Biofilm formation in Staphylococcus epidermidis is dependent upon the ica operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation. The icaR gene, located adjacent to the ica operon, appears to be a member of the tetR family of transcriptional regulators. In the reference strain RP62A, reversible inactivation of the ica operon by IS256 accounts for 25 to 33% of phase variants. In this study, icaA and icaR regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS256 is absent. Predictably, ica operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS256-generated phase variants. In contrast, the icaR gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants. An icaR::Em r insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in ica operon expression but did not significantly alter regulation of the icaR gene itself. Activation of ica operon transcription by ethanol in CSF41498 was icaR dependent. In contrast, a small but significant induction of ica by NaCl and glucose (NaCl-glucose) was observed in the icaR::Em r mutant. In addition, transcription of the icaR gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol. Expression of the ica operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (icaR ؉ ) but not in RP62A variants (icaR deficient). These data indicate that icaR encodes a repressor of ica operon transcription required for ethanol but not NaCl-glucose activation of ica operon expression and biofilm formation.Biofilm-forming coagulase-negative staphylococci (CoNS), particularly Staphylococcus epidermidis, are the etiological agents in a significant proportion of biomaterial-related nosocomial infections. Formation of S. epidermidis biofilms is proposed to occur in a two-step manner (20,24,26,39) in which a cellular accumulation process to form the mature biofilm follows a rapid initial attachment to an inert synthetic surface. Initial adherence is mediated by polysaccharide adhesin (PS/A) (24) and/or one of several proteins (including autolysin [19]), and accumulation of cells is due to production of polysaccharide intercellular adhesin (PIA) (20). The PIA is encoded by the ica (intercellular adhesin) operon (20); however, it has been reported recently that this operon also encodes PS/A and that PS/A and PIA are chemically closely related (27). Production of PIA, which represents the key virulence factor of S. epidermidis, is subject to phase-variable regulation (38, 39). Little is known about the molecular basis of this phase variation process; however, recent evidence has indicated that alternating insertion and excision of an insertion sequence element is responsible for between 25 and 33% of ica operon switching in S. epidermidis RP62A under laboratory conditions (39). The insertion element IS257 has also been observed inserting at the ica locus of a clinical S. epidermidis i...

SummaryMycobacterium abscessus is an emerging pathogen that is increasingly recognized as a relevant cause of human lung infection in cystic fibrosis patients. This highly antibiotic-resistant mycobacterium is an exception within the rapidly growing mycobacteria, which are mainly saprophytic and non-pathogenic organisms. M. abscessus manifests as either a smooth (S) or a rough (R) colony morphotype, which is of clinical importance as R morphotypes are associated with more severe and persistent infections. To better understand the molecular mechanisms behind the S/R alterations, we analysed S and R variants of three isogenic M. abscessus S/R pairs using an unbiased approach involving genome and transcriptome analyses, transcriptional fusions and integrating constructs. This revealed different small insertions, deletions (indels) or single nucleotide polymorphisms within the non-ribosomal peptide synthase gene cluster mps1-mps2-gap or mmpl4b in the three R variants, consistent with the transcriptional differences identified within this genomic locus that is implicated in the synthesis and transport of Glyco-Peptido-Lipids (GPL). In contrast to previous reports, the identification of clearly defined genetic lesions responsible for the loss of GPL-production or transport makes a frequent switching back-and-forth between smooth and rough morphologies in M. abscessus highly unlikely, which is important for our understanding of persistent M. abscessus infections.

Expression of ica operon-mediated biofilm formation in Staphylococcus epidermidis RP62A is subject to phase variable regulation. Reversible transposition of IS256 into icaADBC or downregulation of icaADBC expression are two important mechanisms of biofilm phenotypic variation. Interestingly, the presence of IS256 was generally associated with a more rapid rate of phenotypic variation, suggesting that IS256 insertions outside the ica locus may affect ica transcription. Consistent with this, we identified variants with diminished ica expression, which were associated with IS256 insertions in the B activator rsbU or sarA. Biofilm development and ica expression were activated only by ethanol and not NaCl in rsbU::IS256 insertion variants, which were present in ϳ11% of all variants.B activity was impaired in rsbU::IS256 variants, as evidenced by reduced expression of the B -regulated genes asp23, csb9, and rsbV. Moreover, expression of sarA, which is B regulated, and SarA-regulated RNAIII were also suppressed. A biofilm-forming phenotype was restored to rsbU::IS256 variants only after repeated passage and was not associated with IS256 excision from rsbU. Only one sarA::IS256 insertion mutant was identified among 43 biofilm-negative variants. Both NaCl and ethanolactivated ica expression in this sarA::IS256 variant, but only ethanol increased biofilm development. Unlike rsbU::IS256 variants, reversion of the sarA::IS256 variant to a biofilm-positive phenotype was accompanied by precise excision of IS256 from sarA and restoration of normal ica expression. These data identify new roles for IS256 in ica and biofilm phenotypic variation and demonstrate the capacity of this element to influence the global regulation of transcription in S. epidermidis.Biofilm formation by staphylococci on implanted biomaterials is now recognized as an important virulence factor contributing to the development of a significant proportion of all device-related infections. Enzymes encoded by the icaADBC operon (20,23,27) are required for synthesis of an extracellular polysaccharide termed polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis (37, 58) and poly-Nacetylglucosamine (PNAG) in S. aureus (12,31,41,43,44), which plays an important role in both the initial attachment and cellular proliferation processes characteristic of staphylococcal biofilm development (27,38,38,40).Much effort has been focused on understanding the regulation of ica operon expression, and it is now known that increased transcription of the ica operon can be observed under anaerobic growth conditions (13), in the presence of subinhibitory concentrations of certain antibiotics, and in response to osmotic stress (32, 52). We have recently reported that the icaR gene encodes a transcriptional repressor with a central role in the environmental regulation of ica operon expression in S. epidermidis (10, 11). Jefferson et al. (29) demonstrated that purified IcaR protein from S. aureus bound the ica operon promoter region close to the icaA start codon. Co...