CHARACTERIZATION OF TN917 INSERTION MUTANTS OF STAPHYLOCOCCUS-EPIDERMIDIS AFFECTED IN BIOFILM FORMATION

Biofilm formation is thought to result from the concerted action of pr imary attachment to a specific surface and accumulation in multilayere d cell clusters, Here we describe the isolation and characterization o f transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay, Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolat ed, Each mutant carried one copy of Tn917, The mutants were divided in to two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a), Mutants of phenotypic class A lacked four cell surface pro teins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters . They were able to form a biofilm on a glass surface, a trait that wa s even more pronounced than in the wild-type strain O-47, Loss of seve ral surface proteins might have led to the reduced surface hydrophobic ity by unmasking hydrophilic structures, thus favoring primary attachm ent to a glass surface and leading to subsequent biofilm formation, Mu tants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a g lass surface, mut1 and mut2 could be complemented by wild-type DNA fra gments containing the Tn917 insertion sites of mut1 and mut2, respecti vely, The complemented biofilm-positive clone mut1(pRC20) produced a 6 0-kDa protein which is postulated to function as the adhesin for bindi ng to plastic, The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically di stinct.