ABSTRACTThe genome of the denitrifying bacteriumParacoccus denitrificanspredicts the expression of a small heme-containing nitric oxide (NO) binding protein, H-NOX. The genome organization and prior work in other bacteria suggest that H-NOX interacts with a diguanylate cyclase that cyclizes GTP to make cyclic di-GMP (cdGMP). Since cdGMP frequently regulates attached growth as a biofilm, we first established conditions for biofilm development byP. denitrificans. We found that adhesion to a polystyrene surface is strongly stimulated by the addition of 10 mM Ca2+to rich media. The genome encodes at least 11 repeats-in-toxin family proteins that are predicted to be secreted by the type I secretion system (TISS). We deleted the genes encoding the TISS and found that the mutant is almost completely deficient for attached growth. Adjacent to the TISS genes there is a potential open reading frame encoding a 2,211-residue protein with 891 Asp-Ala repeats. This protein is also predicted to bind calcium and to be a TISS substrate, and a mutant specifically lacking this protein is deficient in biofilm formation. By analysis of mutants and promoter reporter fusions, we show that biofilm formation is stimulated by NO generated endogenously by the respiratory reduction of nitrite. A mutant lacking both predicted diguanylate cyclases encoded in the genome overproduces biofilm, implying that cdGMP is a negative regulator of attached growth. Our data are consistent with a model in which there are H-NOX-dependent and -independent pathways by which NO stimulates biofilm formation.IMPORTANCEThe bacteriumParacoccus denitrificansis a model for the process of denitrification, by which nitrate is reduced to dinitrogen during anaerobic growth. Denitrification is important for soil fertility and greenhouse gas emission and in waste and water treatment processes. The ability of bacteria to grow as a biofilm attached to a solid surface is important in many different contexts. In this paper, we report that attached growth ofP. denitrificansis stimulated by nitric oxide, an intermediate in the denitrification pathway. We also show that calcium ions stimulate attached growth, and we identify a large calcium binding protein that is required for growth on a polystyrene surface. We identify components of a signaling pathway through which nitric oxide may regulate biofilm formation. Our results point to an intimate link between metabolic processes and the ability ofP. denitrificansto grow attached to a surface.
Biofilm formation byExiguobacteriumsp. DR11 and DR14 alter polystyrene surface properties and initiate biodegradation
