MINIREVIEW
Intramembrane-sensing histidine kinases: a new family of cell
envelope stress sensors in Firmicutes bacteria
Thorsten Mascher
Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, Göttingen, Germany
Correspondence: Thorsten Mascher,
Institute of Microbiology and Genetics,
Department of General Microbiology, GeorgAugust-University, Grisebachstr. 8, D-37077
Göttingen, Germany. Tel.: 149 551 3919862;
fax: 149 551 393808; e-mail:
[email protected]
Received 6 June 2006; revised 9 August 2006;
accepted 14 August 2006.
First published online 7 September 2006.
DOI:10.1111/j.1574-6968.2006.00444.x
Editor: Ian Henderson
Key words
signal transduction; two-component system;
histidine kinase; cell envelope stress.
Abstract
Two-component signal-transducing systems (TCS) consist of a histidine kinase
(HK) that senses a specific environmental stimulus, and a cognate response
regulator (RR) that mediates the cellular response. Most HK are membraneanchored proteins harboring two domains: An extracytoplasmic input and a
cytoplasmic transmitter (or kinase) domain, separated by transmembrane helices
that are crucial for the intramolecular information flow. In contrast to the
cytoplasmic domain, the input domain is highly variable, reflecting the plethora
of different signals sensed. Intramembrane-sensing HK (IM-HK) are characterized
by their short input domain, consisting solely of two putative transmembane
helices. They lack an extracytoplasmic domain, indicative for a sensing process at
or from within the membrane interface. Most proteins sharing this domain
architecture are found in Firmicutes bacteria. Two major groups can be differentiated based on sequence similarity and genomic context: (1) BceS-like IM-HK
that are functionally and genetically linked to ABC transporters, and (2) LiaS-like
IM-HK, as part of three-component systems. Most IM-HK sense cell envelope
stress, and identified target genes are often involved in maintaining cell envelope
integrity, mediating antibiotic resistance, or detoxification processes. Therefore,
IM-HK seem to constitute an important mechanism of cell envelope stress
response in low G1C Gram-positive bacteria.
Introduction
Life in the microbial world is characterized by constant
interactions between the bacterial cell and its environment.
A prerequisite for survival in a complex habitat is the ability
of a bacterium to ‘know’ its state by closely monitoring
critical parameters: osmotic activity, ionic strength, composition and concentration of nutrients and presence of
harmful compounds are among a plethora of variables that
are part of the equation that each cell has solve in order to
find the ideal niche to thrive and prosper.
Two-component signal transduction (TCS) is a ubiquitously distributed regulatory principle in bacteria, but can
also be found in lower eukaryotes such as fungi, slime molds
and plants (Hoch & Silhavy, 1995; Hwang et al., 2002;
Inouye & Dutta, 2003). It is a versatile system that allows
adaptational response to a huge variety of environmental
stimuli, based on a simple modular system: a membranebound histidine kinase (HK) that acts as a sensor and a
FEMS Microbiol Lett 264 (2006) 133–144
response regulator (RR) that mediates the cellular response,
most often by regulating differential gene expression. The
activity of, as well as the communication between these
two components is mediated by three phospho-transfer
reactions: (1) the autophosphorylation of a conserved
histidine in the sensor, (2) the phospho-transfer to a
conserved aspartate in the RR, and (3) dephosphorylation
of the RR to set back the system to the prestimulus state
(Parkinson, 1993). With a few exceptions, all bacterial
genomes sequenced so far harbor multiple copies of genes
encoding TCS. While some systems have been studied in
great detail (most notably the paradigms EnvZ/OmpR and
CheA/CheY in Escherichia coli), and transcriptome approaches allowed initial genome-wide investigations on
TCS, most of these systems are still uncharacterized.
A classification of histidine kinases, based on the H-box of
the kinase domain, was proposed by Fabret et al. (1999) and
found widespread use. A more comprehensive and detailed
sequence analysis, based on all six conserved boxes in the
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134
transmitter domain (Grebe & Stock, 1999), allows a more
accurate subgrouping of histidine kinases. In contrast,
investigations of the input (sensing) domains of histidine
kinases are much more difficult, due to the great sequence
diversity reflecting the range of different input signals sensed
(Hoch, 2000). Comparative genomics analyses – applying
sophisticated bioinformatics algorithms – have been performed only recently, resulting in the identification of a
number of novel conserved input domains (Anantharaman
& Aravind, 2000, 2001, 2003; Galperin et al., 2001a, b;
Zhulin et al., 2003; Galperin, 2004). Such analyses are crucial
to understand the individual physiological role of a TCS,
which is defined by the input domain of HK and the output
domain of the RR, rather than the highly conserved transmitter–receiver module, which facilitates the communication between HK and RR. With regard to the amount of data
generated by genome sequencing projects, new approaches
are necessary to predict the biological function of uncharacterized signal-transducing systems. Here, we present such
an alternative approach that is based on domain architecture
and genomic context (instead of sequence similarity alone),
to identify HK with potential roles in sensing bacterial cell
envelope stress response. Our comparative genomics prediction will be evaluated – and is supported – by the
published function of some of the corresponding TCS.
Definition and identification of
intramembrane-sensing histidine kinases
We recently identified three histidine kinases – LiaS, BceS
and YvcQ – as part of the cell envelope stress stimulon of
Bacillus subtilis (Mascher et al., 2003). These proteins share
striking similarities in their overall domain organization:
they are small sensor kinases with no more than 400 amino
acids total length. The N-terminal sensing domain consists
of two deduced transmembrane helices with a spacing of less
than 25 amino acids that is therefore buried almost entirely
in the cytoplasmic membrane, indicating that no extracellular stimulus is detected (Fig. 1). The cytoplasmic transmitter domain harbors only the standard features
characteristic for all HK (HisKA, HATPase_c for kinase
activity, and – sometimes – dimerization domains such as
HAMP, Fig. 1), but lacks any additional domains that
would allow signal detection within the cytoplasm. Therefore, it was proposed that these proteins sense their stimulus
either directly inside or at the surface of the cytoplasmic
membrane. Accordingly, these proteins were named intramembrane-sensing histidine kinases (IM-HK; Mascher
et al., 2003).
This review is based on a comprehensive database analysis
of IM-HK that included domain organization, genomic
context conservation, and sequence homology, in order to
define and classify this new subfamily of sensor kinases. For
2006 Federation of European Microbiological Societies
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c
T. Mascher
Fig. 1. Domain organization of intramembrane-sensing histidine
kinases. Basic types are shown and compared with EnvZ. Scale bar in
amino acids. The figure is based on the graphical output of the SMART
web interface (http://smart.embl-heidelberg.de). The protein is represented by the grey line. Blue vertical bars represent putative transmembrane helices. Size and position of conserved domains is indicated by the
labeled symbols (HAMP, green pentagon; HisKA, turquoise square;
HATPase_c, turquoise triangle). SMART/Pfam accession numbers for the
domains are: HAMP (SM00304/PF00672), HATPase_c (SM00387/
PF02518), HisKA (SM00388/PF00512, or PF07730 (Pfam:HisKA_3) in
case of LiaS-like HK). Abbreviations of bacterial species: Eco, Escherichia
coli; Bsu, Bacillus subtilis.
that purpose, the ‘Simple Modular Architecture Research
Tool’ SMART (Schultz et al., 1998) (URL: http://smart.embl-heidelberg.de/) and the recently released ‘Microbial
Signal Transduction database’ MiST (Ulrich et al., 2005)
(URL: http://genomics.ornl.gov/mist/) were screened for
histidine protein kinases fulfilling the criteria mentioned
above. The resulting dataset was manually analyzed to
identify and omit duplicated sequences (i.e. those derived
from sequencing more than one strain of a given species). A
total of 147 proteins (out of almost 5000 HK in the
databases, based on more than 350 completely sequenced
microbial genomes with about twice as much under way, as
of July 2006) share the domain architecture of IM-HK, as
described above. The vast majority of IM-HK are derived
from genomes of Gram-positive bacteria, primarily with a
low G1C content (110 proteins, with 79 finished genomes
in MiST), and some also from Actinobacteria (11 proteins,
24 finished genomes). Only 17 IM-HK can be found in
Proteobacteria (171 finished genomes!), and four in Cyanobacteria (Tables 1–3). Two phylogenetically distinct subgroups of IM-HK, found almost exclusively in Firmicutes
bacteria, are genetically linked to genes encoding ABC
transporters or a conserved transmembrane protein, respectively (Figs 2 and 3). The functional connection between
IM-HK and these proteins, and the signal-sensing
FEMS Microbiol Lett 264 (2006) 133–144
135
Intramembrane-sensing histidine kinases
Table 1. List of intramembrane-sensing histidine kinases associated with ABC transporters
Protein
Accession no.
Organism
Phylum
Length
Classw
TMRz
Reference
BceS-like IM-HK
BA5088
YvcQ
YxdK
BC4836
BCE2618
BCE4530
ABC0255
ABC3228
BH3912
BH2700
BH0754
BH0289
BH0272
YtsB
YxdK
BLi04270
BE01181
BceS
YvcQ
YxdK
BT9727_2379
BT9727_4172
BT9727_4568
CAC1517
CAC0372
CAC0225
CB03066
CB03117
DF02597
CPE0120
CPE0841
CTC00393
CTP22
DSY0573
DSY3679
EF0927
EFA03839
GK2341
LsaHPK1
LlaKinG
LIN1852
LMO1741
OB0832
SA2417
SA0615
SE2194
SE0428
SH0405
SH2234
SEQ00482
SAG0977‰
MbrD
str1335(hk07)
STH3213
TDE0656
AAP28764
NP_834144
AAP09499
AAP11737
NP_978924
NP_980823
YP_173759
YP_176723
BAB07631
BAB06419
BAB04473
BAB04008
BAB03991
AAU43025
AAU42958
AAU43083
NA
CAB15017
CAB15476
CAB16001
YP_036705
YP_038489
YP_038880
AAK79484
AAK78352
AAK78206
NA
NA
NA
BAB79826
BAB80547
AAO35029
AAO37418
YP_516806
YP_519912
AAO80735
NA
YP_148194
AAD10259
AAK05844
CAC97083
CAC99819
BAC12788
BAB58786
BAB94487
AAO05836
AAO04025
YP_252320
YP_254149
NA
AAM99860
BAB83946
YP_141691
YP_077039
NP_971269
B. anthracis
B. cereus
B. cereus
B. cereus
B. cereus
B. cereus
B. clausii
B. clausii
B. halodurans
B. halodurans
B. halodurans
B. halodurans
B. halodurans
B. licheniformis
B. licheniformis
B. licheniformis
B. stearothermoph.
B. subtilis
B. subtilis
B. subtilis
B. thuringiensis
B. thuringiensis
B. thuringiensis
C. acetobutylicum
C. acetobutylicum
C. acetobutylicum
C. botulinum
C. botulinum
C. difficile
C. perfringens
C. perfringens
C. tetani
C. tetani
D. hafniense
D. hafniense
E. faecalis
E faecium
G. kaustophilus
L. sakei
L. lactis
L. innocua
L. monocytogenes
O. iheyensis
S. aureus
S. aureus
S. epidermidis
S. epidermidis
S. haemolyticus
S. haemolyticus
S. equi
S. agalactiae
S. mutans
S. thermophilus
S. thermophilum
T. denticola
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Actinobact.
Spirochaet.
340
360
327
340
334
369
345
350
334
343
353
346
331
334
326
335
334
334
356
325
334
370
340
349
334
339
352
326
334
337
336
352
314
331
328
341
341
334
339
291
346
346
334
295
346
298
346
298
344
325
312
317
324
371
357
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
HPK3i
09–26, 37–56
12–34, 44–61
12–29, 34–56
09–26, 37–56
17–36, 41–63
21–43, 53–71
17–36, 41–63
12–34, 38–60
12–29, 33–55
13–35, 41–63
13–32, 42–64
12–34, 44–63
09–26, 36–53
12–29, 33–55
07–29, 34–53
09–28, 38–57
12–29, 33–55
12–29, 33–55
10–32, 44–63
07–29, 34–56
17–36, 41–63
21–43, 53–71
09–26, 37–56
12–34, 39–61
15–34, 41–60
13–30, 40–62
15–37, 42–64
12–34, 38–57
13–30, 40–61
12–31, 36–55
12–31, 36–58
15–37, 42–64
02–16, 26–48
15–32, 39–61
20–37, 42–59
13–30, 40–62
13–30, 40–62
12–29, 33–55
12–30, 40–62
13–32, 36–58
12–30, 40–62
12–30, 40–62
12–34, 38–55
13–30, 35–57
17–34, 44–63
13–32, 36–54
15–34, 41–63
13–30, 34–56
15–34, 41–63
09–31, 35–57
10–32, 37–56
12–31, 35–57
18–36, 45–63
14–35, 41–61
23–42, 55–73
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Ohki et al., 2003)
(Joseph et al., 2002)
(Joseph et al., 2004)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Teng et al., 2002)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Tsuda et al., 2002)
Genome sequence
Genome sequence
Genome sequence
FEMS Microbiol Lett 264 (2006) 133–144
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Published by Blackwell Publishing Ltd. All rights reserved
c
136
T. Mascher
Table 1. Continued.
Protein
Accession no.
Organism
Additional IM-HK associated with ABC transporters
BAS0272
YP_026553
B. anthracis
BCZK0257
YP_081868
B. cereus
BCZK1773
YP_083368
B. cereus
ABC3642
YP_177136
B. clausii
BacS
AAD21212
B. licheniformis
SubS
ABB80127
B. subtilis
BT9727_0254
YP_034606
B. thuringiensis
CAC3516
AAK81442
C. acetobutylicum
CTC01716
AAO36431
C. tetani
GK0907
YP_146760
G. kaustophilus
OB0358
NP_691279
O. iheyensis
AAM99032
S. agalactiae
SAG0124‰
ALR3155
BAB74854
Anabaena sp.
SLL1590
Tr|P73865
Synechocystis sp.
NPU03400
NA
N. punctiforme
Phylum
Length
Classw
TMRz
Reference
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Cyanobact.
Cyanobact.
Cyanobact.
340
340
347
372
348
347
340
350
339
354
333
356
344
350
367
–
HPK1b
HPK2a
–
–
–
HPK1a
–
HPK1b
–
HPK1b
HPK1a
HPK1a
HPK1a
09–26, 31–48
10–25, 32–54
13–35, 45–67
07–29, 49–71
10–27, 32–54
10–28, 36–54
09–26, 31–48
07–29, 49–71
20–37, 41–60
07–29, 49–71
07–29, 33–50
13–35, 50–72
29–51, 56–78
29–47, 52–71
21–43, 63–85
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Neumüller et al., 2001)
(Wu et al., 2006)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Length in amino acids (aa).
w
Assignment is based on the histidine kinase classification system of Grebe & Stock (1999). Some remarks concerning this classification: BacS (Bli) and
YbdK (Bsu) lack a G-Box. ‘–’ Some kinases could not be assigned to any of the HK subgroups described so far.
z
Putative transmembrane regions (TMR) were identified by the TMHMM2 algorithm (Krogh et al., 2001).
‰
The two proteins from S. agalactiae were listed under a different name in our initial analysis (Mascher et al., 2003). GBS0122 and GBS0964 were later
renamed to SAG0124 and SAG0977, respectively.
NA, not available.
mechanism of IM-HK will be discussed, based on initial
investigations and the available literature.
BceS-like histidine kinases: a twocomponent system -- ABC transporter
connection
The largest distinct group of IM-HK is characterized by a
topological link of the corresponding TCS to genes encoding
ABC transporters. A total of 70 proteins belong to that
subgroup, 65 of which are derived from Gram-positive
bacteria with a low G1C content. 55 of these HK (53 from
Firmicutes) gather closely in a distinct branch of the
phylogenetic tree of IM-HK (Fig. 2). These proteins all
belong to the HPK3i subfamily (Table 1). A genetic and
functional link between TCS and ABC transporters in the
Bacillus/Clostridium group of low G1C Gram-positive
bacteria was described and exemplarily verified for three
examples from B. subtilis: YvcPQ-YvcRS, YtsAB-YtsCD and
YxdJK-YxdLM (Joseph et al., 2002, 2004). In this group of
IM-HK, the genes encoding TCS are located next to (mostly
upstream of) genes encoding ABC-transporters (Fig. 3a).
The latter are organized in a separate operon and are
expressed from a promoter that completely depends on the
activity of the neighboring TCS. These systems represent
tightly regulated detoxification units: The TCS is expressed
constitutively and senses the presence of toxic compounds,
such as the cell wall antibiotic bacitracin, at sublethal
concentrations. Upon induction, the RR specifically acti2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
vates the expression of the ABC transporter, thereby facilitating removal of the antibiotic (Fig. 3a). Such an inducible
resistance mechanism has been demonstrated for BceRSBceAB and BacRS-BcrABC in case of the bacitracin resistance in B. subtilis and Bacillus licheniformis, respectively
(Neumüller et al., 2001; Mascher et al., 2003; Ohki et al.,
2003). A homologous system, MbrABCD, has been described in Streptococcus mutans only with regard to bacitracin resistance. No gene expression studies were performed.
In this organism, the genes for the TCS are located downstream of the genes encoding the bacitracin exporter (Tsuda
et al., 2002), with MbrD encoding the IM-HK (Fig. 3a).
From low G1C Gram-positive bacteria associated with
ABC transporters 12 of the 67 IM-HK show significant
sequence variations and do not group with the remaining
proteins in the phylogenetic tree (Fig. 2 and Table 1).
Amongst those are a number of systems that are located next
to transporters mediating resistance to cell wall-active nonribosomal peptide antibiotics, such as bacitracin (BacS from
B. licheniformis ATCC10716) or subpeptin (SubS from
B. subtilis JM4). Both corresponding TCS, BacRS and SubRS,
are associated with antibiotic ‘self-resistance’ in these producing strains (Neumüller et al., 2001; Wu et al., 2006). They
are homologous to each other and show the same topology
as all the other TCS-ABC loci mentioned so far. A functional
link between TCS and ABC transporter has been demonstrated in case of BacRS-BcrABC (Neumüller et al., 2001).
The three remaining IM-HK (ALR3155, SLL1590 and
NPU03400) were found in the genome sequences of
FEMS Microbiol Lett 264 (2006) 133–144
137
Intramembrane-sensing histidine kinases
Table 2. List of LiaS-like intramembrane-sensing histidine kinases
Protein
Accession no.
Genomic context liaIH(G)FSR-like
BA1456
AAP25398
YvqE
AAP08419
ABC3375
YP_176869
BH1199
BAB04918
YvqE
AAU24951
BE02643
NA
LiaS
CAB15299
BT9727_1321
YP_035655
LIN1020
CAC96251
LMO1021
CAC99099
OB2823
BAC14779
Genomic context liaFSR-like
EF2912
AAO82600
KinD
AAG53722
LSA1370
OB1161
VraS
SE1570
SH1070
SSP0908
SAG0321
SEQ00811
SMU.486
HK03(spr0343)
SPy1622
str1421
Organism
Phylum
Length
Classw
TMRz
Reference
B. anthracis
B. cereus
B. clausii
B. halodurans
B. licheniformis
B. stearothermoph.
B. subtilis
B. thuringiensis
L. innocua
L. monocytogenes
O. iheyensis
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
351
351
354
351
353
344
360
351
352
352
350
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
10–32, 53–75
10–32, 53–75
07–29, 49–71
12–34, 49–71
13–35, 50–72
05–27, 42–64
13–35, 50–72
10–32, 53–75
07–29, 49–71
07–29, 49–71
07–29, 52–74
Genome sequence
Genome sequence
genome sequence
Genome sequence
Genome sequence
Genome sequence
(Mascher et al., 2004)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
E. faecalis
L. lactis
Firmicutes
Firmicutes
367
332
HPK7
HPK7
09–31, 51–73
05–27, 50–72
L. sakei
O. iheyensis
S. aureus
S. epidermidis
S. haemolyticus
S. saprophyticus
S. agalactiae
S. equi
S. mutans
S. pneumoniae
S. pyogenes
S. thermophilus
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
354
356
347
348
348
347
339
334
334
331
334
336
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
HPK7
07–29, 52–77
16–38, 48–70
13–35, 45–67
13–35, 45–67
13–35, 45–67
07–25, 43–69
07–29, 47–69
07–25, 45–67
07–25, 45–67
07–25, 45–67
07–25, 45–67
05–25, 45–64
Genome sequence
(O’Connell-Motherway
et al., 2000)
Genome sequence
Genome sequence
(Kuroda et al., 2003)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Lange et al., 1999)
Genome sequence
Genome sequence
‰
YP_395981
BAC13117
CAG40962
NP_765125
YP_252985
YP_300998
CAD45954
NA
NP_720926
AAK74553
NP_269673
YP_141773
Length in amino acids (aa).
w
Assignment is based on the histidine kinase classification system of Grebe & Stock (1999).
Putative transmembrane regions (TMR) were identified by the TMHMM2 algorithm (Krogh et al., 2001).
‰
A liaG homolog is only present in B. subtilis, B. licheniformis and B. halodurans. In the two Listeria species, the liaIH homologs form an independent
transcriptional unit, but are predicted to be under control of LiaR-homologous response regulators (Jordan et al., 2006).
NA, not available.
z
cyanobacteria and share little sequence similarities with the
kinases described above. They are closely related to each other
but belong to a different subclass of HK (Fig. 2 and Table 1).
They also show a different genomic clustering: The genes for
the ABC transporter and the TCS are transcribed divergently
and share a common intergenic region (Fig. 3a). A regulatory
and/or functional link between these TCS and their neighboring ABC transporters has not yet been demonstrated.
LiaS-like histidine kinases: threecomponent signal transduction
LiaS and its 24 homologs form a phylogenetically clearly
distinct group of IM-HK (Fig. 2). They can only be found in
Gram-positive bacteria with a low G1C content (Firmicutes). These proteins belong to HPK7, a subclass of
histidine kinases that also contains B. subtilis DegS (Msadek
et al., 1990) and E. coli NarQ (Chiang et al., 1992).
FEMS Microbiol Lett 264 (2006) 133–144
Most database entries on LiaS-homologs are derived from
genome sequencing projects (Table 2). So far, only two
members of this subgroup of IM-HK have been described
in more detail: the eponymous protein from B. subtilis and
VraS from Staphylococcus aureus. The kinases of both TCS
sense the presence of cell wall antibiotics (Kuroda et al.,
2003; Mascher et al., 2003; Mascher et al., 2004).
The VraSR system responds to the inhibition of cell wall
synthesis. It is induced by the presence of diverse cell wall
antibiotics such as glycopeptides, b-lactams, bacitracin and
D-cycloserine. Transcriptome analysis of VraR-dependent
gene expression revealed that this TCS controls a large
regulon. The known or assumed functions of some target
genes indicate that the VraSR system is involved in coordinating important steps of cell wall biosynthesis (Kuroda
et al., 2003; Yin et al., 2006).
In contrast, the biological function of the LiaRS system
is still largely unclear, despite significant progress in
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138
T. Mascher
Table 3. Miscellaneous intramembrane-sensing histidine kinases
Protein
Accession no.
Organism
IM-HK associated with multidrug-efflux pumps
Bcep_A4348
YP_368588
Burkholderia sp.
Bcep_B3051
YP_373805
Burkholderia sp.
Bcep_C6826
YP_366516
Burkholderia sp.
RS05453
CAD17466
R. solanacearum
RPA2367
CAE27808
R. palustris
RS03089
CAD18605
R. solanacearum
Rru_A2930
YP_428014
R. rubrum
IM-HK without genomic context conservation
BA1956
AAP25850
B. anthracis
BA3066
AAT32182
B. anthracis
GtcS
CAA55265
B. brevis
BC1957
AAP08928
B. cereus
BC1801
AAP08775
B. cereus
BC3042
NP_832788
B. cereus
BH1809
BAB05528
B. halodurans
YbdK
CAB11995
B. subtilis
BT9727_1791
YP_036123
B. thuringiensis
BT9727_2824
YP_037148
B. thuringiensis
CAC0831
AAK78807
C. acetobutylicum
DSY2529
YP_518762
D. hafniense
Moth_1622
YP_430467
M. thermoacetica
SaeS
AAD48403
S. aureus
SE0121
AAO03718
S. epidermidis
SH0335
YP_252250
S. haemolyticus
AAM99300
S. agalactiae
SAG0394‰
HK08
CAB54579
S. pneumoniae
HK01
CAB54567
S. pneumoniae
TTE0562
NP_622234
T. tengongensis
BL1001
AAN24809
B. longum
SAV2971
BAC70682
S. avermitidis
SAV7391
BAC75102
S. avermitidis
SCO3740
CAB76987
S. coelicolor
SCO5282
CAC04497
S. coelicolor
SCO6424
CAA18911
S. coelicolor
SCO5784
CAA18321
S. coelicolor
SCO6163
CAA22397
S. coelicolor
STH920
YP_074749
S. thermophilum
STH2729
YP_076558
S. thermophilum
Adeh_0410
YP_463623
A. dehalogenans
Bd3450
NP_970185
B. bacteriovorus
BLL3558
BAC48823
B. japonicum
BLL7277
BAC52542
B. japonicum
Bcen_5050
YP_624898
B. cenocepacia
Rfer_3574
YP_524809
R. ferrireducens
Rru_A1884
YP_426971
R. rubrum
VF0524
YP_203907
V. fischeri
WS2200
CAE11190
W. succinogenes
EnvZ
AAB36612
X. nematophila
Acid345_4169
YP_593243
acidobacterium
CYB_0712
YP_476959
Synechococcus sp.
Dgeo_1625
YP_605089
D. geothermalis
RB11668
NP_870094
Pirellula sp.
NP_227943
T. maritima
TM0127‰
Phylum
Length
Classw
TMRz
Reference
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
330
361
379
360
367
365
352
HPK2b
HPK2a
HPK2a
HPK2a
HPK2a
HPK2b
HPK1a
13–35, 50–72
15–37, 56–78
15–37, 56–78
19–41, 56–78
16–38, 53–75
13–35, 45–67
21–43, 58–80
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Firmicutes
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Acidobacteria
Cyanobacteria
Deinococci
Planctomycetes
Thermotogae
351
333
370
348
358
331
351
320
351
331
366
282
275
353
363
332
345
350
324
289
358
368
388
370
375
331
358
303
346
368
351
291
366
363
361
342
346
320
298
342
302
353
357
317
285
HPK1b
–
–
HPK1b
–
–
HPK2a
–
HPK1b
–
HPK2b
HPK5?
HPK5?
HPK2a
–
–
HPK1a
HPK1a
HPK3i
HPK5?
HPK1a
HPK1a
HPK7
HPK1a
HPK1a
HPK7
HPK7
HPK1a
HPK7
HPK7
HPK2a
–
HPK2a
HPK2a
HPK2a
–
–
–
–
HPK2b
HPK2a
HPK2a
HPK2a
HPK5?
–
13–35, 45–67
15–37, 49–71
05–22, 42–64
07–29, 44–66
13–35, 50–67
15–37, 49–71
04–26, 47–66
03–25, 40–62
13–35, 45–67
15–37, 49–71
13–34, 63–80
09–31, 41–62
07–26, 36–58
09–31, 41–63
13–35, 55–77
13–35, 55–77
07–26, 36–58
15–37, 42–64
07–26, 36–58
08–29, 42–62
32–51, 53–75
28–47, 54–76
53–74, 84–106
05–27, 40–62
35–54, 61–83
12–31, 46–68
34–56, 69–91
05–27, 40–62
17–40, 51–69
17–40, 49–67
10–32, 53–75
13–35, 55–77
13–35, 55–77
12–34, 58–80
10–32, 56–75
13–35, 50–72
13–35, 55–74
13–33, 40–59
12–31, 41–63
17–39, 49–71
11–36, 41–66
24–46, 56–78
18–37, 48–71
13–35, 39–56
06–22, 31–50
Genome sequence
Genome sequence
(Turgay & Marahiel, 1995)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Fabret et al., 1999)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Giraudo et al., 1997)
Genome sequence
Genome sequence
Genome sequence
(Lange et al., 1999)
(Lange et al., 1999)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Hutchings et al., 2004)
(Hutchings et al., 2004)
(Hutchings et al., 2004)
(Hutchings et al., 2004)
(Hutchings et al., 2004)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
(Tabatabai & Forst, 1995)
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Genome sequence
Length in amino acids (aa).
w
Assignment is based on the histidine kinase classification system of Grebe & Stock (1999). Some remarks concerning this classification: In SCO6424,
the phosphoryl-accepting histidine residue is replaced by a tyrosine residue. The D/F-Box is only weakly conserved in BL1001 (Blo), SCO3740 and
SCO6163. YbdK (Bsu) lacks a G-Box. ‘–’ Some HK could not be assigned to any of the HK subgroups described so far.
z
Putative transmembrane regions (TMR) were identified by the TMHMM2 algorithm (Krogh et al., 2001).
‰
Our initial analysis (Mascher et al., 2003) contained one additional IM-HK, ArcB from Haemophilus influenzae, which was based on the published
genome sequence. We later noticed that this protein, HI0220, was a sequencing artifact of the full-length ArcB protein (Manukhov et al., 2000) and
does not belong to the IM-HK. Two proteins were listed under a different name: GBS0430 was renamed SAG0394 after completion of the genome
sequence. A second IM-HK from Thermotoga maritima was originally mis-labeled as TM1258 and is listed here with its correct protein ID, TM0127.
2006 Federation of European Microbiological Societies
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FEMS Microbiol Lett 264 (2006) 133–144
139
Intramembrane-sensing histidine kinases
Fig. 2. Phylogenetic tree of intramembrane-sensing histidine kinases. The tree was generated using ClustalW and Phylip algorithms, implemented into
the BioEdit sequence alignment tool (Hall, 1999). The different groups of IM-HK are highlighted: LiaS-like (light blue), IM-HK associated with ABC
transporters (green), IM-HK located next to multidrug-efflux pumps (orange). Orphan HK are underlined. Proteins derived from Firmicutes bacteria are
indicated by black lines and text color, non-Firmicutes proteins are given in grey. HK that have been characterized in more detail and are referred to in
the text are shown with large and bold letters. For reasons of clarity, not all IM-HK from Tables 1–3 are represented in this figure. In case of high
sequence similarity (i.e. orthologous IM-HK from Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis), only one protein is shown (the rest is not
shown): BA1456 (BT9727_1321, BceYvqE), BA3066 (BC3042, BT9727_2824), BA1956 (BT9727_1791, BCZK1773, BC1957), BA5088 (BT9727_4568,
BC4836), BCE2618 (BT9727_2379), BceYvcQ (BCE4530, BT9727_4172), Bcen_5050 (Bcep18194_B3051), BCZK0257 (BT9727_0254), BH0289
(ABC0255), CTC00393 (CB03066), DSY0573 (DHA03376), EF0927 (EFA03839), LMO1021 (Lin1020), LMO1741 (Lin1852), RPA2367 (BLL3558,
RPC_2736), RPB_0413 (RPD_0407), SauVraS (SE1570, SH1070, SSP0908), SA0615 (SE0428, SH2234), SCO3740 (SCO6163), SCO5282 (SAV2971),
SCO5784 (SAV7391), Spy1622 (SEQ00811), See Table 1–3 for protein descriptions and details. Abbreviations of bacterial species: Bbr, B. brevis; Bce, B.
cereus; Bha, B. halodurans; Bli, B. licheniformis; Bsu, B. subtilis; Sau, S. aureus; Smu, S. mutans; Xne, X. nematophilus.
elucidating its signal transduction mechanism. The LiaRS
TCS was originally identified as a component of the regulatory network that orchestrates the cell envelope stress
response in B. subtilis (Mascher et al., 2003). It is encoded
FEMS Microbiol Lett 264 (2006) 133–144
by the last two genes of the hexa-cistronic liaIHGFSR
operon. In the presence of sublethal concentrations of lipid
II-interacting antibiotics – such as bacitracin, vancomycin,
ramoplanin or nisin – the LiaRS TCS is activated and
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140
T. Mascher
Fig. 3. Genomic context, membrane topology and working model of (a) BceS-, and (b) LiaS-like IM-HK. The loci are drawn to scale, with the lines each
corresponding to 7 kb. The genes are labeled differently for clarity. Thick-hatched and dotted arrows represent genes coding for histidine kinases and
response regulators, respectively. The names of sensory proteins are highlighted. Additional arrow labeling: ABC transporters (white, vertically striped),
liaF homologs (black), additional genes within loci (grey), unrelated flanking genes (white). Putative terminators are marked by black vertical bars. (a)
Working model for BceS-like IM-HK based on the published work (Joseph et al., 2002; Mascher et al., 2003; Ohki et al., 2003): BceS detects the
presence of bacitracin and in turn activates its cognate RR BceR, ultimately resulting in the strong induction of bceAB expression (red arrows). This
operon encodes an ABC transporter that then facilitates removal of bacitracin, which therefore no longer acts as an inducer of BceS: the system shuts
down again. Note that neither the exact site of bacitracin action nor the mode of its removal by the ABC transporter BceAB are known. A direct
interaction of bacitracin with undecaprenol-pyrophosphate has been demonstrated, indicating that it is sensed by BceS either at the inner or outer
surface of the cytoplasmic membrane. Origin of the genomic regions (from top to bottom): Bacillus subtilis, Streptococcus mutans, Anabaena sp. (b)
Working model for LiaS-like IM-HK: In the absence of cell envelope stress, the LiaRS TCS is kept inactive by LiaF (black T-shaped line). In the presence of
envelope stress, LiaS is induced and activates its cognate RR LiaR, which then binds to its target promoters, including its own promoter (positive
autoregulatory feedback loop, red arrows). This autoregulation could be demonstrated for B. subtilis LiaRS, S. aureus VraSR, and S. pneumoniae TCS03
(Kuroda et al., 2003; Mascher et al., 2003; Haas et al., 2005). Origin of the genomic regions (from top to bottom): Bacillus subtilis, Enterococcus faecalis.
The lia locus from Bacillus species consists of five core genes, homologs to liaIHFSR, whereas only liaFSR-homologous genes encoding the threecomponent system are conserved in more distantly related cocci. See text and Table 2 for details.
strongly induces expression of its own locus (positive
autoregulatory feedback loop) from a promoter upstream
of liaI (PliaI) (Mascher et al., 2004). Additionally, the LiaRS
system responds to cationic antimicrobial peptides, alkaline
shock, exposure to organic solvents, detergents, ethanol and
secrection stress (Petersohn et al., 2001; Wiegert et al., 2001;
Hyyryläinen et al., 2005; Pietiäinen et al., 2005). Recently, it
was demonstrated that the product of liaF, a gene that is
topologically linked to liaSR in the genomes of all species
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harboring LiaS homologs, is involved in the signal-sensing
mechanism of LiaS (Fig. 3b): In a liaF-deletion mutant, the
LiaRS system is constitutively ‘ON’, thereby no longer
responding to or necessitating a stimulus for full activity.
Therefore, LiaF together with LiaRS forms a three-component system (Jordan et al., 2006). LiaF is a conserved
membrane anchored protein with an N-terminus consisting
of four TMR that is completely buried within the membrane
and a cytoplasmic C-terminus crucial for LiaF function
FEMS Microbiol Lett 264 (2006) 133–144
141
Intramembrane-sensing histidine kinases
(Jordan et al., 2006; S. Jordan and T. Mascher, unpublished
observation; see Fig. 3b for details). While an important role
of LiaF for signal detection is unquestionable, the biochemical mechanism of the LiaS–LiaF interaction remains to be
elucidated. Is LiaF a negative modulator of the intramembrane-sensing mechanism mediated by the IM-HK LiaS – or
is it itself the sensor of the LiaF–LiaRS three-component
system (due to its membrane topology again necessitating
an intramembane signal perception mechanism)? Likewise,
the LiaF–LiaS interaction could occur in the membrane
interface through the TMR of both proteins or, alternatively,
involve the cytoplasmic domain of LiaF and LiaS.
Miscellaneous IM-HK
The remaining 52 proteins (Table 3) do not form a defined
phylogenetic group, nor do most of them share any other
common features. Twenty proteins were identified in genomes of Firmicutes bacteria, 17 in Proteobacteria, and
another 10 from Actinobacteria. Again, most of the proteins
are found in Gram-positive bacteria (30), but here a
significant portion is also derived from Actinobacteria (high
G1C). Some of these proteins have been identified as
IM-HK in a recent publication on TCS in S. coelicolor
(Hutchings et al., 2004). But so far, no further analyses have
been carried out on any of the IM-HK from Actinobacteria.
Two of the 20 miscellaneous IM-HK from Firmicutes
bacteria have been further investigated.
The GtcRS TCS is located adjacent to the grsAB operon of
Bacillus brevis. It encodes multienzymes involved in the
biosynthesis of the peptide antibiotic gramicidin S (Turgay
& Marahiel, 1995). A putative regulatory link between TCS
and antibiotic biosynthesis/immunity seems indicative,
but remains to be demonstrated.
The SaeRS TCS is part of a complex regulatory network
that controls the expression of virulence determinants in
S. aureus. It transcriptionally activates the production of
several exoproteins, such as a- and b-hemolysins and
coagulase (Giraudo et al., 1997; Giraudo et al., 1999). The
saeRS genes are preceded by a third gene, saeQ, encoding a
hydrophobic protein of unknown function (157 amino acid
length), directly upstream and partially overlapping with
saeR. Further upstream is a forth gene, designated saeP,
encoding a cytoplasmic protein of unknown function. The
saePQRS locus is expressed from an autoregulated promoter
upstream of saeP and is partially terminated by a stem loop,
giving rise to a major short transcript saeP and a much
weaker full-length transcript saePQRS (Novick & Jiang,
2003; Steinhuber et al., 2003). Organization and expression
of this locus is, therefore, somehow reminescent of the lia
locus in B. subtilis and its homologs in other bacilli
(Mascher et al., 2004; Jordan et al., 2006).
FEMS Microbiol Lett 264 (2006) 133–144
Of the 17 IM-HK from Proteobacteria, seven are located
next to putative multidrug-efflux pumps, but a functional
connection has not been established so far. Only one of the
remaining 10 proteins has been investigated in more detail.
EnvZ of Xenorhabdus nematophila is homologous to the
‘classical’ osmo-sensor EnvZ of E. coli in its cytoplasmic
C-terminal domain, but lacks its extracytoplasmic domain.
While the periplasmic domains have diverged extensively,
EnvZ from X. nematophila was still able to complement an
DenvZ mutant of E. coli. It was shown that EnvZ of
X. nematophila was able to sense changes in environmental
osmolarity and properly regulate the levels of the cognate
response regulator OmpR of E. coli (Tabatabai & Forst,
1995). Therefore, the periplasmic domain of E. coli EnvZ is
not necessary for sensing osmolarity. This observation could
be viewed as an indication that an unknown number of HK
with large extracytoplasmic domains might also be IM-HK.
These domains could be obsolete for signal perception,
evolutionary remnants whose sole function is to keep the
TMR in place for signal perception, as has been suggested
before (Hoch, 2000). The EnvZ-OmpR TCS of X. nematophila is involved in the regulation of porine biosynthesis,
swarming motility, exoenzyme and antibiotic production
(Forst & Boylan, 2002; Kim et al., 2003; Park & Forst, 2006).
Sensor kinases with similarities to IM-HK
During our analysis, we noticed two phylogenetically unrelated groups of HK that show striking similarity to LiaS-/
BceS-like HK, but do not belong to the group of IM-HK,
due to their different mode of signal perception.
VanS proteins of the VanB-type are small sensor kinases
that have two putative TMR at the N-terminus with a
spacing of 25–30 aa. The corresponding TCS are involved
in mediating vancomycin resistance. While there is no
sequence conservation in the extracellular spacer between
the two TMR, there is recent evidence from VanS of S.
coelicolor that these kinases sense vancomycin (and related
glycopeptide antibiotics) directly through this short extracellular sensing domain, presumably by binding the drug
directly (Hong et al., 2004; Hutchings et al., 2006).
PmrB-BasS-like proteins are also very similar to IM-HK
in all respects but their periplasmic spacer between the two
TMR, which has a length of 30–35 aa. This short linker
contains two highly conserved ExxE motifs that are involved
in sensing ferric ions (Wösten et al., 2000). The corresponding TCS mediates resistance to cationic antimicrobial peptides by lowering the overall negative charge of the cell
surface through modifications of the lipopolysaccharides
(Gunn et al., 1998; Wösten & Groisman, 1999; Gunn et al.,
2000).
Both groups of HK – while being involved in responding
to cell envelope stress – sense their stimuli directly through
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142
the extracytoplasmic spacer between the two TMR, and do
therefore not belong to the IM-HK. In this sense, the 25
residue cut-off point used in this study for the extracytoplasmic spacer in IM-HK might indeed be critical.
Conclusions and outlook
This review describes a novel subclass of signal-transducing
histidine kinases that were identified based on the domain
architecture of their sensor domain and the genomic context
of the corresponding gene, rather than sequence similarity.
The proteins included here share some overall architectural
features with regard to domain organization, size of protein
and input domain. They are small HK with an overall length
of less than 400 amino acids. The N-terminal ‘sensing’
domains of IM-HK consist of two putative membranespanning helices separated by no more than 25 amino acids
(Tables 1–3).
In our case, the use of the domain organization of the
sensing domains (nonconserved by sequence) as a filter
criterion has proven a suitable and useful approach that
allowed insights into the phylogeny and (putative) function
of a novel subgroup of signal-transducing histidine kinases.
IM-HK were predominantly found in the phylum Firmicutes (110 out of the 147 proteins listed in Tables 1–3). The
two major groups of IM-HK, LiaS- and BceS-like HK, are
restricted to Gram-positive bacteria with a low G1C content. One striking feature of these IM-HK is their common
physiological role, based on the examples that have already
been studied to some extent: they all seem to be involved in
sensing cell envelope stress (very often exerted by cell wall
active antibiotics) and regulate genes important for cell
envelope integrity, detoxification, and virulence. In the light
of their phylogenetic distribution, this observation provokes
the question, if the mechanism of signal perception for IMHK is a direct consequence of the difference in cell wall
structure between Gram-positive and -negative bacteria. But
the most important question – and a big challenge to be
addressed experimentally – remains unanswered at the
moment: ‘Do so-called intramembrane-sensing histidine
kinases sense their stimuli inside the membrane interface?’
This review was written with the hope that it might
stimulate further investigations in this direction.
Acknowledgements
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (MA 3269/1-1) and the Fonds der
Chemischen Industrie. The help of Luke Ulrich with data
extraction of IM-HK from the MiST database is gratefully
acknowledged. I am indebted to John D. Helmann for
invaluable discussions and his continuous support. I would
also like to thank Sina Jordan, Tina Wecke and Anna Staroń
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T. Mascher
for critical reading of the manuscript and their helpful
comments.
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