Search

Lyme disease, caused by spirochetes in the Borrelia burgdorferi sensu lato clade within the Borrelia genus, is transmitted by Ixodes ticks and is currently the most prevalent and rapidly expanding tick-borne disease in Europe and North America. We report complete genome sequences of 47 isolates that encompass all established species in this clade while highlighting the diversity of the widespread human pathogenic species B. burgdorferi . A similar set of plasmids has been maintained throughout Borrelia divergence, indicating that they are a key adaptive feature of this genus. Phylogenetic reconstruction of all sequenced Borrelia genomes revealed the original divergence of Eurasian and North American lineages and subsequent dispersals that introduced B. garinii, B. bavariensis, B. lusitaniae, B. valaisiana, and B. afzelii from East Asia to Europe and B. burgdorferi and B. finlandensis from North America to Europe. Molecular phylogenies of the universally present core replicons (chromosome and cp26 and lp54 plasmids) are highly consistent, revealing a strong clonal structure. Nonetheless, numerous inconsistencies between the genome and gene phylogenies indicate species dispersal, genetic exchanges, and rapid sequence evolution at plasmid-borne loci, including key host-interacting lipoprotein genes. While localized recombination occurs uniformly on the main chromosome at a rate comparable to mutation, lipoprotein-encoding loci are recombination hotspots on the plasmids, suggesting adaptive maintenance of recombinant alleles at loci directly interacting with the host. We conclude that within- and between-species recombination facilitates adaptive sequence evolution of host-interacting lipoprotein loci and contributes to human virulence despite a genome-wide clonal structure of its natural populations., Importance: Lyme disease (also called Lyme borreliosis in Europe), a condition caused by spirochete bacteria of the genus Borrelia , transmitted by hard-bodied Ixodes ticks, is currently the most prevalent and rapidly expanding tick-borne disease in the United States and Europe. Borrelia interspecies and intraspecies genome comparisons of Lyme disease-related bacteria are essential to reconstruct their evolutionary origins, track epidemiological spread, identify molecular mechanisms of human pathogenicity, and design molecular and ecological approaches to disease prevention, diagnosis, and treatment. These Lyme disease-associated bacteria harbor complex genomes that encode many genes that do not have homologs in other organisms and are distributed across multiple linear and circular plasmids. The functional significance of most of the plasmid-borne genes and the multipartite genome organization itself remains unknown. Here we sequenced, assembled, and analyzed whole genomes of 47 Borrelia isolates from around the world, including multiple isolates of the human pathogenic species. Our analysis elucidates the evolutionary origins, historical migration, and sources of genomic variability of these clinically important pathogens. We have developed web-based software tools (BorreliaBase.org) to facilitate dissemination and continued comparative analysis of Borrelia genomes to identify determinants of human pathogenicity., Competing Interests: The authors declare no conflict of interest.

Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. Salmonella phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the c2 (repressor), gtrABC , sieA , and sieB genes. Here we report that the P22-encoded SieA protein is necessary and sufficient for exclusion by the SieA system and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants that overcome the SieA block were isolated, and they have amino acid changes in the C-terminal regions of the gene 16 and 20 encoded ejection proteins. Three different single-amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in phage target specificity. Our data strongly suggest a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.IMPORTANCEThe ongoing evolutionary battle between bacteria and the viruses that infect them is a critical feature of bacterial ecology on Earth. Viruses can kill bacteria by infecting them. However, when their chromosomes are integrated into a bacterial genome as a prophage, viruses can also protect the host bacterium by expressing genes whose products defend against infection by other viruses. This defense property is called "superinfection exclusion." A significant fraction of bacteria harbor prophages that encode such protective systems, and there are many different molecular strategies by which superinfection exclusion is mediated. This report is the first to describe the mechanism by which bacteriophage P22 SieA superinfection exclusion protein protects its host bacterium from infection by other P22-like phages. The P22 prophage-encoded inner membrane SieA protein prevents infection by blocking transport of superinfecting phage DNA across the inner membrane during injection., Competing Interests: The authors declare no conflict of interest.

Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted β-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We present a novel method that provides a measurement of DNA pressure in viral capsids using small angle X-ray scattering (SAXS). This method, unlike our previous assay, does not require triggering genome release with a viral receptor. Thus, it can be used to determine the existence of a pressurized genome state in a wide range of virus systems, even if the receptor is not known, leading to a better understanding of the processes of viral genome uncoating and encapsidation in the course of infection. Furthermore, by measuring DNA pressure for a collection of bacteriophages with varying DNA packing densities, we derived an empirical equation of state (EOS) that accurately predicts the relation between the capsid pressure and the packaged DNA density and includes the contribution of both DNA-DNA interaction energy and DNA bending stress to the total DNA pressure. We believe that our SAXS-osmometer method and the EOS, combined, provide the necessary tools to investigate physico-chemical properties of confined DNA condensates and mechanisms of infection, and may also provide essential data for the design of viral vectors in gene therapy applications and development of antivirals that target the pressurized genome state., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Anderson phage typing scheme has been successfully used worldwide for epidemiological surveillance of Salmonella enterica serovar Typhimurium. Although the scheme is being replaced by whole genome sequence subtyping methods, it can provide a valuable model system for study of phage-host interaction. The phage typing scheme distinguishes more than 300 definitive types of Salmonella Typhimurium based on their patterns of lysis to a unique collection of 30 specific Salmonella phages. In this study, we sequenced the genomes of 28 Anderson typing phages of Salmonella Typhimurium to begin to characterize the genetic determinants that are responsible for the differences in these phage type profiles. Genomic analysis of typing phages reveals that Anderson phages can be classified into three different groups, the P22-like, ES18-like and SETP3-like clusters. Most Anderson phages are short tailed P22-like viruses (genus Lederbergvirus); but phages STMP8 and STMP18 are very closely related to the lambdoid long tailed phage ES18, and phages STMP12 and STMP13 are related to the long noncontractile tailed, virulent phage SETP3. Most of these typing phages have complex genome relationships, but interestingly, two phage pairs STMP5 and STMP16 as well as STMP12 and STMP13 differ by a single nucleotide. The former affects a P22-like protein involved in DNA passage through the periplasm during its injection, and the latter affects a gene whose function is unknown. Using the Anderson phage typing scheme would provide insights into phage biology and the development of phage therapy for the treatment of antibiotic resistant bacterial infections., (© 2023. The Author(s).)

Bacteriophage KL-2146 is a lytic virus isolated to infect Klebsiella pneumoniae BAA2146, a pathogen carrying the broad range antibiotic resistance gene New Delhi metallo-betalactamase-1 (NDM-1). Upon complete characterization, the virus is shown to belong to the Drexlerviridae family and is a member of the Webervirus genus located within the (formerly) T1-like cluster of phages. Its double-stranded (dsDNA) genome is 47,844 bp long and is predicted to have 74 protein-coding sequences (CDS). After challenging a variety of K. pneumoniae strains with phage KL-2146, grown on the NDM-1 positive strain BAA-2146, polyvalence was shown for a single antibiotic-sensitive strain, K. pneumoniae 13,883, with a very low initial infection efficiency in liquid culture. However, after one or more cycles of infection in K. pneumoniae 13,883, nearly 100% infection efficiency was achieved, while infection efficiency toward its original host, K. pneumoniae BAA-2146, was decreased. This change in host specificity is reversible upon re-infection of the NDM-1 positive strain (BAA-2146) using phages grown on the NDM-1 negative strain (13883). In biofilm infectivity experiments, the polyvalent nature of KL-2146 was demonstrated with the killing of both the multidrug-resistant K. pneumoniae BAA-2146 and drug-sensitive 13,883 in a multi-strain biofilm. The ability to infect an alternate, antibiotic-sensitive strain makes KL-2146 a useful model for studying phages infecting the NDM-1+ strain, K. pneumoniae BAA-2146. GRAPHICAL ABSTRACT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gilcrease, Casjens, Bhattacharjee and Goel.)

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ imm21, λ imm434 , and λ h434 imm21. These sequences show that the replacements of λ DNA by nonhomologous segments of 21 or 434 DNA occurred through homologous recombination in adjacent sequences that are nearly identical in the parental phages. These five genome sequences correct a number of errors in published sequence fragments of the 21 and 434 genomes, and they point out nine nucleotide differences from Sanger's original λ sequence that are likely present in most extant λ strains in laboratory use today. We discuss the historical importance of these hybrid phages in the development of fundamental tenets of molecular biology and in some of the earliest gene cloning vectors. The 434 and 21 genomes reinforce the conclusion that the genomes of essentially all natural λ-like phages are mosaics of sequence modules from a pool of exchangeable segments.

Tailed bacteriophages are abundant and extremely diverse. Understanding this diversity is a challenge, and here we examine a small slice of that diversity in some detail. We contrast and compare the small genome, virulent, non-contractile tailed phages that infect the bacterial order Enterobacteriales. These phages, with genomes in the 35-60 kbp range, have very similar virions that are often difficult to distinguish by negative stain electron microscopy. There are currently 651 genome sequences of such phages in the public database. We show that these can be robustly parsed into fifteen well-defined clusters that have very different nucleotide sequences. We examine the similarities and differences among these clusters, as well as genetic exchange among clusters and the relationships between host species and phage clusters., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Recombinational hybrids between phage λ and its relatives were instrumental in the beginnings of molecular biology. Here, we report the complete genome sequences of lambdoid phages 21 and 434 and three of their λ hybrids. In addition, we describe 434B, where the entire lysis gene region was replaced by cryptic prophage sequences.

Here, the full genome sequences of 22 T1-like bacteriophages isolated from wastewater are reported. Eight (BlueShadow, Brooksby, Devorator, ElisaCorrea, Reinasaurus, SorkZaugg, Supreme284, ZeroToHero) were isolated on Citrobacter , six on Klebsiella (Chell, FairDinkum, HazelMika, Opt-817, P528, PeteCarol), and eight on Escherichia (Fulano1, Mishu, Opt-719, PhleaSolo, Punny, Poky, Phunderstruck, Sadiya).

We announce the complete genome sequences of 14 Serratia bacteriophages isolated from wastewater treatment plants. These phages define two previously undescribed types which we call the Carrot-like phage cluster (phages Carrot, BigDog, LittleDog, Niamh, Opt-148, Opt-169, PhooPhighters, Rovert, Serratianator, Stoker, Swain, and Ulliraptor) and Tlacuache-like phage cluster (Tlacuache and Opt-155).

The Enterobacteriales order is composed of Gram-negative bacteria that range from harmless symbionts to well-studied pathogens. We announce complete genome sequences of five related SO-1-like Enterobacteriales bacteriophages (also known as the Dhillonvirus genus) isolated from wastewater that infect Escherichia coli (Opt-212, Over9000, Pubbukkers, and Teewinot) or Shigella boydii (StarDew).

Proteus mirabilis and Klebsiella aerogenes are Gram-negative opportunistic pathogens that are responsible for nosocomial and health care-associated infections, including urinary tract infections. Here, the full genome sequences of six Chi-like Proteus (DanisaurMW, DoubleBarrel, Inception, Jing313, and NotEvenPhaged) or Klebsiella (Phraden) bacteriophages are announced, contributing to the understanding of Chi-like phages.

Full genome sequences of five bacteriophages that were isolated from raw sewage samples and infect Enterobacteriales hosts are presented. Brookers is a P22-like Proteus phage, OddieOddie is a 9g-like Escherichia coli phage, Diencephelon is a Kp3-like Klebsiella phage, and Rgz1 and Lilpapawes are classic T4-like and T7-like virulent Proteus phages, respectively.

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7 , 16 and 20 , respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites)., (© The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America.)

All members of the Borrelia genus that have been examined harbour a linear chromosome that is about 900 kbp in length, as well as a plethora of both linear and circular plasmids in the 5-220 kbp size range. Genome sequences for 27 Lyme disease Borrelia isolates have been determined since the elucidation of the B. burgdorferi B31 genome sequence in 1997. The chromosomes, which carry the vast majority of the housekeeping genes, appear to be very constant in gene content and organization across all Lyme disease Borrelia species. The content of the plasmids, which carry most of the genes that encode the differentially expressed surface proteins that interact with the spirochete's arthropod and vertebrate hosts, is much more variable. Lyme disease Borrelia isolates carry between 7-21 different plasmids, ranging in size from 5-84 kbp. All strains analyzed to date harbor three plasmids, cp26, lp54 and lp17. The plasmids are unusual, as compared to most bacterial plasmids, in that they contain many paralogous sequences, a large number of pseudogenes, and, in some cases, essential genes. In addition, a number of the plasmids have features indicating that they are prophages. Numerous methods have been developed for Lyme disease Borrelia strain typing. These have proven valuable for clinical and epidemiological studies, as well as phylogenomic and population genetic analyses. Increasingly, these approaches have been displaced by whole genome sequencing techniques. Some correlations between genome content and pathogenicity have been deduced, and comparative whole genome analyses promise future progress in this arena.

We report the complete genome sequence of P22-like Salmonella enterica serovar Typhimurium phage MG40, whose prophage repressor specificity is different from that of other known temperate phages., (Copyright © 2020 Gilcrease et al.)

Klebsiella pneumoniae is a pathogen responsible for significant proportions of nosocomial and health care-associated infections and is known to acquire multiple antibiotic resistance genes. Here, we announce the full genome sequences of 12 K. pneumoniae bacteriophages from samples collected in wastewater treatment facilities across the western United States., (Copyright © 2020 Thurgood et al.)

We present the genome sequences of Salmonella enterica tailed phages Sasha, Sergei, and Solent. These phages, along with Salmonella phages 9NA, FSL_SP-062, and FSL_SP-069 and the more distantly related Proteus phage PmiS-Isfahan, have similarly sized genomes of between 52 and 57 kbp in length that are largely syntenic. Their genomes also show substantial genome mosaicism relative to one another, which is common within tailed phage clusters. Their gene content ranges from 80 to 99 predicted genes, of which 40 are common to all seven and form the core genome, which includes all identifiable virion assembly and DNA replication genes. The total number of gene types (pangenome) in the seven phages is 176, and 59 of these are unique to individual phages. Their core genomes are much more closely related to one another than to the genome of any other known phage, and they comprise a well-defined cluster within the family Siphoviridae To begin to characterize this group of phages in more experimental detail, we identified the genes that encode the major virion proteins and examined the DNA packaging of the prototypic member, phage 9NA. We show that it uses a pac site-directed headful packaging mechanism that results in virion chromosomes that are circularly permuted and about 13% terminally redundant. We also show that its packaging series initiates with double-stranded DNA cleavages that are scattered across a 170-bp region and that its headful measuring device has a precision of ±1.8%. IMPORTANCE The 9NA-like phages are clearly highly related to each other but are not closely related to any other known phage type. This work describes the genomes of three new 9NA-like phages and the results of experimental analysis of the proteome of the 9NA virion and DNA packaging into the 9NA phage head. There is increasing interest in the biology of phages because of their potential for use as antibacterial agents and for their ecological roles in bacterial communities. 9NA-like phages that infect two bacterial genera have been identified to date, and related phages infecting additional Gram-negative bacterial hosts are likely to be found in the future. This work provides a foundation for the study of these phages, which will facilitate their study and potential use., (Copyright © 2019 American Society for Microbiology.)

Erwinia and Pantoea are closely related bacterial plant pathogens in the Gram negative Enterobacteriales order. Sixty tailed bacteriophages capable of infecting these pathogens have been completely sequenced by investigators around the world and are in the current databases, 30 of which were sequenced by our lab. These 60 were compared to 991 other Enterobacteriales bacteriophage genomes and found to be, on average, just over twice the overall average length. These Erwinia and Pantoea phages comprise 20 clusters based on nucleotide and protein sequences. Five clusters contain only phages that infect the Erwinia and Pantoea genera, the other 15 clusters are closely related to bacteriophages that infect other Enterobacteriales; however, within these clusters the Erwinia and Pantoea phages tend to be distinct, suggesting ecological niche may play a diversification role. The failure of many of their encoded proteins to have predicted functions highlights the need for further study of these phages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The major coat proteins of dsDNA tailed phages (order Caudovirales ) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary 'decoration' (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis., Competing Interests: RN, JS, EG, SC, MF, CT, AA, KP No competing interests declared, (© 2019, Newcomer et al.)

Bacteriophages are the most abundant members of the microbiota and have the potential to shape gut bacterial communities. Changes to bacteriophage composition are associated with disease, but how phages impact mammalian health remains unclear. We noted an induction of host immunity when experimentally treating bacterially driven cancer, leading us to test whether bacteriophages alter immune responses. Treating germ-free mice with bacteriophages leads to immune cell expansion in the gut. Lactobacillus, Escherichia, and Bacteroides bacteriophages and phage DNA stimulated IFN-γ via the nucleotide-sensing receptor TLR9. The resultant immune responses were both phage and bacteria specific. Additionally, increasing bacteriophage levels exacerbated colitis via TLR9 and IFN-γ. Similarly, ulcerative colitis (UC) patients responsive to fecal microbiota transplantation (FMT) have reduced phages compared to non-responders, and mucosal IFN-γ positively correlates with bacteriophage levels. Bacteriophages from active UC patients induced more IFN-γ compared to healthy individuals. Collectively, these results indicate that bacteriophages can alter mucosal immunity to impact mammalian health., (Published by Elsevier Inc.)

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.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane., (© 2018 John Wiley & Sons Ltd.)

Roger W. Hendrix was at the forefront of bacteriophage biology for nearly 50 years and was central to our understanding of both viral capsid assembly and phage genomic diversity and evolution. Roger's warm and gentle demeanor belied a razor-sharp mind and warmed him to numerous highly productive collaborations that amplified his scientific impact. Roger was always completely open with scientific ideas while at the same time quietly agitating with a stream of new ways of thinking about problems and nudging our communities to search for innovative solutions: a gentle but highly effective provocateur., (Copyright © 2018 American Society for Microbiology.)

Genome ejection proteins are required to facilitate transport of bacteriophage P22 double-stranded DNA safely through membranes of Salmonella. The structures and locations of all proteins in the context of the mature virion are known, with the exception of three ejection proteins. Furthermore, the changes that occur to the proteins residing in the mature virion upon DNA release are not fully understood. We used cryogenic electron microscopy to obtain what is, to our knowledge, the first asymmetric reconstruction of mature bacteriophage P22 after double-stranded DNA has been extruded from the capsid-a state representative of one step during viral infection. Results of icosahedral and asymmetric reconstructions at estimated resolutions of 7.8 and 12.5 Å resolutions, respectively, are presented. The reconstruction shows tube-like protein density extending from the center of the tail assembly. The portal protein does not revert to the more contracted, procapsid state, but instead maintains an extended and splayed barrel structure. These structural details contribute to our understanding of the molecular mechanism of P22 phage infection and also set the foundation for future exploitation serving engineering purposes., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species., Results: We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types., Conclusions: The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

The temperate Escherichia coli bacteriophage D6 can exist as a circular plasmid prophage, and we report here its 91,159bp complete genome sequence. It is a distant relative of the well-studied phage P1, but it is sufficiently different that it typifies a previously undescribed tailed phage type or cluster. Examination of the database of bacterial genome sequences revealed that phage P1 and D6 prophage plasmids are common in the Enterobacteriales, and in addition, previously described Salmonella phage SSU5 represents a different type of temperate tailed phage with a circular plasmid prophage that is also very common in this host order. This analysis also discovered additional divergent clusters of putative circular plasmid prophages within the two larger P1 and SSU5 groups (superclusters) that inhabit the Enterobacteriales as well as bacteria in several other orders in the Gamma-proteobacteria class. Very few of these sequences are annotated as putative prophages., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah., (Copyright © 2017 Leavitt et al.)

Bacteriophages, as the most abundant biological entities on Earth, place significant predation pressure on their hosts. This pressure plays a critical role in the evolution, diversity, and abundance of bacteria. In addition, phages modulate the genetic diversity of prokaryotic communities through the transfer of auxiliary metabolic genes. Various studies have been conducted in diverse ecosystems to understand phage-host interactions and their effects on prokaryote metabolism and community composition. However, hypersaline environments remain among the least studied ecosystems and the interaction between the phages and prokaryotes in these habitats is poorly understood. This study begins to fill this knowledge gap by analyzing bacteriophage-host interactions in the Great Salt Lake, the largest prehistoric hypersaline lake in the Western Hemisphere. Our metagenomics analyses allowed us to comprehensively identify the bacterial and phage communities with Proteobacteria, Firmicutes , and Bacteroidetes as the most dominant bacterial species and Siphoviridae, Myoviridae , and Podoviridae as the most dominant viral families found in the metagenomic sequences. We also characterized interactions between the phage and prokaryotic communities of Great Salt Lake and determined how these interactions possibly influence the community diversity, structure, and biogeochemical cycles. In addition, presence of prophages and their interaction with the prokaryotic host was studied and showed the possibility of prophage induction and subsequent infection of prokaryotic community present in the Great Salt Lake environment under different environmental stress factors. We found that carbon cycle was the most susceptible nutrient cycling pathways to prophage induction in the presence of environmental stresses. This study gives an enhanced snapshot of phage and prokaryote abundance and diversity as well as their interactions in a hypersaline complex ecosystem, which can pave the way for further research studies.

Background: Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi., Results: We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes)., Conclusions: Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

We identified 9371 tailed phage prophages of 20 known types in reported complete genome sequences of 3298 bacteria in the Salmonella genus. These include 4758 P2 type and 744 P22 type prophages. The latter prophage types were found in the genome sequences of 127 and 24 bacterial host genera, increasing the known host ranges of phages in these groups by 114 and 20 genera, respectively. These prophage nucleotide sequences displayed much more diversity than was previously known from the 48 P2 and 24 P22 type authentic phages whose genomes have been sequenced. More detailed analysis of these prophage sequences indicated that major capsid protein (MCP) gene exchange between tailed phage clusters or types is extremely rare and that P22 prophage-encoded tailspikes correspond perfectly with their hosts' surface polysaccharide structure; thus, MCP and tailspike sequences accurately predict tailed phage type (and thus lifestyle) and host cell surface polysaccharide structure, respectively., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three "ejection proteins" (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins' locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection., Importance: While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes., (Copyright © 2016 Wu et al.)

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal "trimer of hairpins" tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages λ and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals., Importance: A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage λ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Streptolysin S (SLS) is a cytolytic virulence factor produced by the human pathogen Streptococcus pyogenes and other Streptococcus species. Related "SLS-like" toxins have been characterized in select strains of Clostridium and Listeria, with homologous clusters bioinformatically identified in a variety of other species. SLS is a member of the thiazole/oxazole-modified microcin (TOMM) family of natural products. The structure of SLS has yet to be deciphered and many questions remain regarding its structure-activity relationships., Results: In this work, we assessed the hemolytic activity of a series of C-terminally truncated SLS peptides expressed in SLS-deficient S. pyogenes. Our data indicate that while the N-terminal poly-heterocyclizable (NPH) region of SLS substantially contributes to its bioactivity, the variable C-terminal region of the toxin is largely dispensable. Through genome mining we identified additional SLS-like clusters in diverse Firmicutes, Spirochaetes and Actinobacteria. Among the Spirochaete clusters, naturally truncated SLS-like precursors were found in the genomes of three Lyme disease-causing Borrelia burgdorferi sensu lato (Bbsl) strains. Although unable to restore hemolysis in SLS-deficient S. pyogenes, a Bbsl SLS-like precursor peptide was converted to a cytolysin using purified SLS biosynthetic enzymes. A PCR-based screen demonstrated that SLS-like clusters are substantially more prevalent in Bbsl than inferred from publicly available genome sequences., Conclusions: The mutagenesis data described herein indicate that the minimal cytolytic unit of SLS encompasses the NPH region of the core peptide. Interestingly, this region is found in all characterized TOMM cytolysins, as well as the novel putative TOMM cytolysins we discovered. We propose that this conserved region represents the defining feature of the SLS-like TOMM family. We demonstrate the cytolytic potential of a Bbsl SLS-like precursor peptide, which has a core region of similar length to the SLS minimal cytolytic unit, when modified with purified SLS biosynthetic enzymes. As such, we speculate that some Borrelia have the potential to produce a TOMM cytolysin, although the biological significance of this finding remains to be determined. In addition to providing new insight into the structure-activity relationships of SLS, this study greatly expands the cytolysin group of TOMMs.

Det7 is a Myoviridae bacteriophage that gains entry into its Salmonella enterica serovar Typhimurium host cells by adsorbing to O-antigen polysaccharide. We report here the complete 157,498-bp sequence of its genome. Det7, together with its Vi01-like relatives, are distantly related to phage T4., (Copyright © 2015 Casjens et al.)

Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives has continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Phylogenomic footprinting is an approach for ab initio identification of genome-wide regulatory elements in bacterial species based on sequence conservation. The statistical power of the phylogenomic approach depends on the degree of sequence conservation, the length of regulatory elements, and the level of phylogenetic divergence among genomes. Building on an earlier model, we propose a binomial model that uses synonymous tree lengths as neutral expectations for determining the statistical significance of conserved intergenic spacer (IGS) sequences. Simulations show that the binomial model is robust to variations in the value of evolutionary parameters, including base frequencies and the transition-to-transversion ratio. We used the model to search for regulatory sequences in the Lyme disease species group (Borrelia burgdorferi sensu lato) using 23 genomes. The model indicates that the currently available set of Borrelia genomes would not yield regulatory sequences shorter than five bases, suggesting that genome sequences of additional B. burgdorferi sensu lato species are needed. Nevertheless, we show that previously known regulatory elements are indeed strongly conserved in sequence or structure across these Borrelia species. Further, we predict with sufficient confidence two new RpoS binding sites, 39 promoters, 19 transcription terminators, 28 noncoding RNAs, and four sets of coregulated genes. These putative cis- and trans-regulatory elements suggest novel, Borrelia-specific mechanisms regulating the transition between the tick and host environments, a key adaptation and virulence mechanism of B. burgdorferi. Alignments of IGS sequences are available on BorreliaBase.org, an online database of orthologous open reading frame (ORF) and IGS sequences in Borrelia., Importance: While bacterial genomes contain mostly protein-coding genes, they also house DNA sequences regulating the expression of these genes. Gene regulatory sequences tend to be conserved during evolution. By sequencing and comparing related genomes, one can therefore identify regulatory sequences in bacteria based on sequence conservation. Here, we describe a statistical framework by which one may determine how many genomes need to be sequenced and at what level of evolutionary relatedness in order to achieve a high level of statistical significance. We applied the framework to Borrelia burgdorferi, the Lyme disease agent, and identified a large number of candidate regulatory sequences, many of which are known to be involved in regulating the phase transition between the tick vector and mammalian hosts., (Copyright © 2015 Martin et al.)

Salmonella bacteriophage χ is a member of the Siphoviridae family that gains entry into its host cells by adsorbing to their flagella. We report the complete 59,578-bp sequence of the genome of phage χ, which together with its relatives, exemplifies a largely unexplored type of tailed bacteriophage., (Copyright © 2015 Hendrix et al.)

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships.

CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the "HK97-fold" shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain ("I-domain"), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently., (Copyright © 2014 Elsevier Inc. All rights reserved.)