Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa

Abstract

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.The biofilm mode of bacterial growth is a surface-attached state in which cells are closely packed and encased in an extracellular polymeric matrix (10, 27). Biofilms are abundant in nature and are of clinical, environmental, and industrial importance. Biofilm development is known to follow a series of complex but discrete and tightly regulated steps (18, 27), including (i) microbial attachment to the surface, (ii) growth and aggregation of cells into microcolonies, (iii) maturation, and (iv) dissemination of progeny cells that can colonize new niches. Over the last decade, several key processes important for biofilm formation have been identified, including quorum sensing (12) and surface motility (28).One of the best-studied model organisms for biofilm development is the bacterium Pseudomonas aeruginosa (10), a notorious opportunistic pathogen which causes many types of infections, including biofilm-associated chronic lung infections in individuals with cystic fibrosis (10, 24, 41). Like most organisms, P. aeruginosa requires iron for growth, as iron serves as a cofactor for enzymes that are involved in many basic cellular functions and metabolic pathways. Recent work has shown that at iron concentrations that are not limiting for growth, this metal serves as a signal for biofilm development (40). Iron limitation imposed, for example, by the mammalian iron chelator lactoferrin blocks the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular mushroom-like biofilm structures (40). By chelating iron, lactoferrin induces twitching motility (a specialized form of surface motility), which causes the cells to move across the surface instead of settling down to form structured communities (39,40). In a recent paper, Berlutti et al. (5) provided further support for the role of iron in cell aggregation and biofilm formation. They reported that in t...

Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

“…The regulation of the rhlAB genes is very complex and is affected by a number of factors and conditions, including the Rhl quorum-sensing system (60,79), nitrogen metabolism (15), the sigma factors RpoN (80), RpoD (41), and RpoS (81), DksA (82), iron (83), phosphate levels (8), and RsmA (84). AlgR repression of rhamnolipids in a 6-day biofilm is attributed to AlgR repression on the rhlI promoter (28).…”

Stenotrophomonas maltophilia: an Emerging Global Opportunistic Pathogen

“…Rhamnolipids may be important for maintaining water channels in biofilms (124). Further studies are needed to establish if the biofilms of S. maltophilia are similarly altered by iron limitation.…”

“…Confocal microscopy and flow chamber studies with P. aeruginosa containing green fluorescent protein and rhlA-gfp reporter fusions have shown that iron limitation induces rhamnolipid synthesis, promotes twitching motility, and alters biofilm structure, resulting in the formation of thin flat biofilms (124). Rhamnolipids may be important for maintaining water channels in biofilms (124).…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…The role of twitching motility in biofilm biology is complex, however, as stimuli that increase twitching motility during early development can reduce the extent of irreversible attachment, which is necessary for further stages to proceed. Studies of P. aeruginosa showed that stimulation of twitching through increased rhamnolipid synthesis, caused by the chelation of iron or treatment with small molecules, peptides, or complex natural products such as ginseng, decreased biofilm formation (118,158,356,412,417).…”