RESUMEN
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.
RESUMEN
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.
Asunto(s)
Bacteriófago P22 , Bacteriófagos , Sobreinfección , Humanos , Bacteriófago P22/genética , Bacteriófagos/genética , Profagos/genética , Profagos/metabolismo , Proteínas de la Membrana/metabolismo , ADN/metabolismo , Aminoácidos/metabolismoRESUMEN
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 â¼6° 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 ß-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.
Asunto(s)
Bacteriófago P22 , Salmonella typhimurium , Bacteriófago P22/genética , Bacteriófago P22/química , Bacteriófago P22/metabolismo , Proteínas de la Cápside/química , Salmonella typhimurium/virología , Proteínas de la Cola de los Virus/genéticaRESUMEN
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.
Asunto(s)
Bacteriófagos , Cápside , Cápside/química , ADN Viral/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Bacteriófagos/genéticaRESUMEN
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 the only phage protein required 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 were isolated that overcome the SieA block, 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 target specificity. Our data are consistent with 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.
RESUMEN
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.
Asunto(s)
Bacteriófagos , Bacteriófagos/genética , Genómica , Bacterias , Salmonella typhimurium/genética , Tipificación de BacteriófagosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Enfermedad de Lyme , Animales , Estados Unidos/epidemiología , Grupo Borrelia Burgdorferi/genética , Filogenia , Enfermedad de Lyme/epidemiología , Peromyscus , GenómicaRESUMEN
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.
Asunto(s)
Bacteriófago lambda , Vigor Híbrido , Bacteriófago lambda/genética , Biología MolecularRESUMEN
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.
Asunto(s)
Bacteriófagos , Caudovirales , Bacteriófagos/genética , Caudovirales/genética , Genoma Viral , Especificidad del Huésped , FilogeniaRESUMEN
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).
RESUMEN
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.
RESUMEN
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).
RESUMEN
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).
RESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Bacteriófago P22/genética , ADN Viral/genética , Proteínas Virales/genética , Virión/genética , Bacteriófago P22/química , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Empaquetamiento del ADN , ADN Viral/metabolismo , Técnicas Genéticas , Proteínas Virales/metabolismoRESUMEN
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 â¼35 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).
Asunto(s)
Bacteriófagos , Salmonella typhimurium , Enzimas de Restricción-Modificación del ADN , Proteómica , SerogrupoRESUMEN
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.
Asunto(s)
Borrelia/genética , Genoma Bacteriano , Genómica , Enfermedad de Lyme/microbiología , Borrelia/clasificación , Borrelia/virología , Susceptibilidad a Enfermedades , Regulación Bacteriana de la Expresión Génica , Genómica/métodos , Interacciones Huésped-Patógeno , Humanos , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/transmisión , Tipificación Molecular , Plásmidos/genética , Profagos/genética , Secuenciación Completa del GenomaRESUMEN
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.