The inaugural mBio Junior Editorial Board-lessons learned and the path forward toward improving the peer review process.

The inaugural Junior Editorial Board (JEB) of mBio consisted of 64 early-career researchers active from 2022 to 2023. The goal of the JEB was to train early-career researchers in the art of peer review under the guidance of experienced editors. JEB members gained hands-on experience in peer review by participating in modules detailing the publishing process through the lenses of the journal, editor, and reviewer. Ultimately, JEB members applied this new knowledge by reviewing mBio manuscripts. Here, we summarize the background, the mission, and the achievements of the first mBio JEB. We also include possible trajectories for the future editions of this important program.

Host Range Expansion of Shigella Phage Sf6 Evolves through Point Mutations in the Tailspike.

The first critical step in a virus's infection cycle is attachment to its host. This interaction is precise enough to ensure the virus will be able to productively infect the cell, but some flexibility can be beneficial to enable coevolution and host range switching or expansion. Bacteriophage Sf6 utilizes a two-step process to recognize and attach to its host Shigella flexneri. Sf6 first recognizes the lipopolysaccharide (LPS) of S. flexneri and then binds outer membrane protein (Omp) A or OmpC. This phage infects serotype Y strains but can also form small, turbid plaques on serotype 2a2; turbid plaques appear translucent rather than transparent, indicating greater survival of bacteria. Reduced plating efficiency further suggested inefficient infection. To examine the interactions between Sf6 and this alternate host, phages were experimentally evolved using mixed populations of S. flexneri serotypes Y and 2a2. The recovered mutants could infect serotype 2a2 with greater efficiency than the ancestral Sf6, forming clear plaques on both serotypes. All mutations mapped to two distinct regions of the receptor-binding tailspike protein: (i) adjacent to the LPS binding site near the N terminus; and (ii) at the distal, C-terminal tip of the protein. Although we anticipated interactions between the Sf6 tailspike and 2a2 O-antigen to be weak, LPS of this serotype appears to inhibit infection through strong binding of particles, effectively removing them from the environment. The mutations of the evolved strains reduce the inhibitory effect by either reducing electrostatic interactions with the O-antigen or increasing reliance on the Omp secondary receptors. IMPORTANCE Viruses depend on host cells to propagate themselves. In mixed populations and communities of host cells, finding these susceptible host cells may have to be balanced with avoiding nonhost cells. Alternatively, being able to infect new cell types can increase the fitness of the virus. Many bacterial viruses use a two-step process to identify their hosts, binding first to an LPS receptor and then to a host protein. For Shigella virus Sf6, the tailspike protein was previously known to bind the LPS receptor. Genetic data from this work imply the tailspike also binds to the protein receptor. By experimentally evolving Sf6, we also show that point mutations in this protein can dramatically affect the binding of one or both receptors. This may provide Sf6 flexibility in identifying host cells and the ability to rapidly alter its host range under selective pressure.

Shigella viruses Sf22 and KRT47 require outer membrane protein C for infection.

Viruses rely on hosts for their replication: thus, a critical step in the infection process is identifying a suitable host cell. Bacterial viruses, known as bacteriophages or phages, often use receptor binding proteins to discriminate between susceptible and non-susceptible hosts. By being able to evade predation, bacteria with modified or deleted receptor-encoding genes often undergo positive selection during growth in the presence of phage. Depending on the specific receptor(s) a phage uses, this may subsequently affect the bacteria's ability to form biofilms, its resistance to antibiotics, pathogenicity, or its phenotype in various environments. In this study, we characterize the interactions between two T4-like phages, Sf22 and KRT47, and their host receptor S. flexneri outer membrane protein C (OmpC). Results indicate that these phages use a variety of surface features on the protein, and that complete resistance most frequently occurs when hosts delete the ompC gene in full, encode premature stop codons to prevent OmpC synthesis, or eliminate specific regions encoding exterior loops.

Large metabolic rewiring from small genomic changes between strains of Shigella flexneri.

The instability of Shigella genomes has been described, but how this instability causes phenotypic differences within the Shigella flexneri species is largely unknown and likely variable. We describe herein the genome of S. flexneri strain PE577, originally a clinical isolate, which exhibits several phenotypic differences compared to the model strain 2457T. Like many previously described strains of S. flexneri, PE577 lacks discernible, functional CRISPR and restriction-modification systems. Its phenotypic differences when compared to 2457T include lower transformation efficiency, higher oxygen sensitivity, altered carbon metabolism, and greater susceptibility to a wide variety of lytic bacteriophage isolates. Since relatively few Shigella phages have been isolated on 2457T or the previously characterized strain M90T, developing a more universal model strain for isolating and studying Shigella phages is critical to understanding both phages and phage-host interactions. In addition to phage biology, the genome sequence of PE577 was used to generate and test hypotheses of how pseudogenes in this strain-whether interrupted by degraded prophages, transposases, frameshifts, or point mutations-have led to metabolic rewiring compared to the model strain 2457T. Results indicate that PE577 can utilise the less-efficient pyruvate oxidase/acetyl-CoA synthetase (PoxB/Acs) pathway to produce acetyl-CoA, while strain 2457T cannot due to a nonsense mutation in acs, rendering it a pseudogene in this strain. Both strains also utilize pyruvate-formate lyase to oxidize formate but cannot survive with this pathway alone, possibly because a component of the formate-hydrogen lyase (fdhF) is a pseudogene in both strains.Importance Shigella causes millions of dysentery cases worldwide, primarily affecting children under five years old. Despite active research in developing vaccines and new antibiotics, relatively little is known about the variation of physiology or metabolism across multiple isolates. In this work, we investigate two strains of S. flexneri that share 98.9% genetic identity but exhibit drastic differences in metabolism, ultimately affecting the growth of the two strains. Results suggest additional strains within the S. flexneri species utilize different metabolic pathways to process pyruvate. Metabolic differences between these closely-related isolates suggest an even wider variety of differences in growth across S. flexneri and Shigella in general. Exploring this variation further may assist the development or application of vaccines and therapeutics to combat Shigella infections.

Ecology, Structure, and Evolution of Shigella Phages.

Numerous bacteriophages-viruses of bacteria, also known as phages-have been described for hundreds of bacterial species. The Gram-negative Shigella species are close relatives of Escherichia coli, yet relatively few previously described phages appear to exclusively infect this genus. Recent efforts to isolate Shigella phages have indicated these viruses are surprisingly abundant in the environment and have distinct genomic and structural properties. In addition, at least one model system used for experimental evolution studies has revealed a unique mechanism for developing faster infection cycles. Differences between these bacteriophages and other well-described model systems may mirror differences between their hosts' ecology and defense mechanisms. In this review, we discuss the history of Shigella phages and recent developments in their isolation and characterization and the structural information available for three model systems, Sf6, Sf14, and HRP29; we also provide an overview of potential selective pressures guiding both Shigella phage and host evolution.

Finally, a Role Befitting Astar: Strongly Conserved, Unessential Microvirus A* Proteins Ensure the Product Fidelity of Packaging Reactions.

In microviruses, 60 copies of the positively charged DNA binding protein J guide the single-stranded DNA genome into the icosahedral capsid. Consequently, ∼12% of the genome is icosahedrally ordered within virions. Although the internal volume of the ϕX174, G4, and α3 capsids are nearly identical, their genome lengths vary widely from 5,386 (ϕX174) to 6,067 (α3) nucleotides. As the genome size increases, the J protein's length and charge decreases. The ϕX174 J protein is 37 amino acids long and has a charge of +12, whereas the 23-residue G4 and α3 proteins have respective +6 and +8 charges. While the large ϕX174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, ϕX174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (ϕXG4J) was generated by replacing the indigenous ϕX174 J gene with that of G4. The resulting mutant, ϕXG4J, was not viable on the level of plaque formation without ϕX174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than the wild type. Complementation-independent ϕXG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, ϕXG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene.IMPORTANCE Double-stranded DNA viruses typically package their genomes into a preformed capsid. In contrast, single-stranded RNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. Single-stranded DNA (ssDNA) viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling-circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential, and/or functionally undefined in-frame versions of A/A*-like proteins.

A cornucopia of Shigella phages from the Cornhusker State.

Bacteriophages are abundant in the environment, yet the vast majority have not been discovered or described. Many characterized bacteriophages infect a small subset of Enterobacteriaceae hosts. Despite its similarity to Escherichia coli, the pathogenic Shigella flexneri has relatively few known phages, which exhibit significant differences from many E. coli phages. This suggests that isolating additional Shigella phages is necessary to further explore these differences. To address questions of novelty and prevalence, high school students isolated bacteriophages on non-pathogenic strains of enteric bacteria. Results indicate that Shigella phages are abundant in the environment and continue to differ significantly from E. coli phages. Our findings suggest that Shigella-infecting members of the Ounavirinae subfamily continue to be over-represented and show surprisingly low diversity within and between sampling sites. Additionally, a podophage with distinct genomic and structural properties suggests that continued isolation on non-model species of bacteria is necessary to truly understand bacteriophage diversity.

Shigella Phages Isolated during a Dysentery Outbreak Reveal Uncommon Structures and Broad Species Diversity.

In 2016, Michigan experienced the largest outbreak of shigellosis, a type of bacillary dysentery caused by Shigella spp., since 1988. Following this outbreak, we isolated 16 novel Shigella-infecting bacteriophages (viruses that infect bacteria) from environmental water sources. Most well-known bacteriophages infect the common laboratory species Escherichia coli and Salmonella enterica, and these phages have built the foundation of molecular and bacteriophage biology. Until now, comparatively few bacteriophages were known to infect Shigella spp., which are close relatives of E. coli We present a comprehensive analysis of these phages' host ranges, genomes, and structures, revealing genome sizes and capsid properties that are shared by very few previously described phages. After sequencing, a majority of the Shigella phages were found to have genomes of an uncommon size, shared by only 2% of all reported phage genomes. To investigate the structural implications of this unusual genome size, we used cryo-electron microscopy to resolve their capsid structures. We determined that these bacteriophage capsids have similarly uncommon geometry. Only two other viruses with this capsid structure have been described. Since most well-known bacteriophages infect Escherichia or Salmonella, our understanding of bacteriophages has been limited to a subset of well-described systems. Continuing to isolate phages using nontraditional strains of bacteria can fill gaps that currently exist in bacteriophage biology. In addition, the prevalence of Shigella phages during a shigellosis outbreak may suggest a potential impact of human health epidemics on local microbial communities.IMPORTANCEShigella spp. bacteria are causative agents of dysentery and affect more than 164 million people worldwide every year. Despite the need to combat antibiotic-resistant Shigella strains, relatively few Shigella-infecting bacteriophages have been described. By specifically looking for Shigella-infecting phages, this work has identified new isolates that (i) may be useful to combat Shigella infections and (ii) fill gaps in our knowledge of bacteriophage biology. The rare qualities of these new isolates emphasize the importance of isolating phages on "nontraditional" laboratory strains of bacteria to more fully understand both the basic biology and diversity of bacteriophages.

Elevating fitness after a horizontal gene exchange in bacteriophage φX174.

In an earlier study, protein-based barriers to horizontal gene transfer were investigated by placing the bacteriophage G4 G gene, encoding the major spike protein, into the φX174 genome. The foreign G protein promoted off-pathway assembly reactions, resulting in a lethal phenotype. After three targeted genetic selections, one of two foreign spike proteins was productively integrated into the φX174 system: the complete G4 or a recombinant G4/φX174 protein (94% G4:6% φX174). However, strain fitness was very low. In this study, the chimeras were characterized and experimentally evolved. Inefficient assembly was the primary contributor to low fitness: accordingly, mutations affecting assembly restored fitness. The spike protein preference of the ancestral and evolved strains was determined in competition experiments between the foreign and φX174G proteins. Before adaptation, both G proteins were incorporated into virions; afterwards, the foreign proteins were strongly preferred. Thus, a previously inhibitory protein became the preferred substrate during assembly.

The microviridae: Diversity, assembly, and experimental evolution.

The Microviridae, comprised of ssDNA, icosahedral bacteriophages, are a model system for studying morphogenesis and the evolution of assembly. Historically limited to the φX174-like viruses, recent results demonstrate that this richly diverse family is broadly divided into two groups. The defining feature appears to be whether one or two scaffolding proteins are required for assembly. The single-scaffolding systems contain an internal scaffolding protein, similar to many dsDNA viruses, and have a more complex coat protein fold. The two-scaffolding protein systems (φX174-like) encode an internal and external species, as well as an additional structural protein: a spike on the icosahedral vertices. Here, we discuss recent in silico and in vivo evolutionary analyses conducted with chimeric viruses and/or chimeric proteins. The results suggest 1) how double scaffolding systems can evolve into single and triple scaffolding systems; and 2) how assembly is the critical factor governing adaptation and the maintenance of species boundaries.

The Kinetic and Thermodynamic Aftermath of Horizontal Gene Transfer Governs Evolutionary Recovery.

Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein-protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera's path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.

The evolution of genes within genes and the control of DNA replication in microviruses.

Single-stranded DNA(ssDNA) viral life cycles must balance double-stranded DNA (dsDNA) and ssDNA biosynthesis. Previously published in vitro results suggest that microvirus C and host cell SSB proteins play antagonistic roles to achieve this balance. To investigate this in vivo, microvirus DNA replication was characterized in cells expressing cloned C or ssb genes, which would presumably alter the C:SSB protein ratios. Representatives of each microvirus clade (φX174, G4, and α3) were used in these studies. α3 DNA replication was significantly more complex. Results suggested that the recognized α3 C gene (C(S): small) is one of two C genes. A larger 5' extended gene could be translated from an upstream GTG start codon (C(B): big). Wild-type α3 acquired resistance to elevated SSB levels by mutations that exclusively frameshifted the C(B) reading frame, whereas mutations in the origin of replication conferred resistance to elevated C protein levels. Expression of either the cloned C(B) or C(S) gene complemented am(C) mutants, demonstrating functional redundancy. When the C(S) start codon was eliminated, strains were only viable if an additional amber mutation was placed in gene C and propagated in an informational suppressing host. Thus, C(B) protein likely reaches toxic levels in the absence of C(S) translation. This phenomenon may have driven the evolution of the C(S) gene within the larger C(B) gene and could constitute a unique mechanism of regulation. Furthermore, cross-complementation data suggested that interactions between the α3 C and other viral proteins have evolved enough specificity to biochemically isolate its DNA replication from G4 and φX174.

Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine.

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.