Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at FeCl 3 concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of FeCl 3 that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhlI, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhlI mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.Pseudomonas aeruginosa is an opportunistic human pathogen that causes debilitating infections, particularly in patients with underlying diseases, such as cystic fibrosis (24, 50), where it can cause chronic infections characterized by the formation of biofilms (24,25,42,50,59,77). These surface-associated communities of sessile bacteria embedded in a polysaccharide matrix are important features of many infectious diseases (25,42,45,59), and their characteristic resistance to antimicrobials (antibiotics and biocides) and host immune responses (4, 20, 22, 32, 57, 60, 66) compromises infection control. Details of in vivo P. aeruginosa biofilm formation, structure, and properties are limited, with most of our understanding of these structures coming from the study of model biofilms formed in vitro (3,15,29,30,56,80,81,84). In vitro biofilm development in P. aeruginosa is characterized by bacterial surface attachment, followed by microcolony formation by clonal expansion or motility-driven cell-to-cell aggregation and subsequent formation of a flat, uniform, confluent biofilm or heterogeneous, structured biofilms characterized by cell aggregates or "mushroom" structures separated by channels or ...