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The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pill. Type IV pill are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilusmediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.

The crystallographic analysis of two crystal forms of truncated pilin from Pseudomonas aeruginosa strain K122-4 (Delta K122-4) at 1.54 and 1.8 angstrom resolution is presented. The Delta K122-4 structure is compared to other crystallographically determined type IV pilin structures and an NMR structure of Delta K122-4 pilin.

Results show that the rigid backbone scaffold of the bacterial K122-4 pilin aids in host cell receptor attachment preserving conformation of the antibody recognizing epitope as shown by (sup)15N NMR relaxation techniques.

Research demonstrates that the receptor binding peptide portion of the pilin protein of Pseudomonas aeruginosa exists in trans and cis forms in solution. Results reveal that both forms of the peptide exhibit similar affinity to the Fab fragment and maintain their beta-turn conformational integrity.

Heteronuclear multidimensional nuclear magnetic resonance spectroscopy of the secondary structure of 15N-labeled recombinant PAK 128-144(Hs145) pilin peptide revealed that both major and minor isomer species bind to receptor and antibody at the second type II beta-turn between Pro139-Lys-Gly-Cys142. Data suggest that this second turn may be sufficient for binding since only the second of the two beta turns is preserved in both pilin peptides. Results may lead to studies in the design of an effective synthetic peptide vaccine.

The kinetics underlying the formation and dissociation of a de novo designed heterodimeric coiled-coil protein consisting of D and K peptides (E/K coil) were investigated using surface plasmon resonance. The experiment, which was conducted to validate the SPR technique, revealed the existence of the linker C-nL-G-G-G where nL = norleucine at the N-terminus of the K peptide and the C-terminus of the E peptide.

Pseudomonas aeruginosa strains PAK and KB7 have differences between their adhesintopes, indicating the existence of compensatory mutations among pilin strains. Structural analysis of the pilins' adherence-binding domains showed this. The interactions between the pilin ligand and the A549 cell surface receptors depends on the conformation mediated by the disulphide bridges. The structural aspects of pilin-receptor interactions are discussed.

The binding of Pseudomonas aeruginosa pilin protein strain PAK peptide, corresponding to residues 128-144, to an Fab fragment of the cognate monoclonal antibody partially stabilizes peptide backbone regions but causes greater loss in backbone conformational entropy. Further, the binding leads to a preferential first turn over the secon turn in each isomer.

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached. [ABSTRACT FROM AUTHOR]