Over 300 essential genes are predicted using transposon sequencing in the genome of Pseudomonas aeruginosa. However, methods for reverse genetic analysis of essential genes are scarce. To address this issue, we developed a three-step protocol consisting of integration of deletion plasmid, introduction of temperature-sensitive rescue plasmid, and excision of integrated-deletion plasmid to construct the plasmid-based temperature-sensitive allele of essential genes. Using PA0006 as an example, we showed that PA0006(Ts) exhibited wild-type cell morphology at permissive temperature but filamentous form at restrictive temperatures. We further showed that the glycerol-mannoheptose-bisphosphate phosphatase GmhB in Escherichia coli shared 32.4% identity with that of PA0006p and functionally complemented the defect of PA0006(Ts) at 42°C. SDS-PAGE and Western blotting indicated the presence and absence of the complete core lipopolysaccharide (LPS) and B-band O-antigen in PA0006(Ts) at 30 and 42°C, respectively. An isolated suppressor sup displayed wild-type-like cell morphology but no complete core LPS or O-antigen. Genome resequencing together with comparative transcriptomic profiling identified a candidate suppressor fructose-bisphosphate phosphatase in which the promoter harbored a SNP and the transcription level was not downregulated at 42°C compared to 30°C in sup. It was further validated that fbp overexpression suppressed the lethality of PA0006(Ts) at 42°C. Taken together, our results demonstrate that PA0006 plays a role in regulation of cell morphology and biosynthesis of core LPS. This three-step protocol for construction of conditional lethal allele in P. aeruginosa should be widely applicable for genetic analysis of other essential genes of interest, including analysis of bypass suppressibility. IMPORTANCE Microbial essential genes encode nondispensable function for cell growth and therefore are ideal targets for the development of new drugs. Essential genes are readily identified using transposon-sequencing technology at the genome scale. However, genetic analysis of essential genes of interest was hampered by limited methodologies. To address this issue, we developed a three-step protocol for construction of conditional allele of essential genes in the opportunistic pathogen Pseudomonas aeruginosa. Using PA0006 as an example, we demonstrated that the plasmid-based PA0006(Ts) mutant exhibited defects in regulation of cell morphology, formation of intact core LPS, and attachment of the O-antigen at restrictive temperatures but not at permissive temperatures. A suppressor of PA0006(Ts) was isolated through spontaneous mutations and showed restored cell morphology but not core oligosaccharide or O-antigen. This method should be widely applicable for phenotype and suppressibility analyses of other essential genes of interest in P. aeruginosa.
Keywords: Pseudomonas aeruginosa; conditional lethal allele; essential genes; lipopolysaccharide (LPS); suicide plasmid; suppressor.