Azeredo, Joana

Oliveira, A.

Sereno, R.

English

Universidade do Minho: RepositoriUM

77W O R K S H O P S76W O R K S H O P S10. Emerging features of bacteriophage biology Thursday 6W10.5 - P.VIR.23DnaA and SeqA: regulators of Escherichia coli chromosomereplication that act also as transcription factors and controlbacteriophage λ DNA replication by modulation of transcriptionat the oriλ regionWegrzyn G1, Narajczyk M1, Baranska S1, Szambowska A1, Lyzen R1,Szalewska-Palasz A1, Glinkowska M1 and Wegrzyn A21 Department of Molecular Biology, University of Gdansk, Kladki 24,80-822 Gdansk, Poland2 Laboratory of Molecular Biology (affiliated with University ofGdansk), Institute of Biochemistry and Biophysics, Polish Academy ofSciences, Kladki 24, 80-822 Gdansk, PolandDnaA and SeqA proteins are major positive and negative regulators ofthe replication initiation of Escherichia coli chromosome, respectively.These proteins are also specific transcription factors. They stimulatetranscription from bacteriophage λ pR promoter by bindingdownstream of the promoter and affecting late stages of transcriptioninitiation. There is a specific interplay between DnaA and SeqA at thepR promoter region. Since pR activity is necessary for transcriptionalactivation of the origin of bacteriophage λ DNA replication, DnaA andSeqA influence this process significantly. They affect frequency ofreplication initiation events, directionality of λ DNA replication andthe switch from early  to late mode of the phage genome replication.Involvement of DnaA and SeqA in the regulation of λ DNA replicationis an example how a parasite (bacteriophage λ ) can employ host (E.coli) proteins to a process (transcription) that is different from theirprimary destination in the host cells (replication control), but whichfinally leads to regulation of the same biochemical reaction (DNAreplication).W10.6 - P.VIR.24Triggering of phage SPP1 DNA ejection by the Bacillus subtilisreceptor YueB: the pressure of the virion capsid as DNA ejectionforceSão-José C (1), Lhuillier S (2), Lurz R (3), Melki R (4), Lepault J (2),Santos MA (1) and Tavares P (2)(1) Instituto de Ciência Aplicada e Tecnologia, Faculdade de Ciênciasda Universidade de Lisboa, Lisboa, Portugal.(2) Unité de Virologie Moléculaire et Structurale, CNRS, Gif-sur-Yvette, France.(3) Max-Planck-Institut für Molekulare Genetik, Ihnestr, Berlin,Germany.(4) Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, Francecjsjose@fc.ul.ptInfection of B. subtilis by phage SPP1 starts with the irreversibleadsorption of the virion to the cell membrane receptor YueB (J.Bacteriol. 2004, 186: 8337-8346). We have purified the extracellulardomain of YueB and found that it formed a fiber-like, elongated dimerthat is long enough to span the bacterial cell wall and be exposed at thecellular surface. This fiber had the ability to trigger SPP1 genomeejection in vitro, in a temperature-dependent way (J. Biol. Chem. 2006,in press). As described in other systems we have shown that thepressure built in the virion capsid acts as phage DNA ejection force.When phage/receptor interaction was studied in the presence ofdifferent concentrations of PEG6000, which provides an externalosmotic pressure that opposes the forces carried in the capsid, we couldtrap DNA ejection at different intermediate states. The length of ejectedDNA was dependent on the external osmotic pressure. This workcorresponds to the first purified receptor from a Gram-positivebacterium that is active to trigger phage DNA ejection in vitro.10. Emerging features of bacteriophage biology Thursday 6W10.1 Phages in the 21st centuryAriane Toussaint, Raphaël Leplae, Gipsi Lima-Mendez.Service de Conformation des Macromolécules Biologiques et deBioinformatique, Université Libre de Bruxelles, Belgium.ariane@scmbb.ulb.ac.beAfter several decades of languishing as tools for molecular biology,phages regained the interest they deserve because of their diversity,their role in bacterial evolution and dynamics, emergence of newpathogens and even biological cycles. Health threats raised by multi-resistant bacterial pathogens induced a renewed attention towardsphage therapy. Phages are now appropriately considered as“nanomachines” and their components serve as new drugs, molecularmachines, diagnostic tools and therapeutic agents. Nevertheless, ourappraisal of the phage sequence space remains minimal. New sequenceinformation fuels a recurrent debate on the need to revise phagetaxonomy; routes have been proposed but none has yet receivedgeneral approval. Classical genetic, biochemical and structural analysisgoes on, more and more sophisticated, on very few model phages. Highthroughput methodologies are not commonly used, maybe because notappropriate to phages in their present settings. Widely divergentterminologies adopted for different phages limit comprehensivecomputer analysis of phage sequences, genome organization,regulatory networks and system biology. I shall illustrate howstructuring information on phage proteins can be exploited for globalanalysis of phage genome organization and relationships. Obviously, we have only scratched the fantastic reservoir that thephage world offers for many fundamental biological processes andbiotechnological applications. W10.2 - PVIR.21Characterisation and molecular phylogeny of small ø29-likephages infecting solvent-producing clostridiaJones D. T. and Asano K.Department of Microbiology, University of Otago, Dunedin,New Zealand.david.jones@stonebow.otago.ac.nzBacteriophage infections caused major problems in the commercialAcetone Butanol fermentation process and the various industrialclostridial species utilised were all reported to be susceptible phageattack. However, the majority of phages were never characterised.Small Ø29-like phages belonging to the genus Podoviridae infectingClostridium beijernickii and Clostridium saccharoperbutylacetonicumwere isolated from infections occurring in the industrial process inJapan and South Africa.  The 2 phages CA1 and HM2  exhibit similarmorphologies and their genomes consist of linear double strandedDNA of 17.1 kb and 17.7 kb that utilise protein-primed replicationprocesses. The genomic nucleotide sequences of these phages,encoding 20 and 25 putative ORF’s respectively, were characterisedand compared with the genomic sequences of similar phages infectingbacteria belonging to the genus Bacillus, Staphylococcus andStreptococcus. Conserved genomic structures and nucleotidesequences suggest that these phages evolved from a common ancestor.An analysis was undertaken of possible phylogenetic relationships ofthis group of phages that have amongst the smallest viral genomesknown.W10.3 - P.VIR.22Dynamic of two coliphages in chicken organism towards phagetherapy Oliveira, A (1,2), Azeredo, J (2) and Sereno, R (1)(1)Controlvet, Lda., Tondela, Portugal(2)CEB, Universidade do Minho, 470-057 Braga, Portugalanaoliveira@deb.uminho.ptE.coli can cause severe respiratory and sistemic infections in chickens,responsible for significant economic losses in the poultry industry.Phages are good candidates to treat bacterial infections as alternative toantibiotics. In order to predict the efficacy of phage therapy, it isimportant to know its dissemination in the animal body. In vivo trialswere conducted by infecting chickens with two lytic phages: F258E(Syphoviridae) and F61E (Myoviridae). 106, 107 and 108pfu wereadministered orally, by spray and intramuscularly. Birds wereeuthanised after 3, 10 and 24h and pfu were measured from thesupernatants of emulsified lungs and air sac membranes (LS), liver,duodenum and spleen. Both phages were recovered from LS after 10hwhen administrated orally with 107 phages. Conversely, when phagesadministration was made by spray, it was already possible to recoverboth phages from LS after 3h, indicating that it reach these tissuesfaster when inhalated. After 10 and 24h of challenging by spray andorally, respectively, only phage F258E was found in these tissues,suggesting that the residence times of each of the phages in the animalbody is different. Both phages were found in the animals duodena after3h of challenging by spray. The phages only reached the other organswhen the administration route was intramuscular. The results suggestthat to treat respiratory E.coli infections, nasal and oral are promissingadministration routes.W10.4 Spo0A, the master regulator for the initiation of Bacillus subtilissporulation, suppresses phage ø29 development at the level oftranscription and replicationSalas M1, Castilla-Llorente V1, Muñoz-Espin D1, Villar L1,Errington J2 and Meijer WJJ11Centro de Biología Molecular “Severo Ochoa” (CSIC–UAM),Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain2Sir William Dunn School of Pathology, University of Oxford,Oxford, UK.msalas@cbm.uam.esPhage ø29 is a virulent phage that infects Bacillus subtilis vegetativecells. The lytic cycle is suppressed when the infected cells initiate theprocess of sporulation and the infecting phage genome is trapped intothe highly resistant spore to remain dormant until germination of thespore occurs. Two host-encoded proteins are key players in thisadaptive infection strategy. On the one hand, the chromosomesegregation protein Spo0J is involved in spore entrapment of the ø29genome. On the other hand, Spo0A, the master regulator for initiationof sporulation, suppresses ø29 development by repressing the mainearly ø29 promoters through different mechanisms and also bypreventing activation of the single late ø29 promoter. In addition,Spo0A inhibits ø29 DNA replication. Binding of Spo0A near the ø29DNA ends, that constitute the two origins of replication of the linearø29 genome, prevents formation of the ø29 protein p6-nucleoproteininitiation complex, resulting in inhibition of the initiation of ø29 DNAreplication.

Dynamic of two coliphages in chicken organism towards phage therapy

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Dynamic of two coliphages in chicken organism towards phage therapy

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