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. Numerous potential peptidase encoding genes are present in the genome of Acinetobacter sp. neg1, but in the absence of a comprehensive evaluation of the capacity of members of the genus to degrade OTA, the identification of candidate genes through comparative genomic approaches remains difficult. 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 with an average per base quality score > 30. The RNA seq data were analyzed in the discovery environment of iplant (http://www.iplantcollaborative.org/). In particular, reads were aligned to the reference genome using TopHat2. Then, Cuffdiff was used to calculate RPKM and detect significant differences between the two conditions. This analysis revealed more than 100 differential expressed genes. Among them, we found 4 genes coding for peptidases or hydrolases that could be involved in the reaction of degradation: 2 serine-type endopeptidases were up-regulated at 6 hours, while one aminopeptidase and one hydrolase were up-regolated at 12 hours. The enrichment analysis for Gene Ontology terms revealed the over-representation of molecular functions which could be involved or consequent to OTA degradation, such as serine-type endopeptidase activity, amino acid transmembrane transporter activity, D-amino acid metabolic process and protein maturation. These results confirmed that OTA degradation proceeds through peptidase activities. We hypothesize that this reaction induces the activation of pathways potentially involved in the phenylalanine metabolism. Further analysis are ongoing to verify the OTA degradation activity of the candidate genes.
Transcriptional analysis of Acinetobacter sp. neg1 capable of degrading ochratoxin A
2015
Abstract
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. Numerous potential peptidase encoding genes are present in the genome of Acinetobacter sp. neg1, but in the absence of a comprehensive evaluation of the capacity of members of the genus to degrade OTA, the identification of candidate genes through comparative genomic approaches remains difficult. 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 with an average per base quality score > 30. The RNA seq data were analyzed in the discovery environment of iplant (http://www.iplantcollaborative.org/). In particular, reads were aligned to the reference genome using TopHat2. Then, Cuffdiff was used to calculate RPKM and detect significant differences between the two conditions. This analysis revealed more than 100 differential expressed genes. Among them, we found 4 genes coding for peptidases or hydrolases that could be involved in the reaction of degradation: 2 serine-type endopeptidases were up-regulated at 6 hours, while one aminopeptidase and one hydrolase were up-regolated at 12 hours. The enrichment analysis for Gene Ontology terms revealed the over-representation of molecular functions which could be involved or consequent to OTA degradation, such as serine-type endopeptidase activity, amino acid transmembrane transporter activity, D-amino acid metabolic process and protein maturation. These results confirmed that OTA degradation proceeds through peptidase activities. We hypothesize that this reaction induces the activation of pathways potentially involved in the phenylalanine metabolism. Further analysis are ongoing to verify the OTA degradation activity of the candidate genes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
