Biochemical, biophysical and structural characterization of Lactococcus lactis bacteriophage proteins involved in Homologous Recombination and Abortive Infection mechanisms

I batteriofagi virulenti del gruppo 936 e P335 sono predominanti nelle infezioni verso Lactococcus lactis, un batterio Gram-positivo molto diffuso nell’industria casearia. Tra i meccanismi adottati dal batterio contro l’infezione virale, ritroviamo i meccanismi abortivi antivirali (Abi), di cui poco si conosce riguardo alla loro modalità d’azione. Sotto la pressione selettiva dei sistemi Abi, la ricombinazione omologa con sequenze presenti nel cromosoma batterico (Bouchard et al 2000 et 2002, Moineau et al 1995) produce fagi mutati che sono stati ritrovati in fermentazioni ad esito negativo e perciò recanti enormi perdite economiche. Riguardo il sistema AbiK, Bouchard et al (Bouchard et al 2004) hanno identificato quattro proteine denominate Sak, a bassa similarità di sequenza, ma tutte implicate nella sensibilità verso AbiK. Tra queste proteine, Ploquin et al (Ploquin et al 2008) hanno dimostrato che la ORF252 del fago ul36 (specie P335), denominata Sak, é una “DNA Single Strand Annealing Protein (SSAP)“, omologa della proteina eucariotica RAD52. Sak3 del fago p2 (specie 936) é invece la proteina di riferimento del terzo gruppo di geni sak che non mostrano alcuna similarità di sequenza con le SSAPs. Indipendentemente da questo, le proteine Sak sono sicuramente coinvolte nel meccanismo di ricombinazione omologa poiché i loro geni sono localizzati in « recombination cassette » insieme a geni di altre proteine chiaramente implicate in questi meccanismi (Bouchard and Moineau, 2004). La prima parte dei nostri studi é stata focalizzata sulla caratterizzazione biochimica e strutturale delle due proteine Sak : Sak del fago UL36 e Sak3 del fago p2. Di queste ultime abbiamo studiato lo stato di oligomerizzazione attraverso un approccio multi-tecnica, abbiamo visualizzato le tipiche strutture ad anello (Cryo Microscopie Electronique) ed allestito test funzionali per chiarire la loro appartenenza alla famiglia delle SSAP. La loro capacità di legare il DNA é stata inoltre testata e studiata attraverso « Electrophoretic Mobility Shift Assays, EMSA », Microscopia a Forza Atomica (AFM) e « Surface Plasmon Resonance, SPR » ; la relazione con altri partners nel processo di ricombinazione omologa é stata inoltre testata tramite SPR. Nella seconda parte del nostro studio, abbiamo caratterizzato la “Single-Strand Binding protein (SSB)” del fago p2, partner di Sak3 nella ricombinazione omologa. A questo proposito sono stati studiati il livello di espressione di SSB durante l’infezione fagica, la sua capacità di legare il DNA a singolo filamento, le sue implicazioni nel meccanismo di ricombinazione omologa ed abbiamo determinato la sua struttura tridimensionale. I nostri risultati costituiscono gli elementi che permetteranno la comprensione di alcune funzioni sconosciute delle proteine Sak, in particolare del legame tra gli avvenimenti del processamento del DNA ed il sistema AbiK, dell’implicazione di altre proteine correlate come le SSBs in questi meccanismi.Virulent phages of the 936 and P335 group are predominant in infections to Lactococcus lactis, a Gram-positive bacterium widely used in the dairy industry. Among the mechanisms adopted by the bacterium against virus infection, abortive infection mechanisms (Abi) were found, but little is known about their molecular way of action. Under the selective pressure of Abi systems, homologous recombination with phage-related sequences present in the host chromosome (Bouchard et al 2000 and 2002, Moineau et al 1995) produces phage mutants which are found in unsuccessful fermentations that lead to huge economical looses. In particular for the AbiK system, Bouchard et al (Bouchard et al 2004) have identified four non similar proteins involved in the sensibility to AbiK named Sak. Among these proteins, Ploquin et al (Ploquin et al 2008) demonstrated that ORF252 of phage ul36 (species P335), renamed Sak, is a DNA Single-Strand Annealing Protein (SSAP), homologue of the eukaryotic protein RAD52. Sak3 from phage p2 (species 936) is reported to be the reference for the third group of identified sak genes that do not show significant sequence similatity to SSAPs. However, Sak proteins were suspected to be involved in homologous recombination due to their genetic localization in “recombination cassette” with other genes of proteins clearly involved in these processes (Bouchard and Moineau, 2004).The first part of our studies present the biochemical and structural characterization of two Sak proteins: the Sak of phage UL36 and the Sak3 of phage p2. In particular, multi-technique approaches were performed to investigate the oligomerization state and to visualise ring structures (Cryo Electron Microscopy), while functional tests were used to elucidate the SSAP family membership. DNA binding properties were also tested by gel mobility shift assays (EMSA), atomic force microscopy (AFM) and surface plasmon resonance (SPR), and the relationship with their partners in homologous recombination was determined. In the second part of our studies the phage p2 single-strand binding protein (SSB), a Sak3 partner in homologous recombination/replication, was studied. Expression level during phage infection, binding properties to ssDNA, involvement in homologous recombination processes and tridimensional structure were elucidated. Our studies might complete and help to understand some aspects of the Sak function leading to the definition of a linkage between DNA processing events and the antiviral system AbiK, and might elucidate the implication of correlated recombinant proteins like SSBs in these processes

UBVRI photopolarimetry of the long period eclipsing AM Herculis binary V1309

We report simultaneous UBVRI photo-polarimetric observations of the long period (7.98 h) AM Her binary V1309 Ori. The length and shape of the eclipse ingress and egress varies from night to night. We suggest this is due to the variation in the brightness of the accretion stream. By comparing the phases of circular polarization zero-crossovers with previous observations, we confirm that V1309 Ori is well synchronized, and find an upper limit of 0.002 percent for the difference between the spin and orbital periods. We model the polarimetry data using a model consisting of two cyclotron emission regions at almost diametrically opposite locations, and centered at colatitude 35 (deg) and 145 (deg) on the surface of the white dwarf. We also present archive X-ray observations which show that the negatively polarised accretion region is X-ray bright.Comment: 11 pages, 12 figures (2 colour), Fig1 and Fig 4 are in lower resolution than in original paper, accepted for publication in Monthly Notices of the Royal Astronomical Societ

Real Time Power Control in a High Voltage Power Supply for Dielectric Barrier Discharge Reactors: Implementation Strategy and Load Thermal Analysis

Atmospheric-pressure plasma treatments for industrial and biomedical applications are often performed using Dielectric Barrier Discharge reactors. Dedicated power supplies are needed to provide the high voltage frequency waveforms to operate these nonlinear and time-dependent loads. Moreover, there is a growing technical need for reliable and reproducible treatments, which require the discharge parameters to be actively controlled. In this work, we illustrate a low-cost power supply topology based on a push-pull converter. We perform experimental measurements on two different reactor topologies (surface and volumetric), showing that open loop operation of the power supply leads to a temperature and average power increase over time. The temperature increases by Delta T-vol similar to 120 degrees C and Delta T-sup similar to 70 degrees C, while the power increases by Delta P-vol similar to 78% and Delta P-sup similar to 60% for the volumetric (40 s) and superficial reactors (120 s), respectively. We discuss how these changes are often unwanted in practical applications. A simplified circuital model of the power supply-reactor system is used to infer the physical relation between the observed reactor thermal behavior and its electrical characteristics. We then show a control strategy for the power supply voltage to ensure constant average power operation of the device based on real-time power measurements on the high voltage side of the power supply and an empirical expression relating the delivered power to the power supply output voltage. These are performed with an Arduino Due microcontroller unit, also used to control the power supply. In a controlled operation the measured power stays within 5% of the reference value for both configurations, reducing the temperature increments to Delta T-vol similar to 80 degrees C and Delta T-sup similar to 44 degrees C, respectively. The obtained results show that the proposed novel control strategy is capable of following the transient temperature behavior, achieving a constant average power operation and subsequently limiting the reactor thermal stress

Extreme Kuiper Belt Object 2001 QG298 and the Fraction of Contact Binaries

Extensive time-resolved observations of Kuiper Belt object 2001 QG298 show a lightcurve with a peak-to-peak variation of 1.14 +-0.04 magnitudes and single-peaked period of 6.8872 +- 0.0002 hr. The mean absolute magnitude is 6.85 magnitudes which corresponds to a mean effective radius of 122 (77) km if an albedo of 0.04 (0.10) is assumed. This is the first known Kuiper Belt object and only the third minor planet with a radius > 25 km to display a lightcurve with a range in excess of 1 magnitude. We find the colors to be typical for a Kuiper Belt object (B-V = 1.00 +- 0.04, V-R = 0.60 +- 0.02) with no variation in color between minimum and maximum light. The large light variation, relatively long double-peaked period and absence of rotational color change argue against explanations due to albedo markings or elongation due to high angular momentum. Instead, we suggest that 2001 QG298 may be a very close or contact binary similar in structure to what has been independently proposed for the Trojan asteroid 624 Hektor. If so, its rotational period would be twice the lightcurve period or 13.7744 +- 0.0004 hr. By correcting for the effects of projection, we estimate that the fraction of similar objects in the Kuiper Belt is at least 10% to 20% with the true fraction probably much higher. A high abundance of close and contact binaries is expected in some scenarios for the evolution of binary Kuiper Belt objects.Comment: 15 text pages,6 figures(Color),5 Tables, Accepted to AJ for May 200

Close Binaries with Infrared Excess: Destroyers of Worlds?

We present the results of a Spitzer photometric investigation into the IR excesses of close binary systems. In a sample of 10 objects, excesses in IRAC and MIPS24 bands implying the presence of warm dust are found for 3. For 2 objects we do not find excesses reported in earlier IRAS studies. We discuss the results in the context of the scenario suggested by Rhee and co-workers, in which warm dust is continuously created by destructive collisions between planetary bodies. A simple numerical model for the steady-state distribution of dust in one IR excess system shows a central clearing of radius 0.22 AU caused by dynamical perturbations from the binary star. This is consistent with the size of the central clearing derived from the Spitzer spectral energy distribution. We conclude that close binaries could be efficient "destroyers of worlds", lead to destabilize the orbits of their planetary progeny by magnetically-driven angular momentum loss and secular shrinkage of the binary separation.Comment: Accepted by The Astrophysical Journa

Linear polarization of a group of symbiotic systems

We report linear polarization measurements of a set of symbiotic stars, made at several epochs during the period 1994-1998. Evidence of intrinsic polarization is looked for from the wavelength dependence of the polarization degree and position angle in UBVRI bands. The results have also been analysed to search for temporal variability of polarization. Several objects have shown a polarization spectrum different from that produced by interstellar dust grains and/or polarimetric variations on time scales as short as several days or months, indicating the presence of polarization component of circumstellar origin.Facultad de Ciencias Astronómicas y Geofísica

Linear polarization of a group of symbiotic systems

We report linear polarization measurements of a set of symbiotic stars, made at several epochs during the period 1994-1998. Evidence of intrinsic polarization is looked for from the wavelength dependence of the polarization degree and position angle in UBVRI bands. The results have also been analysed to search for temporal variability of polarization. Several objects have shown a polarization spectrum different from that produced by interstellar dust grains and/or polarimetric variations on time scales as short as several days or months, indicating the presence of polarization component of circumstellar origin.Facultad de Ciencias Astronómicas y Geofísica

Polarimetry of an Intermediate-age Open Cluster: NGC 5617

We present polarimetric observations in the UBVRI bands of 72 stars located in the direction of the medium age open cluster NGC 5617. Our intention is to use polarimetry as a tool membership identification, by building on previous investigations intended mainly to determine the cluster's general characteristics rather than provide membership suitable for studies such as stellar content and metallicity, as well as study the characteristics of the dust lying between the Sun and the cluster. The obsevations were carried out using the five-channel photopolarimeter of the Torino Astronomical Observatory attached to the 2.15m telescope at the Complejo Astron\'omico El Leoncito (CASLEO; Argentina. We are able to add 32 stars to the list of members of NGC 5617, and review the situation for others listed in the literature. In particular, we find that five blue straggler stars in the region of the cluster are located behind the same dust as the member stars are and we confirm the membership of two red giants. The proposed polarimetric memberships are compared with those derived by photometric and kinematical methods, with excellent results. Among the observed stars, we identify 10 with intrinsic polarization in their light. NGC 5617 can be polarimetrically characterized with $P_{max}= 4.40$ and $\theta_{v}= 73^\circ.1$. The spread in polarization values for the stars observed in the direction of the cluster seems to be caused by the uneven distribution of dust in front of the cluster's face. Finally, we find that in the direction of the cluster, the interstellar medium is apparently free of dust, from the Sun's position up to the Carina-Sagittarius arm, where NGC 5617 seems to be located at its farthest border