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Chapter 24 : Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn by the VanR-VanS Two-Component Regulatory System

Authors: Michel Arthur1, Florence Depardieu1, Patrice Courvalin1, Theodore Holman2, Zhen Wu2, Gerard Wright2, Christopher T. Walsh2
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Affiliations: 1: Unité des Agents Antibacteriéns, Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France; 2: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Content Type: Monograph
Publication Year: 1995

Category: Microbial Genetics and Molecular Biology

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Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn by the VanR-VanS Two-Component Regulatory System, Page 1 of 2

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Abstract:

Glycopeptide antibiotics vancomycin and teicoplanin are used to treat severe infections caused by gram-positive cocci. Strains displaying the so-called VanA resistance phenotype are inducibly resistant to high levels of vancomycin and teicoplanin. Production of the depsipeptide D-Ala-D-Lac is controlled by the VanR-VanS two-component regulatory system that activates transcription of vancomycin resistance genes in response to the presence of glycopeptides in the culture medium. Response regulators (RRs) of this subclass regulate transcription at specific promoters thought to be recognized by the main form of RNA polymerase holoenzyme, corresponding to Eσ in . The first 122 amino acids at the N terminus of VanS are not related in sequence to other HPKs. This region of VanS contains two clusters of hydrophobic amino acids that could correspond to membrane-spanning regions. Validation of the predicted roles of VanS and VanR in sequential phosphoryl group transfer was obtained by overproduction, purification, and assay of the two proteins. The vanR and vanS genes were introduced into the chromosome of a susceptible strain of using an integrative vector. -Activation of transcriptional fusions carried by plasmids were analyzed based on determination of chloramphenicol acetyltransferase (CAT) activity. Mapping of the 5’ end of mRNA by S1 nuclease protection and by primer extension assays identified one transcriptional start site in the vanS-vanH intergenic region. Cloning of , , , , and upstream from the gene in a multicopy vector resulted in high-level transcription of the reporter gene.

Citation: Arthur M, Depardieu F, Courvalin P, Holman T, Wu Z, Wright G, Walsh C. 1995. Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn by the VanR-VanS Two-Component Regulatory System, p 387-391. In Hoch J, Silhavy T (ed), Two-Component Signal Transduction. ASM Press, Washington, DC. doi: 10.1128/9781555818319.ch24
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Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn1546 by the VanR-VanS Two-Component Regulatory System
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Citation: Arthur M, Depardieu F, Courvalin P, Holman T, Wu Z, Wright G, Walsh C. 1995. Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn by the VanR-VanS Two-Component Regulatory System, p 387-391. In Hoch J, Silhavy T (ed), Two-Component Signal Transduction. ASM Press, Washington, DC. doi: 10.1128/9781555818319.ch24
/content/10.1128/9781555818319.chap24.fig24-1
FIGURE 1

Map of Tn and analysis of transcriptional fusions. Open arrows represent coding sequences. Closed and open arrowheads labeled IR and IR. indicate the left and right inverted repeats of the transposon, respectively. Genes , and are cotranscribed from a promoter located in the intergenic region ( ). Restriction sites: B, HI;, II; Bm, I; EI, RI;EV, RV; H, III; He, II;P, I; X, I. The inserts cloned into the multicopy promoter probing vector pAT78 are indicated by solid bars. CAT activity is expressed in nanomoles per minute per milligram of protein. Expression of the cat reporter gene was studied in JH2-2 and in a derivative of JH2-2 harboring a chromosomal copy of . Induction of vancomycin-resistant and -susceptible strains was performed with 50 and 1 µg of vancomycin per milliliter, respectively. R, resistant; S, susceptible; triangles, insertional inactivation of .

10.1128/9781555818319/fig24-1_thmb.gif
10.1128/9781555818319/fig24-1.gif
Image of FIGURE 1
FIGURE 1

Map of Tn and analysis of transcriptional fusions. Open arrows represent coding sequences. Closed and open arrowheads labeled IR and IR. indicate the left and right inverted repeats of the transposon, respectively. Genes , and are cotranscribed from a promoter located in the intergenic region ( ). Restriction sites: B, HI;, II; Bm, I; EI, RI;EV, RV; H, III; He, II;P, I; X, I. The inserts cloned into the multicopy promoter probing vector pAT78 are indicated by solid bars. CAT activity is expressed in nanomoles per minute per milligram of protein. Expression of the cat reporter gene was studied in JH2-2 and in a derivative of JH2-2 harboring a chromosomal copy of . Induction of vancomycin-resistant and -susceptible strains was performed with 50 and 1 µg of vancomycin per milliliter, respectively. R, resistant; S, susceptible; triangles, insertional inactivation of .

Citation: Arthur M, Depardieu F, Courvalin P, Holman T, Wu Z, Wright G, Walsh C. 1995. Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn by the VanR-VanS Two-Component Regulatory System, p 387-391. In Hoch J, Silhavy T (ed), Two-Component Signal Transduction. ASM Press, Washington, DC. doi: 10.1128/9781555818319.ch24
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References

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