Pseudomonas aeruginosa is an opportunistic human pathogen that is especially adept at forming surface-associated biofilms. P. aeruginosa causes catheter-associated urinary tract infections (CAUTIs) through biofilm formation on the surface of indwelling catheters. P. aeruginosa encodes three extracellular polysaccharides, PEL, PSL, and alginate, and utilizes the PEL and PSL polysaccharides to form biofilms in vitro; however, the requirement of these polysaccharides during in vivo infections is not well understood. Here we show in a murine model of CAUTI that PAO1, a strain harboring pel, psl, and alg genes, and PA14, a strain harboring pel and alg genes, form biofilms on the implanted catheters. To determine the requirement of exopolysaccharide during in vivo biofilm infections, we tested isogenic mutants lacking the pel, psl, and alg operons and showed that PA14 mutants lacking these operons can successfully form biofilms on catheters in the CAUTI model. To determine the host factor(s) that induces the ΔpelD mutant to form biofilm, we tested mouse, human, and artificial urine and show that urine can induce biofilm formation by the PA14 ΔpelD mutant. By testing the major constituents of urine, we show that urea can induce a pel-, psl-, and alg-independent biofilm. These pel-, psl-, and alg-independent biofilms are mediated by the release of extracellular DNA. Treatment of biofilms formed in urea with DNase I reduced the biofilm, indicating that extracellular DNA supports biofilm formation. Our results indicate that the opportunistic pathogen P. aeruginosa utilizes a distinct program to form biofilms that are independent of exopolysaccharides during CAUTI.

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttlemediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act as oxidants to balance the intracellular redox state of cells in anoxic biofilm subzones. PA14 colony biofilms show a profound morphogenic response to phenazines resulting from electron acceptor-dependent inhibition of ECM production. This effect is reminiscent of the developmental responses of some eukaryotic systems to redox control, but for bacterial systems its mechanistic basis has not been well defined. Here, we identify the regulatory protein RmcA and show that it links redox conditions to PA14 colony morphogenesis by modulating levels of bis-(3′,5′)-cyclic-dimeric-guanosine (c-di-GMP), a second messenger that stimulates matrix production, in response to phenazine availability. RmcA contains four Per-Arnt-Sim (PAS) domains and domains with the potential to catalyze the synthesis and degradation of c-di-GMP. Our results suggest that phenazine production modulates RmcA activity such that the protein degrades c-di-GMP and thereby inhibits matrix production during oxidizing conditions. RmcA thus forms a mechanistic link between cellular redox sensing and community morphogenesis analogous to the functions performed by PAS-domain-containing regulatory proteins found in complex eukaryotes.W hen microbial cells cannot import or are physically separated from metabolic electron donors or acceptors, diffusible compounds can act as electron carriers and support survival on these substrates (1, 2). These conditions arise in the presence of poised-potential electrodes or insoluble minerals, such as iron oxides (3-5), and in multicellular communities (biofilms) where the formation of chemical gradients leads to oxidant limitation for cells at depth (6-9). Diverse microbes secrete redoxactive compounds with the capacity to function as electron shuttles (10-12). In the pathogenic bacterium Pseudomonas aeruginosa PA14, electron-shuttling antibiotics called phenazines support survival on poised-potential electrodes and balance the intracellular redox state of cells in anoxic biofilm subzones (1, 9).Similar to those formed by many species of microbes, colonies of PA14 develop intricate wrinkle structures on agar-solidified growth media (13). Phenazines profoundly alter PA14 colony morphogenesis, inhibiting the onset of wrinkle formation and changing the organization of wrinkles (12) (Fig. 1A). Modeling of resource availability within colonies suggests that the earlier increase in the colony surface area-to-volume ratio in phenazine-null (Δphz) mutants maximizes access to oxygen for cells that would otherwise become limited for oxidant (14). Measurements of...

Summary Three pathogenic forms, or formae speciales, of Fusarium oxysporum infect the roots of Arabidopsis thaliana belowground, instigating symptoms of wilt disease in leaves aboveground. In prior reports, Arabidopsis mutants that are deficient in the biosynthesis of abscisic acid or salicylic acid or insensitive to ethylene or jasmonates exhibit more or less wilt disease than wild type, implicating the involvement of hormones in the normal host response to F. oxysporum. Our analysis of hormone-related mutants finds no evidence that endogenous hormones contribute to infection in roots. Mutants that are deficient in abscisic acid and insensitive to ethylene have no less infection than wild type, though they exhibit less disease. Whether a mutant that is insensitive to jasmonates affects infection depends on which forma specialis is infecting roots. Insensitivity to jasmonates suppresses infection by F. oxysporum forma specialis conglutinans and F. oxysporum forma specialis matthioli, which produce isoleucine- and leucine-conjugated jasmonate (JA-Ile/Leu) in culture filtrates; whereas, insensitivity to jasmonates has no effect on infection by F. oxysporum forma specialis raphani, which produces no detectable JA-Ile/Leu. Furthermore, insensitivity to jasmonates has no effect on wilt disease of tomato, and the tomato pathogen F. oxysporum forma specialis lycopersici produces no detectable jasmonates. Thus, some but not all F. oxysporum pathogens appear to utilize jasmonates as effectors, promoting infection in roots and/or development of symptoms in shoots. Only when infection of roots is promoted by jasmonates is wilt disease enhanced in a mutant deficient in salicylic acid biosynthesis.

SummaryResistance to wilt fungus Fusarium oxysporum f.sp. matthioli (FOM) is a polygenic trait in Arabidopsis thaliana. RFO3 is one of six quantitative trait loci accounting for the complete resistance of accession Columbia-0 (Col-0) and susceptibility of accession Taynuilt-0 (Ty-0).We find that Col-0 and Ty-0 alleles of RFO3 are representative of two common variants in wild Arabidopsis accessions, that resistance and susceptibility to FOM are ancestral features of the two variants and that resistance from RFO3 is unrivalled by other genes in a genome-wide survey of diversity in accessions. A single receptor-like kinase (RLK) gene in Col-0 is responsible for the resistance of RFO3, although the susceptible Ty-0 allele codes for two RLK homologs.Expression of RFO3 is highest in vascular tissue, which F. oxysporum infects, and rootexpressed RFO3 restricts FOM infection of the vascular system. RFO3 confers specific resistance to FOM and provides no resistance to two other crucifer-infecting F. oxysporum pathogens.RFO3's identity, expression and specificity suggest that RFO3 represents diversity in pattern-recognition receptor (PRR) genes. The characteristics of RFO3 and the previously published RFO1 suggest that diversity in RLK PRRs is a major determinant of quantitative resistance in wild plant populations.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals.