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

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 the liquid phase, iron limitation induced motility and transition to the free-living (i.e., planktonic) mode of growth, while increased iron concentrations facilitated cell aggregation and biofilm formation. We recently demonstrated that iron limitation-induced twitching motility is regulated by quorum sensing (31). Quorum sensing allows bacteria to sense and respond to their population density via the production of small diffusible signal molecules. In P. aeruginosa and many other Gram-negative bacteria, these signal molecules are N-acyl homoserine lactones (acyl-HSLs), which have specific receptors (R proteins) (16, 30). P. aeruginosa possesses two acyl-HSL quorum-sensing systems, one for production of and response to N-3-oxo-dodecanoyl homoserine lactone (3OC₁₂-HSL) (LasR-LasI) and the other for production of and response to N-butanoyl homoserine lactone (C₄-HSL) (RhlR-RhlI) (35, 37). We have reported that an rhlI mutant unable to synthesize the C₄-HSL signal was impaired in iron limitation-induced twitching motility and formed structured biofilms under iron-limiting conditions (31).The correlation between twitching motility, the RhlR-RhlI quorum-sensing system, and iron-regulated biofilm formation led us to hypothesize that rhamnolipids are involved in mediating this process. Rhamnolipids are surface-active amphipathic molecules composed of a hydrophobic lipid and a hydrophilic sugar moiety and compose the main constituents of the biosurfactant produced by P. aeruginosa (reviewed in reference 42). The biosurfactant is required for a form of surface motility called swarming, where it functions as a wetting agent and reduces surface tension (8, 14). Furthermore, elements constituting the biosurfactant were recently shown to modulate the swarming behavior by acting as chemotactic-like stimuli (43). Rhamnolipids are also important in maintaining biofilm structure and inducing biofilm dispersion (6, 11, 29). Their synthesis requires the expression of the rhlAB operon, which is regulated by the RhlR-RhlI quorum-sensing system (14, 25, 32) and is also induced under iron-limiting conditions (14).In this study, we test this hypothesis and demonstrate that rhamnolipid production is induced under iron-limiting conditions and that this promotes twitching motility. We found that increased expression of rhamnolipid synthesis genes during early biofilm development under iron-limiting conditions induces surface motility and results in formation of a thin flat biofilm. Furthermore, a mutant that is incapable of synthesizing rhamnolipids does not display twitching motility under iron-limiting conditions and thus forms structured biofilms under these conditions. These results highlight the importance of biosurfactant production in determining the architecture of mature P. aeruginosa biofilms under iron-limiting conditions.