A Novel Repressor of the ica Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating Staphylococcus aureus

Staphylococcus aureus TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148-159, 2016, https://doi.org/10.1111/1348-0421.12359). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (satf2580 to satf2586) and the ica operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated rob had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with rob from FK300, an rsbU-repaired derivative of S. aureus strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for rob in this process. The deletion of rob in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob, we identified open reading frame (ORF) SAOUHSC_2898 (satf2584). Our results suggest that ORF SAOUHSC_2898 (satf2584) and icaADBC are required for enhanced biofilm elaboration and PIA/PNAG production in the rob deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in S. aureus IMPORTANCE: During the search for molecular mechanisms underlying biofilm overproduction of Staphylococcus aureus TF2758, we found a putative transcriptional regulator gene designated rob within a 7-gene cluster showing a high transcriptional expression level by microarray analysis. The deletion of rob in non-biofilm-producing FK300, an rsbU-repaired derivative of NCTC8325-4, significantly increased biofilm elaboration and PIA/PNAG production. The search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob identified ORF SAOUHSC_2898. Besides binding to its own promoter region to control ORF SAOUHSC_2898 expression, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is a new important regulator of biofilm elaboration through its control of SAOUHSC_2898 and Ica protein expression in S. aureus.