Biofilms are adherent aggregates of bacterial cells that form on biotic and abiotic surfaces, including human tissues. Biofilms resist antibiotic treatment and contribute to bacterial persistence in chronic infections. Hence, the elucidation of the mechanisms by which biofilms are formed may assist in the treatment of chronic infections, such as Pseudomonas aeruginosa in the airways of patients with cystic fibrosis. Here we show that subinhibitory concentrations of aminoglycoside antibiotics induce biofilm formation in P. aeruginosa and Escherichia coli. In P. aeruginosa, a gene, which we designated aminoglycoside response regulator (arr), was essential for this induction and contributed to biofilm-specific aminoglycoside resistance. The arr gene is predicted to encode an inner-membrane phosphodiesterase whose substrate is cyclic di-guanosine monophosphate (c-di-GMP)-a bacterial second messenger that regulates cell surface adhesiveness. We found that membranes from arr mutants had diminished c-di-GMP phosphodiesterase activity, and P. aeruginosa cells with a mutation changing a predicted catalytic residue of Arr were defective in their biofilm response to tobramycin. Furthermore, tobramycin-inducible biofilm formation was inhibited by exogenous GTP, which is known to inhibit c-di-GMP phosphodiesterase activity. Our results demonstrate that biofilm formation can be a specific, defensive reaction to the presence of antibiotics, and indicate that the molecular basis of this response includes alterations in the level of c-di-GMP.

Using UV, CD, and NMR, we demonstrate that the important bacterial signaling molecule involved in biofilm formation, cyclic diguanosine monophosphate (c-di-GMP), exists as a mixture of five different but related structures in an equilibrium that is sensitive both to its concentration and to the metal present. At the lower concentrations used for UV and CD work (0.05 -0.5 mM) Li + , Na + , Cs + , and Mg 2+ favor a bimolecular self-intercalated structure, while K + , Rb + , and NH 4 + favor formation of one or more guanine quartet complexes as well. At the higher NMR concentrations (∼30 mM), the bimolecular structures associate and rearrange to a mixture of all-syn and all-anti tetramolecular and octamolecular quartet complexes. With K + the octamolecular complexes predominate, while with Li + the tetramolecular and octamolecular quartet complexes are present in approximately equal amounts, along with the bimolecular structure. We also find that both guanine amino groups in c-di-GMP are essential for formation of the quartets, since substitution of inosine for one guanosine allows formation of only the bimolecular structure. Further, two molecules of cdi-GMP tethered together are constrained in such a way that limits their ability to form these quartet complexes. The polymorphism we describe may provide different options for this signaling molecule when performing its functions in a bacterial cell, with K + and its own local concentration controlling the equilibrium.

SummarySignature-tagged transposon mutagenesis of Salmonella with differential recovery from wild-type and immunodeficient mice revealed that the gene here named cdgR [for c-diguanylate (c-diGMP) regulator ] is required for the bacterium to resist host phagocyte oxidase in vivo . CdgR consists solely of a glutamatealanine-leucine (EAL) domain, a predicted cyclic diGMP (c-diGMP) phosphodiesterase. Disruption of cdgR decreased bacterial resistance to hydrogen peroxide and accelerated bacterial killing of macrophages. An ultrasensitive assay revealed c-diGMP in wild-type Salmonella with increased levels in the CdgR-deficient mutant. Thus, besides its known role in regulating cellulose synthesis and biofilm formation, bacterial c-diGMP also regulates host-pathogen interactions involving antioxidant defence and cytotoxicity.

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