Genetic dissection of the degradation pathways for the mycotoxin fusaric acid in Burkholderia ambifaria T16.

IMPORTANCE: Fusaric acid (FA) is an important virulence factor produced by several Fusarium species. These fungi are responsible for wilt and rot diseases in a diverse range of crops. FA is toxic for animals, humans and soil-borne microorganisms. This mycotoxin reduces the survival and competition abilities of bacterial species able to antagonize Fusarium spp., due to its negative effects on viability and the production of antibiotics effective against these fungi. FA biodegradation is not a common characteristic among bacteria, and the determinants of FA catabolism have not been identified so far in any microorganism. In this study, we identified genes, enzymes, and metabolic pathways involved in the degradation of FA in the soil bacterium Burkholderia ambifaria T16. Our results provide insights into the catabolism of a pyridine-derivative involved in plant pathogenesis by a rhizosphere bacterium.

Genome mining of Burkholderia ambifaria strain T16, a rhizobacterium able to produce antimicrobial compounds and degrade the mycotoxin fusaric acid.

Burkholderia ambifaria T16 is a bacterium isolated from the rhizosphere of barley plants that showed a remarkable antifungal activity. This strain was also able to degrade fusaric acid (5-Butylpyridine-2-carboxylic acid) and detoxify this mycotoxin in inoculated barley seedlings. Genes and enzymes responsible for fusaric acid degradation have an important biotechnological potential in the control of fungal diseases caused by fusaric acid producers, or in the biodegradation/bio catalysis processes of pyridine derivatives. In this study, the complete genome of B. ambifaria T16 was sequenced and analyzed to identify genes involved in survival and competition in the rhizosphere, plant growth promotion, fungal growth inhibition, and degradation of aromatic compounds. The genomic analysis revealed the presence of several operons for the biosynthesis of antimicrobial compounds, such as pyrrolnitrin, ornibactin, occidiofungin and the membrane-associated AFC-BC11. These compounds were also detected in bacterial culture supernatants by mass spectrometry analysis. In addition, this strain has multiple genes contributing to its plant growth-promoting profile, including those for acetoin, 2,3-butanediol and indole-3-acetic acid production, siderophores biosynthesis, and solubilisation of organic and inorganic phosphate. A pan-genomic analysis demonstrated that the genome of strain T16 possesses large gene clusters that are absent in the genomes of B. ambifaria reference strains. According to predictions, most of these clusters would be involved in aromatic compounds degradation. One genomic region, encoding flavin-dependent monooxygenases of unknown function, is proposed as a candidate responsible for fusaric acid degradation.

Contribution of the Siderophores Pyoverdine and Enantio-Pyochelin to Fitness in Soil of Pseudomonas protegens Pf-5.

Pseudomonas protegens synthesizes two major iron-chelating metabolites (siderophores): pyoverdine (Pvd) and enantio-pyochelin (E-Pch). Although iron sequestration and uptake seem to be the main biological role of these siderophores, other functions including metal homeostasis and antibiotic activity have been proposed. The aim of this study was to evaluate the contribution of Pvd and E-Pch to the survival of P. protegens in soil using wild type and isogenic mutant strains unable to produce Pvd, E-Pch or both siderophores. Survival of these strains in sterile soil microcosms, in soil microcosms containing the native microflora and in sterile soil microcosms containing fusaric acid (a mycotoxin able to chelate iron and other metals), was compared by determination of colony forming units (CFU) per gram dry soil over time. In sterile soil, cell densities of Pvd-producing strains were significantly higher than that of non-producers after 21 days of permanence in the microcosms. In non-sterile soil, viability of all strains declined faster than in sterile soil and Pvd producers showed higher CFU × (g dry weight soil)-1 values than non-producers. The presence of fusaric acid negatively affected viability of strains unable to produce Pvd, while had no effect on the viability of strains able to produce Pvd. Altogether, these results show that the ability to produce Pvd increases survival of P. protegens in soil, while the ability to synthesize E-Pch does not, indicating that under the conditions which prevail in soil, iron scavenging via Pvd is more beneficial than via E-Pch.

A novel Burkholderia ambifaria strain able to degrade the mycotoxin fusaric acid and to inhibit Fusarium spp. growth.

Fusaric acid (FA) is a fungal metabolite produced by several Fusarium species responsible for wilts and root rot diseases of a great variety of plants. Bacillus spp. and Pseudomonas spp. have been considered as promising biocontrol agents against phytopathogenic Fusarium spp., however it has been demonstrated that FA negatively affects growth and production of some antibiotics in these bacteria. Thus, the capability to degrade FA would be a desirable characteristic in bacterial biocontrol agents of Fusarium wilt. Taking this into account, bacteria isolated from the rhizosphere of barley were screened for their ability to use FA as sole carbon and energy source. One strain that fulfilled this requirement was identified according to sequence analysis of 16S rRNA, gyrB and recA genes as Burkholderia ambifaria. This strain, designated T16, was able to grow with FA as sole carbon, nitrogen and energy source and also showed the ability to detoxify FA in barley seedlings. This bacterium also exhibited higher growth rate, higher cell densities, longer survival, higher levels of indole-3-acetic acid (IAA) production, enhanced biofilm formation and increased resistance to different antibiotics when cultivated in Luria Bertani medium at pH 5.3 compared to pH 7.3. Furthermore, B. ambifaria T16 showed distinctive plant growth-promoting features, such as siderophore production, phosphate-solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, in vitro antagonism against Fusarium spp. and improvement of grain yield when inoculated to barley plants grown under greenhouse conditions. This strain might serve as a new source of metabolites or genes for the development of novel FA-detoxification systems.

Production of siderophores increases resistance to fusaric acid in Pseudomonas protegens Pf-5.

Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe2+ and Fe3+, but also Zn2+, Mn2+ and Cu2+, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.

High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

Manipulation of the anoxic metabolism in Escherichia coli by ArcB deletion variants in the ArcBA two-component system.

Bioprocesses conducted under conditions with restricted O(2) supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O(2) and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of d-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, d-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD(+) ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.

Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals.

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level.

The alternative sigma factor, sigmaS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida.

To determine whether the stationary sigma factor, sigma(S), influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS-negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 (phaC1) and polyhydroxyalkanoate depolymerase (phaZ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that sigma(S) might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.

ArcA redox mutants as a source of reduced bioproducts.

Escherichia coli and other facultative anaerobes can adapt their metabolism according to oxygen availability by means of aerobic and anaerobic respiration and fermentation. ArcAB is a two-component signal transduction system that controls, at the transcriptional level, the choice of energy generation pathway according to the intracellular redox state. High throughput studies on different redox regulator mutants, involving transcriptome analysis, RT-PCR and phenotypic arrays enabled the elucidation of a repertoire of operons coordinated by ArcA which extended beyond respiration control including, among others, those which code for survival, chromosome replication and degradation of fatty acids. Flux analysis by (13)C labeling provided new clues to the understanding of the distribution of metabolites mediated by ArcAB. The genetic manipulation of this regulator proved to be useful for the generation of reduced products of commercial value.

dye (arc) Mutants: insights into an unexplained phenotype and its suppression by the synthesis of poly (3-hydroxybutyrate) in Escherichia coli recombinants.

arcA codes for a central regulator in Escherichia coli that responds to redox conditions of growth. Mutations in this gene, originally named dye, confer sensitivity to toluidine blue and other redox dyes. However, the molecular basis for the dye-sensitive phenotype has not been elucidated. In this work, we show that toluidine blue redirects electrons to O2 and causes an increase in the generation of reactive O2 species (ROS). We also demonstrate that synthesis of poly (3-hydroxybutyrate) suppresses the Dye phenotype in E. coli recombinants, as the capacity to synthesize the polymer reduces sensitivity to toluidine blue, O2 consumption and ROS production levels.

A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance.

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.

rpoS gene expression in carbon-starved cultures of the Polyhydroxyalkanoate-accumulating species Pseudomonas oleovorans.

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H2O2 and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.