Comparative Analysis of Three Bradyrhizobium diazoefficiens Genomes Show Specific Mutations Acquired during Selection for a Higher Motility Phenotype and Adaption to Laboratory Conditions.

Microbial genomes are being extensively studied using next-generation sequencing technologies in order to understand the changes that occur under different selection regimes. In this work, the number and type of mutations that have occurred in three Bradyrhizobium diazoefficiens USDA 110T strains under laboratory conditions and during selection for a more motile phenotypic variant were analyzed. Most of the mutations found in both processes consisted of single nucleotide polymorphisms, single nucleotide deletions or insertions. In the case of adaptation to laboratory conditions, half of the changes occurred within intergenic regions, and around 80% were insertions. When the more motile phenotypic variant was evaluated, eight single nucleotide polymorphisms and an 11-bp deletion were found, although none of them was directly related to known motility or chemotaxis genes. Two mutants were constructed to evaluate the 11-bp deletion affecting the alpha subunit of 2-oxoacid:acceptor oxidoreductase (AAV28_RS30705-blr6743). The results showed that this single deletion was not responsible for the enhanced motility phenotype. IMPORTANCE 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. In the present work, we sequenced and compared the genomes of strains derived from a parent strain, B. diazoefficiens USDA 110, that has undergone processes of repeated culture in the laboratory environment, or phenotypic selection toward antibiotic resistance and enhanced motility. Our results represent the first analysis in B. diazoefficiens that provides insights into the specific mutations that are acquired during these processes.

Health-Promoting Properties of Lacticaseibacillus paracasei: A Focus on Kefir Isolates and Exopolysaccharide-Producing Strains.

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.

Dual Control of Flagellar Synthesis and Exopolysaccharide Production by FlbD-FliX Class II Regulatory Proteins in Bradyrhizobium diazoefficiens.

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 to 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 analyzed the gene organization, expression, and function of the lateral flagellar system. Here, we studied the role of the response regulator FlbD and its trans-acting regulator FliX in the regulation of subpolar flagellar genes. We found that the LP-ring, distal rod, and 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, in which bacteria form biofilms. Apart from being essential for locomotion, the flagellum has accessory functions during biofilm formation. The synthesis of flagella is a highly regulated process, and coordination with accessory functions requires the interconnection of various regulatory networks. Here, we show the role of class II regulators involved in the synthesis of the 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 are the main component of the biofilm matrix.

Exopolysaccharides From Lactobacillus paracasei Isolated From Kefir as Potential Bioactive Compounds for Microbiota Modulation.

Microbiota coexists in true symbiosis with the host playing pivotal roles as a key element for well-being and health. Exopolysaccharides from lactic acid bacteria are an alternative as novel potential prebiotics that increase microbiota diversity. Considering this, the aim of the present work was to evaluate the capacity of the EPS produced by two L. paracasei strains isolated from kefir grains, to be metabolized in vitro by fecal microbiota producing short chain fatty acids. For this purpose, fecal samples from healthy children were inoculated in a basal medium with EPS and incubated in anaerobiosis at 37°C for 24, 48, and 72 h. DGGE profiles and the production of SCFA after fermentation were analyzed. Additionally, three selected samples were sequenced by mass sequencing analysis using Ion Torrent PGM. EPS produced by L. paracasei CIDCA 8339 (EPS8339) and CIDCA 83124 (EPS83124) are metabolized by fecal microbiota producing a significant increase in SCFA. EPS8339 fermentation led to an increment of propionate and butyrate, while fermentation of EPS83124 increased butyrate levels. Both EPS led to a profile of SCFA different from the ones obtained with inulin or glucose fermentation. DGGE profiles of 72 h fermentation demonstrated that both EPS showed a different band profile when compared to the controls; EPS profiles grouped in a cluster that have only 65% similarity with glucose or inulin profiles. Mass sequencing analysis demonstrated that the fermentation of EPS8339 leads to an increase in the proportion of the genera Victivallis, Acidaminococcus and Comamonas and a significant drop in the proportion of enterobacteria. In the same direction, the fermentation of EPS83124 also resulted in a marked reduction of Enterobacteriaceae with a significant increase in the genus Comamonas. It was observed that the changes in fecal microbiota and SCFA profile exerted by both polymers are different probably due to 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.

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System.

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.

Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, a soybean N2-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 flbTL , a class III regulator. We observed different requirements for FlbTL in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbTL, the production of both flagellin polypeptides required FlbTL 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 N2-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.