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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). 123 502 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 123 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 123 504 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, 123 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 123 506 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. References Alexandre G, Greer SE, Zhulin IB (2000) Energy taxis is the dominant behavior in Azospirillum brasilense. J Bacteriol 182:6042–6048 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1999) Short protocols in molecular biology. Wiley, New York Barak R, Nur I, Okon Y (1983) Detection of chemotaxis in Azospirillum brasilense. J Appl Bacteriol 53:399–403 123 Arch Microbiol (2009) 191:501–507 Bible AN, Stephens BB, Ortega DR, Xie Z, Alexandre G (2008) Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping and cell length in the alphaproteobacterium Azospirillum brasilense. 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