Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa.

The functional and physiological characterization of bacterial genes required for growth and/or cell survival is limited by the inability to generate deletion mutants lacking the specific gene of interest. This limitation can be circumvented by generating conditional mutants in which the loss of the endogenous copy of the gene is compensated by the introduction of the wild-type allele under the control of an inducible promoter, which allows for tightly regulated expression of the gene of interest. Besides the confirmation and/or functional investigation of essential genes, conditional mutants can also be useful to investigate the effect of finely controlled expression of nonessential genes. In this chapter, we describe a method that can be used to generate stable and unmarked conditional mutants in Pseudomonas aeruginosa.

RecA and Specialized Error-Prone DNA Polymerases Are Not Required for Mutagenesis and Antibiotic Resistance Induced by Fluoroquinolones in Pseudomonas aeruginosa.

To cope with stressful conditions, including antibiotic exposure, bacteria activate the SOS response, a pathway that induces error-prone DNA repair and mutagenesis mechanisms. In most bacteria, the SOS response relies on the transcriptional repressor LexA and the co-protease RecA, the latter being also involved in homologous recombination. The role of the SOS response in stress- and antibiotic-induced mutagenesis has been characterized in detail in the model organism Escherichia coli. However, its effect on antibiotic resistance in the human pathogen Pseudomonas aeruginosa is less clear. Here, we analyzed a recA deletion mutant and confirmed, by conjugation and gene expression assays, that RecA is required for homologous recombination and SOS response induction in P. aeruginosa. MIC assays demonstrated that RecA affects P. aeruginosa resistance only towards fluoroquinolones and genotoxic agents. The comparison of antibiotic-resistant mutant frequency between treated and untreated cultures revealed that, among the antibiotics tested, only fluoroquinolones induced mutagenesis in P. aeruginosa. Notably, both RecA and error-prone DNA polymerases were found to be dispensable for this process. These data demonstrate that the SOS response is not required for antibiotic-induced mutagenesis in P. aeruginosa, suggesting that RecA inhibition is not a suitable strategy to target antibiotic-induced emergence of resistance in this pathogen.

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa.

DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the ß clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

Genetic Basis and Physiological Effects of Lipid A Hydroxylation in Pseudomonas aeruginosa PAO1.

Modifications of the lipid A moiety of lipopolysaccharide influence the physicochemical properties of the outer membrane of Gram-negative bacteria. Some bacteria produce lipid A with a single hydroxylated secondary acyl chain. This hydroxylation is catalyzed by the dioxygenase LpxO, and is important for resistance to cationic antimicrobial peptides (e.g., polymyxins), survival in human blood, and pathogenicity in animal models. The lipid A of the human pathogen Pseudomonas aeruginosa can be hydroxylated in both secondary acyl chains, but the genetic basis and physiological role of these hydroxylations are still unknown. Through the generation of single and double deletion mutants in the lpxO1 and lpxO2 homologs of P. aeruginosa PAO1 and lipid A analysis by mass spectrometry, we demonstrate that both LpxO1 and LpxO2 are responsible for lipid A hydroxylation, likely acting on different secondary acyl chains. Lipid A hydroxylation does not appear to affect in vitro growth, cell wall stability, and resistance to human blood or antibiotics in P. aeruginosa. In contrast, it is required for infectivity in the Galleria mellonella infection model, without relevantly affecting in vivo persistence. Overall, these findings suggest a role for lipid A hydroxylation in P. aeruginosa virulence that could not be directly related to outer membrane integrity.