title: Unphosphorylated CsgD controls biofilm formation in Salmonella enterica serovar Typhimurium.
abstract: The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His(6) binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His(6) is crucial for the expression of csgBA and adrA. CsgD-His(6) is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, FE 280, 17177 Stockholm, Sweden.
authors.forename: Katherina
authors.initials: K
authors.lastname: Zakikhany
authors.forename: Carl R
authors.initials: CR
authors.lastname: Harrington
authors.forename: Manfred
authors.initials: M
authors.lastname: Nimtz
authors.forename: Jay C D
authors.initials: JC
authors.lastname: Hinton
authors.forename: Ute
authors.initials: U
authors.lastname: Römling
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D015534:Trans-Activators