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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.

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.

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.

Plant growth-promoting bacteria (PGPB) have been largely considered as beneficial in harsh and limiting environments given their effects on alleviating plant stress. For practical applications, most of the PGPB are prepared in immobilization matrices to improve the stability and benefits of bacteria. Despite the long list of immobilizing agents/carriers tested to date, a long list of desired requirements is yet to be achieved. Here, lignin stands as a scarcely tested immobilizer for bioinoculants with great potential for this purpose. The aim of this work was to demonstrate the feasibility of lignin as a carrier of the nitrogen-fixing Azospirillum brasilense. These bacteria were cultured in liquid media with recovered organosolv lignin added for bacterial immobilization. Then, lignin was recovered and the immobilized biomass was quantified gravimetrically by DNA extraction and serial dilution plating. Fluorescent microscopy as well as Congo red agar plating showed the immobilization of the bacterial cells in the lignin matrix and crystal violet dyeing showed the biofilms formation in lignin particles. A high number of cells were counted per gram of dried lignin. Lignin can be readily used as low-cost, health-safe bioinoculant carrier to be used in soil and agricultural applications.

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Plant-growth-promoting bacteria belonging to Azospirillum and Pseudomonas genera are major inhabitants of the rhizosphere. Both are increasingly commercialized as crops inoculants. Interspecific interaction in the rhizosphere is critical for inoculants aptness. The objective of this work was to evaluate Azospirillum and Pseudomonas interaction in mixed biofilms by co-cultivation of the model strains Azospirillum brasilense Sp245 and Pseudomonas protegens CHA0. The results revealed enhanced growth of both strains when co-cultured in static conditions. Moreover, Sp245 biofilm formed in plastic surfaces was increased 2-fold in the presence of CHA0. Confocal microscopy revealed highly structured mixed biofilms showing Sp245 mainly on the bottom and CHA0 towards the biofilm surface. In addition, A. brasilense biofilm was thicker and denser when co-cultured with P. protegens. In a colony-colony interaction assay, Sp245 changed nearby CHA0 producing small colony phenotype, which accounts for a diffusible metabolite mediator; though CHA0 spent medium did not affect Sp245 colony phenotype. Altogether, these results point to a cooperative interaction between A. brasilense Sp245 and P. protegens CHA0 in which both strains increase their static growth and produce structured mixed biofilms with a strain-specific distribution. Fil: Pagnussat, Luciana Anabella. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Salcedo, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Maroniche, Guillermo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Keel, Christoph. Universite de Lausanne; Suiza Fil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentina Fil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata; Argentina

The extrusion technology of blends formed by compounds with different physicochemical properties often results in new materials that present properties distinctive from its original individual constituents. Here, we report the use of melt extrusion of blends made from low-cost materials to produce a biodegradable foam suitable for use as an inoculant carrier of plant growth-promoting bacteria (PGPB). Six formulations were prepared with variable proportions of the raw materials; the resulting physicochemical and structural properties are described, as well as formulation performance in the maintenance of bacterial viability during 120 days of storage. Differences in blend composition influenced foam density, porosity, expansion index, and water absorption. Additionally, differences in the capability of sustaining bacterial viability for long periods of time were more related to the foam composition than to the resulting physicochemical characteristics. Microscopic analyses showed that the inoculant bacteria had firmly attached to the extruded material by forming biofilms. Inoculation assays using maize plants demonstrated that the bacteria attached to the extruded foams could survive in the soil for up to 10 days before maize sowing, without diminishing its ability to promote plant growth. The results presented demonstrate the viability of the new matrix as a biotechnological material for bacterial delivery not only in agriculture but also in other biotechnological applications, according to the selected bacterial strains.

Plants support a diverse array of bacteria, including parasites, mutualists, and commensals on and around their roots, in the vasculature, and on aerial tissues. These microbes have a profound influence on plant health and productivity. Bacteria physically interact with surfaces to form complex multicellular and often multispecies assemblies, including biofilms and smaller aggregates. There is growing appreciation that the intensity, duration, and outcome of plant-microbe interactions are significantly influenced by the conformation of adherent microbial populations. Biofilms on different tissues have unique properties, reflecting the prevailing conditions at those sites. Attachment is required for biofilm formation, and bacteria interact with plant tissues through adhesins including polysaccharides and surface proteins, with initial contact often mediated by active motility. Recognition between lectins and their cognate carbohydrates is a common means of specificity. Biofilm development and the resulting intimate interactions with plants often require cell-cell communication between colonizing bacteria.

Bacterial ability to colonize the rhizosphere of plants in arsenic (As) contaminated soils is highly important for symbiotic and free-living plant growth-promoting rhizobacteria (PGPR) used as inoculants, since they can contribute to enhance plant As tolerance and limit metalloid uptake by plants. The aim of this work was to study the effect of As on growth, exopolysaccharide (EPS) production, biofilm formation and motility of two strains used as soybean inoculants, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39. The metabolism of arsenate (As(V)) and arsenite (As(III)) and their removal and/or possible accumulation were also evaluated. The behavior of both bacteria under As treatment was compared and discussed in relation to their potential for colonizing plant rhizosphere with high content of the metalloid. B. japonicum E109 growth was reduced with As(III) concentration from 10. μM while A. brasilense Az39 showed a reduction of growth with As(III) from 500. μM. EPS and biofilm production increased significantly under 25. μM As(III) for both strains. Moreover, this was more notorious for Azospirillum under 500. μM As(III), where motility was seriously affected. Both bacterial strains showed a similar ability to reduce As(V). However, Azospirillum was able to oxidize more As(III) (around 53%) than Bradyrhizobium (17%). In addition, both strains accumulated As in cell biomass. The behavior of Azospirillum under As treatments suggests that this strain would be able to colonize efficiently As contaminated soils. In this way, inoculation with A. brasilense Az39 would positively contribute to promoting growth of different plant species under As treatment. Fil: Armendariz, Ana Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Talano, Melina Andrea. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Wevar Oller, Ana Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Medina, Maria Ines. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Agostini, Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Universidad Nacional de Río Cuarto; Argentina

Pathogenic Leptospira spp. represent one cause of leptospirosis worldwide and have long been regarded as solitary organisms in soil and aquatic environments. However, in the present study, Leptospira interrogans was observed to be associated with environmental biofilms with 21 bacterial isolates belonging to 10 genera. All 21 isolates were examined for their coaggregation and biofilm-forming ability with leptospires in vitro . Among these, Azospirillum brasilense RMRCPB showed maximum interspecies coaggregation with leptospiral strains (>75%, visual score of +4). Other significant coaggregating isolates belonged to the genera Sphingomonas, Micrococcus, Brevundimonas, Acinetobacter and Paracoccus . Biofilms of leptospires in combination with A. brasilense RMRCPB showed high resistance to penicillin G, ampicillin and tetracycline (minimum bactericidal concentration ≥800 μg/mL) and tolerance to UV radiation and high temperature (up to 49°C). This study hypothesized that biofilm formation with A. brasilense protects the pathogenic Leptospira from adverse environmental conditions/stress. This coexistence of pathogenic Leptospira with other bacteria may be the key factor for its persistence and survival. However, the mechanism of biofilm formation by leptospires needs to be explored to help devise an appropriate control strategy and reduce transmission of leptospires.

Flagella are complex surface organelles that allow bacteria to move towards favourable environments and that contribute to the virulence of pathogenic bacteria through adhesion and biofilm formation on host surfaces. There are a few bacteria that possess functional dual flagella systems, such as Vibrio parahaemolyticus, some mesophilic Aeromonas spp., Rhodospirillum centenum and Azospirillum brasilense. These bacteria are able to express both a constitutive polar flagellum required for swimming motility and a separate lateral flagella system that is induced in viscous media or on surfaces and is essential for swarming motility. As flagella synthesis and motility have a high metabolic cost for the bacterium, the expression of the inducible lateral flagella system is highly regulated by a number of environmental factors and regulators.

Azospirillum brasilense is a rhizobacterium that provides beneficial effects on plants when they colonize roots. The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with surfaces in response to appropriate signals. Nitric oxide (NO) is a signaling molecule implicated in numerous processes in bacteria, including biofilm formation or dispersion, depending on genera and lifestyle. Azospirillum brasilense Sp245 produces NO by denitrification having a role in root growth promotion. We analyzed the role of endogenously produced NO on biofilm formation in A. brasilense Sp245 and in a periplasmic nitrate reductase mutant (napA::Tn5; Faj164) affected in NO production. Cells were statically grown in media with nitrate or ammonium as nitrogen sources and examined for biofilm formation using crystal violet and by confocal laser microscopy. Both strains formed bio- films, but the mutant produced less than half compared with the wild type in nitrate medium showing impaired nitrite production in this condition. NO measurements in biofilm confirmed lower values in the mutant strain. The addition of a NO donor showed that NO influences biofilm formation in a dose-dependent manner and reverses the mutant phenotype, indicating that Nap positively regulates the formation of biofilm in A. brasilense Sp245. Fil: Arruebarrena Di Palma, Andrés. Universidad Nacional de Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pereyra, Cintia Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Moreno Ramírez, Lizbeth. Benemérita Universidad Autónoma de Puebla; México Fil: Xiqui Vazquez, Maria L.. Benemérita Universidad Autónoma de Puebla; México Fil: Baca, Beatriz E.. Benemérita Universidad Autónoma de Puebla; México Fil: Pereyra, María Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lamattina, Lamattina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Creus, Cecilia Mónica. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina