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Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Tucker AT, Nowicki EM, Boll JM, Knauf GA, Burdis NC, Trent MS, Davies BW - MBio (2014)

Bottom Line: Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques.Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products.This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.

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Related in: MedlinePlus

Deletion of oxyR homolog A1S_0992 from A. baumannii results in increased sensitivity to hydrogen peroxide (H2O2). The parental A. baumannii strain and oxyR::Kanr mutant were struck on Luria-Bertani (LB) agar with or without 0.25 mM H2O2 (top). The oxyR::Kanr mutant H2O2 sensitivity phenotype is recovered by ectopic expression of the oxyR allele from a plasmid (p_oxyR) but not by the empty plasmid (pABBR_MCS) (bottom).
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fig3: Deletion of oxyR homolog A1S_0992 from A. baumannii results in increased sensitivity to hydrogen peroxide (H2O2). The parental A. baumannii strain and oxyR::Kanr mutant were struck on Luria-Bertani (LB) agar with or without 0.25 mM H2O2 (top). The oxyR::Kanr mutant H2O2 sensitivity phenotype is recovered by ectopic expression of the oxyR allele from a plasmid (p_oxyR) but not by the empty plasmid (pABBR_MCS) (bottom).

Mentions: To establish the relevance of this recombineering system in characterizing gene-phenotype relationships, we first focused on the well-studied response of bacteria to oxidative stress. Response to oxidative stress is important in order for bacteria to adapt and survive in the presence of biocides and antibiotics (15, 16). In E. coli, hydrogen peroxide triggers activation of the transcription factor OxyR, which induces expression of oxidative stress protection genes (17, 18). Deletion of oxyR in E. coli results in increased sensitivity to hydrogen peroxide. The protein A1S_0992 from A. baumannii strain 17978 shows 29% identity (E value, 2e−34) to E. coli OxyR by BLAST bioinformatics analysis, suggesting that A1S_0992 may play an OxyR-like role in A. baumannii. Consistent with studies of OxyR in E. coli, deletion of A1S_0992 using our recombineering system produced an A. baumannii strain that displays increased sensitivity to hydrogen peroxide (Fig. 3, top). Ectopic expression of A1S_0992 (oxyR) from a plasmid complements the mutant phenotype, demonstrating that A1S_0992 is very likely an OxyR homolog that plays an important role in oxidative stress protection in A. baumannii (Fig. 3, bottom).


Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Tucker AT, Nowicki EM, Boll JM, Knauf GA, Burdis NC, Trent MS, Davies BW - MBio (2014)

Deletion of oxyR homolog A1S_0992 from A. baumannii results in increased sensitivity to hydrogen peroxide (H2O2). The parental A. baumannii strain and oxyR::Kanr mutant were struck on Luria-Bertani (LB) agar with or without 0.25 mM H2O2 (top). The oxyR::Kanr mutant H2O2 sensitivity phenotype is recovered by ectopic expression of the oxyR allele from a plasmid (p_oxyR) but not by the empty plasmid (pABBR_MCS) (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128354&req=5

fig3: Deletion of oxyR homolog A1S_0992 from A. baumannii results in increased sensitivity to hydrogen peroxide (H2O2). The parental A. baumannii strain and oxyR::Kanr mutant were struck on Luria-Bertani (LB) agar with or without 0.25 mM H2O2 (top). The oxyR::Kanr mutant H2O2 sensitivity phenotype is recovered by ectopic expression of the oxyR allele from a plasmid (p_oxyR) but not by the empty plasmid (pABBR_MCS) (bottom).
Mentions: To establish the relevance of this recombineering system in characterizing gene-phenotype relationships, we first focused on the well-studied response of bacteria to oxidative stress. Response to oxidative stress is important in order for bacteria to adapt and survive in the presence of biocides and antibiotics (15, 16). In E. coli, hydrogen peroxide triggers activation of the transcription factor OxyR, which induces expression of oxidative stress protection genes (17, 18). Deletion of oxyR in E. coli results in increased sensitivity to hydrogen peroxide. The protein A1S_0992 from A. baumannii strain 17978 shows 29% identity (E value, 2e−34) to E. coli OxyR by BLAST bioinformatics analysis, suggesting that A1S_0992 may play an OxyR-like role in A. baumannii. Consistent with studies of OxyR in E. coli, deletion of A1S_0992 using our recombineering system produced an A. baumannii strain that displays increased sensitivity to hydrogen peroxide (Fig. 3, top). Ectopic expression of A1S_0992 (oxyR) from a plasmid complements the mutant phenotype, demonstrating that A1S_0992 is very likely an OxyR homolog that plays an important role in oxidative stress protection in A. baumannii (Fig. 3, bottom).

Bottom Line: Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques.Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products.This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.

Show MeSH
Related in: MedlinePlus