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 Δ sfhA 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.

Significance: Single-stranded (ss) RNA viruses have ribonucleic acid as their genetic material and infect animals, plants, and bacteria. Here we used cryo-electron microscopy to reveal the genomic RNA (gRNA) of the ssRNA virus Qβ. The asymmetric gRNA adopts a single dominant structure in all virions and binds the capsid of Qβ at each coat protein. At the same time, we determined the structure of the maturation protein, A ₂ , which functions both as the virion’s “tail” and its lysis protein. We see the gRNA is more ordered when interacting with A ₂ . These results provide structural insights into gRNA packaging and host infection in ssRNA viruses.

Significance: Host lysis and virion assembly are essential processes during the infection cycle of single-stranded RNA (ssRNA) viruses. Using single-particle cryoelectron microscopy, we visualized how the ssRNA virus, Qβ, uses its single-molecule “tail protein,” A ₂ , to inhibit MurA, a bacterial enzyme essential for cell wall biosynthesis, leading to lysis of the host cell. We also revealed an extra coat protein dimer, which instead of being a part of the viral capsid, is sequestered within the virion, binding to an RNA hairpin from a five-way junction in the genomic RNA. The same five-way junction also presents hairpins to bind A ₂ and other coat protein dimers in the capsid, potentially supporting a nucleation event for virion assembly.

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 Qβ, 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.