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.

Strategies to display heterologous proteins on the cell surface of lactic acid bacteria using as anchor the C-terminal domain of Lactobacillus acidophilus SlpA.

The surface-layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling subunits, non-covalently bound to the most outer cell wall envelope, which constitutes up to 20% of the total cell protein content. These attributes make S-layer proteins an excellent anchor for the development of microbial cell-surface display systems. In L. acidophilus, the S-layer is formed predominantly by the protein SlpA. We have previously shown that the C-terminal domain of SlpA is responsible for the cell wall anchorage on L. acidophilus ATCC 4356. In the present study, we evaluated the C-terminal domain of SlpA of L. acidophilus ATCC 4356 as a potential anchor domain to display functional proteins on the surface of non-genetically modified lactic acid bacteria (LAB). To this end, green fluorescent protein (GFP)-CTSlpA was firstly produced in Escherichia coli and the recombinant proteins were able to spontaneously bind to the cell wall of LAB in a binding assay. GFP was successfully displayed on the S-layer stripped surface of L. acidophilus. Both the binding stability and cell survival of L. acidophilus decorated with the recombinant protein were then studied in simulated gastrointestinal conditions. Furthermore, NaCl was tested as a safer alternative to LiCl for S-layer removal. This study presents the development of a protein delivery platform involving L. acidophilus, a microorganism generally regarded as safe, which utilizes the contiguous, non-covalently attached S-layer at the cell surface of the bacterium without introducing any genetic modification.