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A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis.

Stirrett KL, Ferreras JA, Rossi SM, Moy RL, Fonseca FV, Quadri LE - BMC Microbiol. (2008)

Bottom Line: Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance.Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA. kls2002@med.cornell.edu

ABSTRACT

Background: Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis.

Results: We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.

Conclusion: Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

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Potential RobA binding sites in the promoter regions of genes upregulated in robAYp-overexpressing Y. pestis. The consensus shown is the 20-bp asymmetric marbox consensus sequence determined by Martin et al., 1999 [37]. R = A or G, Y = C or T, W = A or T, and N = A, T, G, C. Column C is the number of bp's in agreement with the 20-bp consensus sequence. The location of each RobA binding site with respect to the first codon of its cognate gene is indicated by the numbers flanking the putative binding site.
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Figure 5: Potential RobA binding sites in the promoter regions of genes upregulated in robAYp-overexpressing Y. pestis. The consensus shown is the 20-bp asymmetric marbox consensus sequence determined by Martin et al., 1999 [37]. R = A or G, Y = C or T, W = A or T, and N = A, T, G, C. Column C is the number of bp's in agreement with the 20-bp consensus sequence. The location of each RobA binding site with respect to the first codon of its cognate gene is indicated by the numbers flanking the putative binding site.

Mentions: Inspection of the promoter regions upstream of the upregulated genes in Yp pGEM-RobYp revealed the presence of a putative RobAEc binding site in each of these regions (Figure 5). These results suggest that RobAYp may act as a positive regulator for the y0010, y1050-y1049, y2173 and y3392-y3393 systems. This possible regulatory scenario is consistent with the upregulation in the expression levels of these pumps induced by robAYp overexpression in Yp.


A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis.

Stirrett KL, Ferreras JA, Rossi SM, Moy RL, Fonseca FV, Quadri LE - BMC Microbiol. (2008)

Potential RobA binding sites in the promoter regions of genes upregulated in robAYp-overexpressing Y. pestis. The consensus shown is the 20-bp asymmetric marbox consensus sequence determined by Martin et al., 1999 [37]. R = A or G, Y = C or T, W = A or T, and N = A, T, G, C. Column C is the number of bp's in agreement with the 20-bp consensus sequence. The location of each RobA binding site with respect to the first codon of its cognate gene is indicated by the numbers flanking the putative binding site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2500020&req=5

Figure 5: Potential RobA binding sites in the promoter regions of genes upregulated in robAYp-overexpressing Y. pestis. The consensus shown is the 20-bp asymmetric marbox consensus sequence determined by Martin et al., 1999 [37]. R = A or G, Y = C or T, W = A or T, and N = A, T, G, C. Column C is the number of bp's in agreement with the 20-bp consensus sequence. The location of each RobA binding site with respect to the first codon of its cognate gene is indicated by the numbers flanking the putative binding site.
Mentions: Inspection of the promoter regions upstream of the upregulated genes in Yp pGEM-RobYp revealed the presence of a putative RobAEc binding site in each of these regions (Figure 5). These results suggest that RobAYp may act as a positive regulator for the y0010, y1050-y1049, y2173 and y3392-y3393 systems. This possible regulatory scenario is consistent with the upregulation in the expression levels of these pumps induced by robAYp overexpression in Yp.

Bottom Line: Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance.Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA. kls2002@med.cornell.edu

ABSTRACT

Background: Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis.

Results: We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis.

Conclusion: Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

Show MeSH
Related in: MedlinePlus