No abstract

Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.

Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA‐like or TagA‐like proteins with a conserved N‐terminal domain and varying C‐terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N‐terminal domain, their roles in T6SS biogenesis are fundamentally different.

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath–tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath–tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non‐contractile Vibrio cholerae sheaths by cryo‐electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F2/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.

No abstract

Common water disinfectants like chlorine have been reported to select for resistant viruses, yet little attention has been devoted to characterizing disinfection resistance. Here, we investigated the resistance of MS2 coliphage to inactivation by chlorine dioxide (ClO). ClO inactivates MS2 by degrading its structural proteins, thereby disrupting the ability of MS2 to attach to and infect its host. ClO-resistant virus populations emerged not only after repeated cycles of ClO disinfection followed by regrowth but also after dilution-regrowth cycles in the absence of ClO. The resistant populations exhibited several fixed mutations which caused the substitution of ClO-labile by ClO-stable amino acids. On a phenotypic level, these mutations resulted in a more stable host binding during inactivation compared to the wild-type, thus resulting in a greater ability to maintain infectivity. This conclusion was supported by cryo-electron microscopy reconstruction of the virus particle, which demonstrated that most structural modification occurred in the putative A protein, an important binding factor. Resistance was specific to the inactivation mechanism of ClO and did not result in significant cross-resistance to genome-damaging disinfectants. Overall, our data indicate that resistant viruses may emerge even in the absence of ClO pressure but that they can be inactivated by other common disinfectants.

Contractile injection systems (bacteriophage tails, type VI secretions system, R‐type pyocins, etc.) utilize a rigid tube/contractile sheath assembly for breaching the envelope of bacterial and eukaryotic cells. Among contractile injection systems, bacteriophages that infect Gram‐positive bacteria represent the least understood members. Here, we describe the structure of Listeria bacteriophage A511 tail in its pre‐ and post‐host attachment states (extended and contracted, respectively) using cryo‐electron microscopy, cryo‐electron tomography, and X‐ray crystallography. We show that the structure of the tube‐baseplate complex of A511 is similar to that of phage T4, but the A511 baseplate is decorated with different receptor‐binding proteins, which undergo a large structural transformation upon host attachment and switch the symmetry of the baseplate‐tail fiber assembly from threefold to sixfold. For the first time under native conditions, we show that contraction of the phage tail sheath assembly starts at the baseplate and propagates through the sheath in a domino‐like motion.

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryotomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.