Transcriptional and pathway analysis of Acinetobacter sp. neg1 capable of degrading ochratoxin A

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium, contaminating grapes, wine and a variety of food products. We recently isolated from soil a novel free-living Acinetobacter strain, named Acinetobacter sp. neg1, able to degrade OTA. Biochemical studies demonstrated that Acinetobacter sp. neg1 was able to degrade OTA into the not toxic catabolic product OTalpha (OT?), suggesting that the degradation reaction proceeds via peptide bond hydrolysis with phenylalanine release. The identification of the enzymes and biochemical pathway responsible for the degradation is the first step in the development of biotechnology applications for OTA reduction in the food chain. In order to find genes responsible for OTA degradation we made a differential expression analysis of Acinetobacter sp. neg1 grown in the presence or absence of the toxin. Briefly, total RNA was isolated from bacteria grown in the two conditions and mRNA was selectively enriched through rRNA depletion. Samples were then fragmented and used to obtain cDNA libraries that were sequenced with the Illumina MiSeq system, obtaining 4 x 106 paired-end reads. Reads were aligned to the reference genome using GSNAP software. Then, Cuffdiff was used to detect significant differences between the two conditions. This analysis revealed more than 100 differential expressed genes. Among them, 6 genes code for peptidases and resulted up-regulated at 6 hours. The enrichment analysis for Gene Ontology terms by the Java-based tool Bingo (included as a plugin in Cytoscape) revealed the over-representation of pathways which could be involved or consequent to OTA degradation, such as peptidase activity and protein and amino acid metabolic process and transport. These results confirmed that OTA degradation proceeds through peptidase activities. Furthermore, some molecular functions related to phenylalanine catabolism resulted dysregulated, suggesting that phenylalanine is an energy source for Acinetobacter sp. neg1 and the OTA degrading reaction is followed by the modulation of further catabolic activities.