consistently, aminoglycoside phosphotransferases, responsible for inactivation
of aminoglycoside-type antibiotics, also show striking structure similarity with ESTPKs, though the sequence match is
extremely low (Wright, 1999). As well as their primary
enzymic activity they exhibit Ser protein kinase activity, too
(Daigle et al., 1999). None of the putative ESTPKs that we
identified in S. coelicolor fit to the new ESTPK groups as
characterized by Leonard (1998). It seems probable that
with further accumulation of genome data other atypical
kinase classes will be recognized.
Interestingly, it seems that even closely related organisms,
such as different species of a single genus, do not contain
identical sets of ESTPK genes. Available genome sequence
data in Mycobacterium (avium, bovis, tuberculosis, paratuberculosis and leprae) reveal 9–11 ESTPK genes. Of these,
about half are common to all species, but the rest are speciesspecific. Similarly, three PQQ-containing ESTPKs (Pkg2–4)
identified in S. granaticolor do not have close homologues in
S. coelicolor. S. coelicolor contains only two PQQ-containing
ESTPKs, and their catalytic domain sequences, exact
domain structures and gene area arrangements are different.
Apparently, ESTPK involvement in prokaryotic regulatory
pathways has evolved in a species-specific manner,
presumably to satisfy individual lifestyle demands.
Comparison of sets of ESTPK genes found in the genomes
of S. coelicolor and the cyanobacterium Nostoc supports
the idea of species-specific design of ESTPKs. Both prokaryotic organisms have comparably large genomes (8?7
and 10 Mbp, respectively), containing numerous ESTPKs,
and both have incredibly sophisticated life cycles. Nostoc
is a nitrogen-fixing filamentous cyanobacterium which
http://mic.sgmjournals.org
produces several specialized cell types and often establishes
endosymbiosis with the Geosiphon pyriforme fungus
(Castenholz & Waterbury, 1989). Streptomycetes also
exhibit hyphal growth, differentiate into several cell types,
and produce a vast number of secondary metabolites,
extracellular signals and enzymes. The genome of Nostoc
carries over 50 putative ESTPK genes. As in S. coelicolor,
about 70 % of them probably encode transmembrane
enzymes. However, their functional domain architecture
differs remarkably. None of the additional functional
domains found in S. coelicolor kinases is present in the set
of Nostoc kinases. The PQQ and WD-40 protein interactive
domains of S. coelicolor are substituted with TPR repeats
(PF00515) in Nostoc. Many of the Nostoc kinases are huge
proteins with a multidomain structure typical of cyanobacterial regulatory proteins and carry GAF (PF01590), His
kinase A phosphoacceptor (PF00512), histidine-kinase-like
ATPase (PF02518), PAS (PF00989) and PAC (PF00785)
domains in a single molecule (Ohmori et al., 2001). Thus
the overall characteristics of the ESTPK group are different in each species, as are the demands for regulatory
circuit functions.
Concluding remarks
This review collates experimental data on ESTPKs of
S. coelicolor and other related actinomycetes with computer
analysis of the S. coelicolor genome sequence data. The
experimental evidence indicates that Ser/Thr kinases are
involved in cell differentiation, antibiotic production
and stress-response regulation (Umeyama et al., 2002).
Computer analysis of the S. coelicolor genome data led us to
assign putative roles to previously uncharacterized ESTPKs
encoded by the genome.
Comparing the data with those available in M. tuberculosis
(Av-Gay & Everett, 2001), we found only one pair of almost
identical genes. This finding was done with respect to the
level of identity of the ESTPK domains, the presence of
additional functional domains and the relevant gene
regions. Both PknB of M. tuberculosis and PK14 of
S. coelicolor are probably involved in cell growth and
division regulation and both relevant genes lie close to the
origin of chromosome replication. Other S. coelicolor
ESTPKs are more distant from mycobacterial representatives, though some common characteristics can be found,
such as the presence of PQQ domains and proline-rich
regions.
It is difficult to ascribe exact functions to particular ESTPK
genes without analyses of relevant mutants. Interestingly, no
ESTPK genes were detected by classical genetic screening
techniques, including mutagenesis. This may be explained
by either their indispensability for life or their putative
functional redundancy. The latter cause was in some cases
supported by the fact that the disruption of particular
ESTPK genes does not cause any distinct phenotype changes
(Petrickova et al., 2000; Vomastek et al., 1998). In spite of
that, when we consider their abundance in the genome, their
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1619
K. Petřı́čková and M. Petřı́ček
Table 2. Numbers of putative Ser/Thr and His protein kinases in selected bacterial genomes
Streptomyces
coelicolor
Genome size (Mbp)
ESTPK*
His kinase*
8?7
34
86
Mycobacterium
tuberculosis
4?4
11
12
Nostoc
punctiforme
9?2
50
145
Anabaena sp.
PCC 7120
7?2
48
114
Pseudomonas
aeruginosa
6?2
3
64
Bacillus
subtilis
4?2
3
36
*The numbers of putative genes encoding both types of protein kinase are based on the genome annotations.
frequent involvement in the regulation of cell processes in
Streptomyces is quite likely. The presence of 34 putative
genes in the genome, representing about 0?5 % of coding
sequences, cannot be explained just by incidental horizontal
gene transfer. As a comparison, one of the smallest
eukaryotic genomes, that of Saccharomyces cerevisiae,
contains 113 ESTPK genes, representing 2 % of all its
coding sequences (Hunter & Plowman, 1997).
Av-Gay, Y. & Everett, M. (2001). The eukaryotic-like Ser/Thr
Another question may rise: do the ESTPKs functionally
replace ordinary bacterial two-component systems in some
prokaryotes? Table 2 compares the numbers of Ser/Thr- and
His-specific protein-kinase-encoding genes in the genomes
of some selected bacteria: S. coelicolor, M. tuberculosis,
Nostoc punctiforme, Anabaena sp. PCC 7120, Pseudomonas
aeruginosa and Bacillus subtilis. The data do not show any
obvious correlation between the genome size and the
number of two-component systems or ESTPKs. We did not
find any decrease in the number of two-component system
His kinases in those organisms possessing numerous
ESTPKs. In general, we suppose that Ser/Thr phosphorylation has not developed in certain bacteria just to replace
two-component systems. It provides new regulatory circuits
to control various cell processes based on the particular
needs of a species. Moreover, both systems are probably
tightly connected, as was shown in eukaryotes employing
bacterial two-component systems that directly interact with
ESTPK pathways in osmotic regulation and the plant
hormone response (Loomis et al., 1997).
Boch, J., Nau-Wagner, G., Kneip, S. & Bremer, E. (1997). Glycine
Summarizing recent findings, it has become evident that
ESTPKs play as indispensable a role in some prokaryotic
organisms as they do in eukaryotic cells. Newly emerging
experimental and genomic data provide clues to their
functions, which help us to suggest the probable areas of
action of ESTPKs in streptomycetes. Nevertheless, the
complete picture of their exact physiological roles remains
to be revealed.
Gauger, A. K. & Goldstein, L. S. (1993). The Drosophila kinesin light
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