Isolation of human plasma-inducible, growth phase- and temperature-regulated gene fusions in Streptococcus pyogenes using a Tn917-lacZ transposon.
Streptococcus pyogenes is capable of causing a variety of human diseases ranging from superficial or deep tissue infections to non-infectious post-streptococcal infection sequelae. In this paper, we report the use of a Tn917-lacZ transposon to isolate random lacZ transcription fusions in the S. pyogenes chromosome. Libraries of random Tn917-lacZ mutants were generated in a representative opacity factor positive strain CS101 (M49) and an opacity factor negative strain 1881 (M1). Several different mutant phenotypes were isolated. These included: temperature-regulated promoters, growth phase/cell density-regulated promoters and a human plasma-inducible promoter. Expression of the temperature-regulated fusions was 5-10-fold higher when grown at 30 degrees C compared to growth at 37 degrees C. The growth phase-regulated fusions were induced 30-fold at late exponential phase and were repressed by a diffusible S. pyogenes factor(s). Expression of the human plasma-inducible fusion was induced 10-15-fold by human plasma or sera, 4-fold by rabbit sera and was repressed by horse and mouse sera. In addition, hemolysin negative and capsule over expression mutants were isolated. These results demonstrate the utility of Tn917-lacZ mutagenesis for the identification of S. pyogenes promoters.
Sledjeski
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Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA. dsledjeski@mco.edu
Coco
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Corriher
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Boyle
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MD
D019677:Artificial Gene Fusion; D001769:Blood; D003470:Culture Media; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007763:Lac Operon; D016254:Mutagenesis, Insertional; D010447:Peptide Hydrolases; D011401:Promoter Regions, Genetic; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic
D003470:Culture Media; D004251:DNA Transposable Elements; C021908:opacity factor; D010447:Peptide Hydrolases