TY - GEN A1 - Broeker, Nina K. A1 - Kiele, Franziska A1 - Casjens, Sherwood R. A1 - Gilcrease, Eddie B. A1 - Thalhammer, Anja A1 - Koetz, Joachim T1 - In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620 T2 - Viruses N2 - Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 469 KW - O-antigen specific phage KW - podovirus KW - HK620 KW - lipopolysaccharide KW - in vitro particle opening KW - tailspike protein Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-417493 ER - TY - JOUR A1 - Broeker, Nina K. A1 - Kiele, Franziska A1 - Casjens, Sherwood R. A1 - Gilcrease, Eddie B. A1 - Thalhammer, Anja A1 - Koetz, Joachim T1 - In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620 JF - Viruses N2 - Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change. KW - O-antigen specific phage KW - podovirus KW - HK620 KW - lipopolysaccharide KW - in vitro particle opening KW - tailspike protein Y1 - 2018 U6 - https://doi.org/10.3390/v10060289 SN - 1999-4915 VL - 10 IS - 6 SP - 1 EP - 15 PB - Molecular Diversity Preservation International (MDPI) CY - Basel ER - TY - JOUR A1 - Broeker, Nina K. A1 - Roske, Yvette A1 - Valleriani, Angelo A1 - Stephan, Mareike Sophia A1 - Andres, Dorothee A1 - Koetz, Joachim A1 - Heinemann, Udo A1 - Barbirz, Stefanie T1 - Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail JF - The journal of biological chemistry N2 - Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 angstrom resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an E15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches. KW - bacteriophage KW - lipopolysaccharide (YLPS) KW - structural biology KW - DNA viruses KW - glycobiology KW - fluorescence KW - Salmonella enterica KW - contractile tail KW - DNA ejection KW - O-antigen specificity KW - Salmonella myovirus KW - tailspike protein KW - molecular machine Y1 - 2019 U6 - https://doi.org/10.1074/jbc.RA119.008133 SN - 1083-351X VL - 294 IS - 31 SP - 11751 EP - 11761 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER -