Analysis of the Plasmid-Based
            <i>ts</i>
            Allele of
            <i>PA0006</i>
            Reveals Its Function in Regulation of Cell Morphology and Biosynthesis of Core Lipopolysaccharide in Pseudomonas aeruginosa

Yang, Zhili; Zhang, Zengping; Zhu, Jiaqi; Ma, Yunhao; Wang, Jianxin; Liu, Jianhua

doi:10.1128/aem.00480-22

Cited by 3 publications

(23 citation statements)

References 58 publications

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“…To prevent suppressors from occurring, we deleted the fabA chromosomal copy in the presence of a complementary copy in the ts -plasmid. That is, by using a three-step protocol ( 8 ), the chromosomal deletion Δ fabA allele was created under the protection of a native promoter-controlled fabA in the ts -plasmid pTS- fabA ( Fig. 1A ) (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“… Pseudomonas aeruginosa is an opportunistic pathogen for which new drug development is needed ( 1 – 3 ). ts -Plasmid-based conditional alleles of essential genes are useful for deletion analysis of essential genes of interest in P. aeruginosa ( 8 ). However, multicopy ts -plasmids hamper the analysis of the mutant phenotype because cells deplete the ts -plasmid at the onset of the stationary phase after the shift to a restrictive temperature.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, many essential genes are found to be suppressible ( 6 ), partly caused by spontaneously occurring suppressors during construction of essential gene deletion strains ( 6 , 7 ). Spontaneous accumulation of suppressor mutations can be prevented by the presence of a complementary copy in the ts -plasmid during construction of essential gene deletion strains at permissive temperatures ( 8 ). On the other hand, plasmid-based conditional mutant strains are suitable for suppressor screening at a restrictive temperature ( 8 , 9 ).…”

Section: Introductionmentioning

confidence: 99%

“…Spontaneous accumulation of suppressor mutations can be prevented by the presence of a complementary copy in the ts -plasmid during construction of essential gene deletion strains at permissive temperatures ( 8 ). On the other hand, plasmid-based conditional mutant strains are suitable for suppressor screening at a restrictive temperature ( 8 , 9 ).…”

Section: Introductionmentioning

confidence: 99%

“…To address this issue, we have carried out a three-step approach ( 8 ) to construct a plasmid-based ts -mutant strain, Δ fabA / pTS-fabA , that contains a Δ fabA allele on chromosome and a complementary copy of fabA in the ts -plasmid. We show that Δ fabA / pTS-fabA fails to grow at a restrictive temperature and exhibits a terminal phenotype of curved cell morphology under aerobic growth conditions.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Analysis of the Plasmid-Based ts -Mutant Δ fabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa

et al. 2023

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Analysis of the Plasmid-Based ts -Mutant Δ fabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa

et al. 2023

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Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa

Zhu,

Zhao,

Yang

2024

Genes

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Pa0665 in Pseudomonas aeruginosa shares homologous sequences with that of the essential A-type iron–sulfur (Fe-S) cluster insertion protein ErpA in Escherichia coli. However, its essentiality in P. aeruginosa and its complementation with E. coli erpA has not been experimentally examined. To fulfill this task, we constructed plasmid-based ts-mutant Δpa0665/pTS-pa0665 using a three-step protocol. The mutant displayed growth defects at 42 °C, which were complemented by expressing ec.erpA. Microscopic observations indicated a petite cell phenotype for Δpa0665/pTS-pa0665 at 42 °C, correlated with the downregulation of the oprG gene. RNA sequencing revealed significant transcriptional changes in genes associated with the oxidative phosphorylation (OXPHOS) system, aligning with reduced ATP levels in Δpa0665/pTS-pa0665 under 42 °C. Additionally, the ts-mutant showed heightened sensitivity to H2O2 at 42 °C. Overall, our study demonstrates the essential role of pa0665 for OXPHOS function and is complemented by ec.erpA. We propose that the plasmid-based ts-allele is useful for genetic analysis of essential genes of interest in P. aeruginosa.

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Genetic Analysis of the Plasmid-Based Temperature-Lethal Mutant pa1792|lpxH(Ts) in Pseudomonas aeruginosa

Zhang,

Yang,

Liu

2024

Genes

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Many enzymes in the Raetz pathway for lipid A biosynthesis in Escherichia coli are essential. A homologous protein Pa1792|LpxH in Pseudomonas aeruginosa is known to complement the loss of LpxH in E. coli. Genome-wide transposon-insertion sequencing analysis indicates that lpxH is essential in P. aeruginosa. However, genetic analysis of lpxH in P. aeruginosa has not been carried out, partly because the conditional alleles of essential genes are not readily constructed. In this study, we first constructed a plasmid-based temperature-sensitive mutant ΔlpxH/pTS-lpxH or lpxH(Ts) in P. aeruginosa PAO1. Spot-plating assay indicated that lpxH(Ts) was lethal at a restrictive temperature, confirming its essentiality for growth. Microscopic analysis revealed that lpxH(Ts) exhibited an oval-shaped morphology, suggesting that lpxH was required for rod-shape formation. SDS-PAGE and Western blotting analysis showed that lpxH(Ts) failed to synthesize lipid A, consistent with its function in lipid A biosynthesis. Strong expression of lpxH but not the non-homologous isoenzyme lpxI or lpxG impeded growth and caused cell lysis, implying that lpxH-specific cofactors were required for this toxic effect in P. aeruginosa. Together, our results demonstrate that lpxH is essential for lipid A biosynthesis, rod-shaped growth, and viability in P. aeruginosa. We propose that this plasmid-based conditional allele is a useful tool for the genetic study of essential genes in P. aeruginosa.

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Analysis of the Plasmid-Based ts Allele of PA0006 Reveals Its Function in Regulation of Cell Morphology and Biosynthesis of Core Lipopolysaccharide in Pseudomonas aeruginosa

Abstract: 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.

Cited by 3 publications

References 58 publications

Analysis of the Plasmid-Based ts -Mutant Δ fabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa

Analysis of the Plasmid-Based ts -Mutant Δ fabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa

Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa

Genetic Analysis of the Plasmid-Based Temperature-Lethal Mutant pa1792|lpxH(Ts) in Pseudomonas aeruginosa

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