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Applied and Environmental Microbiology, January 2006, p. 334-345, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.334-345.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Construction of Improved Temperature-Sensitive and Mobilizable Vectors and Their Use for Constructing Mutations in the Adhesin-Encoding acm Gene of Poorly Transformable Clinical Enterococcus faecium Strains

Sreedhar R. Nallapareddy,1,2 Kavindra V. Singh,1,2 and Barbara E. Murray1,2,3*

Division of Infectious Diseases, Department of Internal Medicine,1 Center for the Study of Emerging and Re-emerging Pathogens,2 Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 770303

Received 23 July 2005/ Accepted 22 September 2005

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082{Delta}acm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical School at Houston, 6431 Fannin St., MSB 2.112, Houston, TX 77030. Phone: (713) 500-6745. Fax: (713) 500-6766. E-mail: bem.asst{at}uth.tmc.edu

Applied and Environmental Microbiology, January 2006, p. 334-345, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.334-345.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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