Actinorhizal Signaling Molecules: Frankia Root Hair Deforming Factor Shares Properties With NIN Inducing Factor

Actinorhizal plants are able to establish a symbiotic relationship with Frankia bacteria leading to the formation of root nodules. The symbiotic interaction starts with the exchange of symbiotic signals in the soil between the plant and the bacteria. This molecular dialog involves signaling molecules that are responsible for the specific recognition of the plant host and its endosymbiont. Here we studied two factors potentially involved in signaling between Frankia casuarinae and its actinorhizal host Casuarina glauca: (1) the Root Hair Deforming Factor (CgRHDF) detected using a test based on the characteristic deformation of C. glauca root hairs inoculated with F. casuarinae and (2) a NIN activating factor (CgNINA) which is able to activate the expression of CgNIN, a symbiotic gene expressed during preinfection stages of root hair development. We showed that CgRHDF and CgNINA corresponded to small thermoresistant molecules. Both factors were also hydrophilic and resistant to a chitinase digestion indicating structural differences from rhizobial Nod factors (NFs) or mycorrhizal Myc-LCOs. We also investigated the presence of CgNINA and CgRHDF in 16 Frankia strains representative of Frankia diversity. High levels of root hair deformation (RHD) and activation of ProCgNIN were detected for Casuarina-infective strains from clade Ic and closely related strains from clade Ia unable to nodulate C. glauca. Lower levels were present for distantly related strains belonging to clade III. No CgRHDF or CgNINA could be detected for Frankia coriariae (Clade II) or for uninfective strains from clade IV

Signalling in actinorhizal root nodule symbioses

International audiencePlants able to establish a nitrogen-fixing root nodule symbiosis with the actinobacterium Frankia are called actinorhizal. These interactions lead to the formation of new root organs, called actinorhizal nodules, where the bacteria are hosted intracellularly and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. Like other symbiotic interactions, actinorhizal nodulation involves elaborate signalling between both partners of the symbiosis, leading to specific recognition between the plant and its compatible microbial partner, its accommodation inside plant cells and the development of functional root nodules. Actinorhizal nodulation shares many features with rhizobial nodulation but our knowledge on the molecular mechanisms involved in actinorhizal nodulation remains very scarce. However recent technical achievements for several actinorhizal species are allowing major discoveries in this field. In this review, we provide an outline on signalling molecules involved at different stages of actinorhizal nodule formation and the corresponding signalling pathways and gene networks

Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment

International audienceA halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35–45 °C, at pH 6.0–8.0 and 0.05–0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3–5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA–DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB′ genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T)

Characterization of leucocin B-KM432Bz from Leuconostoc pseudomesenteroides isolated from boza, and comparison of its efficiency to pediocin PA-1.

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EII(t) Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1

Inhibitory spectrum of cell-free culture supernatant of <i>Leuc. pseudomesenteroides</i> KM432Bz against various target strains.

a<p>LB, Luria-Bertani broth; MRS, de Man Rogosa Sharpe; BHI, Brain heart infusion; TSYE, Trypticase soy yeast extract,</p>b<p>Inhibition halos were measured by radial diffusion assays (+: Inhibition halo; -: No inhibition).</p

Growth of strain KM432Bz and bacteriocin production in MRS broth.

<p>Antimicrobial activity of cell-free supernatants is evaluated by radial diffusion assay against <i>Lact. sakei</i> subsp. <i>sakei</i> CIP 103139 and measure of the inhibition halos (▪). For the inhibition halos, standard deviation values are less than 2% and are not indicated. Values of optical density (⧫) are presented as the mean of three independent experiments with standard error of the mean.</p

Minimal Inhibitory Concentrations^a (nM) of purified leucocin B-KM432Bz and pediocin PA-1 on sensitive strains.

a<p>The MIC is defined as the lowest bacteriocin concentration for which no growth could be observed,</p>b<p>Pediocin used in this study was purchased from Sigma (P0098).</p

Mass spectrometry analysis of leucocin B-KM432Bz.

<p>(a) MALDI-TOF MS spectrum of the purified leucocin B-KM432Bz showing a single [M+H]⁺ ion at <i>m/z</i> 3932; (b) ESI-MS spectrum of the RP-HPLC fraction containing leucocin B-KM432Bz that exhibits [M+5H]⁵⁺ and [M+4H]⁴⁺ ions.</p

Reversed-phase HPLC elution profile of the 40% isopropanol Sep-Pak fraction.

<p>Separation was performed on an ODS2 Inertsil column under a gradient of 0 to 100% acetonitrile in 0.1% aqueous trifluoroacetic acid (dashed line). Arrow indicates the active fraction against <i>Lact. sakei</i> subsp. <i>sakei</i> CIP 103139 and <i>L. ivanovii</i> subsp. <i>ivanovii</i> CIP 78.42.</p

Alignment of amino acid sequences of class IIa bacteriocin precursors related to leucocin B-432Bz.

<p>Amino acid sequence of leucocin B-KM432Bz precursor (this study) was aligned class IIa bacteriocin precursors from <i>Leuconostoc</i>: leucocin A-UAL187, leucocin A-QU15, leucocin B-Ta11a, mesentericin Y105 and pediocin PA-1 from <i>Ped. acidilactici</i> (UniProtKB database accession numbers P34034, D7UPI7, Q53446, P38577, and P29430). Bold letters show the sequence of the mature bacteriocin and italics indicate the sequence of the leader peptide. The sequence of the leader peptide and the identification of leucine residue of leucocin B-KM432Bz were obtained by translation of the nucleotide sequence. Light grey background highlights the typical class IIa bacteriocin consensus sequence. Cysteines involved in the formation of the disulfide bridges are shown on dark grey background. The amino acid sequence of leucocin B-KM432Bz determined by Edman degradation is underlined. The amino acid sequence obtained by MS/MS fragmentation is indicated by a broken line. Arrow indicates the GluC endoproteinase cleavage site.</p