Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

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 (Ascorbate 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 long-term 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.

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.

The role of broad-host range IncP-1ε 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-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε 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-1ε 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-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

Background Cupriavidus necator JMP134 is a Gram-negative β-proteobacterium able to grow on a variety of aromatic and chloroaromatic compounds as its sole carbon and energy source.Methodology/Principal FindingsIts genome consists of four replicons (two chromosomes and two plasmids) containing a total of 6631 protein coding genes. Comparative analysis identified 1910 core genes common to the four genomes compared (C. necator JMP134, C. necator H16, C. metallidurans CH34, R. solanacearum GMI1000). Although secondary chromosomes found in the Cupriavidus, Ralstonia, and Burkholderia lineages are all derived from plasmids, analyses of the plasmid partition proteins located on those chromosomes indicate that different plasmids gave rise to the secondary chromosomes in each lineage. The C. necator JMP134 genome contains 300 genes putatively involved in the catabolism of aromatic compounds and encodes most of the central ring-cleavage pathways. This strain also shows additional metabolic capabilities towards alicyclic compounds and the potential for catabolism of almost all proteinogenic amino acids. This remarkable catabolic potential seems to be sustained by a high degree of genetic redundancy, most probably enabling this catabolically versatile bacterium with different levels of metabolic responses and alternative regulation necessary to cope with a challenging environment. From the comparison of Cupriavidus genomes, it is possible to state that a broad metabolic capability is a general trait for Cupriavidus genus, however certain specialization towards a nutritional niche (xenobiotics degradation, chemolithoautotrophy or symbiotic nitrogen fixation) seems to be shaped mostly by the acquisition of “specialized” plasmids.Conclusions/SignificanceThe availability of the complete genome sequence for C. necator JMP134 provides the groundwork for further elucidation of the mechanisms and regulation of chloroaromatic compound biodegradation.

Abiotic stress has a growing impact on plant growth and agricultural activity worldwide. Specific plant growth promoting rhizobacteria have been reported to stimulate growth and tolerance to abiotic stress in plants, and molecular mechanisms like phytohormone synthesis and 1-aminocyclopropane-1-carboxylate deamination are usual candidates proposed to mediate these bacterial effects. Paraburkholderia phytofirmans PsJN is able to promote growth of several plant hosts, and improve their tolerance to chilling, drought and salinity. This work investigated bacterial determinants involved in PsJN stimulation of growth and salinity tolerance in Arabidopsis thaliana, showing bacteria enable plants to survive long-term salinity treatment, accumulating less sodium within leaf tissues relative to non-inoculated controls. Inactivation of specific bacterial genes encoding ACC deaminase, auxin catabolism, N-acyl-homoserine-lactone production, and flagellin synthesis showed these functions have little influence on bacterial induction of salinity tolerance. Volatile organic compound emission from strain PsJN was shown to reproduce the effects of direct bacterial inoculation of roots, increasing plant growth rate and tolerance to salinity evaluated both in vitro and in soil. Furthermore, early exposure to VOCs from P. phytofirmans was sufficient to stimulate long-term effects observed in Arabidopsis growth in the presence and absence of salinity. Organic compounds were analyzed in the headspace of PsJN cultures, showing production of 2-undecanone, 7-hexanol, 3-methylbutanol and dimethyl disulfide. Exposure of A. thaliana to different quantities of these molecules showed that they are able to influence growth in a wide range of added amounts. Exposure to a blend of the first three compounds was found to mimic the effects of PsJN on both general growth promotion and salinity tolerance. To our knowledge, this is the first report on volatile compound-mediated induction of plant abiotic stress tolerance by a Paraburkholderia species.

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB I and tfdB II genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB I and tfdB II genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB I contributes to a significantly higher extent than TfdB II . Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.

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