Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1ß Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Poly-3-hydroxybutyrate synthesis by different Azospirillum brasilense strains under varying nitrogen deficiency: A comparative in-situ FTIR spectroscopic analysis.

Monitoring of poly-3-hydroxybutyrate accumulation and changes in its relative contents in biomass of the plant-growth-promoting bacteria Azospirillum brasilense (strains Sp7, Cd and Sp245) was performed during aerobic cultivation for 1 to 8 days at various initial concentrations of bound nitrogen (0.1 to 0.5 g∙L-1 NH4Cl) in the culture medium using in-situ transmission FTIR spectroscopy. A methodology has been proposed based on calculating band areas in FTIR spectra (instead of band intensities commonly used earlier) for determining relative contents of PHB in dry bacterial biomass, using the ester ν(C=O) band as a PHB marker (in the region 1750-1720 cm-1) and amide II band of cellular proteins (at ca. 1540 cm-1). Differences in PHB accumulation levels and their changes in the course of cultivation under various trophic stress for the three strains are discussed in relation to their different ecological niches which they occupy in the rhizosphere.

Plasmid gene for putative integral membrane protein affects formation of lipopolysaccharide and motility in Azospirillum brasilense Sp245.

The bacterium Azospirillum brasilense can swim and swarm owing to the work of polar and lateral flagella. Its major surface glycopolymers consist of lipopolysaccharides (LPS) and Calcofluor-binding polysaccharides (Cal+ phenotype). Motility and surface glycopolymers are important for the interactions of plant-associated bacteria with plants. The facultative plant endophyte A. brasilense Sp245 produces two antigenically different LPS, LpsI, and LpsII, containing identical O-polysaccharides. Previously, using vector pJFF350 for random Omegon-Km mutagenesis, we constructed a mutant of Sp245 named KM018 that still possessed flagella, although paralyzed. The mutant was no longer able to produce Calcofluor-binding polysaccharides and LpsII. Because of the limited experimental data on the genetic aspects of surface glycopolymer production and flagellar motility in azospirilla, the aim of this study was to identify and examine in more detail the coding sequence of strain Sp245, inactivated in the mutant. We found that pJFF350 was integrated into a coding sequence for a putative integral membrane protein of unknown function (AZOBR_p60025) located in the sixth plasmid of Sp245. To clarify the role of the putative protein, we cloned AZOBR_p60025 in the expression vector pRK415 and used it for the genetic complementation of mutant KM018. The SDS-PAGE, immunodiffusion, and linear immunoelectrophoresis analyses showed that in strain KM018 (pRK415-p60025), the wild-type LpsI+ LpsII+ profile was restored. The complemented mutant had a Cal+ phenotype and it was capable of swimming and swarming motility. Thus, the AZOBR_p60025-encoded protein significantly affects the composition of the major cell-surface glycopolymers and the single-cell and social motility of azospirilla.

Polar flagellum of the alphaproteobacterium Azospirillum brasilense Sp245 plays a role in biofilm biomass accumulation and in biofilm maintenance under stationary and dynamic conditions.

Bacteria Azospirillum brasilense may swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf). They also construct sessile biofilms on various interfaces. As compared to the wild-type strain Sp245, the previously characterized Fla- Laf- flhB1 mutant Sp245.1063 accumulated less biomass in mature biofilms, which also were susceptible to the forces of hydrodynamic shear. In this study, we compared biofilms formed by strain Sp245 and its previously constructed derivatives on the interfaces between a minimal (malate-salt medium, or MSM) or rich (LB) liquid growth medium and a hydrophilic (glass) or hydrophobic (polystyrene) solid surface under static or dynamic conditions. In all experimental settings, the alterations in Sp245.1063's mature biofilm traits were partially (in MSM) or completely (in LB) rescued in the complemented mutant Sp245.1063 (pRK415-150177), which received the pRK415-borne coding sequence for the putative FlhB1 protein of the flagellar type III secretion system. Although Laf were not found in the biofilms of azospirilla, Fla was present on the biofilm cells of the complemented mutant Sp245.1063 (pRK415-150177) and other studied strains, which had normal flagellation on liquid and solid nutritional media. Accordingly, mature biofilms of these strains contained more biomass and were significantly more resistant to shaking at 140 rpm, as compared to the biofilms of the flagella-free mutant bacteria. These data proved that the polar flagellum of A. brasilense Sp245 plays a significant positive role in biofilm biomass increase and in biofilm stabilization.

Restoration of polar-flagellum motility and biofilm-forming capacity in the mmsB1 mutant of the alphaproteobacterium Azospirillum brasilense Sp245 points to a new role for a homologue of 3-hydroxyisobutyrate dehydrogenase.

The bacterium Azospirillum brasilense can swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella, respectively. They also form biofilms on various interfaces. Experimental data on flagellar assembly and social behaviours in these bacteria are scarce. Here, for the first time, the chromosomal coding sequence mmsB1 for a homologue of 3-hydroxyisobutyrate dehydrogenase (protein accession Nos. ADT80774 and E7CWE2) was shown to play a role in the assembly of motile Fla and in biofilm biomass accumulation. In the previously obtained mutant SK039 of A. brasilense Sp245, an Omegon-Km insertion in mmsB1 was concurrent with changes in cell-surface properties and with suppression of Fla assembly (partial) and Fla-dependent motility (complete). Here, the immotile leaky Fla- mutant SK039 was complemented with the expression vector pRK415-borne mmsB1 gene of Sp245. In the complemented mutant, the elevated relative cell hydrophobicity and changed relative membrane fluidity of SK039 returned to the wild-type levels; also, biofilm biomass accumulation increased and even reached Sp245's levels under nutritionally rich conditions. In strain SK039 (pRK415-mmsB1), the percentage of cells with Fla became significantly higher than that in mutant SK039, and the Fla-driven swimming velocity was equal to that in strain Sp245.

Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.