Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 317-325
doi:10.1111/j.1745-7270.2007.00282.x
Comparative Analysis of
Two-component Signal Transduction System in Two Streptomycete Genomes
Wu WEI1,3#, Weihua
WANG2#, Zhiwei CAO3#, Hong YU3, Xiaojing WANG1,
Jing ZHAO3, Hao TAN3, Hao XU3, Weihong JIANG2*,
and Yixue LI1,3*
1
Bioinformation Center, Key Lab of Systems Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Graduate School of the
Chinese Academy of Sciences, Shanghai 200031, China;
2 Laboratory of Molecular
Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, Graduate School of the
Chinese Academy of Sciences, Shanghai 200032, China;
3 Shanghai
Center for Bioinformation Technology, Shanghai 200235, China
Received: November
29, 2006
Accepted: February
27, 2007
This study was
supported by the grants from the National Natural Science Foundation of China (30470029,
30500107), Key Program of Basic Research of Shanghai (No. 06PJ14072), and the
Major State Basic Research Development Program of China (2004CB720103,
2006CB910705, and 2007CB707803)
# These authors
contributed equally to this work
*Corresponding
authors:
Yixue LI: Tel,
86-21-64363311; Fax, 86-21-64838882; E-mail, [email protected]
Weihong JIANG: Tel,
86-21-54924172; Fax, 86-21-54924015; E-mail, [email protected]
Abstract Species of the genus Streptomyces are
major bacteria responsible for producing most natural antibiotics. Streptomyces
coelicolor A3(2) and Streptomyces avermitilis were sequenced in
2002 and 2003, respectively. Two-component signal transduction systems (TCSs),
consisting of a histidine sensor kinase (SK) and a cognate response regulator
(RR), form the most common mechanism of transmembrane signal transduction in
prokaryotes. TCSs in S. coelicolor A3(2) have been analyzed in detail.
Here, we identify and classify the SK and RR of S. avermitilis and
compare the TCSs with those of S. coelicolor A3(2) by computational
approaches. Phylogenetic analysis of the cognate SK-RR pairs of the two species
indicated that the cognate SK-RR pairs fall into four classes according to the
distribution of their orthologs in other organisms. In addition to the cognate
SK-RR pairs, some potential partners of non-cognate SK-RR were found, including
those of unpaired SK and orphan RR and the cross-talk between different
components in either strain. Our study provides new clues for further
exploration of the molecular mechanism for regulation of industrially important
streptomycetes.
Key words Streptomyces; two-component system; cross-talk;
phylogenetic analysis
Two-component signal transduction systems (TCSs), consisting of a
histidine (His) sensor kinase (SK) and a cognate response regulator
(RR), serve as a basic stimulus-response coupling mechanism to allow organisms
to sense and respond to changes in many different environmental conditions in
prokaryotes [1]. They are widespread not only in almost all prokaryotes and
many archaea, but also in some eukaryotes, such as fungi and plants, in which
they play an important role in light and hormone signaling. Most of the SKs are membrane-associated His kinases. Extracellular
stimuli are sensed by the periplasmic domain of the SK, and serve to modulate
the activities of the SK. The SK catalyzes ATP-dependent autophosphorylation of
a specific His residue located in its dimerization domain. The phosphoryl group
subsequently transfers from the phosphohistidine of the SK to a specific
aspartate (Asp) residue within the conserved regulatory domain of the RR.
Phosphorylation of the regulatory domain activates a downstream output domain
that elicits the specific cellular response [2].Streptomyces is a genus of Gram-positive
bacteria. Unlike normal bacteria, streptomycetes have a complex
development life cycle such as mycelial growth and spore formation. To adapt
the particularly complex and variable environment, streptomycetes possess a
broad range of metabolic processes and biotransformations [3,4]. The most
interesting property of streptomycetes is their ability to produce most natural
antibiotics used in human and veterinary medicine and agriculture. Therefore it
is quite essential to understand the biological process of remarkable
morphological differentiation and antibiotic production in streptomycetes [5].
Over 20 different pleiotropic genes influence antibiotic production in Streptomyces
coelicolor A3(2), three of which are TCSs, indicating an important role for
protein phosphorylation and phosphorylation cascades in the regulation of
antibiotic production [6].Two complete genomic sequences of the genus Streptomyces are
now available. S. coelicolor A3(2), the best-known representative of
streptomycetes, was sequenced in 2002 and has an 8.7 Mbp linear chromosome
containing 7825 protein-encoding sequences [3,4]. The second streptomycete
genome, S. avermitilis, was published in 2003 [4]. The linear chromosome
of this genome is just over 9 Mbp, which is larger than that of S.
coelicolor A3(2) but contains fewer
open reading frames (7574 instead of 7825). Comparative analysis of the two
streptomycete genomes revealed that there is a common highly conserved 6.5 Mbp
region with respect to gene order and content [4]. SKs and RRs of S.
coelicolor A3(2) have been analyzed in detail [7]. However, to date, a
detailed analysis of TCSs of S. avermitilis has not been reported. In this study, we attempt to conduct a comparative analysis that
will constitute a basis for further exploration of the signal transduction
systems of streptomycetes.
Materials and Methods
Identification and
classification of SKs and RRs of S. avermitilis
The genome sequences of S. avermitilis (http://avermitilis.ls.kitasato-u.ac.jp/)
were searched against the Interpro database [8] using InterproScan on a local
workstation. The SKs were identified by visual inspection of all the proteins
that contain the ATPase domain [7,10]. According to the alignments of the 16
amino acids around the conserved histidine for each of the five groups of SKs
in Bacillus subtilis [11], we made five hidden Markov models (HMMs)
using the hmmbuild program of the HMMER package Version 2.3.2 [12] (http://hmmer.janelia.org/). The
identified SKs were assigned to five groups by searching the five HMMs using
the hmmpfam program of the HMMER suite (E<10–5). The transmembrane (TM) domains of each SK were acquired using
TopPred II (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html)
[13]. The two best matches for each SK were assumed to be the true TM domains,
and all the residues between the end of the first and the start of the second
TM domain were taken as the sensor domain. Similar to SKs, RRs were identified
by checking all the proteins that contain the CheY-like domain. Alignments of
the C-terminal output domains of S. avermitilis RRs with those of Escherichia
coli RRs revealed the five different groups of RRs [11]. All the functional
domains of SKs and RRs were obtained from the InterproScan results.
Comparison of TCSs in S.
coelicolor A3(2) and S. avermitilis
All the orthologs of the TCSs were retrived through KEGG API (http://www.genome.jp/kegg/soap/;
SOAP interface to KEGG) from KEGG SSDB using best-best relations
(Smith-Waterman score>100). The orthologs of SKs in the two streptomycete
genomes with less than 50% amino acid sequence identity in their sensor domains
were ignored.
Sequence alignment and
phylogenetic analysis
In addition to trans-phosphorylation between cognate SK-RR pairs,
cross-talk in trans-phosphorylation between non-adjacent SK-RR pairs was also
reported [14]. To explore those potential cross-talks in S. coelicolor
A3(2) and S. avermitilis, we inferred functional coupling between SKs
and RRs based on conservation of SK-RR pairs between genomes, which is employed
as a classical computational method to predicting functionally coupled genes
[15]. If orthologs of non-cognate SK and RR in S. coelicolor A3(2) and S.
avermitilis are adjacent in some other organisms, we assumed that the
potential cross-talk in signal transduction might take place between them or
they are a potential pair (Fig. 1). We took those non-cognate SK and RR
pairs with more than 10 support ortholog pairs in other organisms as the
potential pairs or the potential cross-talks of TCSs in either streptomycete
genomes. The multiple alignments were obtained by aligning with the program
CLUSTAL W [16] and phylogenetic trees were constructed using PHYLIP based on
the neighbor-joining algorithm [17] and the bootstrap value was 1000.
Results
Identification of TCSs of S.
avermitilis and comparison with S. coelicolor A3(2)
Sixty-seven SKs were obtained after the visual inspection of S.
avermitilis proteins that contain both the ATPase domain and the conserved
His domain. Similarly, 68 RRs were found by checking the CheY-like domain and
the RR output domain. Of these 67 SKs, 53 are adjacent to RRs (one pair is
assumed), and 14 are unpaired. Besides the 53 paired RRs, there are 15 orphan
RRs as well (Table 1). The sensor domains of SKs vary greatly to sense diverse environment stimuli,
and the ATP domains, conserved in all SKs, do little to differentiate them .
The region around the conserved His, the site of autophosphorylation, proved to
be more informative for classification of SKs. Alignment with the region around
the His that becomes phosphorylated revealed that the SKs of S. avermitilis
fell into the five main groups (I, II, IIIa, IIIb and IV), as defined in B.
subtilis [11]. Similar to S. coelicolor A3(2), most of the SKs of
the S. avermitilis fell into group II and IIIa (32 and 30,
respectively), with only one in group I, one in group IIIb and five in group
IV. The RRs were classified to NarL, OmpR, DBD, LytTR and wHtH groups by the
relatedness of their output domains (Table 1). The largest number of RRs
fell into the NarL group, which are all contain the bacterial regulatory
protein LuxR domain (InterPro IPR000792). It was found that the second largest
number of RRs containing the C-terminal transcriptional regulatory domain
(Trans_reg_C; IPR001867) was related to the OmpR group. LuxR domain and the
C-terminal transcriptional regulatory domain are subfamilies of winged helix
repressor DNA-binding domain (IPR011991), which contains the winged
helix-turn-helix DNA-binding motif. It is notable that SAV6219 contains the
ANTAR domain (IPR005561), which is an RNA-binding domain found in bacterial
transcription antitermination regulatory proteins [18]. This domain has been
detected in various RRs of TCSs and also some one-component sensory regulators
from a variety of bacteria. Most activated RRs interact with DNA to activate or
repress the transcription of a series of genes, however, ANTAR-containing RR
might interact with RNA to resist the termination of special gene
transcription.It is interesting that, as in B. subtilis and S. coelicolor
A3(2), all of the SKs in group II are linked with RRs classified as NarL
family, whereas all of the group IIIa SKs are paired with RRs belonging to the
OmpR family without exception. Two SKs of group IV are paired with RRs
containing a typical winged helix-turn-helix domain, while the SKs of group I and IIIb are unpaired.
The conclusion for the conserved relationship is that the catalytic domain of
the SKs and both domains of the RRs might have evolved as a unit from a common
ancestor [11]. Consistent with this conclusion, the gene orders of SK and RR in
the transcription units are preserved within different classes (Table 1).Most SKs contain a variety of extracellular, intracellular and/or
transmembrane functional domains to respond to specific environmental stimuli.
As a result, there is little sequence similarity in the N-terminal domains of
the SKs. The membrane topology predictions of the SKs indicate that over half
of the SKs have three or more TM domains. Three soluble cytoplasmic proteins
were predicted for those SKs that contain no TM domain. They might be activated
by another SK in the transduction pathway, as for E. coli CheA [19]. The
region between the end of the first and the start of the second TM domain was
taken as the sensor domain (Table 1). The size of the sensor domains
ranges from 3 to 275 amino acids. Eight SKs containing sensor domains of less
than 20 amino acids were predicted to belong to a new subfamily of SKs, which
is almost entirely buried in the cytoplasmic membrane and frequently linked to
ABC transporters. SKs of this new subfamily were speculated to sense changes in
membrane structure or topology [20].PAS and GAF domains, as cytosolic sensing modules, have been found
in a large number of SKs. Three SKs that contain PAS domain (IPR000014) were
found in the S. avermitilis genome. PAS domain proteins function to
detect some signals, such as oxygen, redox potential and light, by binding
flavins, haems, chromophores or some other cofactors [21]. The GAF domain
(IPR003018) is found in six SKs of S. avermitilis. GAF domains appear to
act as binding sites for small ligands that induce the autophosphorylation of
the SK and subsequent signal transduction to activate specific gene
transcription [22]. One SK (SAV6889) was detected that contains the nitrate and
nitrite-sensing domain (IPR010910),
which responds to changes in nitrate and nitrite concentrations.
Phylogenetic analysis of the
TCSs in S. avermitilis and S. coelicolor A3(2) genomes
All the potential pairs of the TCSs in two streptomycete genomes
were analyzed and the numbers of all supported pairs of each potential pair
were counted. The organism distribution of each cognate SK-RR pair and the
support pairs consisting of their orthologs showed that the paired TCSs fell
into four classes: present in most bacteria; present in most actinobacteria;
specific to streptomycetes; and specific to either of the two streptomycete
strains.
KdpD-KdpE of S. coelicolor A3(2), have orthologs in S. avermitilis
and 45 other bacteria. KdpD and KdpE, which regulate the expression of the high
affinity K+ transport system most notably under K+ limiting conditions [23], have been extensively studied in E.
coli and appear to be ubiquitous in most bacteria. Independent phylogenetic
analyses were carried out using amino acid sequences of the regions assigned
for classification of KdpD, KdpE and their orthologs (Fig. 2). The
similarity of the two phylogenetic trees could imply the inherent functional
connection between the catalytic domain of the SKs and both the regulatory and
output domains of the RRs.
In the S. coelicolor A3(2) genome, 12 pairs of TCSs are
present in most actinobacteria, and 42 TCS pairs are specific to
streptomycetes. CutRS, which might play roles in the production of actinorhodin
[24], and ChiRS, which is related to chitinase production [25], might be
specific to streptomycete strains. The TCS pairs specific in streptomycetes
exceed more than those present in most bacteria or in the actinobacteria group,
suggesting that this genus might be well equipped to adapt to a wide range of
environmental stimuli and stresses, and to regulate complex multicellular
development, a broad range of metabolic processes and biotransformation.Phylogenetic analysis using all orthologs of available organisms
revealed that 26 pairs of TCSs are specific to S. coelicolor A3(2). As
mentioned above, the S. coelicolor A3(2) genome contains
31 paired TCSs that are specific compared to the S. avermitilis genome. Of
these paired TCSs, five have orthologs in some other organisms but not in S.
avermitilis. While in the 17 paired TCSs specific to S. avermitilis,
only one pair has orthologs in other bacteria.
Discussion
The basic TCS elements can be combined to produce a His-Asp-His-Asp
phosphorelay. Central to this phosphorelay pathway is a hybrid-type SK that
contains both an SK core and an RR receiver domain in a single protein [26].
Two hybrid-type SKs and their orthologs (SAV1085 with SCO7327, SAV2512 with
SCO5748) were identified in S. avermitilis and S. coelicolor
A3(2). The third hybrid-type SK in S. avermitilis, SAV 5564, has no
ortholog in S. coelicolor A3(2). The complexity of phosphorelay systems
permits the integration of multiple check points and regulatory steps into the
pathway [27]. In addition to the cognate TCS pairs we have discussed in detail,
some potential pairs that are not adjacent in the genome were found by the
ortholog analysis. The SK SAV1990 and RR SAV1988 are not back-to-back in the
S. avermitilis genome. However, our analysis suggested that they are
cognate, as their orthologs (SCO6253 and SCO6254) are adjacent and they accord
with the rule that all SKs falling into group II are linked with an RR
classified to the NarL family (Table 1). Orthologs of S. avermitilis
TCSs, SAV7118 (SK) and SAV7115 (RR) are adjacent in 47 organisms (Table 2),
and orthologs of SAV3017 (SK) and SAV6219 (RR) are adjacent in 10 organisms.
These four TCSs had been supposed to be unpaired SKs or orphan RRs in S.
avermitilis because they did not have an adjacent pair in the genome, but
the cases in other organisms indicated that they might be two paired TCSs.Microarray analysis for the TCS mutants of E. coli has
represented that TCSs functionally interact with each other, at least for
certain combinations, to expand the signal transduction network so as to allow
some genes to respond to a wide range of environmental stimuli [28].
Trans-phosphorylation in vitro was detected between non-cognate SK-RR
pairs in E. coli at a rate of approximately 3% [14], raising the
possibility that the cross-talk in signal transduction takes place between
non-cognate SKs and RRs. In this study, some non-cognate SK-RR pairs were found
in both the S. avermitilis and S. coelicolor A3(2) genomes that
have cognate ortholog SK-RR pairs in other organisms (Table 2),
suggesting that cross-talk might take place between them. TCSs, such as
SCO7534, SCO3741, SCO1136 and SCO1801 in S. coelicolor A3(2), and
SAV2430, SAV2971, SAV4416 and SAV4047 in S. avermitilis might be
inclined to take part in the cross-talk. The streptomycete strain-specific TCSs
have insufficient support pairs, so that the cross-talk referencing to these
TCSs might be omitted. While our results accord with the corollary that if
cross-talk between SK-RR pairs is of regulatory significance, it is likely to
occur only within a group [11]. The identification and classification of the TCSs in the S.
avermitilis genome provide the foundation to understand the signal
transduction system of the strain. Approximately 80% of the commercially
available antibiotics are produced by the streptomycetes, therefore comparison
analysis of the TCSs of the two streptomycete strains can improve our understanding
of the two-component systems of this organism and also provide insight into the
molecular mechanisms of regulation in industrially important strains of
streptomycetes.
Acknowledgements
We are grateful
to Drs. Govind CHANDRA and Matthew I. HUTCHINGS from the John Innes Centre
(Nowich, UK), for their useful discussion and valuable advice.
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