Abstract: Background Rhizosphere microorganisms play a crucial role in plant health and development. Plant root exudates (PRE) are a complex mixture of organic molecules and provide nutritional and signaling information to rhizosphere microorganisms. Burkholderiaceae species are non-abundant in the rhizosphere but exhibit a wide range of plant-growth-promoting and plant-health-protection effects. Most of these plant-associated microorganisms have been studied in isolation under laboratory conditions, whereas in nature, they interact in competition or cooperation with each other. To improve our understanding of the factors driving growth dynamics of low-abundant bacterial species in the rhizosphere, we hypothesized that the growth and survival of four Burkholderiaceae strains (Paraburkholderia phytofirmans PsJN, Cupriavidus metallidurans CH34, C. pinatubonensis JMP134 and C. taiwanensis LMG19424) in Arabidopsis thaliana PRE is affected by the presence of each other. Results Differential growth abilities of each strain were found depending on plant age and whether PRE was obtained after growth on N limitation conditions. The best-adapted strain to grow in PRE was P. phytofirmans PsJN, with C. pinatubonensis JMP134 growing better than the other two Cupriavidus strains. Individual strain behavior changed when they succeeded in combinations. Clustering analysis showed that the 4-member co-culture grouped with one of the best-adapted strains, either P. phytofirmans PsJN or C. pinatubonensis JMP134, depending on the PRE used. Sequential transference experiments showed that the behavior of the 4-member co-culture relies on the type of PRE provided for growth. Conclusions The results suggest that individual strain behavior changed when they grew in combinations of two, three, or four members, and those changes are determined first by the inherent characteristics of each strain and secondly by the environment.

Abstract: The role of broad-host range IncP-1 epsilon plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1 epsilon plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1 epsilon plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1 epsilon plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1 epsilon plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1 epsilon plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS 1326, and in three of the plasmids the tetracycline resistance transposon In 1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1 epsilon plasmids in soil microcosms. Our data suggest that IncP-1 epsilon plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

Abstract: Cupriavidus necator JMP134(pJP4) is able to grow on 3-chlorobenzoate (3-CB), a model chloroaromatic pollutant. Catabolism of 3-CB is achieved via the expression of the chromosomally encoded benABCD genes and the tfd genes from plasmid pJP4. Since passive diffusion of benzoic acid derivatives at physiological pH is negligible, the uptake of this compound should be facilitated by a transport system. However, no transporter has so far been described to perform this function, and identification of chloroaromatic compound transporters has been limited. In this work, uptake experiments using 3-[ring-UL-C-14]CB showed an inducible transport system in strain JMP134, whose expression is activated by 3-CB and benzoate. A similar level of 3-CB uptake was found for a mutant strain of JMP134, defective in chlorobenzoate degradation, indicating that metabolic drag is not an important component of the measured uptake rate. Competitive inhibitor assays showed that uptake of 3-CB was inhibited by benzoate and, to a lesser degree, by 3-CB and 3,5-dichlorobenzoate, but not by any of 12 other substituted benzoates tested. The expression of several gene candidates for this transport function was analysed by RT-PCR, including both permease-type and ABC-type ATP-dependent transporters. Induction of a chromosomally encoded putative permease transporter (benP gene) was found specifically in the presence of 3-CB or benzoate. A benP knockout mutant of strain JMP134 displayed an almost complete loss of 3-CB transport activity. This is to our knowledge the first report of a 3-CB transporter.

Abstract: Plant rhizosphere and internal tissues may constitute a relevant habitat for soil bacteria displaying high catabolic versatility towards xenobiotic aromatic compounds. Root exudates contain various molecules that are structurally related to aromatic xenobiotics and have been shown to stimulate bacterial degradation of aromatic pollutants in the rhizosphere. The ability to degrade specific aromatic components of root exudates could thus provide versatile catabolic bacteria with an advantage for rhizosphere colonization and growth. In this work, Cupriavidus pinatubonensis JMP134, a well-known aromatic compound degrader (including the herbicide 2,4-dichlorophenoxyacetate, 2,4-D), was shown to stably colonize Arabidopsis thaliana and Acacia caven plants both at the rhizoplane and endorhizosphere levels and to use root exudates as a sole carbon and energy source. No deleterious effects were detected on these colonized plants. When a toxic concentration of 2,4-D was applied to colonized A. caven, a marked resistance was induced in the plant, showing that strain JMP134 was both metabolically active and potentially beneficial to its host. The role for the beta-ketoadipate aromatic degradation pathway during plant root colonization by C. pinatubonensis JMP134 was investigated by gene inactivation. A C. pinatubonensis mutant derivative strain displayed a reduced ability to catabolise root exudates isolated from either plant host. In this mutant strain, a lower competence in the rhizosphere of A. caven was also shown, both in gnotobiotic in vitro cultures and in plant/soil microcosms.

Abstract: In Veliz-Cuba and Stigler 2011, Boolean models were proposed for the lac operon in Escherichia coli capable of reproducing the operon being OFF, ON and bistable for three (low, medium and high) and two (low and high) parameters, representing the concentration ranges of lactose and glucose, respectively. Of these 6 possible combinations of parameters, 5 produce results that match with the biological experiments of Ozbudak et al., 2004. In the remaining one, the models predict the operon being OFF while biological experiments show a bistable behavior. In this paper, we first explore the robustness of two such models in the sense of how much its attractors change against any deterministic update schedule. We prove mathematically that, in cases where there is no bistability, all the dynamics in both models lack limit cycles while, when bistability appears, one model presents 30% of its dynamics with limit cycles while the other only 23%. Secondly, we propose two alternative improvements consisting of biologically supported modifications; one in which both models match with Ozbudak et al., 2004 in all 6 combinations of parameters and, the other one, where we increase the number of parameters to 9, matching in all these cases with the biological experiments of Ozbudak et al., 2004.

Abstract: Salinity is one of the major limitations for food production worldwide. Improvement of plant salt-stress tolerance using plant-growth promoting rhizobacteria (PGPR) has arisen as a promising strategy to help overcome this limitation. However, the molecular and biochemical mechanisms controlling PGPR/plant interactions under salt-stress remain unclear. The main objective of this study was to obtain new insights into the mechanisms underlying salt-stress tolerance enhancement in the salt-sensitive Arabidopsis thaliana Col-0 plants, when inoculated with the well-known PGPR strain Burkholderia phytofirmans PsJN. To tackle this, different life history traits, together with the spatiotemporal accumulation patterns for key metabolites and salt-stress related transcripts, were analyzed in inoculated plants under short and long-term salt-stress. Inoculated plants displayed faster recovery and increased tolerance after sustained salt-stress. PsJN treatment accelerated the accumulation of proline and transcription of genes related to abscisic acid signaling (Relative to Dessication, RD29A and RD29B), ROS scavenging Oscorbate Peroxidase 2), and detoxification (Glyoxalase I 7), and down-regulated the expression of Lipoxygenase 2 (related to jasmonic acid biosynthesis). Among the general transcriptional effects of this bacterium, the expression pattern of important ion-homeostasis related genes was altered after short and longterm stress (Arabidopsis K Transporter 1, High-Affinity K Transporter 1, Sodium Hydrogen Exchanger 2, and Arabidopsis Salt Overly Sensitive 1). In all, the faster and stronger molecular changes induced by the inoculation suggest a PsJN-priming effect, which may explain the observed tolerance after short-term and sustained salt-stress in plants. This study provides novel information about possible mechanisms involved in salt-stress tolerance induced by PGPR in plants, showing that certain changes are maintained over time. This opens up new venues to study these relevant biological associations, as well as new approaches to a better understanding of the spatiotemporal mechanisms involved in stress tolerance in plants.

Abstract: There have been over 25 independent unicellular to multicellular evolutionary transitions, which have been transformational in the complexity of life. All of these transitions likely occurred in communities numerically dominated by unicellular organisms, mostly bacteria. Hence, it is reasonable to expect that bacteria were involved in generating the ecological conditions that promoted the stability and proliferation of the first multicellular forms as protective units. In this study, we addressed this problem by analyzing the occurrence of multicellularity in an experimental phylogeny of yeasts (Sacharomyces cerevisiae) a model organism that is unicellular but can generate multicellular clusters under some conditions. We exposed a single ancestral population to periodic divergences, coevolving with a cocktail of environmental bacteria that were inoculated to the environment of the ancestor, and compared to a control (no bacteria). We quantified culturable microorganisms to the level of genera, finding up to 20 taxa (all bacteria) that competed with the yeasts during diversification. After 600 generations of coevolution, the yeasts produced two types of multicellular clusters: clonal and aggregative. Whereas clonal clusters were present in both treatments, aggregative clusters were only present under the bacteria treatment and showed significant phylogenetic signal. However, clonal clusters showed different properties if bacteria were present as follows: They were more abundant and significantly smaller than in the control. These results indicate that bacteria are important modulators of the occurrence of multicellularity, providing support to the idea that they generated the ecological conditions-promoting multicellularity.

Abstract: Although not fully understood, molecular communication in the rhizosphere plays an important role regulating traits involved in plant-bacteria association. Burkholderia phytofirmans PsJN is a well-known plant-growth-promoting bacterium, which establishes rhizospheric and endophytic colonization in different plants. A competent colonization is essential for plant-growth-promoting effects produced by bacteria. Using appropriate mutant strains of B. phytofirmans, we obtained evidence for the importance of N-acyl homoserine lactone-mediated (quorum sensing) cell-to-cell communication in efficient colonization of Arabidopsis thaliana plants and the establishment of a beneficial interaction. We also observed that bacterial degradation of the auxin indole-3-acetic acid (IAA) plays a key role in plant-growth-promoting traits and is necessary for efficient rhizosphere colonization. Wildtype B. phytofirmans but not the iacC mutant in IAA mineralization is able to restore promotion effects in roots of A. thaliana in the presence of exogenously added IAA, indicating the importance of this trait for promoting primary root length. Using a transgenic A. thaliana line with suppressed auxin signaling (miR393) and analyzing the expression of auxin receptors in wild-type inoculated plants, we provide evidence that auxin signaling in plants is necessary for the growth promotion effects produced by B. phytofirmans. The interplay between ethylene and auxin signaling was also confirmed by the response of the plant to a 1-aminocyclopropane-1-carboxylate deaminase bacterial mutant strain.