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Arch Microbiol (2009) 191:501–507
DOI 10.1007/s00203-009-0475-x
ORIGINAL PAPER
Characterization of chsA, a new gene controlling the chemotactic
response in Azospirillum brasilense Sp7
Ricardo Carreño-López · Araceli Sánchez ·
Nohemí Camargo · Claudine Elmerich ·
Beatriz Eugenia Baca
Received: 16 September 2008 / Revised: 7 March 2009 / Accepted: 27 March 2009 / Published online: 24 April 2009
© Springer-Verlag 2009
Abstract We report, here, the characterization of a
mutant strain of Azospirillum brasilense Sp7 impaired in
surface motility and chemotactic response. Presence of
Xagella in the mutant strain was conWrmed by western blot
analysis, using antisera raised against the polar and lateral
Xagellins, and by electron microscopy. Genetic complementation and nucleotide sequencing led to the identiWcation of a new gene, named chsA. The deduced translation
product, ChsA protein, contained a PAS sensory domain
and an EAL domain. As ChsA displayed characteristic signaling protein architecture, it is thought that this protein is a
component of the signaling pathway controlling chemotaxis in Azospirillum.
Keywords Azospirillum brasilense ·
ChsA signaling protein · Chemotactic response
Introduction
Azospirillum brasilense, a nitrogen-Wxing -proteobacterium promotes growth of various grasses and cereals
(Dobbelaere et al. 2001). The bacterium possesses a single
Communicated by Ursula Priefer.
R. Carreño-López · A. Sánchez · N. Camargo · B. E. Baca (&)
Centro de Investigaciones en Ciencias Microbiológicas,
Benemérita Universidad Autónoma de Puebla,
72000 Puebla, Mexico
e-mail: [email protected]
C. Elmerich
Département de Microbiologie, BMGE,
Institut Pasteur, Paris, France
polar Xagellum and multiple lateral Xagella. The polar
Xagellum is responsible for the swimming motility in liquid media, whereas the lateral Xagella are responsible for
surface motility also referred to as swarming motility
(Hall and Krieg 1983; Moens et al. 1995). A. brasilense
exhibits chemotaxis toward a variety of oxidizable substrates including sugars, amino acids and organic acids,
oxygen, and redox molecules (Alexandre et al. 2000;
Barak et al. 1983). Non-Xagellated mutants as well as a
mutant strain impaired in chemotactic response showed
reduced colonization of the root system suggesting that
active movement of Azospirillum is important for the initiation of root colonization (Vande Broek et al. 1998).
Several of the che genes encoding central transduction
pathways in chemotaxis in other bacterial species (cheA,
-W, -Y, -B and -R) were identiWed in Azospirillum (Hauwaerts et al. 2002; Bible et al. 2008). Evidence for the existence of a putative methyl-accepting chemotaxis protein
(Potrich et al. 2001), as well as the identiWcation of a chemoreceptor-like protein (Tlp1), which mediates energy
taxis in A. brasilense was also reported (Greer-Phillips
et al. 2004).
The present study reports the genetic characterization
of a mutant strain impaired in surface motility and
chemotaxis. This led to the identiWcation of chsA, diVerent from genes previously characterized as being
involved in chemotaxis in Azospirillum. The ChsA
deduced translation product contains a sensory PAS
domain and an EAL domain suggesting it may be part of
the signaling pathway controlling chemotaxis in
Azospirillum.
An abstract of this work presented at the 15th International nitrogen-Wxation conference has been published in
the conference proceedings book (Carreño-López et al.
2008).
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Arch Microbiol (2009) 191:501–507
Materials and methods
Bacterial strain, plasmids, and growth conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. Escherichia coli was grown at 37°C and
A. brasilense was grown at 30°C. Rich medium was LB.
A. brasilense minimal medium was medium K (CarreñoLópez et al. 2000) supplemented with 20 mM lactate or
with one of the following carbon sources at 10 mM: succinate, malate, pyruvate, glutamate, and proline as indicated.
Antibiotics were used at the Wnal concentration (g/ml):
kanamycin 20, tetracycline 10, and ampicillin 100 g/ml.
SalI site of pVK100 and subsequently a 0.8 kb fragment
was deleted to yield pABPst. A 6.8 kb EcoRI-KpnI fragment was cloned into the pBS vector for nucleotide
sequencing. The same fragment was also cloned into
pVK100 to yield pAB6.8. The PvuI fragments, carrying
Tn5 insertions in chsA (insertion 31) and purK (insertion
88) in pAB7115, were subsequently cloned into pSUP202
for mutagenesis of strain Sp7, by homologous recombination, to yield Sp74031 (chsA-Tn5) and Sp74088 (purKTn5), respectively (Table 1). Successful recombination was
conWrmed by hybridization as previously described
(Carreño-López et al. 2000).
Nucleotide sequence accession number
DNA manipulations
Standard methods were used for DNA puriWcation, digestion with restriction enzymes, agarose gel electrophoresis,
DNA ligation, hybridization, transformation, and molecular
cloning (Ausubel et al. 1999). To amplify the structural
gene of the lateral Xagella laf1 (Moens et al. 1995), we have
designed the following oligonucleotides: forward primer
5⬘-TGACCGACGATCTGGCGACCACCCA-3⬘, reverse
primer 5⬘-CTCGTTCATGTCGGCGTCCACCAGG-3⬘.
Plasmid construction and mutagenesis
Plasmid pAB7.4 (Fig. 1) contains a 7.4-kb SalI fragment
puriWed from pAB7115 and then cloned into the XhoI site
of pVK100. The same fragment was also cloned into the
The chsA, purK and pfkA1 sequence, have been deposited
in the GenBank database with accession number
AM050060.
Chemotaxis assay
The chemotaxis assay was performed as described by
Alexandre et al. (2000). Bacteria grown in K-lactate medium
were collected by centrifugation at 10,000g at 4°C for
5 min and suspended in medium K supplemented with a
carbon source to be tested as an attractant. Five l of suspension containing 5 £ 106 CFU were spotted onto semisolid medium, of the same composition, containing 0.25%
agar. The size of the bacterial chemotactic ring was
recorded after incubation at 30°C for 48 h.
Table 1 Bacterial strains and plasmids used in this study
Strain or plasmids
Relevant properties
Reference or source
Azospirillum brasilense
Sp7
Wild type ATCC29145
Sp7S
Sp7 derivative impaired in Xocculation
Katupitiya et al. (1995)
Sp74031
Derivative of Sp7, chsA-Tn5, KmR
This work
Sp74088
Derivative of Sp7, purK-Tn5, KmR
This work
7201
Derivative of Sp7, rpoN
Elmerich et al. (1997)
Cloning vector, ApR, lacZ
Stratagene
Plasmids
pBSK
R
pTZ18R
Cloning vector, Ap
Pharmacia
pSUP202
Suicide vector, pBR325 mob Tc Ap Cm
Simon et al. (1983)
pVK100
RK2 replicon, IncP, Tc Km
Knauf and Nester (1982)
pAB7115
pVK100 derivative containing a 19 kb insert that restored motility to Sp7S
Pereg-Gerk et al. (1998)
pAB7.4
pVK100 derivative containing a 7.4 kb SalI fragment carrying chsA and purK
This work
pABPst
pAB7.4 with a PstI
This work
pAB6.8
pVK100 derivative containing a 6.8 kb EcoRI-KpnI fragment carrying chsA
This work
pAB1.2
pBSK derived containing a EcoRI 1.2 fragment from pAB7115
This work
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Arch Microbiol (2009) 191:501–507
Fig. 1 Map of the DNA region
on pAB7115 carrying the chsA,
purK and pfkA1 genes in
A. brasilense Sp7. Orientation of
transcription is indicated by
arrows. Black triangles show the
location of the Tn5 (KmR)
insertion 31 and 88. The
approximate location of PAS
and EAL domains in ChsA
protein is shown in boxes.
Restriction sites: S SalI, K KpnI,
E EcoRI, P PstI, X XhoI, B BglII
503
X/S SE
E PE P
P
S
P
KX K
P K
S
B
P
pAB7115
1 kb
pAB7.4
S/X
K
E
pAB6.8
pAB1.2
Sp74031
Sp74088
Tn5
Tn5
E
E
586
purK
chsA
hmp
P
500
pfkA1
K
S
400
300
200
100
1
1 kb
ChsA
EAL
PAS
Preparation of antibodies against polar and lateral Xagellins
Electron microscopy
Polar Xagellum Wlaments were puriWed from bacteria
grown in LB liquid medium according to Croes et al.
(1993). A crude preparation of a mixture of polar and lateral Wlaments was obtained from bacteria grown on LB
plates solidiWed with 0.75% agar. Polar and lateral Wlaments were separated by CsCl gradient (Croes et al. 1993).
Homogeneity of both Xagellin preparations was checked by
SDS-PAGE and speciWc antibodies were obtained by
immunization of two rabbits, according to standard procedures (Ausubel et al. 1999).
Bacteria collected from liquid medium or from semi-solid
medium at the periphery of the chemotactic ring were suspended into a drop of water on a carbon-coated copper grid
(mesh 200). The grids were washed once with distilled
water and stained with 0.5% uranyl acetate. The negative
stained cells were visualized with a transmission electron
microscope (Phillips TEM, CM 100).
Western immunoblots
IdentiWcation of chsA, a locus involved in surface motility
Bacteria were harvested from liquid medium or plates (as
above) and sonicated in Tris–HCl 10 mM buVer, pH 8.0,
centrifuged at 10,000g, at 4°C for 10 min to obtain crude
extracts. Approximately 50 g of soluble protein was separated by 12% SDS-PAGE (Laemmli 1970) and blotted onto
polyvinylidene diXuoride membranes (Millipore) in transfer buVer (Tris 15 mM, glycine 120 mM, 10% methanol).
Membranes were incubated in blocking buVer [TBS (Tris–
HCl 20 mM, pH 7.6, NaCl 130 mM) containing 3% bovine
serum albumin and 0.1% Tween 20] and then incubated
with shaking for 2 h, at 25°C with anti-polar Xagellin or
anti-lateral-Xagellin antibodies at a dilution of 1:1,000.
Membranes were incubated with secondary horseradish
peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical Co.) at 20°C for 30 min at a dilution of 1:2,000. The
reaction was developed by 5-bromide-4-chlore-3-indole
phosphate and blue tetrazolium (BCIP/NTB).
A. brasilense strain Sp7S, which was originally characterized as a Xocculation mutant of Sp7 wild type strain
(Katupitiya et al. 1995), was also found to carry another
independent mutation that impaired its motility properties
when inoculated in semi-solid medium containing lactate as
the growth substrate, but not when inoculated in liquid
medium (Pereg-Gerk et al. 1998). Preliminary experiments
led to the isolation of plasmid pAB7115 that restored full
motility to Sp7S (Pereg-Gerk et al. 1998). It was then
reported that the motility defect of Sp7S resulted from the
absence of lateral Xagella (Pereg-Gerk et al. 2000). Therefore, the presence of laf1 gene on pAB7115 was checked
by PCR ampliWcation. No ampliWcation of laf1 was
obtained using pAB7115 DNA, while ampliWcation was
achieved using the total DNA of Sp7 as a template (data not
shown). Consequently, the laf1 gene is not carried by
pAB7115, and the genetic complementation of Sp7S
Results
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504
observed cannot be due to complementation of a mutation
in laf1 structural gene, in contrast to former conclusion
(Pereg-Gerk et al. 1998).
Isolation of Tn5 insertions in pAB7115 allowed to
roughly map the DNA region required for genetic complementation of the motility defect in Sp7S (Carreño-López
et al. 2002). Tn5 insertion 31 (Fig. 1) in pAB7115, that did
not complement Sp7S, mapped within a 7.4 kb SalI fragment. It was then found that plasmid pAB7.4 (Fig. 1) containing the SalI fragment restored the wild type phenotype,
while its derivative pABPst carrying a short deletion
abolished the complementation. Recombination of insertion
31 into the wild type genome led to a mutant strain, named
Sp74031, displaying the same growth rate as the wild type
in liquid medium but showing a defect in motility in semisolid lactate medium.
Nucleotide sequencing of the complementing region led
to the identiWcation chsA gene coding for a putative signal
transduction protein designated ChsA. The inferred ChsA
protein (63,714 Da, 586 residues) has characteristics similar to cytoplasmic signaling proteins. It contains two
domains: a PAS sensory domain near the N terminus region
(from residues 50 to 160) and an EAL transmitter domain
in the C-terminus part (from residues 330 to 560) (Fig. 1).
The EAL domain is present in proteins with phosphodiesterase activity (PDE-A) involved in the hydrolysis of c-diGMP (cyclic-bis (3⬘-5⬘) dimeric GMP), a compound known
to function as a second messenger in a broad spectrum of
cellular processes including motility, bioWlm formation,
and cellular diVerentiation (Römling et al. 2005; Jenal and
Malone 2006). Proteins containing an EAL domain often
possess another domain GGDEF, carrying guanylate
cyclase activity, but this domain is not present in ChsA.
The PAS sensory domain is found in a variety of proteins,
with redox functions, including NifL, Dos and Aer, which
typically bind heme, and Xavines (Taylor and Zhulin 1999;
Greer-Phillips et al 2003). While many proteins carry
GGDEF/EAL/PAS domains, very few contain only the
PAS and EAL domains. Databases searches using the
BLASTP program revealed that ChsA shared similarity
with a limited number of uncharacterized ORFs from Proteobacteria, phylogenetically close to Azospirillum, such as
Magnetospirillum magnetotacticum (YP_421664.1) and
Rhodospirillum rubrum (YP_426135.1).
The nucleotide sequencing of the DNA region upstream
of chsA revealed a second ORF, in the same orientation
(Fig. 1). This ORF was similar to the purK gene encoding
for a putative phosphoribosyl amine imidazol carboxylase
synthase involved in purine synthesis. However, inactivation of purK (strain Sp74088) did not lead to purine auxotrophy and the mutant strain was not impaired in its motility
properties. Another ORF that shares sequence identity with
the phosphofructokinase pfkA1 gene was also identiWed,
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Arch Microbiol (2009) 191:501–507
which catalyze the phosphorylation of fructose-6-phosphate to fructose-1, 6-biphosphate. This ORF was in the
opposite orientation of chsA, and was upstream of purK.
After an intergenic region of 366 nts was identiWed a gene
which was downstream and divergent to chsA, that coded
for a putative membrane protein, likely not involved in the
quimiotactic response (Fig. 1).
Analysis of Xagella and Xagellin contents in the chsA
mutant
Transmission electron microscopy was used to determine if
both Xagella types were present in Sp7S and in the chsA
Sp74031 mutant strain. The wild type, and the non-motile
rpoN mutant were used as controls. The rpoN mutant was
chosen because rpoN controls the synthesis of polar and lateral Xagella in Azospirillum (Milcamps et al. 1996). TEM
showed that all strains, except the rpoN mutant, had a polar
Xagellum in liquid medium (not shown) and both polar
Xagellum and lateral Xagella when grown on solid surface
(Fig. 2). Presence of Xagella was also found with the
Sp74088 purK mutant (not shown). Then, Xagellins proteins were detected by Western blot analysis using polyclonal antibodies raised against polar and lateral Xagellin
proteins. Cell-free extracts from Sp7, Sp7S, 74031, and
74031(pAB6.8) (Fig. 3a) reacted to antibodies speciWc for
the polar Xagellin, and a protein with a molecular mass of
100 kDa was detected corresponding to the size previously
reported by Croes et al. (1993). Antibody speciWc for the
lateral Xagellin protein reacted with a 45 kDa protein
(Fig. 3b), in agreement with the size reported by Moens
et al. (1995). As expected, cell-free extracts from the rpoN
mutant did not react to any antibodies (Fig. 3a, b, lane 6).
This favors the hypothesis that ChsA protein does not play
a role in the synthesis of Xagella in A. brasilense.
Chemotactic properties of the chsA mutant
Because the motility defect of the chsA mutant was not due
to lack of Xagella, we assayed diVerent substrates involved
in the chemotactic response. Strain Sp7 showed a strong
chemotactic response to three organic acids, namely
malate, succinate and pyruvate, and the amino acids glutamate and proline. As expected strain rpoN was completely
non-chemotactic. We observed a signiWcant decrease (ca.
of 20%) in the chemotactic response, with all the substrates
used, in the case of chsA-Tn5 mutant strain (Sp74031) as
compared to the wild type (Fig. 4). Statistical analysis
revealed a P value < 0.05. Chemotactic ability was restored
in the 74031 strain by introducing the wild type chsA gene
harbored by pAB7115 and pAB6.8 plasmids. It was
checked that pVK100 plasmid vector as well as pAB6.8chsA-Tn5 did not restore the chemotactic response when
Arch Microbiol (2009) 191:501–507
505
Fig. 2 Transmission electron
microscopy of A. brasilense wild
type and mutant strain showing
polar and lateral Xagella. Sp7
wild type, 7201 rpoN non-Xagellated control, Sp7S surface
motility mutant, 74031 chsATn5 mutant. Bacteria were
grown in semi-solid media with
0.25% agar and the photographs
represent the most predominant
cell type observed. Bar 1 m
1
2
3
4
5
7
6
6
100 kDa
1 2
3 4
5 6
45 kDa
Migration cm
5
4
3
2
1
Fig. 3 Western immunoblot of cell-free extract probed with polyclonal antibodies speciWc for polar Xagellin (a) or polyclonal antibodies for lateral Xagellin (b). Lane 1 A. brasilense Sp7, pre immune
serum, lane 2 A. brasilense Sp7, lane 3 74031, lane 4 74031(pAB6.8),
lane 5 Sp7S, lane 6 RpoN
introduced into Sp74031 mutant (data not shown). The chemotactic response of the purK mutant strain (Sp74088) did
not diVer from the wild type (data not shown).
Discussion
Genetic complementation of a mutant strain, Sp7S,
impaired in surface motility led to the cloning and identiWcation of a new gene, designated chsA. Construction of a
0
a b c d e
Wild type
a b c d e
chsA
a b c d e
a b c d e
a b c d e
chsA(pAB7115) chsA(pAB6.8) rpoN
Fig. 4 Chemotactic response of A. brasilense Sp7 and mutant strains.
Chemotaxis was evaluated by the size of the swarming halo with on
semi-solid media containing the 10 mM of the indicated substrate:
a malate, b succinate, c pyruvate, d glutamate, e proline. Error bars
represent standard deviation from three independent experiments
chsA-Tn5 mutant strain (74031) isogenic to the wild type
Sp7 led to a mutant strain not impaired in its growth properties but showing reduced motility in soft-agar plates. Electron microscopy and immunochemistry using polyclonal
antibodies raised against both polar and lateral Xagellins
revealed that Sp7, Sp7S, and 74031 possessed both polar
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506
and lateral Xagella (Figs. 2, 3) suggesting that the motility
defect was not due to lack of lateral Xagella but instead it
may be due to a diVerence in chemotactic response. Partially reduced chemotactic response was also reported for
cheB and cheR mutants of the adaptation pathway suggesting multiple chemotaxis systems in Azospirillum (Stephens
et al. 2006; Bible et al. 2008).
Deduced translation of chsA revealed that ChsA product
contains a PAS and an EAL domain. Many of the proteins
involved in the metabolism of the second messenger compound, c-di-GMP, contains both EAL and GGDEF
domains responsible, respectively, for phosphodiesterase
and diguanylate cyclase activities (Römling et al. 2005;
Jenal and Malone 2006; Schmidt et al. 2005). As ChsA protein does not contain GGDEF domain, its involvement in
the metabolism of c-di-GMP remains hypothetical. However, a recent study has shown that indeed the EAL domain
of the Vibrio cholerae VieA protein, which does not contain a GGDEF domain, displayed cyclic diguanylate phosphodiesterase activity (Tamayo et al. 2005). VieA protein
controls the shift between bioWlm formation and virulence
(Tamayo et al. 2005) and another EAL protein also devoid
of GGDEF domain, YhjH protein from Salmonella enterica
serovar Typhimurium is implicated in the transition
between sessility to motility (Simm et al. 2004). Survey of
bacterial genomes revealed that the number of genes encoding proteins with GGDEF or EAL domains could vary from
none to up to 99 suggesting that the role of each individual
protein may be extremely complex and that their inactivation is unlikely to result in clear-cut phenotype in case of
high multiplicity (Galperin 2004). Proteins containing a
PAS domain are present in numerous bacteria and are
involved in sensing a variety of environmental signals
(Taylor and Zhulin 1999). It is tempting to speculate that
the PAS domain in ChsA protein sense the redox state of
the cell through a cytoplasmic signaling molecule directly
coupled to the transmitter EAL module. However, the relationship between ChsA and the regulation of the chemotactic machinery is unknown.
Acknowledgments The authors wish to thank Ms N. Desnoues and
Ma. L. Xiqui for skillful technical assistance and Ms Jerri Bram for
improving the language. This work was partially supported by a grant
of VIEP-SEP.
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