Microbiology (2005), 151, 2159–2173
DOI 10.1099/mic.0.27987-0
Distribution and evolution of multiple-step
phosphorelay in prokaryotes: lateral domain
recruitment involved in the formation of hybrid-type
histidine kinases
Weiwen Zhang and Liang Shi
Microbiology Department, Pacific Northwest National Laboratory, 902 Battelle Blvd,
PO Box 999, Mail Stop P7-50, Richland, WA 99352, USA
Correspondence
Weiwen Zhang
[email protected]
Liang Shi
[email protected]
Received 22 February 2005
Revised 5 April 2005
Accepted 18 April 2005
Although most two-component signal transduction systems use a simple phosphotransfer pathway
from one histidine kinase (HK) to one response regulator (RR), a multiple-step phosphorelay
involving a phosphotransfer scheme of His–Asp–His–Asp was also discovered. Central to this
multiple-step-type signal transduction pathway are a hybrid-type HK, containing both an HK
domain and an RR receiver domain in a single protein, and a histidine-containing phosphotransfer
(HPT) that can exist either as a domain in hybrid-type HKs or as a separate protein. Although
multiple-step phosphorelay systems are predominant in eukaryotes, it has been previously
suggested that they are less common in prokaryotes. In this study, it was found that putative
hybrid-type HKs were present in 56 of 156 complete prokaryotic genomes, indicating that
multiple-step phosphorelay systems are more common in prokaryotes than previously appreciated.
Large expansions of hybrid-type HKs were observed in 26 prokaryotic species, including
photosynthetic cyanobacteria such as Nostoc sp. PCC 7120, and several pathogenic bacteria
such as Coxiella burnetii. Phylogenetic analysis indicated that there was no common ancestor
for hybrid-type HKs, and their origin and expansion was achieved by lateral recruitment of a receiver
domain into an HK molecule and then duplication as one unit. Lateral recruitment of additional
sensory domains such as PAS was also evident. HPT domains or proteins were identified in 32
of the genomes with hybrid-type HKs; however, no significant gene expansion was observed for
HPTs even in a genome with a large number of hybrid-type HKs. In addition, fewer HPTs than
hybrid-type HKs were identified in all prokaryotic genomes.
INTRODUCTION
Micro-organisms must modulate their gene expression
repertoire in order to adapt to changing environments.
One of the predominant signal transduction mechanisms
employed by microbes is the phosphotransfer pathway
commonly referred to as ‘two-component’ signal transduction systems (TCSTSs), which typically consist of a sensor
histidine kinase (HK) and a response regulator (RR), and
have been found across all three domains of life, the Bacteria,
Archaea and Eukarya (Hoch, 2000). The sensor HK, generally an integral membrane protein, consists of a signal
recognition domain, with unique specificity, coupled to an
autokinase domain. In most cases, binding of extracellular
Abbreviations: GAF, cyclic nucleotide-binding domain of GAF; HAMP,
HAMP domain for histidine kinases, adenylyl cyclases, methyl-binding
proteins and phosphatases; HK, histitidine kinase; HPT, histidinecontaining phosphotransfer domain (or protein); PAS, PAS domain; RR,
response regulator; TCSTS, two-component signal transduction system.
0002-7987 G 2005 SGM
signalling molecules to the signal recognition domain
causes activation of the autokinase domain, resulting in
phosphorylation of a conserved histidine (His) residue on
the His-containing subdomain of the autokinase. The
His-containing subdomain is associated with the receiver
domain of the cognate RR, to which another phosphotransfer to an aspartate (Asp) on the RR occurs. Regulator
domains normally inhibit the output domain of RRs and
phosphorylation relieves this inhibition, freeing the output
domain to carry out its function, which is usually transcription activation (Stock et al., 1989; Parkinson & Kofoid,
1992).
In addition to the prototypical TCSTSs described above, a
more complex version of this phosphotransfer scheme was
also discovered in both prokaryotic and eukaryotic cells
(Appleby et al., 1996). This system involves multiple phosphotransfer steps with often more than two proteins. Three
types of multiple-step phosphorelay systems have been
revealed. The first pathway is exemplified by the system that
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2159
W. Zhang and L. Shi
governs the initiation of sporulation in Bacillus subtilis. This
phosphorelay cascade begins with the autophosphorylation
of one of the three sensor kinases, KinA, KinB or KinC. The
phosphoryl group is then transferred to a receiver domain
in the regulator Spo0F. The Spo0F then serves as a phosphodonor for Spo0B, which is phosphorylated on a His
residue. Finally, the phosphoryl group completes its course
by transfer to an Asp in Spo0A (Burbulys et al., 1991). The
second and third type of multiple-step phosphorelay involve
a hybrid-type HK in which both the HK domain and the
RR receiver domain are present within a single protein. The
second type has an intermediate His-containing phosphotransfer (HPT) domain in the same molecule, whereas in
the third type, the HPT domain is contained on a separate
protein. In the hybrid-type HK systems the phosphoryl
group is first transferred from a His to an Asp residue within
the hybrid HKs, then through the HPT domain or protein
and is subsequently transferred to a cytoplasmic RR (Fig. 1).
It has also been suggested that protein phosphatases, such as
the sixA gene in Escherichia coli, may be implicated in the
His–Asp phosphorelay through regulating the phosphorylation state of the HPT domain (Ogino et al., 1998). There
are three well-studied cases where these hybrid HK mechanisms are utilized. The first is the BvgS–BvgA system
controlling the transcriptional regulation of virulence factors in Bordetella pertussis, in which the BvgS protein
contains the HK domain, the receiver domain and the HPT
domain (Uhl & Miller, 1996) (Fig. 1a). The second is the
Sln1p–Ypd1p–Ssk1p system governing osmoregulation in
the yeast Saccharomyces cerevisiae. In this system the HPT
domain is contained on Ypd1p, a separate protein of 167
residues (Posas et al., 1996) (Fig. 1b). The third is the RcsC–
YojN–RcsB signalling pathway, implicated in capsular
synthesis and swarming behaviour in E. coli (Takeda et al.,
2001; Clarke et al., 2002). In this system, the HPT domain is
present at the C terminus of the protein YojN, which shows a
similarity to RcsC, particularly in the HK domain, although
the crucial autophosphorylation His site is missing (Takeda
et al., 2001; Clarke et al., 2002) (Fig. 1c). Early results from
the analysis of HK domain architecture from a limited
number of prokaryotic and eukaryotic genomes showed that
most eukaryotic HKs are of the hybrid type, while only a
small proportion of prokaryotic HKs contain both the
kinase and receiver domains in a single HK molecule. It has
thus been suggested that TCSTSs in prokaryotes generally
use a simple two-component phosphotransfer scheme,
whereas phosphorelays and hybrid HKs dominate twocomponent signalling in eukaryotes (West & Stock, 2001;
Oka et al., 2002; Catlett et al., 2003). However, in recent
years more hybrid-type HKs have been identified from
various bacterial genomes (Xu et al., 2003; Rabus et al., 2004;
W. Zhang and others, unpublished data), suggesting that the
Fig. 1. Schemes of multiple-step phosphorelay signal transduction systems containing a hybrid-type HK and an HPT protein.
(a) The scheme with hybrid-type HK containing all autokinase, receiver and HPT domains in one protein, as in the case of the
BvgS–BvgA system of Bortedella pertussis; (b) the scheme with a separate HPT protein as in the case of Sln1p–Ypd1p–
Ssk1p system of the yeast Sac. cerevisiae; (c) the scheme with an HPT domain located in another protein as in the case of
RcsC–YojN–RcsB of E. coli.
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Microbiology 151
Evolution of multiple-step phosphorelay in prokaryotes
role of multiple-step phosphorelay systems in prokaryotes
might have been underestimated.
TCSTSs were first identified decades ago and several model
systems, including sporulation in Bac. subtilis, chemotaxis
and osmoregulation in E. coli, have been extensively studied
(Forst et al., 1989; Bourret et al., 1989; Leonardo & Forst,
1996; Stephenson & Hoch, 2002). However, until recently,
few studies have been conducted on their origin and evolution. In an extensive study conducted recently, the TCSTSs
from 14 complete and six partial genomes were phylogenetically analysed by distance methods (Koretke et al., 2000).
The results suggested that the TCSTSs are of bacterial origin,
and may share a common ancestor with heat-shock protein
Hsp90, DNA-mismatch repair protein MutL and type II
topoisomerases (Dutta et al., 1999). Phylogenetic analysis also indicated that the hybrid-type HKs in eukaryotes
were acquired from bacteria through lateral gene transfer
(Koretke et al., 2000). However, little information is available on how hybrid-type HKs evolved in bacteria other than
that they cluster together in phylogenetic trees of HKs
(Koretke et al., 2000). With the progress of bacterial genome
sequencing programmes, more than 150 microbial genomes
from almost all major phylogenetic lineages have been fully
sequenced. The availability of complete genome sequences
from numerous microbial species allows us to perform a
detailed evaluation of the presence of multiple-step phosphorelay systems in prokaryotes and their evolutionary
relationships, which was not previously possible. In this
paper, we describe the results of a detailed survey and
phylogenetic analysis of the hybrid-type HK and HPT
proteins involved in multiple-step phosphorelay systems
from 156 complete genomes. Our survey results show that
more than one-third of bacterial genomes possessed hybridtype HKs. In addition, the hybrid-type HKs were also
identified from several archaeal genomes (Koretke et al.,
2000; Kim & Forst, 2001). The results indicated that the
role of multiple-step phosphorelay systems in prokaryotes
may have been underestimated. Phylogenetic analysis indicated that there was no common ancestor for hybrid-type
HKs, and that their origin and expansion were achieved by
lateral recruitment of a receiver domain into an HK molecule and then duplication as one unit. In addition, survey and
phylogenetic analysis of HPT proteins from prokaryotic
sources were performed to infer their evolutionary course.
METHODS
Identification of hybrid-type HKs. Sequences of all of the puta-
tive HKs from 156 complete microbial genomes (as of 22 September
2004) were extracted from the Comprehensive Microbial Resource
(CMR) database of the Institute for Genomic Research (TIGR)
(http://www.tigr.org), and then subjected to domain identification using the molecular architecture research tools provided by
SMART (http://smart.embl-heidelberg.de/) with an E value of <0?01
(Letunic et al., 2002). The Che protein kinases involved in chemotaxis were excluded from this study because they have different
domain constitution and organization where their His-containing
subdomain has lost the catalytic His and is used exclusively for
http://mic.sgmjournals.org
dimerization, while a HPT domain that may have originally served
a regulatory function is now used for phosphorelay to CheY and
CheB (Dutta et al., 1999; Koretke et al., 2000). A hybrid-type HK
was determined by the presence of complete kinase and receiver
domains in a single protein according to the definition by Catlett
et al. (2003). The protein sequences of kinase domains (including
both autokinase and His-containing subdomains), and the receiver
domains from RR, HPT and various sensory domains were extracted
separately. To confirm tentatively identified domains, the protein
sequences of these domains were used to search for conserved
domains from the Conserved Domain Databases, Pfam and COG
using BLAST with a cut-off E value of <0?01 (Marchler-Bauer et al.,
2003). In addition, visual inspection was used to eliminate domains
that lacked the minimum complement of conserved sequence features considered necessary for each type of domain (Stock et al.,
1989; Kato et al., 1997; Taylor & Zhulin, 1999).
Identification of HPT. All known HPT proteins from micro-
bial sources, along with HPT domains from proteins identified as
hybrid-type HKs by SMART in this study, were used as query
sequences in two separate searches for HPT gene homologues. The
first search was performed against the 156 complete microbial
genomes contained in the OMINOME Pep database of TIGR
using BLASTP (http://tigrblast.tigr.org/cmr-blast/) and the second was
against the NCBI sequence database using BLASTP (http://www.ncbi.
nih.gov/blast). Both searches used an E value threshold of <0?01.
Sequence alignment and phylogenetic analysis. Sequence
alignments were performed using the default parameters of the
program originally developed by Higgins & Sharp (1988),
available from the LaserGene software package (DNAStar) and
PAUP* 4.0 beta version (Blumenberg, 1988) with an alignment gap
penalty of 10?00 and a gap length penalty of 0?1. Confidence levels
were determined by analysing 100 bootstrap replicates. For phylogenetic classification of kinase and receiver domains, functional
domains of all known HKs and RRs in E. coli and Bac. subtilis were
extracted and used as indicators for each phylogenetic group according to the method described previously (Koretke et al., 2000).
CLUSTALW
RESULTS
Distribution of hybrid-type HKs in prokaryotic
genomes
From 156 complete microbial genomes listed in the CMR of
TIGR (representing 18 archaea and 138 bacteria as of 22
September 2004), a total of 505 protein sequences encoding putative hybrid-type HKs were identified from a total
of 2041 putative HKs present in the genomes surveyed.
Hybrid-type HKs were more widely spread in prokaryotes
than previously expected, with representatives found in four
archaeal and 52 bacterial genomes (Table 1). Genes encoding hybrid-type HKs have previously been identified in
Bacteria and Eukarya (Koretke et al., 2000; Kim & Forst,
2001; West & Stock, 2001). Our survey revealed that
putative hybrid-type HKs were also present in four of 18
archaeal genomes, with Met. acetivorans containing five, and
Halobacterium sp. NRC-1 containing three hybrid-type
HKs, Met. mazei Goe1 containing two, and Arc. fulgidus
DSM 4304 containing one. Among the Bacteria surveyed,
the largest number of hybrid-type HKs were found in the
Nostoc sp. PCC 7120 genome (49 of 122 putative HKs);
followed by Bact. thetaiotaomicron (41 hybrid-type HKs of
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2161
Species
Archaea
Methanosarcina acetivorans
Halobacterium sp. NRC-1
Methanosarcina mazei Goe1
Archaeoglobus fulgidus DSM 4304
Bacteria
Nostoc sp. PCC 7120
Total Hybrid Percentage
HK*
HK
HPT domain
Inside HK Independent
molecule HPT protein
Additional domains
PAS
GAF
HAMP
–
64
10
28
23
5
3
2
1
7?8
30?0
7?1
4?3
1
–
–
–
–
–
–
–
12
13
4
–
–
2
–
–
–
–
122
49
40?2
3
–
48
18
12
85
41
48?2
–
–
–
–
–
CharacteristicsD
An extreme halophile
Formerly known as Anabaena sp. PCC 7120; a multicellular
cyanobacterium that forms heterocysts to fix nitrogen
Dominant member of human normal distal intestinal
microbiota
Nitrogen-fixing symbiotic bacterium
Microbiology 151
Bacteroides thetaiotaomicron
VPI 5482
Bradyrhizobium japonicum
USDA 110
Caulobacter crescentus CB15
90
28
31?1
1
–
15
1
14
55
25
45?5
–
–
9
3
–
Geobacter sulfurreducens PCA
88
22
25?0
1
1
11
4
3
Pseudomonas syringae DC3000
Desulfovibrio vulgaris Hildenborough
Xanthomonas axonopodis pv. citri 306
Vibrio vulnificus CMCP6
Rhodopseudomonas palustris CGA009
64
59
61
47
66
20
19
19
19
19
31?3
32?2
31?1
40?4
28?8
2
–
–
4
–
1
1
–
4
1
7
–
8
4
7
1
–
3
1
2
2
–
–
5
–
Xanthomonas campestris pv.
campestris ATCC 33913
Pseudomonas putida KT2440
Pseudomonas aeruginosa PAO1
Vibrio parahaemolyticus RIMD
2210633
Pirellula sp. strain 1
63
18
28?6
4
–
9
3
–
Grows in a dilute aquatic environment; coordinates the cell
division cycle and multiple cell differentiation events
Multiple metabolic capabilities, e.g. aerobic metabolism,
one-carbon and complex carbon metabolism, motility
and chemotactic behaviour
Phytopathogen to tomato and Arabidopsis thaliana
Ability to reduce pollutants such as uranium and chromium
Bacterial phytopathogen causing citrus canker
Pathogenic bacterium
The most metabolically versatile bacteria known; uses light,
inorganic compounds or organic compounds, for energy;
acquires carbon from many types of green plant-derived
compounds or by carbon dioxide fixation, and fixes
nitrogen
Bacterial phytopathogen causing citrus canker
59
58
33
17
16
16
28?8
27?6
48?5
3
5
2
1
3
3
2
6
3
2
–
–
2
1
3
Metabolically versatile soil bacterium
Pathogenic bacterium
Worldwide cause of food-borne gastroenteritis
39
16
41?0
4
1
22
2
–
43
12
27?9
–
–
4
1
–
A marine, aerobic, heterotrophic representative of the
globally distributed and environmentally important
bacterial order Planctomycetales
Pathogenic bacterium
Leptospira interrogans serovar Lai str.
56601
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W. Zhang and L. Shi
2162
Table 1. List of prokaryotic genomes with hybrid-type HKs
http://mic.sgmjournals.org
Table 1. cont.
Species
Total Hybrid Percentage
HK*
HK
HPT domain
Inside HK Independent
molecule HPT protein
Additional domains
PAS
GAF
HAMP
37
37
11
10
29?7
27?0
3
2
–
1
9
–
4
–
–
1
Mesorhizobium loti MAFF303099
Agrobacterium tumefaciens C58
Magnetococcus sp. MC-1
Chromobacterium violaceum ATCC 12472
Sinorhizobium meliloti 1021
Shewanella oneidensis MR-1
Wolinella succinogenes DSM 1740
Chlorobium tepidum TLS
42
42
47
30
42
34
36
8
8
8
7
7
7
7
6
6
19?0
19?0
14?9
23?3
16?7
20?6
16?7
75?0
–
–
5
4
–
2
1
–
1
1
1
–
1
–
–
–
5
7
5
6
2
5
1
–
4
–
2
1
–
–
1
–
8
–
5
1
1
2
–
–
Yersinia pestis
Vibrio cholerae el tor N16961
Gloeobacter violaceus PCC 7421
Escherichia coli
Burkholderia mallei ATCC 23344
Coxiella burnetii RSA 493
Shigella flexneri 2a 2457T
Thermosynechococcus elongatus BP-1
Xylella fastidiosa Temecula1
Salmonella enterica serovar Typhi CT18
Deinococcus radiodurans R1
Salmonella typhimurium LT2 SGSC1412
Streptomyces avermitilis
Ralstonia solanacearum GMI1000
Prevotella intermedia 17
Brucella suis 1330
Wolbachia pipientis wMel
Borrelia burgdorferi B31
Porphyromonas gingivalis W83
Treponema denticola ATCC 35405
Bordetella bronchiseptica RB50
Bordetella parapertussis 12822
Brucella melitensis 16M
Nitrosomonas europaea ATCC 19718
Clostridium tetani E88
17
25
40
29
33
7
17
18
9
13
17
19
72
26
6
17
2
4
6
8
9
12
16
16
24
6
6
6
5
4
4
4
3
3
3
3
3
3
2
2
2
1
1
1
1
1
1
1
1
1
35?3
24?0
15?0
17?2
12?1
57?1
23?5
16?7
33?3
23?1
17?6
15?8
4?2
7?7
33?3
11?8
50?0
25?0
16?7
12?5
11?1
8?3
6?3
6?3
4?2
3
3
–
4
–
1
3
2
1
–
–
4
–
1
–
–
–
–
–
–
–
–
–
–
–
–
2
1
–
–
1
–
1
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
1
2
4
1
3
–
1
3
–
1
–
1
2
1
–
–
–
–
–
–
1
1
–
1
4
–
–
3
–
–
–
–
4
–
–
–
–
2
–
–
–
–
–
–
–
1
1
–
–
–
2
2
4
2
–
–
2
–
–
2
–
1
13
–
–
–
–
–
–
–
–
–
–
1
–
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Unicellular cyanobacterium
A predatory Gram-negative bacterium that invades and
consumes other Gram-negative bacteria
Capable of performing anoxygenic photosynthesis by the
reductive tricarboxylic acid cycle
Pathogenic bacterium
Pathogenic bacterium
Phytopathogen causing citrus canker
Pathogenic bacterium
Obligate intracellular bacterium of Drosophila melanogaster
Pathogenic bacterium
Evolution of multiple-step phosphorelay in prokaryotes
2163
Synechocystis sp. PCC 6803
Bdellovibrio bacteriovorus HD100
CharacteristicsD
2164
*Number not including Che kinases.
DThe prokaryotes with more than 25 % of their HKs as hybrid-type or more than 10 hybrid-type HKs in single genome are marked in bold and their characteristics presented.
dData from West & Stock (2001) and Catlett et al. (2003).
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
9
11
3
16
21
20
11
1
11
11
3
16
21
20
11
100?0
81?8
100?0
100?0
100?0
100?0
100?0
100?0
–
–
–
–
–
–
–
–
1
5
1
1
1
1
1
1
–
–
–
–
–
–
–
–
5
1
–
1
Streptomyces coelicolor A3(2)
Eukaryotesd
Saccharomyces cerevisiae
Arabidopsis thaliana
Dictyostelium discoideum
Schizosaccharomyces pombe
Gibberella moniliformis
Cochliobolus heterostrophus
Botryotinia fuckeliana
Neurospora crassa
76
1?3
–
–
GAF
PAS
Independent
HPT protein
Inside HK
molecule
Hybrid
HK
Total
HK*
Species
Table 1. cont.
Percentage
HPT domain
Additional domains
HAMP
CharacteristicsD
W. Zhang and L. Shi
85 putative HKs). By proportion, Chl. tepidum was found
to contain the largest percentage of hybrid-type HKs (six
of eight HKs; 75 %), followed by the Cox. burnetii genome
(four of seven HKs; 57 %) (Table 1).
Among the prokaryotic genomes containing hybrid-type
HKs, more than half possessed fewer than five hybrid-type
HKs. However, 26 bacterial species were identified that
contained unexpectedly large numbers of hybrid-type HKs
(more than 10 in each genome or more than 25 % of all
HKs in the genome) (Table 1). This group of species with
enriched hybrid-type HKs consists of Bact. thetaiotaomicron
and Bra. japonicum involved in bacterial–eukaryotic interactions (Xu et al., 2003, 2004; Hagiwara et al., 2004),
photosynthetic cyanobacteria, such as Nostoc sp. PCC
7120 (Kaneko et al., 2001) and Synechocystis sp. PCC 6803
(Kaneko et al., 1996), and several bacterial species that
possess versatile metabolic capabilities, such as Geo. sulfurreducens, which is capable of both anaerobic and aerobic
respiration, one-carbon and complex carbon metabolism,
motility and chemotactic behaviour (Methe et al., 2003),
and Des. vulgaris, with an extraordinary ability to reduce
(and bioremediate) multiple pollutants, including uranium
and chromium (Heidelberg et al., 2004).
Phylogenetic analysis of kinase and receiver
domains of hybrid-type HKs
The finding that hybrid-type HKs were unevenly distributed
across microbial species leads to several immediate questions. First, did all hybrid-type HKs share the same ancestor or were they formed as the result of lateral events
independently occurring in each species under specific
selective pressure? Second, in 26 bacterial genomes with
large numbers of hybrid-type HKs, what evolutionary
mechanism was involved in their expansion? Is the same
mechanism shared by all species? To address these questions, independent phylogenetic analyses were performed
using sequences of functional kinase domains from HKs,
and using the functional receiver domains from RRs,
respectively. To help define the phylogenetic subfamilies to
which these domains belong, the functional domains of
all known TCSTSs from E. coli and Bac. subtilis were also
extracted and included in the phylogenetic analysis. Phylogenetic trees of kinase and receiver domains were generated
for each species from the aligned sequences and the confidence of the tree topology was evaluated.
In an earlier study (Koretke et al., 2000), the phylogeny
constructed from a limited number of genomes showed that
hybrid-type HKs were clustered together in one clade. In
addition, they all shared the same root in the phylogenetic
tree, implying that the hybrid-type HKs may have been
generated before the divergence of microbial species, and
that the kinase and receiver domains then evolved as a single
unit into the present-day hybrid-type HKs. The group was
thus conveniently named as ‘Hybrid’ phylogenetic group
(Pao & Saier, 1997; Koretke et al., 2000).
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Microbiology 151
Evolution of multiple-step phosphorelay in prokaryotes
In this study, we included all the kinase and receiver
domains from the hybrid-type HKs we identified, and were
therefore able to perform phylogenetic analyses in more
detail. The domains were assigned to several known phylogenetic subfamilies (Cit, Nar, Ntr, Pho and Hybrid groups)
according to their clustering characteristics. For those
domains not showing a clear clustering pattern with any
of the above subfamilies, they were classified as the ‘Other’
category. The results showed that although most of the
kinase and receiver domains belonged to the ‘Hybrid’
phylogenetic subfamily, some of them were clustered into
the Ntr phylogenetic subfamily (containing systems regulating nitrogen assimilation, acetoacetate metabolism and
hydrogenase activity in E. coli) (Stoker et al., 1989), and the
Pho phylogenetic subfamily (containing systems involved
in phosphate regulation, virulence, osmoregulation and
anaerobic nitrite reduction in E. coli) (Stock et al., 1989),
suggesting that the members of hybrid-type HKs could be
phylogenetically different.
Further analysis showed that the kinase and receiver
domains from the same hybrid-type HKs were not necessarily located in the corresponding phylogenetic subfamily.
For example, in Nostoc sp. PCC 7120, 49 kinase domains
were clustered into Cit (1, number of kinase domains), Ntr
(11), Pho (4), ‘Hybrid’ (29) and ‘Other’ (4) subfamilies,
while its 59 receiver domains were clustered into Ntr (21),
Pho (1), ‘Hybrid’ (32) and ‘Other’ (5) subfamilies, respectively (Table 2). In Bact. thetaiotaomicron all 41 kinase
domains were clustered into the ‘Hybrid’ clade, but only 8
of the receiver domains located to their cognate phylogenetic clade, while the other 33 actually belonged to the Pho
phylogenetic subfamily (Table 2). Examination of our data
suggests that species from the same genus may have similar,
but not identical, patterns of their HK and receiver domains
in term of the subfamilies to which they belong. This is
exemplified in the cases of Xan. axonopodis and Xan.
campestris, or of Ps. aeruginosa, Ps. putida and Ps. syringae.
Differences become even more obvious when comparing
species across higher classification groups to determine to
which subfamilies their domains belong. For example, two
cyanobacteria have very different phylogenetic origination
patterns for their kinase and receiver domains: in Nostoc
sp. PCC 7120 the HK and receiver domains are mainly from
the ‘Hybrid’ and Ntr phylogenetic groups, while those in
Synechocystis sp. PCC 6803 are mainly from the ‘Hybrid’ and
Pho phylogenetic groups (Table 2). These results suggest
that hybrid-type HKs might not have originated from a
common ancestor and that domain recruitment events
occurred as lateral events during evolution. In addition, we
found that some of the hybrid-type HKs contain more than
one receiver domain (Table 2).
Several types of bacteria with more complex metabolic
activities and/or complex cell–cell interactions possessed a
larger number of hybrid-type HKs. To explore the evolutionary mechanism operating in these bacteria, detailed
phylogenetic analysis of kinase and receiver domains from
http://mic.sgmjournals.org
each species was performed. The results from Bra. japonicum are presented as an example (Fig. 2). There are 28
hybrid-type HKs in Bra. japonicum and domain phylogeny analysis showed that they can be divided into five
categories: Ntr (kinase domain)–Hybrid (receiver domain),
Hybrid–Hybrid, Hybrid–Cit, Hybrid–Ntr and an Unknown–
Unknown domain combination. The Ntr–Hybrid, Hybrid–
Hybrid and Hybrid–Cit types were found to be the major
categories and contained 14, six and six hybrid-type HKs,
respectively (Fig. 2). Most hybrid-type HKs in the same
category shared very high sequence similarity for both their
kinase and receiver domains. This implies that each category
might be a result of a single domain recruitment event at an
early evolutionary stage, and that the genes for the hybrid
proteins were then duplicated and mutated to provide new
functional specializations (Fig. 2). Using the same approach, phylogenetic analysis of the genomes with large
numbers of hybrid-type HKs, including Nostoc sp. PCC
7120, Cau. crescentus CB15 and V. vulnificus CMCP6, was
performed and the results showed that a similar mechanism
was operating in the expansion of hybrid-type HKs in these
species as well.
Evolution of HPT proteins/domains
With both kinase and receiver domains in a single protein, one obvious question is whether hybrid-type HKs can
act directly on target proteins in a manner similar to the
chemotaxis paradigm (Koretke et al., 2000). However, the
analysis of protein sequences of hybrid-type HKs showed
that, except in Bact. thetaiotaomicron, where 32 hybridtype HKs were found to contain a putative DNA-binding
domain in addition to the kinase and receiver domains
and might be able to accomplish all the signal transduction processing within one protein (Xu et al., 2004), no
DNA-binding domain was detected in any hybrid-type HKs
from other bacterial species, suggesting that there must
be intermediate proteins that can transfer signal to their
cognate RRs. Although the possibility cannot be excluded
that the hybrid-type HKs can also work other types of
intermediate proteins, so far almost all hybrid-type HKs
from both prokaryotic and eukaryotic sources worked
with an intermediate HPT protein/domain, which mediates
phosphotransfer reaction from the receiver domain in
hybrid-type HKs to their cognate RRs (Kato et al., 1997;
Chang & Stewart, 1998; West & Stock, 2001; Catlett et al.,
2003) (Fig. 1). A total of 67 HPT domains were identified
from hybrid-type HKs by SMART using an E value of <0?1.
These sequences, along with previously identified bacterial HPT proteins, were used as queries to search multiple
protein databases using an E value threshold of <0?01. The
approach identified 26 separate HPT proteins from 156
prokaryotic genomes (Table 1). The results showed that
among the 56 genomes with hybrid-type HKs, only 32
possessed HPT(s). Surprisingly, the genomes without any
identifiable HPTs included those with a large number of
hybrid-type HKs, such as Bact. thetaiotaomicron and Cau.
crescentus. In addition, the number of HPTs found in each
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2165
Species
Cit
Nar
Ntr
Pho
Hybrid
Other
Total*
Kinase Receiver Kinase Receiver Kinase Receiver Kinase Receiver Kinase Receiver Kinase Receiver Kinase Receiver
Microbiology 151
Nostoc sp. PCC 7120
Bacteroides thetaiotaomicron VPI 5482
Bradyrhizobium japonicum USDA 110
Caulobacter crescentus CB15
Geobacter sulfurreducens PCA
Pseudomonas syringae DC3000
Desulfovibrio vulgaris Hildenborough
Xanthomonas axonopodis pv. citri 306
Vibrio vulnificus CMCP6
Rhodopseudomonas palustris CGA009
Xanthomonas campestris pv. campestris ATCC 33913
Pseudomonas putida KT2440
Vibrio parahaemolyticus RIMD 2210633
Pirellula sp. strain 1
Pseudomonas aeruginosa PAO1
Leptospira interrogans serovar Lai str. 56601
Synechocystis sp. PCC 6803
Bdellovibrio bacteriovorus HD100
Mesorhizobium loti MAFF303099
Agrobacterium tumefaciens C58
Magnetococcus sp. MC-1
Chromobacterium violaceum ATCC 12472
Sinorhizobium meliloti 1021
Shewanella oneidensis MR-1
Wolinella succinogenes DSM 1740
Chlorobium tepidum TLS
Yersinia pestis
Vibrio cholerae el tor N16961
Gloeobacter violaceus PCC 7421
Escherichia coli
Methanosarcina acetivorans
Burkholderia mallei ATCC 23344
Coxiella burnetii RSA 493
Shigella flexneri 2a 2457T
Halobacterium sp. NRC-1
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
7
–
–
–
–
2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
1
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11
–
15
2
11
7
6
5
–
8
5
6
2
–
–
2
–
–
1
3
–
–
2
–
1
5
1
–
–
–
–
–
–
–
–
21
–
–
–
–
–
–
2
1
3
7
1
–
–
–
–
–
–
–
1
–
–
–
2
–
5
–
–
–
–
1
–
–
–
3
4
–
–
2
4
3
–
1
2
1
1
2
5
4
1
5
–
2
–
–
–
–
–
–
–
–
–
–
–
1
1
–
–
3
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1
33
–
–
2
–
2
–
2
2
1
1
1
1
–
–
2
–
–
3
–
–
1
–
–
–
–
1
–
–
5
2
–
–
–
29
41
12
21
7
10
13
13
17
8
12
11
12
11
12
8
6
10
5
4
7
7
5
6
5
–
5
5
6
5
4
2
4
4
–
32
8
22
25
9
23
21
19
17
11
13
18
14
15
16
7
10
10
8
7
2
8
4
5
5
1
6
6
8
5
1
4
4
4
–
4
–
1
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
1
–
–
–
–
–
–
–
5
–
1
–
12
–
–
2
3
3
2
2
2
1
–
5
2
–
4
–
2
1
3
2
3
–
–
–
1
–
–
–
–
–
–
49
41
28
25
22
20
19
19
19
19
18
17
16
16
16
12
11
10
8
8
7
7
7
7
6
6
6
6
6
5
5
4
4
4
3
59
41
31
25
23
23
23
25
23
19
23
22
17
17
16
13
14
10
12
11
9
9
8
9
8
6
6
7
9
5
7
6
4
4
3
W. Zhang and L. Shi
2166
Table 2. Phylogenetic origin of kinase and receiver domains of hybrid HKs from bacteria
http://mic.sgmjournals.org
Table 2. cont.
Species
Cit
Kinase
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Ntr
Pho
Hybrid
Other
Total*
Receiver
Kinase
Receiver
Kinase
Receiver
Kinase
Receiver
Kinase
Receiver
Kinase
Receiver
Kinase
Receiver
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
1
–
1
–
–
–
1
1
1
–
–
1
–
–
–
–
2
–
–
–
–
1
–
–
–
–
–
–
–
–
–
1
–
–
–
2
–
1
–
1
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
–
–
–
–
1
–
1
1
–
–
–
–
–
2
1
3
1
3
2
2
2
–
2
–
1
1
1
–
–
–
1
–
–
1
3
2
3
1
2
2
3
–
–
4
–
1
–
–
–
–
–
1
–
–
1
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
1
–
2
3
1
–
–
1
–
1
–
–
2
–
–
1
–
–
1
–
3
3
3
3
3
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
3
3
3
5
5
3
3
2
2
4
1
1
1
2
1
1
1
1
1
1
1
*In several cases, multiple receiver domains within one hybrid-type HK were identified.
2167
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Evolution of multiple-step phosphorelay in prokaryotes
Salmonella enterica serovar Typhi CT18
Deinococcus radiodurans R1
Salmonella typhimurium LT2 SGSC1412
Xylella fastidiosa Temecula1
Thermosynechococcus elongatus BP-1
Streptomyces avermitilis
Ralstonia solanacearum GMI1000
Prevotella intermedia 17
Brucella suis 1330
Methanosarcina mazei Goe1
Wolbachia pipientis wMel
Borrelia burgdorferi B31
Porphyromonas gingivalis W83
Treponema denticola ATCC 35405
Bordetella bronchiseptica RB50
Bordetella parapertussis 12822
Brucella melitensis 16M
Nitrosomonas europaea ATCC 19718
Archaeoglobus fulgidus DSM 4304
Clostridium tetani E88
Streptomyces coelicolor A3(2)
Nar
W. Zhang and L. Shi
Fig. 2. Phylogenetic analysis of the kinase and receiver domains from Bra. japonicum. Different phylogenetic groups are
indicated by colours and labels beside the clades. The definition of each phylogenetic subfamily was according to Koretke
et al. (2000). The domain sequences isolated from E. coli are those with protein ID starting with ‘Ec’, followed by protein ID.
The domain sequences isolated from Bac. subtilis are those with protein ID starting with ‘Bs’, followed by protein ID. The
domain sequences isolated from Bra. japonicum are those with protein ID starting with ‘BLL’ or ‘BLR’, followed by protein ID.
The kinase and receiver domains located in the same hybrid-type HKs in Bra. japonicum are indicated by lines linking the
phylogenetic trees. The scale bar represents 0?1 expected amino acid replacements per site.
individual genome, ranging from one to eight, is smaller
than that of hybrid-type HKs, and no evidence of significant duplication of HPTs was found even in the genomes
where hybrid-type HKs were significantly duplicated
(Table 1).
Sequence alignments of HPT domains from hybrid-type
HKs or from separate HPT proteins were performed using
the CLUSTALW program (Higgins & Sharp, 1988). The result
showed that overall sequence similarity between various
HPTs was low. The relatively conserved region was a region
2168
of approximately 30 aa in length starting from the forty-first
amino acid in the N termini (the number shown in the case
of TLR0349 from The. elongatus) (Fig. 3), consistent with
an early study with a limited number of HPT sequences
(Rodrigue et al., 2000). Histidine H44 is the only residue
conserved through all HPTs, although several other residues, lysine K47, glycine G48, glycine G54 and glutamic acid
E66 were also conserved in most HPTs (the number shown
in the case of TLR0349) (Fig. 3). Phylogenetic analyses were
conducted with the HPTs identified (Fig. 4). Although the
HPT domains/proteins showed less than 20 % sequence
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Microbiology 151
Evolution of multiple-step phosphorelay in prokaryotes
Fig. 3. Sequence alignments of HPT
domains of prokaryotic sources. (a) Alignment of sequences of HPT domains located
within hybrid-type HK protein. (b) Alignment
of sequences of HPT domains located in
separate HPT proteins. The sequences are
indicated by their protein ID and species
name. The conserved residues are shaded.
identity, several recognizable clusters can be identified.
However, no obvious correlation between the distribution
of HPTs in each cluster and their taxonomic relationship was found, suggesting that the sequence diversification resulted mainly from specialization of function rather
than bacterial speciation. All HPTs shared a single root in
the phylogenetic tree, suggesting that there is a common
ancestor for HPTs. This result is consistent with a previous
observation that all HPTs share a common structural motif
and active site (Kato et al., 1997; Xu & West, 1999).
The HPT proteins from eukaryotes were grouped as a
single subgroup within one cluster of bacterial HPTs, rather
than clustering as a sister group of bacterial HPT (Fig. 4).
This indicates that HPTs are likely to have originated in
bacteria and then later radiated into the eukaryotic lineage through horizontal gene transfer event(s) before the
divergence of eukaryotic species. This finding is consistent with the previously proposed hypothesis that eukaryotic TCSTSs evolved through horizontal transfer of
bacterial hybrid-type HKs (Pao & Saier, 1997; Koretke
et al., 2000).
http://mic.sgmjournals.org
Phylogenetic analysis of additional sensory
domains in hybrid-type HKs
The analysis showed that a fraction of prokaryotic
hybrid-type HKs contained sensory domains (Table 1).
Three types of sensory domains were most frequently found
in these hybrid-type HKs: PAS domains that bind flat
heterocyclic molecules such as haem and flavin and are
involved in sensing energy-related environmental factors
such as oxygen, redox potential or light (Taylor & Zhulin,
1999; Taylor et al., 1999); GAF domains involved in binding cyclic nucleotides (Aravind & Ponting 1997); and the
HAMP domain that is often found in various HKs,
adenylyl cyclases, methyl-binding proteins and phosphatases (Galperin et al., 2001). A total of 245 PAS domain
sequences (mean of 65–100 aa in length) were identified
from hybrid-type HKs in prokaryotes using the SMART
program. These sequences were then used in the construction of a phylogenetic tree. To help in the classification and
definition of each phylogenetic cluster, a few dozen PAS
domains with known function, obtained from other bacterial sources, were also used in the phylogenetic tree
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2169
W. Zhang and L. Shi
Fig. 4. Phylogenetic analysis of HPT
domains/proteins. The domain sequences
are indicated by protein ID, followed by species name. The HPTs present as a separate
protein are indicated by an S in parentheses
following their species names. Several
obvious clusters were indicated by dashes
beside the tree. The HPT proteins of eukaryotic sources are marked in bold.
2170
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Microbiology 151
Evolution of multiple-step phosphorelay in prokaryotes
construction as described previously (Taylor & Zhulin,
1999; Zhang & Shi, 2004). It is obvious from the phylogenetic analysis that, although individual exceptions are
present and overall bootstrap support was not high, PAS
domains extracted from hybrid-type HKs tend to be clustered based on their putative physiological function rather
than taxonomic relationship (data not shown). This finding
suggested that PAS domains with different functional
specialties were recruited into hybrid-type HKs as lateral
events.
DISCUSSION
Although the multiple-step phosphorelay and hybrid HKs
were found in both prokaryotes and eukaryotes, they were
previously thought to be less common in prokaryotes than
in eukaryotes (Uhl & Miller, 1996; Posas et al., 1996;
Robinson et al., 2000; West & Stock, 2001; Oka et al., 2002;
Catlett et al., 2003). In this paper, a detailed survey and
phylogenetic analysis were performed using the sequences
from 156 complete prokaryotic genomes. The results
showed that more than one-third of the bacterial and
archaeal genomes possessed hybrid-type HKs, demonstrating that multiple-step phosphorelay systems are substantially more common in prokaryotes than previously
thought. Although the functions of these HKs are still
unknown, the existence of large numbers of hybrid-type
HKs certainly indicates that these enzymes play a significant
role in multiple-step phosphorelay systems in prokaryotes.
Hybrid-type HKs were found selectively enriched in very
diverse bacterial species, from photosynthetic cyanobacteria to various pathogenic bacteria (Table 1), indicating
that multiple-step phosphorelay may have special signalling
properties such that the evolution and expansion of this
unique family of signalling molecules occurred in response
to unique challenges that these bacteria faced. Compared
with simple scheme of TCSTSs, multiple-step phosphorelay
has been suggested to have three major advantages:
(i) presence of kinase and receiver domains in one protein
may constrain signal amplification, modularity or cross-talk
between components of TCSTSs (Bijlsma & Groisman,
2003); (ii) because of the involvement of HPT, the
mechanism provides greater versatility in signalling strategies and a greater number of potential sites for regulation (Grossman, 1995; Appleby et al., 1996); and (iii) the
multiple phosphorylation sites of the phosphorelay could
provide more junction points for communicating with
other signalling pathways (Appleby et al., 1996).
Unlike HKs in other phylogenetic subfamilies, such as Pho
and Ntr (Koretke et al., 2000), several observations emerging
from this study suggest that there was no single ancestor
for hybrid-type HKs. First, the survey of hybrid-type HKs
across prokaryotic genomes showed that their distribution
did not follow any taxonomic relationship; species with
very close relationship could be quite different in terms of
the total numbers and percentage of hybrid-type HKs. For
http://mic.sgmjournals.org
example, Synechocystis sp. PCC 6803 contains 11 hybridtype HKs, while Synechococcus sp. WH8102 has none.
Second, independent phylogenetic analysis of kinase and
receiver domains from hybrid-type HKs showed that
hybrid-type HK may have kinase and receiver domains
with different phylogenetic origins. Further support was
also provided by the observation that hybrid-type HKs
from the same species often have multiple combinations
of individual kinase and receiver domains (based on their
phylogenetic origins), indicating that lateral recruitment
events were involved in the evolution of these proteins. The
results demonstrated that domain recruitment followed
by gene duplication may be responsible for the expanding
of hybrid-type HKs in bacteria.
No correlation was found between the number of hybridtype HKs and HPTs in prokaryotic genomes, which was
consistent with a previous study in fungal genomes (Catlett
et al., 2003). Even more interesting, 41 % of the prokaryotic
genomes with hybrid-type HKs do not have any identifiable
HPT sequences. This observation raises questions regarding the mechanism by which prokaryotic multiple-step
phosphorelay systems function and how the specificity
of signal transduction is being controlled. One plausible
explanation might be that the prokaryotic systems are
indeed functioning like the eukaryotic systems, but that
most of the bacterial HPT proteins were not identified in
this study because of low sequence similarity to known
HPT proteins (Rodrigue et al., 2000). Another hypothesis
is suggested by a mechanism that has been proposed for
Arabidopsis HPTs involving chaperone-like proteins that
associate with TCSTSs at the membrane/cytoplasm interface and/or guide the phosphorylated HPT into the nucleus
or other subcellular compartments (Pawson & Scott 1997;
Grefen & Harter, 2004). However, it is unclear whether
there is any similar mechanism involved in guiding the
specialization of phosphorylation in prokaryotes. Finally,
although almost all known hybrid-type HKs appear to
function in multiple-step phosphorelays, in which the
phosphate is transferred from the receiver domain of the
hybrid HK to a second His residue in an HPT domain and
then to RR (Chang & Stewart, 1998; West & Stock, 2001;
Catlett et al., 2003), it is still possible that phosphorelay
may not be the only use of this architectural design and
it is therefore possible that not all of the prokaryotic
hybrid-type HKs are involved in multiple-step phosphorelay. An example of this is seen in Agr. tumefaciens, where
the attached receiver domain of VirA, a transmembrane
hybrid HK, functions as an autoinhibitory domain. In its
unphosphorylated state, this receiver domain interacts with
the transmitter module and prevents the transmitter from
autophosphorylating and serving as a phosphodonor to
its cognate response regulator VirG (Chang et al., 1996;
Appleby et al., 1996).
In conclusion, this study presents a survey of the distribution and evolutionary analysis of the components
involved in multiple-step phosphorelay in prokaryotes, and
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2171
W. Zhang and L. Shi
constitutes a basis for further exploration of their physiological functions.
Hagiwara, D., Yamashino, T. & Mizuno, T. (2004). Genome-wide
comparison of the His-to-Asp phosphorelay signaling components of
three symbiotic genera of Rhizobia. DNA Res 11, 57–65.
Heidelberg, J. F., Seshadri, R., Haveman, S. A. & 32 other authors
(2004). The genome sequence of the anaerobic, sulfate-reducing
ACKNOWLEDGEMENTS
bacterium Desulfovibrio vulgaris Hildenborough. Nat Biotechnol 22,
554–559.
We would like to thank Drs David E. Culley and Brian H. Lower of
Pacific Northwest National Laboratory for their critical reading of this
manuscript. The research described in this paper was conducted under
the LDRD Program at the Pacific Northwest National Laboratory, a
multi-program national laboratory operated by Battelle for the US
Department of Energy under Contract DE-AC06-76RLO1830.
Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL: a package for
performing multiple sequence alignment on a microcomputer. Gene
73, 237–244.
Hoch, J. A. (2000). Two-component and phosphorelay signal
transduction. Curr Opin Microbiol 3, 165–170.
Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996).
Sequence analysis of the genome of the unicellular cyanobacterium
Synechocystis sp. strain PCC6803. II. Sequence determination of the
entire genome and assignment of potential protein-coding regions
(supplement). DNA Res 3, 185–209.
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transduction via the multi-step phosphorelay: not necessarily a road
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Complete genomic sequence of the filamentous nitrogen-fixing
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