Porphyromonas gingivalis requires fur for haem-responsive biofilm formation

Porphyromonas gingivalis has an obligate requirement for iron and protoporphyrin IX which it satisfies by accumulating haem and iron liberated from the human host. P. gingivalis needs to regulate the amount of iron transported into the cell to maintain iron homeostasis, given that the amount of iron in the extracellular environment varies with disease state. Iron homeostasis in Gram negative bacteria is usually mediated at the transcriptional level by the Ferric Uptake Regulator (Fur). Objective: To determine the role of the Fur homologue Pg-Fur in P. gingivalis 33277. Methods: A fur deletion strain ECR455 was constructed in 33277, then a functional fur gene was inserted back into ECR455, making the fur complemented strain ECR475. Microarray analyses of ECR455 compared to 33277 determined the regulome of Pg-Fur whilst cellular metal content was determined by ICP-MS. The role of Pg-Fur in biofilm development was determined by assessing biofilms of 33277, ECR455 and ECR475 using Confocal Laser Scanning Microscopy. Results: Microarray analyses indicated that the Fur homologue of P. gingivalis did not regulate any genes involved in iron/haem homeostasis. This was supported by the lack of difference in cellular metal content between 33277 and ECR455. However mfaI, the gene encoding the minor fimbriae, which is inhibitory to P. gingivalis biofilm development, was up-regulated in ECR455. Both the 33277 and ECR475 biofilms showed a significant decrease in biovolume under haem-limited conditions. The fur mutant ECR455 produced significantly thinner biofilms with lower biovolume than the 33277 or ECR475, however haem availability had no effect on the ECR455 biofilms. ECR455 cells in biofilms were significantly elongated compared to the two control strains. Conclusion: The Fur homologue of P. gingivalis does not regulate iron homeostasis, but does promote P. gingivalis biofilm development, affects cellular morphology in the biofilm, and regulates biofilm development in response to haem availability.