Lysis of the host by bacteriophage 21 requires two proteins: the pinholin S21 (forms pinholes in the cytoplasmic membrane and controls lysis timing) and the endolysin (degrades the cell wall). S21 has a dual-start motif, encoding a holin, S2168, and a weak antiholin, S2171. Both proteins have two transmembrane domains (TMD) and adopt an N-in, C-in topology. The topology of S2168 is dynamic because TMD1 is a signal-anchor-release (SAR) domain which, while initially integrated into the cytoplasmic membrane, is eventually released into the periplasm. TMD1 is dispensable because the truncated protein, S2168?TMD1, retains the holin function. Adding two positive charges to N-terminus of S2168 by an irs tag (RYIRS) prevents the release of TMD1. The irsS2168 protein not only has lost its holin function, but is a potent antiholin and blocks the function of S2168. In this dissertation, the structure of S2168 was suggested by incorporating electron-microscopy, biochemical, and computational approaches. The results suggest that S2168 forms a symmetric heptamer, with the hydrophilic side of TMD2 lining the channel of ~ 15 A in diameter. This model also identifies two interacting surfaces, A and B, of TMD2. A model for the pinhole formation pathway was generated from analyzing phenotypes of an extensive collection of S21 mutants. In this model, the individually folded and inserted S21 molecules first form the inactive dimer, with the membrane-inserted TMD1 inhibiting the lethal function of TMD2 both inter- and intra-molecularly. A second inactive dimer may form, with one TMD1 released. When both TMD1s are released, the activated dimer is formed, with the homotypic interfaces A:A interaction of the TMD2s. However, this interaction might not be stable, which will shift to heterotypic A:B interactions, allowing TMD2 to oligomerize. Finally, the pinhole forms, possibly driven by the hydration of lumenal hydrophilic residues. In addition, the localization of pinholes was visualized by fusing the green fluorescent protein (GFP) to the C-terminus of pinholins. The results showed that pinholins form numerous small aggregates, designated as rafts, spread all over the cell body. The antiholin irsS2168 not only inhibits the triggering of S2168GFP, but inhibits the rafts formation as well.

Bacteriophage holins are a diverse group of proteins that are responsible for the spontaneous and specifically-timed triggering of host cell lysis. The best-studied holin, S105 of phage lambda, is known to form lesions, or “holes”, in the inner membrane of E. coli which are large enough to allow the endolysin through to the periplasm. S105 has been studied extensively by both genetic and biochemical approaches; however, little is known about the mechanism of hole formation or the structure of the lambda holin and its inner membrane lesion. An in vitro system for reconstituting hole formation by S105 was developed in which liposomes containing a self-quenched fluorophore served as artificial cell membranes (1-2). Upon delivery of solubilized S105 to the liposomes, an increase in fluorescence was observed, indicating that the fluorophore within the liposomes had escaped into the surrounding media via an S105-mediated hole in the membrane. This in vitro system, which has been optimized in this work, has been a valuable biochemical tool for analysis and reconstitution of the pathway to S105 hole formation in the cell membrane. Due to the difficulty associated with over-expression and purification of toxic membrane proteins, there are no solved structures of bacteriophage holins. Sample preparation and experimental conditions for NMR spectroscopy were optimized and structural information about a lambda holin mutant protein was obtained. Specifically, micellar contacts of transmembrane domain regions versus water contacts of the C-terminal region, secondary structure, and backbone dynamics were determined. Cryo-electron microscopy was used to visualize the inner membrane lesions formed by phage holins [lambda] S105, P2 Y, and T4 T. Therefore, the large holes initially seen in cells expressing S105 are not specific to the lambda holin, nor to class I holins. The S105 holes average ~340 nm (3), and are the largest membrane lesions ever observed in biology. They are stable at their original size, and are not localized to a specific region of the membrane. In addition, missense mutants of S105 were used to correlate hole size, protein accumulation, and lysis timing in a current model for the S105 hole formation pathway.

Thesis (Ph. D.)--University of California, San Diego, 2002. / Vita. Includes bibliographical references (leaves 146-152).

No description available.

Microviruses (canonical members: øX174, G4, and alpha3) are T=1 icosahedral virions with a two scaffolding protein-mediated assembly pathway. The external scaffolding protein D mainly mediates the assembly of coat protein pentamers into procapsids. The results of previous genetic studies suggest that helix 1 of D protein may act as a substrate specificity domain, mediating the initial coat-scaffolding protein recognition in a species-specific manner. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were initially constructed in a plasmid, which over-expresses the protein, between the closely related phages G4 and øX174. The results of biochemical and genetic analyses identify coat-scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.However, protein concentrations higher than those found in typical infections could drive reactions that may not occur under physiological conditions. In order to elucidate a more detailed mechanistic model, the same chimeric external scaffolding gene was placed directly in the øX174 genome, and the chimeric virus was characterized. The results of the genetic and biochemical analyses indicate that helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production.Finally, by using improved protocols, two novel early assembly intermediates, the 9S* and 12S* particles, have been isolated and characterized. The 9S* particle consists of a coat protein pentamer associated with the internal scaffolding protein. The 12S* intermediate is a complex of a 9S* particle with the major spike protein, and the DNA pilot protein. The existence of internal scaffolding and DNA pilot proteins that were absent in previously characterized intermediates suggest that 9S* and 12S* particles are biologically active intermediates. Moreover, preliminary in vitro assembly experiments performed with the 12S* particle and exogenous external scaffolding protein yield empty capsids-like particle, strongly suggesting that these are the physiologically relevant intermediates.

Tectiviridae šeimos bakteriofagas PRD1, infekuojantis dauginiu atsparumu antibiotikams pasižyminčias gramneigiamąsias bakterijas, patekimui į ląstelę naudoja viriono viduje esančią membraną. Manoma, kad šio bakterijų viruso membrana patekimo metu suliejama su infekuojamų ląstelių plazmine membrana. Deja, tiesiogiai įrodyti, kad faginė membrana susilieja su infekuojamų bakterijų plazmine membrana yra sudėtinga. Registruodami fago sukeliamas TPP+ ir K+ sroves bei tirdami membranose aktyvių antibiotikų poveikį membranos įtampai ir kalio jonų gradientui pabandėme ištirti Salmonella enterica ląstelių plazminės ir išorinės membranų laidumo pokyčius pradiniame infekcijos etape ir nustatyti infekcijos terpės fizikocheminių savybių įtaką bakteriofago PRD1 sąveikai su S. enterica ląstelėmis. Bakteriofagas PRD1, infekuodamas Salmonella enterica ląsteles, didina išorinės membranos laidumą, bet nedepoliarizuoja jų plazminės membranos. 300 OsmM ir didesnis infekavimo terpės osmosinis slėgis bei 22 oC ir žemesnė temperatūra slopina bakteriofago PRD1 poveikį Salmonella enterica ląstelių apvalkalėlio laidumui. Skirtingai nuo giminingo bakteriofago Bam35 infekcijos poreikių, dvivalenčiai katijonai nėra reikalingi fago PRD1 sąveikai su S. enterica ląstelių apvalkalėliu pradiniuose infekcijos etapuose. Dvivalenčių katijonų kompleksonai net skatina membranas laidinantį fago PRD1 poveikį, o Ca2+ ir Mg2+ jonai šiek tiek slopina plazminės membranos laidinimą. Arsenatas slopina PRD1 poveikį... [toliau žr. visą tekstą] / Bacteriophage PRD1 from Tectiviridae family uses its internal membrane to enter the host multiple resistant to antibiotics Gram-negative bacteria. Some indirect evidences indicate that during the entry phage membrane fuses with the plasma membrane of bacterial cell. However, it is very complicated to get the direct evidences or indications of the membrane fusion in this system. Monitoring the phage-induced fluxes of TPP+ and K+ ions and studying the influence of membrane-active antibiotics on membrane voltage and potassium gradient we were investigating PRD1-induced changes in the permeability of the outer and the plasma membranes of Salmonella enterica cells and registering the influence of physicochemical parameters of infection medium on the efficiency of phage-cell interaction. We have shown that phage PRD1 increases permeability of the outer membrane but does not depolarize the plasma membrane during the entry. 300 OsmM and higher osmotic pressure in the infection medium and temperature lower than 22 oC blocks the phage induced changes in bacterial envelope. Differently from the relative bacteriophage Bam35, divalent cations are not needed for PRD1 entry. Complexones of divalent cations EDTA and EGTA stimulate, but Ca2+ ir Mg2+ slightly inhibit the plasma membrane-permeabilizing activity of PRD1. Arsenate inhibits PRD1 induced changes of the cell envelope and the reduced intracellular ATP content could be the reason. Bacteriophage-induced lysis of S. enterica cells is... [to full text]

No description available.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

Resonant microcantilevers are promising transducers for bacteria detection because of their high sensitivities. Surface stress and mass from adsorbates affect the resonant frequency. We developed a novel method for decoupling the frequency contributions of a change in mass and surface stress on a cantilever sensor validated in theoretical, finite element and experimental framework. Bacteria capture was achieved by several different chemical immobilization of T4 phages. The most successful bacteria capturing surface produced bacterial densities of about 11 bacteria/100^m2. The developed theory is then applied to determine captured bacterial mass on the cantilevers. This provides an estimate of the bacteria mass on the cantilever. Two different functionalizations resulted in predicted bacterial densities of 5 bacteria/100^m2 and 3 bacteria/100^m2. Poor densities relative to surface capture experiments is caused by the boundary effects of the cantilever in solution. / Microelectromechanical Systems and Nanosystems