Cell death in Pseudomonas aeruginosa biofilm development. (1/14)

Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.  (+info)

Prediction of charge-induced molecular alignment of biomolecules dissolved in dilute liquid-crystalline phases. (2/14)

Alignment of macromolecules in nearly neutral aqueous lyotropic liquid-crystalline media such as bicelles, commonly used in macromolecular NMR studies, can be predicted accurately by a steric obstruction model (Zweckstetter and Bax, 2000). A simple extension of this model is described that results in improved predictions for both the alignment orientation and magnitude of protein and DNA solutes in charged nematic media, such as the widely used medium of filamentous phage Pf1. The extended model approximates the electrostatic interaction between a solute and an ordered phage particle as that between the solute's surface charges and the electric field of the phage. The model is evaluated for four different proteins and a DNA oligomer. Results indicate that alignment in charged nematic media is a function not only of the solute's shape, but also of its electric multipole moments of net charge, dipole, and quadrupole. The relative importance of these terms varies greatly from one macromolecule to another, and evaluation of the experimental data indicates that these terms scale differently with ionic strength. For several of the proteins, the calculated alignment is sensitive to the precise position of the charged groups on the protein surface. This suggests that NMR alignment measurements can potentially be used to probe protein electrostatics. Inclusion of electrostatic interactions in addition to steric effects makes the extended model applicable to all liquid crystals used in biological NMR to date.  (+info)

Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage. (3/14)

Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.  (+info)

The polybasic region that follows the plant homeodomain zinc finger 1 of Pf1 is necessary and sufficient for specific phosphoinositide binding. (4/14)

The plant homeodomain (PHD) zinc finger is one of 14 known zinc-binding domains. PHD domains have been found in more than 400 eukaryotic proteins and are characterized by a Cys(4)-His-Cys(3) zinc-binding motif that spans 50-80 residues. The precise function of PHD domains is currently unknown; however, the PHD domains of the ING1 and ING2 tumor suppressors have been shown recently to bind phosphoinositides (PIs). We have recently identified a novel PHD-containing protein, Pf1, as a binding partner for the abundant and ubiquitous transcriptional corepressor mSin3A. Pf1 contains two PHD zinc fingers, PHD1 and PHD2, and functions to bridge mSin3A to the TLE1 corepressor. Here, we show that PHD1, but not PHD2, binds several monophosporylated PIs but most strongly to PI(3)P. Surprisingly, a polybasic region that follows the PHD1 is necessary for PI(3)P binding. Furthermore, this polybasic region binds specifically to PI(3)P when fused to maltose-binding protein, PHD2, or as an isolated peptide, demonstrating that it is sufficient for specific PI binding. By exchanging the polybasic regions between different PHD fingers we show that this region is a strong determinant of PI binding specificity. These findings establish the Pf1 polybasic region as a phosphoinositide-binding module and suggest that the PHD domains function down-stream of phosphoinositide signaling triggered by the interaction between polybasic regions and phosphoinositides.  (+info)

Proteomic, microarray, and signature-tagged mutagenesis analyses of anaerobic Pseudomonas aeruginosa at pH 6.5, likely representing chronic, late-stage cystic fibrosis airway conditions. (5/14)

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Solid-state NMR spectroscopy of a membrane protein in biphenyl phospholipid bicelles with the bilayer normal parallel to the magnetic field. (6/14)

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Liquid crystalline phase of G-tetrad DNA for NMR study of detergent-solubilized proteins. (7/14)

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Conformational dynamics of an intact virus: order parameters for the coat protein of Pf1 bacteriophage. (8/14)

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