Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation

The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis of Staphylococcus epider midis biomaterial-related infections. Using biofilm-producing S. epidermidi s 1457 and transposon Tn917 carried on plasmid pTVlts, we isolated nine iso genic biofilm-negative transposon mutants. Transduction by S, epidermidis p hage 71 was used to prove the genetic linkage of transposon insertions and altered phenotypes, Mapping of the different transposon insertions by South ern hybridization and pulsed-field gel electrophoresis indicated that these were inserted in four unlinked genetic loci, According to their phenotypes , including quantitative differences in biofilm production in different gro wth media, in the amount of the polysaccharide intercellular adhesin (PW) p roduced, in the hemagglutination titers, and in the altered colony morpholo gy, the mutants could be separated into four phenotypic classes correspondi ng with the genetic classes. Synthesis of PIA was not detectable with class I and II mutants, whereas the amount of PIA produced reflected the residua l degree of biofilm production of class III and IV mutants in different gro wth media. Chromosomal DNA flanking the transposon insertions of five class I mutants was cloned and sequenced, and the insertions were mapped to diff erent locations of icaADBC, representing the synthetic genes for PIA, Expre ssion of icaADBC from a xylose-dependent promoter in the different Isogenic mutant classes reconstituted biofilm production in all mutants. In a North ern blot analysis no icaADBC-specific transcripts were observed in RNA isol ated from mutants of classes II, III, and IV. Apparently, in addition to ic aADBC, three other gene loci have a direct or indirect regulatory influence on expression of the synthetic genes for PIA on the level of transcription .