ABBS 2005,38(02): Characterization of cheW Genes of Leptospira interrogans and Their Effects in Escherichia coli

*Corresponding authors:

Guo-Ping ZHAO: Tel, 86-21-50801919; Fax, 86-21-50801922; E-mail,
[email protected]

Xiao-Kui GUO: Tel, 86-21-64453285; Fax, 86-21-64453285; Email,
[email protected]

Abstract        The motility and chemotaxis systems are
critical for the virulence of leptospires.
In this study, the phylogenetic profiles method was used to predict the
interaction of chemotaxis proteins. It was shown that CheW1 links to CheA1,
CheY, CheB and CheW2; CheW3 links to CheA2, MCP (LA2426), CheB3 and CheD1; and
CheW2 links only to CheW1. The similarity analysis demonstrated that CheW2 of Leptospira
interrogans strain Lai
had poor homology with CheW of Escherichia coli in the region of
residues 30–50. In order
to verify the function of these proteins, the putative cheW genes were
cloned into pQE31 vector and expressed in wild-type E. coli strain RP437
or cheW defective strain RP4606. The swarming results indicated that
CheW1 and CheW3 could restore swarming of RP4606 while CheW2 could not.
Overexpression of CheW1 and CheW

Key words        Leptospira interrogans;
chemotaxis; cheW

Leptospirosis is a
widely spread zoonosis of global concern. Leptospire infection causes flu-like
symptoms and severe renal and hepatic damages, such as hemorrhage and jaundice.
In more severe cases, massive pulmonary hemorrhages, including fatal sudden
hemoptysis, can ­occur [1]. The genome sequences of L. interrogans strain Lai and Copenhageni
have recently been reported [2,3]. Many molecular and cellular studies have
been conducted on leptospires concerning the dynamics of motility and
chemotaxis, biosynthesis of lipopolysaccharide and ­­outer-membrane proteins,
and other potential virulence factors [4].

Bacteria usually live in
a dynamic, sometimes harsh, environment. To survive, they need to detect the ­concentrations
of toxins, and nutrients and the levels of light and oxygen, and move away from
unfavorable conditions to favorable conditions. This process is known as taxis
[5]. Chemotaxis, the sensing and response to changes in the levels of
environmental chemicals, is regulated by a large family of signal transduction
systems [6–8]. Escherichia
coli chemotaxis is arguably the best understood of all biological
behaviors [9]. E. coli governs its swimming ­behavior in response
to environmental changes by ­controlling the frequency and direction of
flagellar rotation. A signal transduction pathway controls the frequency of
tumbling, biasing the random swimming pattern so that there is a net movement
towards the attractant. ­Numerous proteins are involved in the chemotactic
signal ­transduction network. These include chemoreceptors, also called ­transducers
or methyl-accepting chemotaxis proteins (MCPs), such as Tsr (sensitive to
serine), Tar (sensitive to aspartate and maltose), Trg (sensitive to ribose and
galactose), Tap (sensitive to dipeptides), and cytoplasmic proteins, such as
CheA, CheB, CheR, CheW, CheY and CheZ. MCPs are transmembrane proteins
approximately 550 amino acids in length with a periplasmic input or sensing
domain and a cytoplasmic output or signaling domain [10]. The receptor senses
changes in the periplasmic concentration of an attractant or of an attractant binding
protein. The cytoplasmic proteins (CheA, CheW, CheY and CheZ) relay information
from the receptor to the flagella by a phosphorylation cascade [11]. Chemotaxis
and motility are critical for the virulence of pathogenic leptospires, enabling
the bacteria to move towards attractants and avoid repellents, and thus respond
quickly to different environments. In addition, chemotaxis enables bacteria to
penetrate host tissue barriers during infection [12].

Analysis of the complete
genomic sequences of L. interrogans strain Lai suggested the presence of multiple copies of
chemotaxis homologue genes which were located in its large chromosome (12*MCP,
2*cheA, 3*cheW, 5*cheY, 3*cheB, 2*cheR, 2*cheDa
and 1*cheX, but not cheZ). This implies that L. interrogans
strain Lai employs
and regulates a complex chemosensory pathway. L. interrogans serovar
Lai strains 017 and
KH-1 were shown to chemotax toward hemoglobin and this movement was shown
to be related to their virulence [13]. A genetic approach for further study on
the role of chemotaxis in L. interrogans virulence is hindered by
the lack of adequate genetic systems and by their fastidious cultivation
requirements. There are no reports of successful transformation or transduction
in pathogenic Leptospira. This lack of adequate genetic systems is also
in part responsible for the paucity of information about chemosensory
mechanisms in L. interrogans [14].

CheW, is a coupling
protein, is involved in the interactions of chemotaxis histidine kinases with
methyl-accepting chemotaxis proteins (MCPs). In this study, we tried to analyze
the functions of L. interrogans chemosensory genes using E.
coli as a surrogate host by in silico analysis and in vivo
complementation.

ARKINSON

Materials and Methods

Strains and plasmids

Bacterial strains and
plasmids used in this study are shown in Table 1. L. interrogans
strain Lai was
cultured aerobically in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH)
medium at 28 ºC. E. coli strains were cultured aerobically in LB
broth at 30 ºC. Appropriate antibiotics (50 mg/ml streptomycin, 100 mg/ml ampicillin and 25 mg/ml kanamycin) were added when necessary. pQE31 and prr48 were used to allow the
IPTG-induced expression of desired proteins with an N-terminal tag containing
six histidine residues and the expression of the cloned native proteins,
respectively.

Bioinformatic analysis
of the interaction network of L. interrogans strain Lai
chemotactic proteins

The amino acid sequences of all ORFs in L. interrogans
strain Lai were obtained
from http://www.chgc.sh.cn/lep/.
Multiple-sequence alignments were accomplished online using the T-coffee
program (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi).
Domain prediction was performed using Pfam software (http://pfam.wustl.edu/hmmsearch.shtml).
Comparisons of the similarity of CheW proteins of L. interrogans strain Lai with those of E.
coli were performed using the plotsimilarity program from the GCG package
(Shanghai Institute of Biological Sciences,

Eq.
1

where all scores of the
four methods were normalized, respectively and the Sn is the normalized
score.

We compiled L.
interrogans strain Lai
proteins from different categories in the first level of the KO in KEGG database
[19] as the negative dataset, and those from the same categories of the fourth
level as the positive dataset. Then the correlation coefficient (CC) for
each method (CCPP, CCGN, CCRS and CCOP) was calculated as Equation 2:

Eq. 2

where the true positive
(TP) is the total number of protein pairs that appear both in the
predicted results and the positive database; the true negative (TN) is
the total number of protein pairs that appear in the negative database but not
in the predicted results; the false negative (FN) is the total number of
protein pairs that appear in the predicted ­results but not in the negative
database; and the false positive (FP) is the total number of
protein pairs that appear both in the ­predicted results and in the negative database.

In order to assess the
performance of computational methods, we calculated the range of accuracy (Equation
3) and ­coverage values of the result of each computational method.

Eq. 3

The coverage was calculated
as the number of proteins involved in predicted linkages appearing in KEGG
divided by the number of proteins of L. interrogans strain Lai in the KEGG
database. Visualization and network analysis were performed using the Pajek
program (http://vlado.fmf.uni-lj.si/pub/networks/pajek/).

Construction of the
recombinant plasmids

L. interrogans
strain Lai was cultured
at 28 ºC in liquid EMJH medium under aerobic conditions and collected at a
density of approximately 108 bacteria per ml. Genomic DNA was prepared
using Bacteria DNA minikit (Watson, Shanghai, China) according to the
manufacturer’s instructions. DNA fragments of 534, 465 and 516 base pairs,
containing the ORFs of cheW1, cheW2 and cheW3,
respectively, from L. interrogans strain Lai genomic DNA, were amplified by polymerase
chain reaction (PCR) ­using the following primers: cheW1 sense primer 5‘-CGCGGATCCATTGAT­TCTGGATTTTTATG-3‘
and antisense primer 5‘-CCCAAGCTTTTAGATAACCTCCTGTTCAA-3‘; cheW2 sense primer 5‘–CGCGGATCCcatgtcatccgaaatagacc-3‘
and antisense primer 5‘–CCCAAGCTTTTCATCCGACATTGTTTCCC-3‘; cheW3 sense primer 5‘–CGCGGATCCgatgagtgttttagaagat-3‘
and antisense primer 5‘–CCCAAGCTTCTGGTTCTAATATCATGTAG-3‘ which contain BamHI and HindIII
recognition sites. PCR products were purified by 1% agarose gel electrophoresis
and Qiaquick gel extraction kit (Qiagen), and digested with appropriate
restriction enzymes (Promega,

Expression of L.
interrogans strain Lai cheW genes

The expression of
recombinant plasmids was under the control of the tac promoter. This
allowed IPTG-induced expression of the desired protein with an N-terminal His
tag. Plasmids with inserts of the correct sequence were introduced into wild
type E. coli strain RP437 and cheW defective strain RP4606
containing the compatible ­plasmid pREP4. Plasmid pREP4 has lacIq for
negatively regulating expression from the tac promoter, thereby reducing
leaky expression. To test CheW1, CheW2 and CheW3 overproduction, 100 ml of overnight culture of RP437(pREP4) containing
the appropriate expression plasmid was added to 5 ml fresh LB, and after 2 h at
30 ºC, IPTG was added to a final concentration of

E. coli motility
assays

Swarming assay    E. coli swarming assays were
carried out using a modification of the method of Wolfe et al. [20]. The
strains carrying the expression plasmids were grown at 30 ºC overnight in LB
broth supplemented with streptomycin (50 mg/ml), ampicillin (100 mg/ml) and kanamycin (25 mg/ml). A 5 ml aliquot of
the appropriate culture (106–107 cells) was inoculated on the surface of an LB
swarm plate containing streptomycin, ampicillin, kanamycin, 0.3% agar and IPTG
at 0, 1, 10 or 100 mM near its
center. The plates were incubated at 30 ºC in a humid environment. RP437 and
mutant swarm sizes were measured after 8 h and 48 h respectively. Three ­independent
experiments were carried out and the ­corresponding swarm radii were averaged
and plotted.

Swimming behavior assay    The free-swimming ­behavior of E. coli
strains carrying expression plasmids was examined with a phase-contrast
microscope. Cells were grown overnight in TB medium (10 mg/ml tryptone, 5 mg/ml
NaCl) containing appropriate antibiotics. The ­stationary culture was diluted
at 1:E. coli growth assay    Fifteen milliliters of LB medium
containing streptomycin, kanamycin and ampicillin was inoculated with 150 ml of stationary phase overnight culture of RP437
(pREP4) containing an overexpression plasmid. A600 was read
immediately after inoculation. The cultures were incubated at 30 ºC for 1 h and
IPTG was added to a final concentration of

Results

In silico interaction
network of L. interrogans strain Lai che genes

There are 12 MCPs and 18
Che proteins in L. interrogans strain Lai [3]. Our predicted protein interaction network
of chemotaxis system includes 11 MCPs and 16 Che proteins (Fig. 1). The
11 MCPs almost link to each other. Among the Che proteins, CheW1 links to
CheA1, CheY, CheB and CheW2; CheW3 links to CheA2, MCP (LA2426), CheB3 and
CheD1; while CheW2 only links to CheW1. CheA1 links to Aer (MCP), CheY, LA4173
(CheY-like protein), CheW1, CheB1 and CheB3 while CheA2 links to LA2426 (MCP),
LA4173, CheW3, CheB1, CheB3 and CheD1.

Multiple-sequence
alignment of amino acid sequence of three CheW homologues from L.
interrogans strain Lai and CheW of E. coli K12 and Thermotoga
maritima

Multiple-sequence alignment
of amino acid sequence between three CheW homologues from L. interrogans
strain Lai and that
of E. coli K12 and Thermotoga ­maritima was accomplished
online using the t-coffee
program. The identity and similarity of CheW1(L), CheW2(L) and CheW3(L) with E.
coli CheW and that of Thermotoga maritima was significant (Figs.
2 and 3). The
identity and similarity with E. coli CheW was 31%, 35%,
36% and 54%, 56%, 57% respectively. The identity and similarity with Thermotoga
maritima CheW was 36%, 25%, 28% and 57%, 52%, 48% respectively.
Comparison of the similarity of CheW proteins of L. interrogans strain Lai with CheW of E.
coli was completed using the Plotsimilarity
program from the GCG package (Shanghai Institute of Biological Sciences). The
window size was 10. Regions of high sequence similarity have higher scores than
those with low similarity on an arbitrary scale. The dotted line is the average
similarity score for the whole protein (Fig. 4). From Fig. 4 we
can see that in the region of residues 30–

Effect of the expression
of L. interrogans strain Lai
cheW genes on swarming phenotypes of wild-type and cheW
mutant in E. coli strains

E. coli
RP437 and mutant RP4606, containing the pREP4 plasmid expressing lacIq, were transformed with plasmids expressing N-terminal
His-tagged cheW1, cheW2 and cheW3, or with pQE31 as a
control. Expressions
of these fused proteins were confirmed by SDS-PAGE gels stained with Coomassie
blue (Fig. 5) and western
blotting (Fig. 6). The effects of the expression of N-terminal
His-tagged CheW1, CheW2 and CheW3 proteins on swarming in E. coli mutant
RP4606 are shown in Fig. 7. CheW1 and CheW3 restored swarming to RP4606,
giving the biggest
bands at an IPTG concentration of 10 mM. For CheW2, no significant increase in swarm size
compared with that of the negative control, RP4606 (pREP4) containing only
pQE31, was seen at any IPTG concentration (Fig. 7). Expression of native
(untagged) CheW2 also failed to
restore swarming of RP4606. The swarming of RP437 was inhibited by a high
expression of CheW1 and CheW3, but to a lesser extent by CheW2 (Fig. 8).
The inhibitory effect was IPTG concentration-dependent. In each case, there was
no significant effect on the growth rate of RP437.

Effect of the expression
of L. interrogans strain
Lai cheW genes on tumbling frequency of E. coli
wild-type and mutant strains

Phase-contrast
microscopy was used to test the effect of the expression of L. interrogans
cheW genes on the ­tumbling frequency. The mutant strain RP4606 is ­­smooth-swimming,
that is, it is locked in CCW rotation and ­exhibits no (or little) tumbling. An
increase in tumbling frequency is observed as decreased smooth swimming occurs,
that is, decreased swimming in one direction without reversal of direction.
Using this approach, it was found that ­expression of CheW1 and CheW

Discussion

The complete genome
sequence of L. interrogans strain
Lai suggested that there are multiple copies of putative
chemotaxis-associated genes located at its large ­chromosome (12*MCP, 2*cheA,
3*cheW, 5*cheY, 3*cheB, 2*cheR, 2*cheD, 1*cheX,
but no cheZ), but their real functions have not been verified. To date,
little is known about the mechanisms involved in sensing of attractants in this
species and suitable genetic tools for studying this bacterium are still not
available. In this study we focused on the characterization of three cheW-like
genes found in the genome of L. interrogans, using a combination
of in silico and in vivo approaches. The prediction of the ­interaction
network of L. interrogans strain
Lai ­chemotaxis and motility associated proteins demonstrated that
the 11 MCPs are likely to link to each other and that CheW2 links solely to
CheW1, suggesting that CheW2 may not ­function alone but in certain
physiological conditions may require CheW1 to function. Multiple amino acid
sequence ­alignment of the three CheW homologues from L. interrogans strain Lai, CheW1(L),
CheW2(L) and CheW3(L), shows >31% identity
and >54% similarity with E.
coli K12 CheW. In addition, all three L. interrogans CheWs
are predicted to have a global CheW-like domain. These analyses suggested that
the L. interrogans CheWs are highly homologous to E. coli
CheW. Our results indicated that CheW1
and CheW3 could restore the
defective swarming phenotype of the cheW mutant E. coli
RP4606, while CheW2 could not. E.
coli CheW was proposed to play a central role in linking the signaling
state of the receptor to the phosphorylation cascade. E. coli
CheW was also a crucial member of a stable functional CheA/CheW complex and was
required for the receptor-mediated regulation of autophosphorylation of the
complex [11,21,22].

A molecular analysis of
the residues involved in E. coli CheA/E. coli CheW
interaction gives an insight into the failure of L. interrogans
CheW2 to complement an E. coli cheW mutant. E.
coli CheW has been shown to interact with the signaling domain of MCPs and
the C-terminus of CheA [22–25]. The
region between residues 50–110 of CheW
shows homology to the P5 domain of CheA ­proteins and may be important in
CheW/CheA interactions [26]. However, mutations in the MCP Tsr protein are
clustered between residues 50–110 [27], implying
that this region may be involved in MCP binding. A similarity plot comparing
the amino acid sequences of L. interrogans strain Lai CheWs with E.
coli CheW (Fig. 4) shows that in the ­region of residues 30–50 CheW2 has much poorer homology to E. coli CheW than L.
interrogans CheW1 or CheW3. ­Although all three CheWs have high homology
to E. coli CheW at residues 50–110, the lack of swarm rings typical of true chemotaxis
[28] implies that the restoration of CheW effect to RP4606 is not subject to
chemosensory regulation by E. coli MCPs. Therefore, even if CheW1
and CheW3 are able to bind to MCPs, they are unable to transmit the signal to E.
coli CheA, but presumably enhance the rate of its autophosphorylation
[5] assuming a similar mechanism in E. coli and L.
interrogans chemotaxis.

The solution
structure of CheW from Thermotoga ­maritima provides a structural
basis for a model in which CheW acts as a molecular bridge between CheA and the
cytoplasmic tails of the receptors and also revealed the probable interaction
sites [29]. To better
define the role of CheW and its binding interactions, Boukhvalova et
al. [30] performed biochemical characterization of six mutant ­variants of
CheW and examined the ability of the purified mutant CheW proteins to bind to
CheA and Tar, to ­promote formation of active ternary complexes and to support
chemotaxis in vivo. The results indicate that mutations with elimination
of CheW binding to Tar (VOverexpression of cheW1
and cheWIn conclusion, this
study suggested that CheW1 and CheW3 of L. interrogans strain Lai are able to ­substitute
for the E. coli CheW proteins in the phosphorelay ­pathway and
thus had an in vivo function analogous to E. coli CheW
function. Again, the lack of swarm ring suggested that L. interrogans
strain Lai CheWs
could not restore ­chemotaxis to the E. coli strains. The
existence of multiple copies of chemotaxis proteins suggests that L. interrogans
strain Lai might
have a complex chemosensory pathway. Our studies demonstrating by
complementation that L. interrogans CheW1 and CheW3 act
differently from CheW2 define one aspect of that complexity. While a ­detailed
explanation for the presence of multiple copies of chemotaxis genes is
presently unknown, it is important to note that multiple chemotaxis operons or
gene homologues have been found in many bacterial genomes. Among these are Borellia
burgdorferi [33], Treponema pallidum [34], Caulobacter crescentus [35], Sinorhizobium meliloti
[36] and Rhodobacter sphaeroides [5]. Our study demonstrates the
feasibility of combining in vivo and in silico approaches to
study protein function. In addition, the recombinant L. interrogans
CheW proteins we expressed will be useful for more detailed studies of function
of the three CheW homologues, for example, for studying interactions with L.
interrogans MCPs.

Acknowledgements

We are extremely
grateful to Dr. S. PARKINSON (University of Utah, Salt Lake City, USA) for
kindly ­providing the E. coli strains, and to Dr. H. Steinman (Albert Einstein College of
Medicine, Bronx, USA) for the critical reading of the manuscript. We also thank
Dr. Wei Wu and Dr. Zhi-Wei Cao for their useful information.

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