New evolving strategies revealed by transcriptomic analysis of a fur− mutant of the cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT 5344

Summary The transcriptomic analysis (RNA‐seq) of a fur mutant of P. pseudoalcaligenes CECT 5344 has revealed that Fur regulates the expression of more than 100 genes in this bacterial strain, most of them negatively. The highest upregulated genes in response to fur deletion, with respect to the wild type, both cultivated in LB medium, corresponded to genes implicated in iron uptake. They include both TonB‐dependent siderophore transporters for the active transport across the outer membrane, and ABC‐type and MSF‐type transporters for the active transport across the cytoplasmic membrane. Therefore, the main response of this bacterium to iron limitation is expressing genes necessary for metabolism of Fe siderophores produced by other microorganisms (xenosiderophores). The number of genes whose expression decreased in the fur− mutant, as well as its normalized expression (fold change), was lower. Among them, it is remarkable the presence of one of the two cas operons of the two CRISP/Cas clusters was detected in the genome of this bacterium. The transcriptome was validated by qPCR, including the decrease in the expression of cas genes (cse1). The expression of cse1 was also decreased by limiting the amount of iron, carbon or nitrogen in the medium, or by adding menadione, a compound that causes oxidative stress. The higher decrease in cse1 expression was triggered by the addition of cyanide in minimal medium. These results suggest that this bacterium responds to stress conditions, and especially to cyanide, taking a reasonable risk with respect to both the uptake of (TonB‐dependent receptors gates) and the tolerance to (reduced immunity) foreign nucleic acids. In conjunction, this can be considered a yet unknown molecular mechanism forcing bacterial evolution. Cyanide triggers iron deprivation that, in a process mediated by the master regulator protein Fur, induce the expression of TonB‐dependent receptors gates that may serve for the entrance of foreign nucleic acids. At the same time, in P. pseudoalcaligenes CECT 5344, cyanide repress the expression of a CRISP‐Cas system. The presence of Fur boxes upstream the TonB dependent receptors and the Cas proteins suggest a direct regulation of these systems by holo‐Fur, repressing the expression of the former but promoting the expression of the CRISPR‐Cas system. Moreover, the presence of cyanide was more effective than iron starvation in repressing the expression of the CRISPR‐Cas system. Therefore, cyanide seems t