A custom microarray based platform to decipher ToxR regulatory network in Photobacterium profundum SS9.

Photobacterium profundum is a psychropiezophilic bacterium, considered a model organism for piezophic adaptation for its capability of living both at hadal and atmospheric pressure. A transcription factor, ToxR, was found to be a pressure sensor that regulates pressure-specific porins. Furthermore, ToxR is a widespread sensor among the Vibrios. To better understand the effect of ToxR in the transcriptional landscape of P. profundum we designed a custom microarray, using the CombiMatrix®”12K CustomArray” technology. This kind of arrays are the ideal device for microbial genomics, as they allow to design up to two different probes per ORF. Array imaging is often a neglected step in expression studies, while enhancing image quality and intensity extraction can give better results. We decided to perform multiple image acquisitions per hybridization, at increasing laser power. We developed a software for integrating multiple acquisitions, in order to extend the dynamic range and discriminate the differences in gene expression for rare transcripts as well for rRNAs. We compared the expression profile of P. profundum wild type with the toxRdeficient strain, grown both at high and atmospheric pressure and in different media. From these comparisons we could obtain a list of putative ToxR-regulated genes. Results confirmed the relevance of nutrient transporters and flagellar proteins in piezoresistance, and for the first time we found cold shock proteins overexpressed and several ribosomal proteins at low pressure. Now we aim to screen the protein-DNA interactions (ToxR with promoters of regulated genes) with an ALPHAScreen(R) based assay.