Improved engineering of Pseudomonas aeruginosa to study the adaptation of pyoverdine production under intra- or inter- specific bacterial competition.

Pseudomonas aeruginosa (PA) is a common cause of chronic infections, particularly feared by cystic fibrosis patients. PA colonizes the lung where it adapts to the local environment, and/or to treatments by drugs. This genotypic and phenotypic adaptation, in turns, influences its interaction with its environment, like bacteria from the microbiota. As an example, to access iron, PA produces and secretes two siderophores, pyoverdine and pyochelin that are iron chelators scavenging iron from the environment and bringing it back into the bacterial cells. Siderophores production depends on the level of iron starvation, on the presence of other bacteria, etc. this latter component being less well investigated. Even if studies on bacterial interactions, and their evolution, have been increasing since several years, we are still facing a lack of tools, for example, to specifically follow the growth of PA isolates in such competitive environments. We thus improved a cloning method to gain time in the cloning steps, to lower the polar effects, and to accurately follow the interactions of any PA isolate with other bacteria. For that, a fluorescent reporter gene was inserted between two genes, the glutamine-fructose-6-phosphate transaminase (glmS) and PA5548. This reporter was efficiently produced either from an inducible or a house-keeping promoter, and its expression did not lead to polar effects. We used this strain to study intra and inter-specific bacterial competitions for iron between different lung pathogens. We thus grew wild-type PA together either with an isogenic PA ΔpvdS variant, that does not produce the most efficient siderophore pyoverdine, or with Klebsiella pneumoniae or Acinetobacter baumanii, two other lung pathogens. We finally monitored the effect of the loss of pvdS on the competition between PA and the other bacterial species. These studies enabled us to differentiate intra from inter specific competitions, both arising in the lung environment, and pinpoint the importance of the bacterial specie for the adaptation of pyoverdine production.