title: A Novel Repressor of the ica Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating Staphylococcus aureus.
abstract: 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.
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan.
authors.forename: Liansheng
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan.|Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, Hiroshima City, Hiroshima, Japan.
authors.forename: Junzo
authors.initials: J
authors.lastname: Hisatsune
authors.affiliation: Research Facility, Hiroshima University Faculty of Dentistry, Hiroshima City, Hiroshima, Japan.
authors.forename: Ikue
authors.initials: I
authors.lastname: Hayashi
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Tatsukawa
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sato'o
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan.
authors.forename: Emiri
authors.initials: E
authors.lastname: Mizumachi
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan.|Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, Hiroshima City, Hiroshima, Japan.
authors.forename: Fuminori
authors.initials: F
authors.lastname: Kato
authors.affiliation: Genome Informatics Group, Department of Technology Development, Kazusa DNA Research Institute, Kisarazu City, Chiba, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hirakawa
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Gerald B
authors.initials: GB
authors.lastname: Pier
authors.affiliation: Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima City, Hiroshima, Japan sugai@hiroshima-u.ac.jp.|Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, Hiroshima City, Hiroshima, Japan.
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Sugai
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D018389:Codon, Nonsense; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D056426:Genetic Loci; D016680:Genome, Bacterial; D046228:Microarray Analysis; D005810:Multigene Family; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009876:Operon; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D018389:Codon, Nonsense; D050505:Mutant Proteins; D011135:Polysaccharides, Bacterial; D012097:Repressor Proteins; C113579:polysaccharide intercellular adhesin