title: Systematic analysis of diguanylate cyclases that promote biofilm formation by Pseudomonas fluorescens Pf0-1.
abstract: Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.
authors.affiliation: Department of Microbiology and Immunology, Dartmouth Medical School, Vail Building, Rm. 505, Hanover, NH 03755, USA.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Newell
authors.forename: Shiro
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Kelli L
authors.initials: KL
authors.lastname: Hvorecny
authors.forename: Russell D
authors.initials: RD
authors.lastname: Monds
authors.forename: George A
authors.initials: GA
authors.lastname: O'Toole
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D016254:Mutagenesis, Insertional; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*
chemical_list: D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP