Raúl Donoso

Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales.

The relevance of the β-proteobacterial Burkholderiales order in the degradation of a vast array of aromatic compounds, including several priority pollutants, has been largely assumed. In this review, the presence and organization of genes encoding oxygenases involved in aromatics biodegradation in 80 Burkholderiales genomes is analysed. This genomic analysis underscores the impressive catabolic potential of this bacterial lineage, comprising nearly all of the central ring-cleavage pathways reported so far in bacteria and most of the peripheral pathways involved in channelling of a broad diversity of aromatic compounds. The more widespread pathways in Burkholderiales include protocatechuate ortho ring-cleavage, catechol ortho ring-cleavage, homogentisate ring-cleavage and phenylacetyl-CoA ring-cleavage pathways found in at least 60% of genomes analysed. In general, a genus-specific pattern of positional ordering of biodegradative genes is observed in the catabolic clusters of these pathways indicating recent events in its evolutionary history. In addition, a significant bias towards secondary chromosomes, now termed chromids, is observed in the distribution of catabolic genes across multipartite genomes, which is consistent with a genus-specific character. Strains isolated from environmental sources such as soil, rhizosphere, sediment or sludge show a higher content of catabolic genes in their genomes compared with strains isolated from human, animal or plant hosts, but no significant difference is found among Alcaligenaceae, Burkholderiaceae and Comamonadaceae families, indicating that habitat is more of a determinant than phylogenetic origin in shaping aromatic catabolic versatility.

Quorum Sensing and Indole-3-Acetic Acid Degradation Play a Role in Colonization and Plant Growth Promotion of Arabidopsis thaliana by Burkholderia phytofirmans PsJN

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.

Genuine genetic redundancy in maleylacetate-reductase-encoding genes involved in degradation of haloaromatic compounds by Cupriavidus necator JMP134.

Maleylacetate reductases (MAR) are required for biodegradation of several substituted aromatic compounds. To date, the functionality of two MAR-encoding genes (tfdF(I) and tfdF(II)) has been reported in Cupriavidus necator JMP134(pJP4), a known degrader of aromatic compounds. These two genes are located in tfd gene clusters involved in the turnover of 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-CB). The C. necator JMP134 genome comprises at least three other genes that putatively encode MAR (tcpD, hqoD and hxqD), but confirmation of their functionality and their role in the catabolism of haloaromatic compounds has not been assessed. RT-PCR expression analyses of C. necator JMP134 cells exposed to 2,4-D, 3-CB, 2,4,6-trichlorophenol (2,4,6-TCP) or 4-fluorobenzoate (4-FB) showed that tfdF(I) and tfdF(II) are induced by haloaromatics channelled to halocatechols as intermediates. In contrast, 2,4,6-TCP only induces tcpD, and any haloaromatic compounds tested did not induce hxqD and hqoD. However, the tcpD, hxqD and hqoD gene products showed MAR activity in cell extracts and provided the MAR function for 2,4-D catabolism when heterologously expressed in MAR-lacking strains. Growth tests for mutants of the five MAR-encoding genes in strain JMP134 showed that none of these genes is essential for degradation of the tested compounds. However, the role of tfdF(I)/tfdF(II) and tcpD genes in the expression of MAR activity during catabolism of 2,4-D and 2,4,6-TCP, respectively, was confirmed by enzyme activity tests in mutants. These results reveal a striking example of genetic redundancy in the degradation of aromatic compounds.

Modified 3-Oxoadipate Pathway for the Biodegradation of Methylaromatics in Pseudomonas reinekei MT1

Catechols are central intermediates in the metabolism of aromatic compounds. Degradation of 4-methylcatechol via intradiol cleavage usually leads to the formation of 4-methylmuconolactone (4-ML) as a dead-end metabolite. Only a few microorganisms are known to mineralize 4-ML. The mml gene cluster of Pseudomonas reinekei MT1, which encodes enzymes involved in the metabolism of 4-ML, is shown here to encode 10 genes found in a 9.4-kb chromosomal region. Reverse transcription assays revealed that these genes form a single operon, where their expression is controlled by two promoters. Promoter fusion assays identified 4-methyl-3-oxoadipate as an inducer. Mineralization of 4-ML is initiated by the 4-methylmuconolactone methylisomerase encoded by mmlI. This reaction produces 3-ML and is followed by a rearrangement of the double bond catalyzed by the methylmuconolactone isomerase encoded by mmlJ. Deletion of mmlL, encoding a protein of the metallo-beta-lactamase superfamily, resulted in a loss of the capability of the strain MT1 to open the lactone ring, suggesting its function as a 4-methyl-3-oxoadipate enol-lactone hydrolase. Further metabolism can be assumed to occur by analogy with reactions known from the 3-oxoadipate pathway. mmlF and mmlG probably encode a 4-methyl-3-oxoadipyl-coenzyme A (CoA) transferase, and the mmlC gene product functions as a thiolase, transforming 4-methyl-3-oxoadipyl-CoA into methylsuccinyl-CoA and acetyl-CoA, as indicated by the accumulation of 4-methyl-3-oxoadipate in the respective deletion mutant. Accumulation of methylsuccinate by an mmlK deletion mutant indicates that the encoded acetyl-CoA hydrolase/transferase is crucial for channeling methylsuccinate into the central metabolism.

Volatile-Mediated Effects Predominate in Paraburkholderia phytofirmans Growth Promotion and Salt Stress Tolerance of Arabidopsis thaliana

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.

Biochemical and genetic basis of indole-3-acetic acid (auxin phytohormone) degradation by the plant growth promoting rhizobacterium Paraburkholderia phytofirmans PsJN.

ABSTRACT Several bacteria use the plant hormone indole-3-acetic acid (IAA) as a sole carbon and energy source. A cluster of genes (named iac) encoding IAA degradation has been reported in Pseudomonas putida 1290, but the functions of these genes are not completely understood. The plant-growth-promoting rhizobacterium Paraburkholderia phytofirmans PsJN harbors iac gene homologues in its genome, but with a different gene organization and context than those of P. putida 1290. The iac gene functions enable P. phytofirmans to use IAA as a sole carbon and energy source. Employing a heterologous expression system approach, P. phytofirmansiac genes with previously undescribed functions were associated with specific biochemical steps. In addition, two uncharacterized genes, previously unreported in P. putida and found to be related to major facilitator and tautomerase superfamilies, are involved in removal of an IAA metabolite called dioxindole-3-acetate. Similar to the case in strain 1290, IAA degradation proceeds through catechol as intermediate, which is subsequently degraded by ortho-ring cleavage. A putative two-component regulatory system and a LysR-type regulator, which apparently respond to IAA and dioxindole-3-acetate, respectively, are involved in iac gene regulation in P. phytofirmans. These results provide new insights about unknown gene functions and complex regulatory mechanisms in IAA bacterial catabolism. IMPORTANCE This study describes indole-3-acetic acid (auxin phytohormone) degradation in the well-known betaproteobacterium P. phytofirmans PsJN and comprises a complete description of genes, some of them with previously unreported functions, and the general basis of their gene regulation. This work contributes to the understanding of how beneficial bacteria interact with plants, helping them to grow and/or to resist environmental stresses, through a complex set of molecular signals, in this case through degradation of a highly relevant plant hormone.

Hierarchy of Carbon Source Utilization in Soil Bacteria: Hegemonic Preference for Benzoate in Complex Aromatic Compound Mixtures Degraded by Cupriavidus pinatubonensis Strain JMP134.

ABSTRACT Cupriavidus pinatubonensis JMP134, like many other environmental bacteria, uses a range of aromatic compounds as carbon sources. Previous reports have shown a preference for benzoate when this bacterium grows on binary mixtures composed of this aromatic compound and 4-hydroxybenzoate or phenol. However, this observation has not been extended to other aromatic mixtures resembling a more archetypal context. We carried out a systematic study on the substrate preference of C. pinatubonensis JMP134 growing on representative aromatic compounds channeled through different catabolic pathways described in aerobic bacteria. Growth tests of nearly the entire set of binary combinations and in mixtures composed of 5 or 6 aromatic components showed that benzoate and phenol were always the preferred and deferred growth substrates, respectively. This pattern was supported by kinetic analyses that showed shorter times to initiate consumption of benzoate in aromatic compound mixtures. Gene expression analysis by real-time reverse transcription-PCR (RT-PCR) showed that, in all mixtures, the repression by benzoate over other catabolic pathways was exerted mainly at the transcriptional level. Additionally, inhibition of benzoate catabolism suggests that its multiple repressive actions are not mediated by a sole mechanism, as suggested by dissimilar requirements of benzoate degradation for effective repression in different aromatic compound mixtures. The hegemonic preference for benzoate over multiple aromatic carbon sources is not explained on the basis of growth rate and/or biomass yield on each single substrate or by obvious chemical or metabolic properties of these aromatic compounds.

Aromatic compounds degradation plays a role in colonization of Arabidopsis thaliana and Acacia caven by Cupriavidus pinatubonensis JMP134

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 β-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.

Quorum-Sensing Systems in the Plant Growth-Promoting Bacterium Paraburkholderia phytofirmans PsJN Exhibit Cross-Regulation and Are Involved in Biofilm Formation

Quorum-sensing systems play important roles in host colonization and host establishment of Burkholderiales species. Beneficial Paraburkholderia species share a conserved quorum-sensing (QS) system, designated BraI/R, that controls different phenotypes. In this context, the plant growth-promoting bacterium Paraburkholderia phytofirmans PsJN possesses two different homoserine lactone QS systems BpI.1/R.1 and BpI.2/R.2 (BraI/R-like QS system). The BpI.1/R.1 QS system was previously reported to be important to colonize and produce beneficial effects in Arabidopsis thaliana plants. Here, we analyzed the temporal variations of the QS gene transcript levels in the wild-type strain colonizing plant roots. The gene expression patterns showed relevant differences in both QS systems compared with the wild-type strain in the unplanted control treatment. The gene expression data were used to reconstruct a regulatory network model of QS systems in P. phytofirmans PsJN, using a Boolean network model. Also, we examined the phenotypic traits and transcript levels of genes involved in QS systems, using P. phytofirmans mutants in homoserine lactone synthases genes. We observed that the BpI.1/R.1 QS system regulates biofilm formation production in strain PsJN and this phenotype was associated with the lower expression of a specific extracytoplasmic function sigma factor ecf26.1 gene (implicated in biofilm formation) in the bpI.1 mutant strain.

Paraburkholderia phytofirmans PsJN Protects Arabidopsis thaliana Against a Virulent Strain of Pseudomonas syringae Through the Activation of Induced Resistance

Paraburkholderia phytofirmans PsJN is a plant growth-promoting rhizobacterium (PGPR) that stimulates plant growth and improves tolerance to abiotic stresses. This study analyzed whether strain PsJN can reduce plant disease severity and proliferation of the virulent strain Pseudomonas syringae pv. tomato DC3000, in Arabidopsis plants, through the activation of induced resistance. Arabidopsis plants previously exposed to strain PsJN showed a reduction in disease severity and pathogen proliferation in leaves compared with noninoculated, infected plants. The plant defense-related genes WRKY54, PR1, ERF1, and PDF1.2 demonstrated increased and more rapid expression in strain PsJN-treated plants compared with noninoculated, infected plants. Transcriptional analyses and functional analysis using signaling mutant plants suggested that resistance to infection by DC3000 in plants treated with strain PsJN involves salicylic acid-, jasmonate-, and ethylene-signaling pathways to activate defense genes. Additionally, activation occurs through a specific PGPR-host recognition, being a necessary metabolically active state of the bacterium to trigger the resistance in Arabidopsis, with a strain PsJN-associated molecular pattern only partially involved in the resistance response. This study provides the first report on the mechanism used by the PGPR P. phytofirmans PsJN to protect A. thaliana against a widespread virulent pathogenic bacterium.