Abstract
Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections, which is largely used as a model organism to study antibiotic resistance and pathogenesis. As other species of the genus, its wide metabolic versatility appears to be attractive to study biotechnological applications. However, its natural resistance to antibiotics and its capacity to produce a wide range of virulence factors argue against its biotechnological potential. By reducing the genome of the reference strain PAO1, we explored the development of four hypovirulent and hypersusceptible recombinant DNA hosts (rDNA hosts). Despite deleting up to 0.8% of the core genome, any of the developed strains presented alterations of fitness when cultured under standard laboratory conditions. Other features such as antibiotic susceptibility, cytotoxicity, in vivo pathogenesis, and expression of heterologous peptides were also explored to highlight the potential applications of these models. This work stands as the first stage of the development of a safe-platform strain of Pseudomonas aeruginosa that will be further optimized for biotechnological applications.
Highlights
Since the pioneering works of Bruce Holloway in the 1950s, Pseudomonas aeruginosa has become a model organism for exploring the genetics and physiological functions of genus Pseudomonas, and more generally Gram-negative non-fermenters
Our results showed that when antibiotic related genes were targeted, as in SMEff, SMRes or SM54, the basal susceptibility to antibiotics was highly increased and, on the same logic, when virulence factor genes were deleted in strains SMVir or SM54 their capacities to produce these factors was abolished
Our results showed that when virulence related genes are deleted, the dry weight and the number of CFUs recovered after 24 h of culture are significantly increased when compared to strain PAO1
Summary
Since the pioneering works of Bruce Holloway in the 1950s, Pseudomonas aeruginosa has become a model organism for exploring the genetics and physiological functions of genus Pseudomonas, and more generally Gram-negative non-fermenters. This natural resistance is principally due to the low permeability of its membrane, the production of enzymes capable to degrade or modify antibiotics and the action of two efflux systems able to export multiple molecules outside the cell (Yonezawa et al, 1995; Poole, 2004; Juan et al, 2005, 2006; Lister et al, 2009; Vettoretti et al, 2009; Berrazeg et al, 2015; Ropy et al, 2015; Jeannot et al, 2017) All these genomic determinants were targeted in this work with the objective to build up four different rDNA hosts by reducing the genome of the reference strain PAO1 without causing any negative impact on bacterial growth very similar as the work done on P. putida (Lieder et al, 2015). This project is placed as a first stage to develop a safe-platform strain of Pseudomonas aeruginosa which will be further optimized for biotechnological applications such as expression of heterologous proteins or secondary metabolites
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