Site-Directed Mutagenesis and Gene Deletion Using Reverse Genetics

Muhl, Daniela; Filloux, Alain

doi:10.1007/978-1-4939-0473-0_40

“…Details of genes were obtained from The Pseudomonas Genome Database (27). In-frame deletions were constructed as previously described (83). Briefly, for construction of deletion mutants of PA14 rclR, rclX, pyeRM-xenB operon, and oxyR a ~500 bp upstream and ~500 bp downstream fragment of the genes was amplified by PCR using primer pairs 1F and 2R, and 3F…”

Section: Construction Of Plasmids and Mutant Strainsmentioning

confidence: 99%

Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

Farrant

¹

,

Spiga

²

,

Davies

³

et al. 2020

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Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system, sulphur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection the host's immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress, and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type III secretion system.

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“…Details of genes were obtained from The Pseudomonas Genome Database (28). In-frame deletions were constructed as previously described (90). Briefly, for construction of deletion mutants of the PA14 rclR and rclX genes, pyeRM-xenB operon, and oxyR, a ~500 bp upstream and ~500 bp downstream fragment of the genes was amplified by PCR using primer pairs 1F and 2R, and 3F and 4R (Table S1).…”

Section: Construction Of Plasmids and Mutant Strainsmentioning

confidence: 99%

Response ofPseudomonas aeruginosato the innate immune system-derived oxidants hypochlorous acid and hypothiocyanous acid

Farrant

¹

,

Spiga

²

,

Davies

³

et al. 2020

Preprint

2

6

0

View full text Add to dashboard Cite

Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite the host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and survives attack from HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. We investigated the P. aeruginosa response to these oxidants by screening 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators involved in antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that were required for HOCl survival. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). While there was specificity in the transcriptional response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system (T3SS), sulphur and taurine transport, and

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“…In many cases, a toxic gene such as ccdB or sacB (Lund et al, 2014; Muhl and Filloux, 2014) in the parent construct is used to increase the likelihood of recovering recombinant clones. These systems generally require specific hosts or selection schemes and can be limited in scope.…”

Section: Discussionmentioning

confidence: 99%

In vivo elimination of parental clones in general and site-directed mutagenesis

Holland

¹

,

Acca

²

,

Belanger

³

et al. 2015

Journal of Immunological Methods

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The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5’-CCGCGG-3’ and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 107 from a single transformation and with greater than 90% recombinant clones.

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Site-Directed Mutagenesis and Gene Deletion Using Reverse Genetics

Cited by 18 publications

References 8 publications

Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

Response ofPseudomonas aeruginosato the innate immune system-derived oxidants hypochlorous acid and hypothiocyanous acid

In vivo elimination of parental clones in general and site-directed mutagenesis

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