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Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different
M Nedelmann1, A Sabottke, R Laufs
1Institut für Medizinische Mikrobiologie und Immunologie, Universitätskrankenhaus Eppendorf, Hamburg, Germany.
Abstract:
Staphylococcus epidermidis phage 48 was used to efficiently transduce plasmid pTV1ts and a chromosomal Tn917 insertion M27 from S. epidermidis 13-1 to biofilm-producing clinical S. epidermidis isolates 1457, 9142, and 8400. The Tn917 insertion leading to the biofilm-negative phenotype of transposon mutant M10 was sequentially transduced to biofilm-producing S. epidermidis 1457 using S. epidermidis phage 48 and then, using the resulting biofilm-negative transductant 1457-M10 as a donor, into several unrelated biofilm-producing clinical S. epidermidis isolates using S. epidermidis phage 71. All resultant transductants displayed a completely biofilm-negative phenotype. In addition, S. epidermidis phage 71 was adapted to S. epidermidis 1457 and 8400, which allowed generalized transduction of transposon insertions in these wild-type strains. As Tn917 predominantly transposed into endogenous plasmids of all three strains used, an efficient system for chromosomal transposon mutagenesis was established by curing of S. epidermidis 1457 of a single endogenous plasmid p1457 by sodium dodecylsulfate treatment. After transduction of the resulting derivative, S. epidermidis 1457c with pTV1ts, insertion of transposon Tn917 to different sites of the chromosome of S. epidermidis 1457c was observed. Biofilm-producing S. epidermidis 1457c x pTV1ts was used to isolate a biofilm-negative transposon mutant (1457c-M3) with a chromosomal insertion apparently different from two previously isolated isogenic biofilm-negative transposon mutants, M10 and M11 (Mack, D., M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs: Infect Immun 62 [1994] 3244-3253). S. epidermidis phage 71 was used to prove genetic linkage between transposon insertion and altered phenotype by generalized transduction. In combination with phage transduction, 1457c x pTV1ts will be a useful tool facilitating the study of bacterial determinants of the pathogenicity of S. epidermidis.
Insights
Bacteriophage transduction enabled the transfer of genetic elements, including transposon Tn917 insertions, between Staphylococcus epidermidis strains. This facilitated the creation of biofilm-negative mutants, offering a new tool to study S. epidermidis pathogenicity.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Staphylococcus epidermidis is a significant opportunistic pathogen, often associated with biofilm formation on medical devices.
- Understanding the genetic basis of S. epidermidis pathogenicity, particularly biofilm production, is crucial for developing effective treatments.
Purpose of the Study:
- To establish and utilize a phage transduction system for efficient genetic manipulation of clinical Staphylococcus epidermidis isolates.
- To generate biofilm-negative mutants of S. epidermidis using transposon mutagenesis and phage transduction.
- To develop a tool for studying bacterial determinants of S. epidermidis pathogenicity.
Main Methods:
- Utilized Staphylococcus epidermidis phage 48 and phage 71 for transduction of plasmid pTV1ts and transposon Tn917 insertions.
- Developed a system for chromosomal transposon mutagenesis by curing endogenous plasmids and subsequent transduction.
- Generated and characterized biofilm-negative S. epidermidis transductants and mutants.
Main Results:
- Successfully transduced plasmid and chromosomal insertions into biofilm-producing S. epidermidis clinical isolates, resulting in biofilm-negative phenotypes.
- Established an efficient system for chromosomal transposon mutagenesis in S. epidermidis.
- Isolated a novel biofilm-negative transposon mutant (1457c-M3) with a distinct chromosomal insertion.
Conclusions:
- Phage transduction is an effective method for transferring genetic elements and generating mutants in clinical S. epidermidis strains.
- The developed system provides a valuable tool for investigating the genetic factors contributing to S. epidermidis pathogenicity and biofilm formation.