Gotowa bibliografia na temat „Gifsy-2”

ABSTRACT Mutations in the Salmonella enterica serovar Typhimurium ompC gene conferred resistance to Gifsy-1 and Gifsy-2 bacteriophages. Selection for complementing plasmids yielded clones of ompC. Introduction of anompC clone into Escherichia coliconferred the ability to adsorb Gifsy phage. These data show that OmpC is the receptor for Gifsy-1 and Gifsy-2 phages.

ABSTRACT A class of mutations that suppress the recombination defects of recB mutants in Salmonella enterica serovar Typhimurium strain LT2 activates the normally silent recET module of the Gifsy-1 prophage. Allele sbcE21 is a 794-bp deletion within the immunity region of the prophage. Concomitant with activating recET, sbcE21 stimulates Gifsy-1 excision, resulting in unstable suppression. Early studies found both recB suppression and its instability to depend on the presence of the related Gifsy-2 prophage elsewhere in the chromosome. In cells lacking Gifsy-2, the sbcE21 allele became stable but no longer corrected recB defects. Here, we show that a single Gifsy-2 gene is required for Gifsy-1 recET activation in the sbcE21 background. This gene encodes GtgR, the Gifsy-2 repressor. Significantly, the sbcE21 deletion has one end point within the corresponding gene in the Gifsy-1 genome, gogR, which in strain LT2 is a perfect duplicate of gtgR. The deletion truncates gogR and places the Gifsy-1 left operon, including the recET and xis genes, under the control of the gogR promoter. The ability of GtgR to trans-activate this promoter therefore implies that GtgR and GogR normally activate the transcription of their own genes. Consistent with the symmetry of the system, a similar deletion in Gifsy-2 results in a Gifsy-1-dependent sbc phenotype (sbcF24). Two additional Gifsy-1 deletions (sbcE23 and sbcE25) were characterized, as well. The latter causes all but the last codon of the gogR gene to fuse, in frame, to the second half of recE. The resulting hybrid protein appears to function as both a transcriptional regulator and a recombination enzyme.

ABSTRACT The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.

ABSTRACT The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.

ABSTRACT The lambdoid phage Gifsy-2 contributes significantly toSalmonella enterica serovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene,sodCI, and other unidentified virulence factors. We have characterized the gene grvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes,grvA null mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-type grvA in single copy complemented this phenotype. However, grvA +on a multicopy plasmid also conferred the antivirulence phenotype. Neither a grvA null mutation nor thegrvA + plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by the grvA null mutation and thegrvA + plasmid required wild-typesodCI but was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such assodCI and antivirulence factors such asgrvA.

ABSTRACT The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.

ABSTRACT The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind−) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.

ABSTRACT Peptide inhibitors of phage lambda site-specific recombination were previously isolated by screening synthetic combinatorial peptide libraries. These inhibitors cause the accumulation of complexes between the recombinase and the Holliday junction intermediate of several highly divergent tyrosine recombinases. Peptide WRWYCR and its d-amino acid derivative bind to the center of protein-free junctions and prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the peptides also bind to branched DNA molecules that mimic replication forks. The peptides are bactericidal to both gram-positive and gram-negative bacteria, presumably because they can interfere with DNA repair and with chromosome dimer resolution by the XerC and XerD tyrosine recombinases. In order to test the correspondence between their mechanism in vivo and in vitro, we have tested and shown peptide wrwycr's ability to inhibit the excision of several prophages (lambda, P22, Gifsy-1, Gifsy-2, Fels-1, Fels-2) and to trap Holliday junction intermediates of phage lambda site-specific recombination in vivo. In addition, we found that the peptide inhibits replication of the Salmonella prophage Fels-1 while integrated in the chromosome. These findings further support the proposed mechanistic basis for the antimicrobial activity of the peptide and its use as a tool to dissect strand exchange-dependent DNA repair within cells.

ABSTRACT Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Les génomes de bactériophages sont connus pour être mosaïques. Avec l'augmentation des structures disponibles sur ces virus, leur morphologie aussi apparaît de plus en plus comme telle. Les structures de 4 différents intermédiaires dans l'assemblage de la capside icosaédrique du bactériophage Gifsy-2, infectant les bactéries Salmonella ont été résolues par cryo-microscopie électronique jusqu'à une résolution de 12Å. Au niveau génétique, un long gène code en partie Cterminale pour la protéine de capside majeure alors que sa partie Nterminale est fortement similaire du point de vue de sa séquence primaire aux protéases de la famille Clp. Ces résultats et ceux obtenus sur d'autres phages suggèrent que les 200 résidus en partie centrale regroupent les fonctions restantes, nécessaire à l'assemblage de la capside. Cette organisation en un gène unique est une nouvelle variante des stratégies développées par les phages pour modifier la voie d'assemblage de leur capside. Nous avons aussi déterminé les structures de la capside et de la queue du phage T5 , infectant la bactérie E. Coli à des résolutions de 19 et 30 Å respectivement confirmant l'inhabituelle symétrie d'ordre 3 de sa queue. Sa capside icosaédrique est composée par des pentamères et hexamères de pb8 et par pb10 s'attachant au centre de ces derniers, le tout étant décrit par une géométrie T=13. La comparaison des séquences primaires de pb8 et gp5 d'HK97 et la modélisation du modèle atomique de gp5 dans la capside de T5 montrent que ces 2 protéines adoptent sûrement le même repliement 3D. Nous proposons aussi que le domaine Nterminal de la protéine pb8 soit impliqué dans l'assemblage initial de la capside d'une manière similaire à HK97
Bacteriophage genomes are distinctly mosaic, and as more structures are solved, the phage morphologies also demonstrate a mosaic pattern. In a first project, we have solved capsid structures for several maturation intermediates of Gifsy-2, a phage infecting the bacteria Salmonella, by cryo-electron microscopy to resolutions up to 12 Ångstroms. On the genetic level we observe a long gene encoding the mature gifsy-2 capsid protein at the C-terminal end, and find striking homology with the Clp-P protease at the N-terminus including alignment of the catalytic triad. By analogy with related phage genomes the central domain encoding ~200 amino acids is expected to have scaffolding and/or decoration functions. This unique gene structure gives rise to a capsid maturation pathway that engages features from phages ? and HK97. In a second project, we have determined the E. Coli phage T5 structures of the icosahedral capsid to 19 Ångstroms resolution and the helical tail to ~30Å. We confirm the unusual trimeric symmetry of the tail tube. The capsid is composed of pentamers and hexamers of pb8 arranged with triangulation number t=13 and we localize the pb10 decoration protein to the exterior surface of the hexamers. Sequence comparison between pb8 and the HK97 capsid protein, together with modeling of the T5 capsid with HK97 atomic model, suggests that the T5 capsid likely adopts the same capsid protein fold as its smaller sibling, differing in size by the inclusion of additional hexamers. We propose that the precursor N-terminal domain of pb8 is involved in initial capsid assembly, as for HK97, specifically directing construction of the appropriately sized capsid

Les génomes de bactériophages sont connus pour être mosaïques. Avec l'augmentation des structures disponibles sur ces virus, leur morphologie aussi apparaît de plus en plus comme telle. Les structures de 4 différents intermédiaires dans l'assemblage de la capside icosaédrique du bactériophage Gifsy-2, infectant les bactéries Salmonella ont été résolues par cryo-microscopie électronique jusqu'à une résolution de 12Å. Au niveau génétique, un long gène code en partie Cterminale pour la protéine de capside majeure alors que sa partie Nterminale est fortement similaire du point de vue de sa séquence primaire aux protéases de la famille Clp. Ces résultats et ceux obtenus sur d'autres phages suggèrent que les 200 résidus en partie centrale regroupent les fonctions restantes, nécessaire à l'assemblage de la capside. Cette organisation en un gène unique est une nouvelle variante des stratégies développées par les phages pour modifier la voie d'assemblage de leur capside.
Nous avons aussi déterminé les structures de la capside et de la queue du phage T5 , infectant la bactérie E.Coli à des résolutions de 19 et 30 Å respectivement confirmant l'inhabituelle symétrie d'ordre 3 de sa queue. Sa capside icosaédrique est composée par des pentamères et hexamères de pb8 et par pb10 s'attachant au centre de ces derniers, le tout étant décrit par une géométrie T=13. La comparaison des séquences primaires de pb8 et gp5 d'HK97 et la modélisation du modèle atomique de gp5 dans la capside de T5 montrent que ces 2 protéines adoptent sûrement le même repliement 3D. Nous proposons aussi que le domaine Nterminal de la protéine pb8 soit impliqué dans l'assemblage initial de la capside d'une manière similaire à HK97.

Systems understanding of the biological data is required to capture the complex biochemical information when a pathogen interacts with its host. Such interactions are dynamic and complex, especially because various components of the pathogen interact with the host at multiple levels. Understanding this complex data has been made somewhat feasible with the advent of systems biology. System biology involves studying the complex interactions which take place between genes, proteins, and other components in a biological network, compared to traditional biological research, where the focus is only on a small number of components. For instance, a systems biology study of host-pathogen interactions investigates the interaction between the components of two distinct organisms, a pathogen, and its animal host. The only way this is accomplished is through the integration and interpretation of the high-throughput data made available at various levels of detail. However, high-throughput techniques like sequencing and multi-omics not only generate a massive volume of data but also pose challenges to meaningfully extract and interpret the data. Salmonella sp. is a Gram-negative, intracellular pathogen and causes severe complications contributing significantly to the global burden of foodborne illnesses. Salmonella Typhi is a human-restricted pathogen that causes a systemic disease called Typhoid (enteric fever). On the other hand, Salmonella Typhimurium, a zoonotic pathogen, causes a less severe form of the disease called gastroenteritis in humans but a systemic typhoid-like disease in susceptible mice strains (typhoid model). The genomes of most organisms, including Salmonella, as they exist today are the result of millions of years of evolution - the acquisition or elimination of certain key genetic determinants, genomic rearrangements, and the insertion of novel genes into existing genetic circuitries. Reverse genetics has proven extremely useful in deciphering the function of such key genes and their protein products required during Salmonella pathogenesis. In the first part of this study, we sought to identify proteins that are exclusive to Salmonella sp. using comparative genome sequence analysis. Comparing the proteome of Salmonella with a non-redundant protein sequence database allowed us to not only detect sequences unique to the organism but also identify its conservation among the Salmonella serovars. We argue that such proteins with a unique sequence architecture might be acquired and evolutionarily retained in the genome of the organism only to serve some essential function. By careful genetic manipulation and phenotypic analysis, we were able to shed functional insights in a novel protein sequence, GtgF and demonstrate its role in S. Typhimurium adaptation in oxidative and genotoxic stress response mechanisms. Using a mouse typhoid model, the second part of our study assessed and compared the virulence function of GtgF in WT S. Typhimurium 14028s. In a susceptible mouse strain, like BALB/c, WT S. Typhimurium 14028s administered through the orogastric route is known to cause lethal infection. However, we observed that S. Typhimurium ΔgtgF was significantly more lethal (approx. two times) than the WT in an oral typhoid model. A marginally higher bacterial burden in tissues with increased inflammatory markers in the blood, and exacerbated tissue inflammation explained why the mice were more susceptible to infection with ΔgtgF strain. In addition to grvA, we believe gtgF might be an additional anti-virulence genetic determinant encoded by the same phage, Gifsy-2. Although it is unclear why bacterial pathogens possess anti-virulence genes and what evolutionary advantage they provide to their host, it is believed that certain intracellular pathogens evolve toward a less virulent form in order to attain a chronic carrier state, a phenomenon well-established in the intracellular pathogen, Salmonella. In the third and concluding part of the study, we developed a drug-repurposing strategy to design antibiotic adjuvants that can target the MsgA-like family of proteins in Salmonella. As these proteins are mostly conserved in pathogenic organisms, targeting this family of proteins has the selective advantage of having negligible and unintentional anti-commensal activity. It is well-known that targeting conditionally essential proteins can impede the rate of anti-microbial resistance. While discovering novel drugs or even new classes of antimicrobials might be an extremely time-consuming and resource-intensive affair, orthogonal approaches such as designing antibiotic potentiators can be implemented simultaneously to preserve our existing arsenal of antimicrobials. Through high-throughput virtual screening with an FDA-approved drug subset, we identified potential drugs which can be repurposed as antibiotic adjuvants against the intended targets. The computational findings were validated to determine whether these drugs can indeed potentiate the action of the co-administered antibiotic. Some true validations obtained in this study clearly demonstrate that the proposed methodology holds promise; however, it remains to be seen whether these observations hold true for drug-resistant isolates. Consequently, the mechanism of action of the drugs must also be established. However, as a proposed methodology, these observations has significant ramifications in the antibiotic resistance problem. In summary, the entire workflow presented here is extremely generic and can be adapted for any pathogenic microorganism to tackle the rising concern of anti-microbial resistance.

Chapter 2 looks at jump scares and the fad of “screamers” found across the internet: brief videos or gifs designed to cause an unsuspecting browser to scream and jump. They appeared when jump scares were ascendant within the horror genre thanks to video games; screamers’ online popularity led to importation into the cinema through films such as the Paranormal Activity series. The chapter develops the importance of shock to the horror genre and the similarities between engagement across mediums. Shock is a challenge to viewer/browser/gamer mastery and self-control. Screamers offer the opportunity to reassert one’s own self through repetition and sharing, showing how similar processes are central to the experience of horror film viewing and gameplay. It discusses “elevated horror” and the tendency to disparage sensation in opposition to traditional virtues of narrative cinema. The chapter’s counterpoint for this assumption is with close readings of “elevated” horror films, The Witch (2015) and The Babadook (2014).

Machining process modeling requires cutter/workpiece engagement geometry in order to predict cutting forces. The calculation of these engagements is challenging due to the complicated and changing intersection geometry that occurs between the cutter and the in-process workpiece. Solid modelers can be used to perform these calculations by executing intersection operations between cutter and workpiece surfaces at successive cutter locations. These operations utilize parametric surface/surface intersection (SSI) algorithms. For the large number of engagements that can occur in machining a complicated workpiece this can be a time-consuming and sometimes unreliable process. In this paper, in-process machining features are introduced into machining process modeling for 2 1/2 D end milling, and a feature based approach is presented for addressing the computational complexity and robustness issues in the cutter/workpiece engagement calculations. Geometric Invariant (giF) and Form Invariant Machining Features (fiF) are modeled to help represent engagement conditions analytically. Volume decomposition and composition algorithms are described that extract these two types of machining features from the removal volumes generated at each tool pass. Cutter/workpiece engagements can be analytically extracted from giFs and fiFs without applying repetitive SSI operations. This paper presents one part of ongoing collaborative research into developing Virtual Machining Systems. The engagement conditions that are found are inputs to machining process models that identify cutting forces, predict stability and that optimize the process.