Bradyrhizobium diazoefficiens, a soybean N 2 -fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbT L , a class III regulator. We observed different requirements for FlbT L in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbT L , the production of both flagellin polypeptides required FlbT L . Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.IMPORTANCE Bacterial motility seems essential for the free-living style in the environment, and therefore these microorganisms allocate a great deal of their energetic resources to the biosynthesis and functioning of flagella. Despite energetic costs, some bacterial species possess dual flagellar systems, one of which is a primary system normally polar or subpolar, and the other is a secondary, lateral system that is produced only under special circumstances. Bradyrhizobium diazoefficiens, an N 2 -fixing symbiont of soybean plants, possesses dual flagellar systems, including the lateral system that contributes to swimming in wet soil and competition for nodulation and is expressed under high energy availability, as well as under requirement for high torque by the flagella. The structural organization and transcriptional regulation of the 41 genes that comprise this secondary flagellar system seem adapted to adjust bacterial energy expenditures for motility to the soil's environmental dynamics.KEYWORDS Bradyrhizobium, flagella, expression, lafR, flbT, FlbT, LafR F lagellum-driven swimming motility-a characteristic trait of many bacterial species-is essential for the colonization of diverse niches in environments such as seas, freshwaters, sediments, soils, and the organs of plant or animal hosts. This form of bacterial locomotion requires the propulsion provided by flagella, as well as a guidance system mediated by chemotaxis (1).Flagella are complex organelles formed by three main structures: a basal body that anchors the flagellum to the cell envelope...

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction. IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system’s fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

Bengoa et al. EPS From Lactobacillus paracasei as Microbiota Modulators differences in their structural characteristics. It can be concluded that EPS synthesized by both L. paracasei strains, could be potentially used as bioactive compound that modify the microbiota increasing the production of propionic and butyric acid, two metabolites highly associated with beneficial effects both at the gastrointestinal and extra-intestinal level.

Among artisanal fermented beverages, kefir (fermented milk drink) and water kefir (fermented nondairy beverage) are of special interest because their grains can be considered natural reservoirs of safe and potentially probiotic strains. In the last years, several reports on Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) isolated from both artisanal fermented beverages were published focusing on their health-promoting properties. Although this is not the predominant species in kefir or water kefir, it may contribute to the health benefits associated to the consumption of the fermented beverage. Since the classification of L. paracasei has been a difficult task, the selection of an adequate method for identification, which is essential to avoid mislabeling in products, publications, and some publicly available DNA sequences, is discussed in the present work. The last findings in health promoting properties of L. paracasei and the bioactive compounds are described and compared to strains isolated from kefir, providing a special focus on exopolysaccharides as effector molecules. The knowledge of the state of the art of Lacticaseibacillus paracasei from kefir and water kefir can help to understand the contribution of these microorganisms to the health benefits of artisanal beverages as well as to discover new probiotic strains for applications in food industry.

SUMMARY Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, has two independent flagellar systems: a single, subpolar flagellum and several lateral flagella. Each flagellum is a very complex organelle composed of 30-40 different proteins located inside and outside the cell, whereby flagellar gene expression must be tightly controlled. Such control is achieved by a hierarchy of regulators that ensure the timing of synthesis and the allocation of the different flagellar substructures. Previously, we analysed the gene organization, expression, and function of the lateral flagellar system. Here, we studied the role of response regulator FlbD and its trans-acting regulator FliX in the regulation of subpolar flagellar genes. We found that the LP-ring, the distal rod and the hook of the subpolar flagellum were tightly controlled by FlbD and FliX. Furthermore, we obtained evidence for the existence of cross-regulation between these gene products and the expression of LafR, the master regulator of lateral flagella. In addition, we observed that extracellular polysaccharide production and biofilm formation also responded to these flagellar regulators. In this regard, FlbD might contribute to the switch between the planktonic and sessile states. IMPORTANCE: Most environmental bacteria switch between two free-living states: planktonic, in which individual cells swim propelled by flagella, and sessile, where bacteria form biofilms. Apart from being essential for locomotion, the flagellum also has accessory functions during biofilm formation. The synthesis of flagella is a highly regulated process and the coordination with accessory functions requires interconnection of various regulatory networks. Here we show the role of class-II regulators involved in the synthesis of B. diazoefficiens subpolar flagellum, and their possible participation in cross-regulation with the lateral flagellar system and exopolysaccharide production. These findings highlight the coordination of the synthetic processes of external structures such as subpolar and lateral flagella with exopolysaccharides, which constitute the main component of the biofilm matrix.

The genetic and genomic changes that occur under laboratory conditions in Bradyrhizobium diazoefficiens genomes remain poorly studied. Only a few genome sequences of this important nitrogen-fixing species are available, and there are no genome-wide comparative analyses of related strains.

La capacidad de moverse es una propiedad muy importante para cualquier especie bacteriana. Esta característica le permite colonizar nuevos nichos, alejarse de sustancias nocivas y acercarse en busca de nuevos nutrientes, entre otras. Particularmente, B. diazoefficiens es un rizobio que puede hallarse en los suelos (naturales y antrópicos) y vivir en vida libre -estado planctónico (movilidad) o estado sésil (formando biofilms)- o interaccionando con su par simbiótico. Para buscar y colonizar plantas de soja, una bacteria móvil debe desplazarse hacia sus raíces, ser capaz de adherirse y formar microcolonias, infectar las raíces más pequeñas y diferenciarse a bacteroide dentro de los nódulos. Este cambio radical entre los diferentes estadíos se debe a un gran cambio en la transcripción genética y expresión proteica en cada uno de ellos (Jiménez-Guerrero et al., 2017). B. diazoefficiens utiliza dos sistemas de flagelos (lateral y subpolar) que le permiten desplazarse. La síntesis flagelar es un proceso que se encuentra altamente regulado dado la gran cantidad de proteínas necesarias y el alto costo energético que implica su formación (Macnab, 2003). En general, la síntesis de los flagelos ocurre en etapas escalonadas, que, dependiendo del microorganismo, puede ser dividida en tres o cuatro, siendo cada una de ellas desencadenada por una señal específica (Figura I.7). A través de estudios bioinformáticos, hemos podido asignar dos modelos de regulación diferentes a cada uno de los sistemas flagelares de B. diazoefficiens. Para los flagelos laterales, nuestro grupo de investigación demostró, mediante estrategias de mutagénesis dirigida y ensayos de PCR cuantitativa en tiempo real (qRT-PCR) que el sistema de regulación depende del par LafR/FlbTL, siendo el sistema regulatorio similar al descripto en E. meliloti. Por el contrario, la regulación de la síntesis del sistema subpolar, el cual es necesario en los procesos de natación lineal, adhesión a superficies y quimiotaxis, aún no ha sido estudiado en este microorganismo. Objetivo general En este trabajo de Tesis nos planteamos como objetivo principal la caracterización de la cascada regulatoria de la síntesis del flagelo subpolar de B. diazoefficiens USDA 110 y las señales involucradas en este proceso. Objetivos específicos: 1) Caracterización genómica detallada de los genes que codifican el flagelo subpolar mediante técnicas bioinformáticas. 2) Identificar y caracterizar los genes relacionados con la regulación de la síntesis del flagelo subpolar de B. diazoefficiens USDA 110. 3) Evaluar posibles señales involucradas en el control de la síntesis del flagelo subpolar. 4) Evaluar el rol del c-di-GMP sobre la regulación del flagelo subpolar de B. diazoefficiens USDA 110.