Analysis of the lambdoid prophage element e14 in the E. coli K-12 genome

BACKGROUND: Many sequenced bacterial genomes harbor phage-like elements or cryptic prophages. These elements have been implicated in pathogenesis, serotype conversion and phage immunity. The e14 element is a defective lambdoid prophage element present at 25 min in the E. coli K-12 genome. This prophage encodes important functional genes such as lit (T4 exclusion), mcrA (modified cytosine restriction activity) and pin (recombinase). RESULTS: Bioinformatic analysis of the e14 prophage sequence shows the modular nature of the e14 element which shares a large part of its sequence with the Shigella flexneri phage SfV. Based on this similarity, the regulatory region including the repressor and Cro proteins and their binding sites were identified. The protein product of b1149 was found to be a fusion of a replication protein and a terminase. The genes b1143, b1151 and b1152 were identified as putative pseudogenes. A number of duplications of the stfE tail fibre gene of the e14 are seen in plasmid p15B. A protein based comparative approach using the COG database as a starting point helped detect lambdoid prophage like elements in a representative set of completely sequenced genomes. CONCLUSIONS: The e14 element was characterized for the function of its encoded genes, the regulatory regions, replication origin and homology with other phage and bacterial sequences. Comparative analysis at nucleotide and protein levels suggest that a number of important phage related functions are missing in the e14 genome including parts of the early left operon, early right operon and late operon. The loss of these genes is the result of at least three major deletions that have occurred on e14 since its integration. A comparative protein level approach using the COG database can be effectively used to detect defective lambdoid prophage like elements in bacterial genomes

Exploring the atomic structure and conformational flexibility of a 320 Å long engineered viral fiber using X-ray crystallography.

Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Å resolution crystal structure of this engineered fiber, which measures ∼320 Å in length and is only 20-35 Å wide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility

The protein interaction map of bacteriophage lambda

<p>Abstract</p> <p>Background</p> <p>Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage.</p> <p>Results</p> <p>In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda.</p> <p>Conclusions</p> <p>Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.</p

Adaption und Vergleich evolutionärer mehrkriterieller Algorithmen mit Hilfe von Variablenwichtigkeitsmaßen

Bei der Herleitung eines Klassifikationsmodells ist neben der Vorhersagegüte auch die Güte der Variablenauswahl ein wichtiges Kriterium. Bei Einflussvariablen mit unterschiedlichen Kosten ist eine kostensensitive Klassifikation erstrebenswert, bei der ein Kompromiss aus hoher Vorhersagegüte und geringen Kosten getroffen werden kann. Werden konfliktäre Ziele, wie etwa hier die Vorhersagegüte und die Kosten, gleichzeitig optimiert, entsteht ein mehrkriterielles Optimierungsproblem, für das keine einzelne sondern eine Menge unvergleichbarer Lösungen existieren. Für das Auffinden der unvergleichbaren Lösungen sind evolutionäre mehrkriterielle Optimierungsalgorithmen (EMOAs) gut geeignet, da sie unter anderem nach verschiedenen Lösungen parallel suchen können und unabhängig von der zugrunde liegenden Datenverteilung sind. Häufig werden EMOAs für die Lösung mehrkriterieller Klassifikationsprobleme in Form von Wrapper-Ansätzen verwendet, wobei die EMOA-Individuen als binäre Zeichenketten (Bitstrings) codiert sind und jedes Bit die Verfügbarkeit der entsprechenden Einflussvariable beschreibt. Basierend auf diesen Variablenteilmengen und gegebenen Daten erstellt der umhüllte (wrapped) Klassifikationsalgorithmus ein Klassifikationsmodell, mit dem Ziel die Vorhersagegüte zu optimieren. Erst nach der Konstruktion des Klassifikationsmodells können weitere Zielkriterien, wie etwa die Kosten der selektierten Variablen, ausgewertet werden. Damit entsteht eine Hierarchie der zu optimierenden Zielkriterien mit Vorteil für die Vorhersagegüte, sodass durch einen mehrkriteriellen Wrapper-Ansatz keine nicht-hierarchischen Lösungen gefunden werden können. Diese Hierarchie der Zielfunktionen wird erstmals in Rahmen dieser Arbeit beschrieben und untersucht. Als Alternative zum mehrkriteriellen Wrapper-Ansatz wird in dieser Arbeit ein nicht-hierarchischer evolutionärer mehrkriterieller Optimierungsalgorithmus mit Baum-Repräsentation (NHEMOtree) entwickelt, um mehrkriterielle Optimierungsprobleme mit gleichberechtigten Optimierungszielen zu lösen. NHEMOtree basiert auf einem EMOA mit Baum-Repräsentation, der ohne internen Klassifikationsalgorithmus die Variablenselektion vollzieht und ohne Hierarchie in den Zielfunktionen mehrkriteriell optimierte binäre Entscheidungsbäume erstellt. Des Weiteren werden ein auf mehrkriteriellen Variablenwichtigkeitsmaßen (VIMs) basierter Rekombinationsoperator für NHEMOtree und eine NHEMOtree-Version mit lokaler Cutoff-Optimierung entwickelt. In dieser Arbeit werden erstmalig die Lösungen einer mehrkriteriellen Optimierung durch einen mehrkriteriellen Wrapper-Ansatz und durch einen EMOA mit Baum-Repräsentation (NHEMOtree) miteinander verglichen. Die Bewertung der Lösungen erfolgt dabei sowohl mittels der bekannten S-Metrik als auch durch den hier entwickelten Dominanzquotienten. Die Güte des VIM-basierten Rekombinationsoperators wird im Vergleich zum Standard-Rekombinationsoperator für EMOAs mit Baum-Repräsentation untersucht. Die mehrkriteriellen Optimierungsansätze und Operatoren werden auf medizinische und simulierte Daten angewendet. Die Ergebnisse zeigen, dass NHEMOtree bessere Lösungen als der mehrkriterielle Wrapper-Ansatz findet. Die Verwendung des VIM-basierten Rekombinationsoperators führt im Gegensatz zum Standard-Operator zu nochmals besseren Lösungen des mehrkriteriellen Optimierungsproblems und zu einer schnelleren Konvergenz des NHEMOtrees

Genomic analysis of Anderson typing phages of Salmonella Typhimrium: towards understanding the basis of bacteria-phage interaction

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

Elasticity Theory and Shape Transitions of Viral Shells

Recently, continuum elasticity theory has been applied to explain the shape transition of icosahedral viral capsids - single-protein-thick crystalline shells - from spherical to buckled/faceted as their radius increases through a critical value determined by the competition between stretching and bending energies of a closed 2D elastic network. In the present work we generalize this approach to capsids with non-icosahedral symmetries, e.g., spherocylindrical and conical shells. One key new physical ingredient is the role played by nonzero spontaneous curvature. Another is associated with the special way in which the energy of the twelve topologically-required five-fold sites depends on the background local curvature of the shell in which they are embedded. Systematic evaluation of these contributions leads to a shape phase diagram in which transitions are observed from icosahedral to spherocylindrical capsids as a function of the ratio of stretching to bending energies and of the spontaneous curvature of the 2D protein network. We find that the transition from icosahedral to spherocylindrical symmetry is continuous or weakly first-order near the onset of buckling, leading to extensive shape degeneracy. These results are discussed in the context of experimentally observed variations in the shapes of a variety of viral capsids.Comment: 53 pages, 17 figure

The tip of the tail needle affects the rate of DNA delivery by bacteriophage P22.

The P22-like bacteriophages have short tails. Their virions bind to their polysaccharide receptors through six trimeric tailspike proteins that surround the tail tip. These short tails also have a trimeric needle protein that extends beyond the tailspikes from the center of the tail tip, in a position that suggests that it should make first contact with the host\u27s outer membrane during the infection process. The base of the needle serves as a plug that keeps the DNA in the virion, but role of the needle during adsorption and DNA injection is not well understood. Among the P22-like phages are needle types with two completely different C-terminal distal tip domains. In the phage Sf6-type needle, unlike the other P22-type needle, the distal tip folds into a knob with a TNF-like fold, similar to the fiber knobs of bacteriophage PRD1 and Adenovirus. The phage HS1 knob is very similar to that of Sf6, and we report here its crystal structure which, like the Sf6 knob, contains three bound L-glutamate molecules. A chimeric P22 phage with a tail needle that contains the HS1 terminal knob efficiently infects the P22 host, Salmonella enterica, suggesting the knob does not confer host specificity. Likewise, mutations that should abrogate the binding of L-glutamate to the needle do not appear to affect virion function, but several different other genetic changes to the tip of the needle slow down potassium release from the host during infection. These findings suggest that the needle plays a role in phage P22 DNA delivery by controlling the kinetics of DNA ejection into the host

Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging

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

Primordial origin and diversification of plasmids in Lyme disease agent bacteria

Abstract 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 nonhomologous rearrangements

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈50 nm). In this work we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly