Structures of Qß virions, virus-like particles, and the Qß-MurA complex reveal internal coat proteins and the mechanism of host lysis.

In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the canonical Allolevivirus Qß the maturation protein, A2, has an additional role as the lysis protein, by its ability to bind and inhibit MurA, which is involved in peptidoglycan biosynthesis. Here, we determined structures of Qß virions, virus-like particles, and the Qß-MurA complex using single-particle cryoelectron microscopy, at 4.7-Å, 3.3-Å, and 6.1-Å resolutions, respectively. We identified the outer surface of the ß-region in A2 as the MurA-binding interface. Moreover, the pattern of MurA mutations that block Qß lysis and the conformational changes of MurA that facilitate A2 binding were found to be due to the intimate fit between A2 and the region encompassing the closed catalytic cleft of substrate-liganded MurA. Additionally, by comparing the Qß virion with Qß virus-like particles that lack a maturation protein, we observed a structural rearrangement in the capsid coat proteins that is required to package the viral gRNA in its dominant conformation. Unexpectedly, we found a coat protein dimer sequestered in the interior of the virion. This coat protein dimer binds to the gRNA and interacts with the buried α-region of A2, suggesting that it is sequestered during the early stage of capsid formation to promote the gRNA condensation required for genome packaging. These internalized coat proteins are the most asymmetrically arranged major capsid proteins yet observed in virus structures.

Asymmetric cryo-EM structure of the canonical Allolevivirus Qß reveals a single maturation protein and the genomic ssRNA in situ.

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qß, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qß with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA-coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.

Enzymatic resistance to the lipopeptide surfactin as identified through imaging mass spectrometry of bacterial competition.

Many species of bacteria secrete natural products that inhibit the growth or development of competing species. In turn, competitors may develop or acquire resistance to antagonistic molecules. Few studies have investigated the interplay of these countervailing forces in direct competition between two species. We have used an imaging mass spectrometry (IMS) approach to track metabolites exchanged between Bacillus subtilis and Streptomyces sp. Mg1 cultured together. Surfactin is a cyclic lipopeptide produced by B. subtilis that inhibits the formation of aerial hyphae by streptomycetes. IMS analysis exposed an addition of 18 mass units to surfactin in the agar proximal to Streptomyces sp. Mg1 but not other streptomycetes tested. The spatially resolved change in the mass of surfactin indicated hydrolysis of the molecule. We observed that the aerial growth of Streptomyces sp. Mg1 was resistant to inhibition by surfactin, which suggests that hydrolysis was a mechanism of resistance. To identify possible enzymes from Streptomyces sp. Mg1 with surfactin hydrolase activity, we isolated secreted proteins and identified candidates by mass spectrometry. We purified one candidate enzyme that hydrolyzed surfactin in vitro. We tested the role of this enzyme in surfactin resistance by deleting the corresponding gene from the S. Mg1 genome. We observed that aerial growth by the ΔsfhA mutant strain was now sensitive to surfactin. Our results identify an enzyme that hydrolyzes surfactin and confers resistance to aerial growth inhibition, which demonstrates the effective use of an IMS approach to track natural product modifications during interspecies competition.

Structural Assembly of Qß Virion and Its Diverse Forms of Virus-like Particles.

The coat proteins (CPs) of single-stranded RNA bacteriophages (ssRNA phages) directly assemble around the genomic RNA (gRNA) to form a near-icosahedral capsid with a single maturation protein (Mat) that binds the gRNA and interacts with the retractile pilus during infection of the host. Understanding the assembly of ssRNA phages is essential for their use in biotechnology, such as RNA protection and delivery. Here, we present the complete gRNA model of the ssRNA phage Qß, revealing that the 3' untranslated region binds to the Mat and the 4127 nucleotides fold domain-by-domain, and is connected through long-range RNA-RNA interactions, such as kissing loops. Thirty-three operator-like RNA stem-loops are located and primarily interact with the asymmetric A/B CP-dimers, suggesting a pathway for the assembly of the virions. Additionally, we have discovered various forms of the virus-like particles (VLPs), including the canonical T = 3 icosahedral, larger T = 4 icosahedral, prolate, oblate forms, and a small prolate form elongated along the 3-fold axis. These particles are all produced during a normal infection, as well as when overexpressing the CPs. When overexpressing the shorter RNA fragments encoding only the CPs, we observed an increased percentage of the smaller VLPs, which may be sufficient to encapsidate a shorter RNA.