Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, ⌬nirS) did not disperse, whereas a NO reductase mutant (⌬norCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

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Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

Bacterial biofilms are highly dynamic communities which display a range of differentiated phenotypes during the course of development. By exchange of cell-cell signals, subpopulations of cells can coordinate their activity and undertake particular metabolic tasks or defense strategies (56). At times, the bacterial community releases single cells that escape from the biofilm and revert to a free-swimming, planktonic mode of growth, leaving behind hollow voids in the biofilm architecture (5, 37, 57). This process, referred to as dispersal, completes the biofilm life cycle and is thought to be important for successful colonization of new surfaces. Although the mechanisms underlying these events remain to be fully elucidated, previous studies of various species, including the opportunistic pathogen Pseudomonas aeruginosa, have revealed that dispersal events correlate with the induction of a specific phenotype that involves cellular motility (37, 42).In P. aeruginosa, biofilm dispersal can be triggered by environmental factors, including nutrient (42, 45) and iron (4, 36) availability, and has recently been linked to the intracellular second messenger cyclic di-GMP (c-di-GMP) (45, 47). Numerous studies revealed that decreased c-di-GMP levels are related to a motile mode of growth and to cell dispersal in eubacteria. In this second messenger system, diguanylate cyclases (DGCs) and specific phosphodiesterases (PDEs) are responsible for the biosynthesis and the degradation of c-di-GMP, respectively. DGCs and PDEs contribute to a genetic network that responds to a broad range of environmental cues and/or cell-cell signals and modulate intracellular levels of c-di-GMP, which has been shown to regulate various cellular functions, including biofilm formation, virulence, and dispersal, in many bacterial species (47,(51)(52)(53). Recently, we identified the gas nitric oxide (NO) as an important factor in the regulation of dispersal in P. aeruginosa biofilms (5). Exogenous addition of nontoxic concentrations of NO, typically in the low nanomolar range, was found to stimulate motility and biofilm dispersal in P. aeruginosa. A role for anaerobic metabolism and NO in biofilm dispersal and survival was further supported by other studies of P. aeruginosa (54, 61), Staphylococcus aureus (44), and various single and multispecies biofilms (6).NO is a water-soluble, hydrophobic free radical that can freely diffuse in biological systems. At high concentrations (micromolar to millimolar range), NO

Francisella tularensis is able to survive and grow within macrophages, a trait that contributes to pathogenesis. Several genes have been identified that are important for intramacrophage survival, including mglA and iglC. F. tularensis is also able to survive within amoebae. It is shown here that F. tularensis mglA and iglC mutant strains are not only defective for survival and replication within the macrophage-like cell line J774, but also within Acanthamoebae castellanii. Moreover, these strains are highly attenuated for virulence in mice, suggesting that a common mechanism underlies intramacrophage and intraamoebae survival and virulence. A 2D gel analysis of cell extracts of wild-type and mglA mutant strains revealed that at least seven prominent proteins were at low levels in the mglA mutant, and one MglA-regulated protein was identified as the IglC protein. RT-PCR analysis demonstrated reduced transcription of iglC and several other known and suspected virulence genes in the mglA mutant. Thus, MglA regulates the transcription of virulence factors of F. tularensis that contribute to intramacrophage and intraamoebae survival.