 |
   |
           |
|
 | |
|
Biochemistry Cell
Biology Computational
Biology Developmental
Biology Ecology Evolution Genetics Microbiology Molecular
Biology Plant
Biology Science
Education Structural
Biology |
Pham, T.T., D. Jacobs-Sera, M.L. Pedulla, R.W. Hendrix, and G.F. Hatfull (2007) Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. Microbiology 153:2711-2723 Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNA(Lys) gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions. Conway, J.F., N. Cheng, P.D. Ross, R.W. Hendrix, R.L. Duda, and A.C. Steven (2007) A thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged. J. Struct. Biol. 158:224-232 Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450A-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Delta-domain (11kDa) and gp5* (31 kDa): gp5* forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5* hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53 degrees, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation. Pope, W.H., P.R. Weigele, J. Chang, M.L. Pedulla, M.E. Ford, J.M. Houtz, W. Jiang, W. Chiu, G.F. Hatfull, R.W. Hendrix, and J. King (2007) Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a "horned" bacteriophage of marine synechococcus. J. Mol. Biol. 368:966-981 Marine Synechococcus spp and marine Prochlorococcus spp are numerically dominant photoautotrophs in the open oceans and contributors to the global carbon cycle. Syn5 is a short-tailed cyanophage isolated from the Sargasso Sea on Synechococcus strain WH8109. Syn5 has been grown in WH8109 to high titer in the laboratory and purified and concentrated retaining infectivity. Genome sequencing and annotation of Syn5 revealed that the linear genome is 46,214 bp with a 237 bp terminal direct repeat. Sixty-one open reading frames (ORFs) were identified. Based on genomic organization and sequence similarity to known protein sequences within GenBank, Syn5 shares features with T7-like phages. The presence of a putative integrase suggests access to a temperate life cycle. Assignment of 11 ORFs to structural proteins found within the phage virion was confirmed by mass-spectrometry and N-terminal sequencing. Eight of these identified structural proteins exhibited amino acid sequence similarity to enteric phage proteins. The remaining three virion proteins did not resemble any known phage sequences in GenBank as of August 2006. Cryo-electron micrographs of purified Syn5 virions revealed that the capsid has a single "horn", a novel fibrous structure protruding from the opposing end of the capsid from the tail of the virion. The tail appendage displayed an apparent 3-fold rather than 6-fold symmetry. An 18 A resolution icosahedral reconstruction of the capsid revealed a T=7 lattice, but with an unusual pattern of surface knobs. This phage/host system should allow detailed investigation of the physiology and biochemistry of phage propagation in marine photosynthetic bacteria. Weigele, P.R., W.H. Pope, M.L. Pedulla, J.M. Houtz, A.L. Smith, J.F. Conway, J. King, G.F. Hatfull, J.G. Lawrence, and R.W. Hendrix (2007) Genomic and structural analysis of Syn9, a cyanophage infecting marine Prochlorococcus and Synechococcus. Environ. Microbiol. 9:1675-1695 Cyanobacteriophage Syn9 is a large, contractile-tailed bacteriophage infecting the widespread, numerically dominant marine cyanobacteria of the genera Prochlorococcus and Synechococcus. Its 177,300 bp genome sequence encodes 226 putative proteins and six tRNAs. Experimental and computational analyses identified genes likely involved in virion formation, nucleotide synthesis, and DNA replication and repair. Syn9 shows significant mosaicism when compared with related cyanophages S-PM2, P-SSM2 and P-SSM4, although shared genes show strong purifying selection and evidence for large population sizes relative to other phages. Related to coliphage T4 - which shares 19% of Syn9's genes - Syn9 shows evidence for different patterns of DNA replication and uses homologous proteins to assemble capsids with a different overall structure that shares topology with phage SPO1 and herpes virus. Noteworthy bacteria-related sequences in the Syn9 genome potentially encode subunits of the photosynthetic reaction centre, electron transport proteins, three pentose pathway enzymes and two tryptophan halogenases. These genes suggest that Syn9 is well adapted to the physiology of its photosynthetic hosts and may affect the evolution of these sequences within marine cyanobacteria. Pham, T.T., D. Jacobs-Sera, M.L. Pedulla, R.W. Hendrix, and G.F. Hatfull (2007) Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. Microbiology 153:2711-2723 Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNA(Lys) gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions. Gan, L., J.A. Speir, J.F. Conway, G. Lander, N. Cheng, B.A. Firek, R.W. Hendrix, R.L. Duda, L. Liljas, and J.E. Johnson (2006) Capsid conformational sampling in HK97 maturation visualized by X-ray crystallography and cryo-EM. Structure 14:1655-1665 Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 A radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.
Hanauer, D.I., D. Jacobs-Sera, M.L. Pedulla, S.G. Cresawn, R.W. Hendrix, and G.F. Hatfull (2006) Inquiry learning. Teaching scientific inquiry. Science 314:1880-1881 Conway, J.F., N. Cheng, P.D. Ross, R.W. Hendrix, R.L. Duda, and A.C. Steven (2006) A thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged. J. Struct. Biol. 0: Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450A-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Delta-domain (11kDa) and gp5( *) (31kDa): gp5( *) forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5( *) hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5( *) rings from switching to a lower free energy, EI-I-like, state; above 53 degrees , this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation. Ross, P.D., J.F. Conway, N. Cheng, L. Dierkes, B.A. Firek, R.W. Hendrix, A.C. Steven, and R.L. Duda (2006) A free energy cascade with locks drives assembly and maturation of bacteriophage HK97 capsid. J. Mol. Biol. 364:512-525 We investigated the thermodynamic basis of HK97 assembly by scanning calorimetry and cryo-electron microscopy. This pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their N-terminal Delta-domains; expansion, a major conformational change; and covalent crosslinking. The thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble.
Hatfull, G.F., M.L. Pedulla, D. Jacobs-Sera, P.M. Cichon, A. Foley, M.E. Ford, R.M. Gonda, J.M. Houtz, A.J. Hryckowian, V.A. Kelchner, S. Namburi, K.V. Pajcini, M.G. Popovich, D.T. Schleicher, B.Z. Simanek, A.L. Smith, G.M. Zdanowicz, V. Kumar, C.L. Peebles, W.R. .J.r. Jacobs, J.G. Lawrence, and R.W. Hendrix (2006) Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet. 2:e92 Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.
Duda, R.L., R.W. Hendrix, W.M. Huang, and J.F. Conway (2006) Shared architecture of bacteriophage SPO1 and herpesvirus capsids. Curr. Biol. 16:440 Twarock, R., and R.W. Hendrix (2006) Crosslinking in viral capsids via tiling theory. J. Theor. Biol. 240:419-424 A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.
Wikoff, W.R., J.F. Conway, J. Tang, K.K. Lee, L. Gan, N. Cheng, R.L. Duda, R.W. Hendrix, A.C. Steven, and J.E. Johnson (2006) Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography. J. Struct. Biol. 153:300-306 The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.
Baranov, P.V., O. Fayet, R.W. Hendrix, and J.F. Atkins (2006) Recoding in bacteriophages and bacterial IS elements. Trends Genet. 22:174-181 Dynamic shifts between open reading frames and the redefinition of codon meaning at specific sites, programmed by signals in mRNA, permits versatility of gene expression. Such alterations are characteristic of organisms in all domains of life and serve a variety of functional purposes. In this article, we concentrate on programmed ribosomal frameshifting, stop codon read-through and transcriptional slippage in the decoding of phage genes and bacterial mobile elements. Together with their eukaryotic counterparts, the genes encoding these elements are the richest known source of nonstandard decoding. Recent analyses revealed several novel sequences encoding programmed alterations in gene decoding and provide a glimpse of the emerging picture.
Ross, P.D., N. Cheng, J.F. Conway, B.A. Firek, R.W. Hendrix, R.L. Duda, and A.C. Steven (2005) Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization. EMBO J 24:1352-1363 In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.
Li, Y., J.F. Conway, N. Cheng, A.C. Steven, R.W. Hendrix, and R.L. Duda (2005) Control of virus assembly: HK97 "Whiffleball" mutant capsids without pentons. J. Mol. Biol. 348:167-182 The capsid of Escherichia coli bacteriophage HK97 assembles as a 420 subunit icosahedral shell called Prohead I which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature Head II shell. Prohead I have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. We report here the properties of a mutant of the capsid protein, E219K, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wild-type Prohead I, when the mutant structure is dissociated into capsomers in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into "Whiffleballs", particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer.
Lee, K.K., H. Tsuruta, R.W. Hendrix, R.L. Duda, and J.E. Johnson (2005) Cooperative reorganization of a 420 subunit virus capsid. J. Mol. Biol. 352:723-735 The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, large-scale conformational maturation of the 420 subunit, 13 MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60 A larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles "hop" over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.
Hendrix, R.W. (2005) Bacteriophage HK97: assembly of the capsid and evolutionary connections. Adv. Virus Res. 64:1-14 Casjens, S.R., E.B. Gilcrease, D.A. Winn-Stapley, P. Schicklmaier, H. Schmieger, M.L. Pedulla, M.E. Ford, J.M. Houtz, G.F. Hatfull, and R.W. Hendrix (2005) The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J. Bacteriol. 187:1091-1104 The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.
Casjens, S., and R.W. Hendrix (2004) Bacteriophages and the bacterial genome. Pp 39-52 in The Bacterial Chromosome, Higgins, N.P., Ed. ASM Press, Washington, DC Gan, L., J.F. Conway, B.A. Firek, N. Cheng, R.W. Hendrix, A.C. Steven, J.E. Johnson, and R.L. Duda (2004) Control of crosslinking by quaternary structure changes during bacteriophage HK97 maturation. Mol. Cell 14:559-569 Radical structural changes drive the maturation of the capsid of HK97, a lambda-like, dsDNA bacteriophage of Escherichia coli. These include expansion from approximately 560 to approximately 660 A in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers. Analogous transformations also occur in unrelated viruses and protein complexes. We find that expansion and crosslinking happen concurrently during maturation at low pH. Expansion causes residues on three different subunits to move up to 35 A to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change. Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates. A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice.
Gibbs, K.A., D.D. Isaac, J. Xu, R.W. Hendrix, T.J. Silhavy, and J.A. Theriot (2004) Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB. Mol. Microbiol. 53:1771-1783 The global decline in amphibian diversity has become an international environmental problem with a multitude of possible causes. There is evidence that pesticides may play a role, yet few pesticides have been tested on amphibians. For example, Roundup® is a globally-common herbicide that is conventionally thought to be nonlethal to amphibians. However, Roundup® has been tested on few amphibian species, with existing tests conducted mostly under laboratory conditions and on larval amphibians. Recent laboratory studies have indicated that Roundup® may be highly lethal to North American tadpoles, but we need to determine whether this effect occurs under more natural conditions and in post-metamorphic amphibians. I assembled communities of three species of North American tadpoles in outdoor pond mesocosms that contained different types of soil (which can absorb the pesticide) and I applied Roundup® as a direct overspray. After 3 wks, Roundup® killed 96-100% of larval amphibians (regardless of soil presence). I then exposed three species of juvenile (post-metamorphic) anurans to a direct overspray of Roundup in laboratory containers. After 1 d, Roundup killed 68-86% of juvenile amphibians. These results suggest that Roundup&re
Casjens, S.R., E.B. Gilcrease, W.M. Huang, K.L. Bunny, M.L. Pedulla, M.E. Ford, J.M. Houtz, G.F. Hatfull, and R.W. Hendrix (2004) The pKO2 linear plasmid prophage of Klebsiella oxytoca. J. Bacteriol. 186:1818-1832 Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.
Dobbins, A.T., M. George Jr., D.A. Basham, M.E. Ford, J.M. Houtz, M.L. Pedulla, J.G. Lawrence, G.F. Hatfull, and R.W. Hendrix (2004) Complete genomic sequence of the virulent Salmonella bacteriophage SP6. J. Bacteriol. 186:1933-1944 We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is approximately 5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.
Benevides, J.M., P. Bondre, R.L. Duda, R.W. Hendrix, and G.J. .J.r. Thomas (2004) Domain structures and roles in bacteriophage HK97 capsid assembly and maturation. Biochemistry 43:5428-5436 Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.
Hendrix, R.W. (2004) Hot new virus, deep connections. Proc. Natl. Acad. Sci., USA 101:7495-7496
Lee, K.K., L. Gan, H. Tsuruta, R.W. Hendrix, R.L. Duda, and J.E. Johnson (2004) Evidence that a local refolding event triggers maturation of HK97 bacteriophage capsid. J. Mol. Biol. 340:419-433 Bacteriophage capsids are a striking example of a robust yet dynamic genome delivery vehicle. Like most phages, HK97 undergoes a conformational maturation that converts a metastable Prohead into the mature Head state. In the case of HK97, maturation involves a significant expansion of the capsid and concomitant cross-linking of capsid subunits. The final state, termed Head-II, is a 600 angstroms diameter icosahedral structure with catenated subunit rings. Cryo-EM, small angle X-ray scattering (SAXS), and biochemical assays were used previously to characterize the initial (Prohead-II) and final states (Head-II) as well as four maturation intermediates. Here we extend the characterization of the acid-induced expansion of HK97 in vitro by monitoring changes in intrinsic fluorescence, circular dichroism (CD), and SAXS. We find that the greatest changes in all observables occur at an early stage of maturation. Upon acidification, fluorescence emissions from HK97 exhibit a blueshift and decrease in intensity. These spectral changes reveal two kinetic phases of the expansion reaction. The early phase exhibits sensitivity to pH, increasing in rate nearly 200-fold when acidification pH is lowered from 4.5 to 3.9. The second, slower phase reported by fluorescence is relatively insensitive to pH. Time-resolved SAXS experiments report an increase in overall particle dimension that parallels the fluorescence changes for the early phase. Native agarose gel assays corroborated this finding. By contrast, probes of CD at far-UV indicate that secondary structural changes precede the early expansion phase reported by SAXS and fluorescence. Based on the crystallographic structure of Head-II and the pseudo-atomic model of Prohead-II, we interpret these changes as reflecting the conversion of subunit N-terminal arms (N-arm) from unstructured polypeptide to the mixture of beta-strand and beta-turn observed in the Head-II crystal structure. Refolding of the N-arm may thus represent the conformational trigger that initiates the irreversible expansion of the phage capsid.
Xu, J., R.W. Hendrix, and R.L. Duda (2004) Conserved translational frameshift in dsDNA bacteriophage tail assembly genes. Mol. Cell 16:11-21 A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.
Helgstrand, C., W.R. Wikoff, R.L. Duda, R.W. Hendrix, J.E. Johnson, and L. Liljas (2003) The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution. J. Mol. Biol. 334:885-899 The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits. The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18A and the maximum diameter is 550A. The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization. The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion. Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar. The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits. The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation. A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.
Wikoff, W.R., Z. Che, R.L. Duda, R.W. Hendrix, and J.E. Johnson (2003) Crystallization and preliminary analysis of a dsDNA bacteriophage capsid intermediate: Prohead II of HK97. Acta Crystallogr. D 59:2060-2064 HK97 Prohead II is an early intermediate in the maturation of HK97, a T = 7 dsDNA-tailed bacteriophage related to bacteriophage lambda. Previously, selected capsid-protein genes of HK97 were expressed in Escherichia coli and spontaneously assembled to form an icosahedral capsid that followed a maturation pathway closely similar to the authentic virion. The crystal structure of the mature HK97 capsid (Head II) made in this way was reported at 3.5 A resolution. Additional high-resolution structures of intermediates are needed to understand the maturation mechanism. The crystal structure of expressed Prohead II will elucidate the early steps of HK97 assembly. Crystals of the Prohead II mutant W336F were grown in 0.1 M HEPES pH 7.5, 0.2 M CaCl(2) and 2-3% PEG 4000 at a Prohead II concentration of 16.5 mg ml(-1). It was not possible to grow high-quality crystals of wild-type Prohead II. Diffraction was observed to 5 A resolution from these crystals on beamline 14BM-C at the Advanced Photon Source and data were collected to 5.5 A with a completeness of 77%. The space group was P2(1)3, with unit-cell parameter a = 707.0 A and four particles in the unit cell. The particles are on the body diagonals of the cubic cell, with icosahedral threefold axes coincident with crystallographic threefold axes. Self-rotation function and locked-rotation function analysis determined the particle orientation and a one-dimensional R-factor search along the body diagonal indicated that the particle centers were close to (1/4, 1/4, 1/4) and symmetry-related positions. Molecular-replacement averaging and phase extension are under way.
Hendrix, R.W. (2003) Bacteriophage genomics. Curr. Opin. Microbiol. 6:506-511 Comparative genomic studies of bacteriophages, especially the tailed phages, together with environmental studies, give a dramatic new picture of the size, genetic structure and dynamics of this population. Sequence comparisons reveal some of the detailed mechanisms by which these viruses evolve and influence the evolution of their bacterial and archaeal hosts. We see rampant horizontal exchange of sequences among genomes, mediated by both homologous and nonhomologous recombination. High frequency exchange among phages occupying similar ecological niches leads to a high degree of mosaic diversity in local populations. Horizontal exchange also takes place at lower frequency across the entire span of phage sequence space.
Hendrix, R.W., G.F. Hatfull, and M.C. Smith (2003) Bacteriophages with tails: chasing their origins and evolution. Res. Microbiol. 154:253-257 Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution. Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages. It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons. Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages.
Pedulla, M.L., M.E. Ford, T. Karthikeyan, J.M. Houtz, R.W. Hendrix, G.F. Hatfull, A.R. Poteete, E.B. Gilcrease, D.A. Winn-Stapley, and S.R. Casjens (2003) Corrected sequence of the bacteriophage P22 genome. J. Bacteriol. 185:1475-1477 We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences. DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources.
Pedulla, M.L., M.E. Ford, J.M. Houtz, T. Karthikeyan, C. Wadsworth, J.A. Lewis, D. Jacobs-Sera, J. Falbo, J. Gross, N.R. Pannunzio, W. Brucker, V. Kumar, J. Kandasamy, L. Keenan, S. Bardarov, J. Kriakov, J.G. Lawrence, W.R. Jacobs, R.W. Hendrix, and G.F. Hatfull (2003) Origins of highly mosaic mycobacteriophage genomes. Cell 113:171-182 Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.
Hendrix, R.W. (2002) Bacteriophage lambda and its relatives. Pp 127-143 in Modern Microbial Genetics, Streips, U., and R. Yasbin, Ed. Wiley-Liss, New York Hendrix, R.W. (2002) Bacteriophage : Evolution of the majority. Theor. Popul. Biol. 61:471-480
Morgan, G.J., G.F. Hatfull, and R.W. Hendrix (2002) Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus. J. Mol. Biol. 317:337-359 We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.
Lawrence, J.G., G.F. Hatfull, and R.W. Hendrix (2002) Imbroglios of viral taxonomy: Genetic exchange and the failings of phenetic approaches. J. Bacteriol. 184:4891-4905 The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linneaus. The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification. While long established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task, and has proven exceedingly difficult for describing viruses and bacteriophages. Moreover, the assembly of viral "species" into higher-order taxonomic groupings has been even more tenuous, based initially on limited numbers of morphological features and more recently on overall genomic similarities. The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms obligates a reëvaluation of the concept of viral species, genera, families and higher levels of classification. Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes. For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnean paradigm that necessitate a new view of viral evolution, classification and taxonomy.
Brussow, H., and R.W. Hendrix (2002) Phage genomics: small is beautiful. Cell 108:13-16 The Age of Genomics dawned only gradually for bacteriophages. It was 1977 when the genome of phage phi X174 was published and 1983 when the "large" genome of phage lambda hit the streets. More recently, the pace has quickened, so that we now have over 100 complete phage genomes and can expect thousands in a very few years. These sequences have been marvelously informative for the biology of the individual phages, but with the advent of high volume sequencing technology, the real excitement for phage biology is that it is now possible to analyze the sequences together and thereby address--for the first time at whole genome resolution--a set of fundamental biological questions related to populations: What is the structure of the global phage population? What are its dynamics? How do phages evolve? This is Comparative Genomics with a capital "C".
Lawrence, J.G., R.W. Hendrix, and S. Casjens (2001) Where are the pseudogenes in bacterial genomes? Trends Microbiol. 9:535-540 Most bacterial genomes have very few pseudogenes; notable exceptions include the genomes of the intracellular parasites Rickettsia prowezekii and Mycobacterium leprae. We propose that the influx of dangerous genetic elements (transposons and bacteriophages) selects for the maintenance of relatively high deletion rates in most bacteria; the sheltered lifestyle of intracellular parasites removes this threat, leading to reduced deletion rates and larger pseudogene loads.
Conway, J.F., W.R. Wikoff, N. Cheng, R.L. Duda, R.W. Hendrix, J.E. Johnson, and A.C. Steven (2001) Virus maturation involving large subunit rotations and local refolding. Science 292:744-748 Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head- II, was recently solved by crystallography, revealing a catenated cross- linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.
Saint Girons, I., P. Bourhy, C. Ottone, M. Picardeau, D. Yelton, R.W. Hendrix, P. Glaser, and N. Charon (2000) The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector. J. Bacteriol. 182:5700-5705 We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.
Mediavilla, J., S. Jain, J. Kriakov, M.E. Ford, R.L. Duda, W.R. Jacobs Jr., R.W. Hendrix, and G.F. Hatfull (2000) Genome organization and characterization of mycobacteriophage Bxb1. Mol. Microbiol. 38:955-970 Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow-growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.
Hendrix, R.W., J.G. Lawrence, G.F. Hatfull, and S. Casjens (2000) The origins and ongoing evolution of viruses. Trends Microbiol. 8:504-508 Genome analyses of dsDNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes, and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.
Wikoff, W.R., L. Liljas, R.L. Duda, H. Tsuruta, R.W. Hendrix, and J.E. Johnson (2000) Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289:2129-2133 The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution. The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry. Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid. Ravin, V., N. Ravin, S. Casjens, M.E. Ford, G.F. Hatfull, and R.W. Hendrix (2000) Genomic sequence and analysis of the atypical temperate bacteriophage N15. J. Mol. Biol. 299:53-73 N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. We describe here the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely-related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.
Lata, R., J.F. Conway, N. Cheng, R.L. Duda, R.W. Hendrix, W.R. Wikoff, J.E. Johnson, H. Tsuruta, and A.C. Steven (2000) Maturation dynamics of a viral capsid: visualization of transitional intermediate states. Cell 100:253-263 Typical of DNA bacteriophages and herpesviruses, HK97 assembles in two stages: polymerization and maturation. First, capsid protein polymerizes into closed shells; then, these precursors mature into larger, stabler particles. Maturation is initiated by proteolysis, producing a metastable particle primed for expansion-the major structural transition. We induced expansion in vitro by acidic pH and monitored the resulting changes by time-resolved X-ray diffraction and cryo-electron microscopy. The transition, which is not synchronized over the population, proceeds in a series of stochastically triggered subtransitions. Three distinct intermediates were identified, which are comparable to transitional states in protein folding. The intermediates' structures reveal the molecular events occurring during expansion. Integrated into a movie (see Dynamic Visualization below), they show capsid maturation as a dynamic process.
Juhala, R.J., M.E. Ford, R.L. Duda, A. Youlton, G.F. Hatfull, and R.W. Hendrix (2000) Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages. J. Mol. Biol. 299:27-52
We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), dsDNA bacteriophages of Escherichia coli and members of the lambdoid or l-like group of phages. We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E. coli phage lambda and Salmonella typhimurium phage P22. The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence. The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses. The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or in some cases at protein domain boundaries within genes. Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome.
Weisberg, R.A., M.E. Gottesman, R.W. Hendrix, and J.W. Little (1999) Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022. Annu. Rev. Genet. 33:565-602 HK022 is a temperate coliphage related to phage lambda. Its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied. In the overall arrangement, expression, and function of most of its genes, HK022 broadly resembles lambda and other members of the lambda family. Upon closer view, significant differences emerge. The differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown regulatory and structural motifs. HK022 prophages protect lysogens from superinfection by producing a sequence-specific RNA binding protein that prematurely terminates nascent transcripts of infecting phage. It uses a novel RNA-based mechanism to antiterminate its own early transcription. The HK022 protein shell is strengthened by a complex pattern of covalent subunit interlinking to form a unitary structure that resembles chain-mail armour. Its integrase and repressor proteins are similar to those of lambda, but the differences provide insights into the evolution of biological specificity and the elements needed for construction of a stable genetic switch. Hendrix, R.W. (1999) The long evolutionary reach of viruses. Curr. Biol. 9:R914-R917 The structure of a phage capsid protein provides good evidence this phage shares ancestry with an animal virus. In this and similar cases, either the viral lineages predate the emergence of the three contemporary domains of life, or viruses have been leaping the phylogenetic chasms that separate the domains. Hendrix, R.W., M.C.M. Smith, R.N. Burns, M.E. Ford, and G.F. Hatfull (1999) Evolutionary relationships among diverse bacteriophages and prophages: All the world's a phage. Proc. Natl. Acad. Sci., USA 96:2192-2197 We report DNA and predicted protein sequence similarities, implying homology, among genes of double stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage fC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, fFlu, in the Haemophilus influenzae genome, and two small prophage-like elements, fRv1 and fRv2, in the genome of M. tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phages are mosaices with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phages.
Wikoff, W.R., R.L. Duda, R.W. Hendrix, and J.E. Johnson (1999) Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid. Acta Crystallogr. D. 55:763-771 HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is similar in architecture to bacteriophage lambda, the type member of this family, with an icosahedral capsid of triangulation number T = 7. No high-resolution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-rays. At 650 A in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. The empty Head II (mature) particles were expressed in Escherichia coli and assembled in vitro, but they have the same morphology as the mature HK97 capsid. Previously reported Head II crystals diffracting to 3.5 A resolution are examined here in detail. Although the cell dimensions suggest an orthorhombic lattice, further analysis demonstrated that the space group was monoclinic. This has been confirmed by the present study. Images were recorded on the F1 beamline at CHESS and they were processed and scaled, resulting in a data set with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 A. The cell dimensions after post-refinement were a = 580, b = 626, c = 788 A, beta = 90.0 degrees. From the particle dimensions determined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even reflections along the 0k0 lattice line indicate that the space group is P21. The rotation function was used to determine the orientation of the particles in the unit cell and to confirm the space group. An icosahedral twofold axis is approximately, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to the b axis, is rotated 18 degrees away from the a axis. The centers of the two particles must be positioned close to the minimum-energy packing arrangement for spheres, which places one particle at ((1/4), 0, (1/4)) and the other particle at ((3/4), (1/2), (3/4)). The particle position and orientation were confirmed by calculating a Patterson function. The particles interact closely along icosahedral threefold axes, which occurs both along the crystallographic a axis and along the b axis. The particle dimensions derived from this packing arrangement agree well with those determined by cryo-EM and image reconstruction. The cryo-EM reconstruction will be used as a model to initiate phase determination; structure determination at 7 A is under way. Ford, M.E., C. Stenstrom, R.W. Hendrix, and G.F. Hatfull (1998) Mycobacteriophage TM4: Genome structure and gene expression. Tubercule Lung Dis. 79:63-73 Mycobacteriophage TM4 is a dsDNA-tailed phage that infects both fast-growing and slow-growing strains of mycobacteria. While TM4 has been used extensively for the construction of mycobacterial shuttle phasmids and for the delivery of reporter genes and transposons into mycobacterial cells, little is known about its genetics or molecular biology. We describe here the complete 52,797 bp genome sequence of TM4 and a map of its genome organization. While not a close relative of other mycobacteriophages, TM4 encodes several proteins with sequence similarity to those of other bacteriophages--including L5 and D29--indicating that they have common ancestry. In addition, TM4 encodes proteins with similarity to haloperoxidases, glutaredoxins and the WhiB family of transcriptional regulators. Following infection, TM4 genes are expressed in a defined temporal pattern, with the virion structural proteins expressed late in the phage growth cycle. Understanding the genetics of TM4 will greatly facilitate its use as a tool for the genetic manipulation of the mycobacteria. Smith, M.C.M., N. Burns, J.R. Sayers, J.A. Sorrell, S.R. Casjens, and R.W. Hendrix (1998) Bacteriophage collagen. Science 279:1834 Wikoff, W.R., R.L. Duda, R.W. Hendrix, and J.E. Johnson (1998) Crystallization and preliminary X-ray analysis of the dsDNA bacteriophage HK97 mature empty capsid. Virology 243:113-118 HK97 is a temperate dsDNA bacteriophage of Escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7. Although the capsids of several large dsDNA phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. We have grown crystals of mature but empty bacteriophage HK97 capsids that diffract to at least 3.5 A using synchrotron radiation. The HK97 Head II crystals are the first capsid crystals from a dsDNA bacteriophage that diffract X-rays to high resolution. A capsid precursor (prohead) was made in vivo by expressing capsid proteins in E. coli. This prohead was purified, converted to Head II in vitro, and used to grow crystals. The empty Head II has the same form as the mature HK97 capsid, but without DNA. The crystals were grown in a mixture of ammonium sulfate and PEG 8000, directly in an X-ray capillary to minimize crystal handling. The unit cell is monoclinic, with dimensions a = 580 A, b = 625 A, c = 790 A, beta = 90.0 degrees and two particles per unit cell. Hendrix, R.W., and R.L. Duda (1998) Bacteriophage HK97 head assembly: a protein ballet. Adv. Virus Res. 50:235-288 Ford, M.E., G.J. Sarkis, A.E. Belanger, R.W. Hendrix, and G.F. Hatfull (1998) Genome structure of mycobacteriophage D29: implications for phage evolution. J. Mol. Biol. 279:143-164 Mycobacteriophage D29 is a lytic phage that infects both fast and slow-growing mycobacterial species. The complete genome sequence of D29 reveals that it is a close relative of the temperate mycobacteriophage L5, whose sequence has been described previously. The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens. Comparison of the two genomes shows that they are punctuated by a large number of insertions, deletions, and substitutions of genes, consistent with the genetic mosaicism of lambdoid phages.
Hendrix, R.W. (1998) Bacteriophage DNA packaging: RNA gears in a DNA transport machine. Cell 94:147-150
Duda, R.L., K. Maartinic, Z. Xie, and R.W. Hendrix (1995) Bacteriophage HK97 head assembly. FEMS Microbiol. Rev. 17:41-46 The head assembly pathway of bacteriophage HK97 shares many features with head assembly pathways determined for other dsDNA phages, and it also provides examples of novel variations on the basic theme. We describe aspects of two specific steps in the assembly pathway, the covalent cross-linking among the assembled head protein subunits and the cleavage of those subunits that takes place earlier in the pathway. Comparisons of head assembly pathways among different phages, as well as comparisons of the organization of the genes that specify those pathways, suggest the range of different solutions phages have found to common assembly problems and give insight into the evolutionary histories of these assembly processes. Ding, Y., R.L. Duda, R.W. Hendrix, and J.M. Rosenberg (1995) Complexes between chaperonin GroEL and the capsid protein of bacteriophage HK97. Biochemistry 34:14918-14931 The 42 kDa capsid protein of bacteriophage HK97 requires the GroEL and GroES chaperonin proteins of its Escherichia coli host to facilitate correct folding, both in vivo and in vitro. In the absence of GroES and ATP, denatured gp5 forms a stable complex with the 14 subunit GroEL molecule. We characterized the electrophoretic and biochemical properties of this complex. In electrophoresis on a native (nondenaturing) gel, the band of the gp5-GroEL complex shifts to a slower migrating position relative to uncomplexed GroEL. The results show that there is only one subunit of gp5 bound to each GroEL 14-mer and that the shift in band position is due primarily to a change in the overall charge of the complex relative to uncomplexed GroEL, and not to a change in size or shape. GroEL forms similar complexes with proteolytic fragments of gp5, with a series of sequence duplication derivatives of gp5, and with other proteins. Electrophoretic examination of these complexes shows that a band shift occurs with proteins larger than 31-33 kDa but not with smaller proteins. For those proteins that cause a band shift upon complex formation, the magnitude of the shift is correlated with the predicted if the charge of the complex were simply the sum of the charge of GroEL and the charge of the substrate protein. We suggest that binding of a substrate protein to GroEL is accompanied by a net binding of solution cations to the complex, but only in the case of proteins above a minimum size of 31-33 kDa. The gp5-GroEL complex is in an association/dissociation equilibrium, with a binding constant measured in the range of 11-17 microM-1. Conway, J., R. Duda, N. Cheng, R. Hendrix, and A. Steven (1995) Proteolytic and conformational control of virus capsid mautration: The bacteriophage HK97 system. J. Mol. Biol. 253:86-99 Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes. These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling. Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion. In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation. To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II. Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers. The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 Å. Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein. Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation. No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 Å, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites").
Xie, Z., and R.W. Hendrix (1995) Assembly in vitro of bacteriophage HK97 proheads. J. Mol. Biol. 253:74-85 Bacteriophage HK97 is a lambdoid phage with a head assembled from 415 copies of a 42 kDa subunit arranged in an icosahedrally symmetrical lattice with a triangulation number of 7. Prohead I, the first shell structure in the assembly pathway, is composed of 42 kDa coat protein subunits that have not yet undergone the proteolytic cleavage, conformational changes, and covalent cross-linking steps that occur later in the assembly of mature heads. Prohead I can be efficiently dissociated into capsomeres by treatment with 2 M KCl. The resulting capsomeres are a mixture of two species, identified as pentamers and hexamers of the 42 kDa subunit. These capsomeres were also detected as the products of chaperonin-assisted renaturation of 42 kDa polypeptide in vitro at room temperature or in the course of self folding and assembly in vitro at 0 degrees C. Pentamer and hexamer capsomeres can be interconverted in vitro by manipulating solvent conditions, and this makes it possible to carry out the in vitro shell assembly reaction at different input ratios of hexamer to pentamer. The Prohead I structures produced are always the normal (T = 7) size regardless of the input pentamer to hexamer ratio. Assembly is most efficient when the pentamer to hexamer ratio is 1:5 (a mass ratio of 1:6), or the same as the capsomere ratio in a T = 7 shell.
Duda, R.L., K. Martincic, and R.W. Hendrix (1995) Genetic basis of bacteriophage HK97 prohead assembly. J. Mol. Biol. 247:636-647 We report studies to determine which bacteriophage genes are required for assembly of phage HK97 proheads and what roles they play. We identify the gene encoding the major capsid protein of phage HK97 and report its DNA sequence, together with the DNA sequences of the two genes immediately upstream from it. When the capsid protein is expressed from a plasmid in the absence of other phage-encoded proteins, it assembles, with good efficiency and accuracy into prohead-like structures composed of the unprocessed 42 kDa capsid protein. No separately encoded scaffolding protein is required for this assembly. If the 25 kDa product of the next gene upstream is co-expressed with the capsid protein, the prohead structures that are produced undergo the normal morphogenetic cleavage, which removes 102 amino acids from the N terminus of each subunit, leaving 31 kDa subunits. The 25 kDa protein is therefore probably a phage-encoded protease. The third gene, upstream from the protease gene, encodes the portal protein. Presence of the portal protein is not required for assembly of the capsid protein. Analysis of the phenotypes of four single amino acid-substitution mutants in the capsid-protein gene leads to several insights into the functions of the capsid protein and its interactions with the putative protease.
Duda, R.L., J. Hempel, H. Michael, J. Shabanowitz, D. Hunt, and R.W. Hendrix (1995) Structural transitions during bacteriophage HK97 head assembly. J. Mol. Biol. 247:618-635 Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.
Hendrix, R.W., and R.L. Garcea (1994) Capsid assembly of dsDNA viruses. Seminars in Virol. 5:15-26 Hendrix, R.W. (1993) Virus shell game. Biophys. J. 64:836-837
Levin, M.E., R.W. Hendrix, and S.R. Casjens (1993) A programmed translational frameshift is required for the synthesis of a bacteriophage lambda tail assembly protein. J. Mol. Biol. 234:124-139 Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage lambda tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a translational frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys- in both the zero and -1 reading frames. The frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance frameshifting, as is seen in other well-characterized frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the frameshifting rate. Our results together with those of Katsura and Kuhl show that the frameshift product, gpG-T, has an essential role in lambda tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.
Casjens, S., G. Hatfull, and R. Hendrix (1992) Evolution of dsDNA tailed-bacteriophage genomes,. Seminars in Virol. 3:383-397 Hendrix, R.W., and R.L. Duda (1992) Bacteriophage lambda PaPa: not the mother of all lambda phages. Science 258:1145-1148 The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly. Hendrix, R.W. (1991) Protein carpentry. Curr. Biol. 1:71-73 Popa, M.P., T.A. McKelvey, J. Hempel, and R.W. Hendrix (1991) Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits. J. Virol. 65:3227-3237 We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation.
Casjens, S., and R.W. Hendrix (1988) Control mechanisms in dsDNA bacteriophage assembly. Pp 15-91 in The Bacteriophages, Calendar, R., Ed. Plenum Publishers, New York Hendrix, R.W. (1988) Tail length determination in double-stranded DNA bacteriophages. Curr. Top. Microbiol. Immunol. 136:21-29 Hemmingsen, S.M., C. Woolford, S.M. vander Vies, K. Tilly, D.T. Dennis, C.P. Georgopolos, R.W. Hendrix, and R.J. Ellis (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333:330-334 An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon. Sampson, L.L., R.W. Hendrix, W.M. Huang, and S.R. Casjens (1988) Translation initiation controls the relative rates of expression of the bacteriophage lambda late genes. Proc. Natl. Acad. Sci., USA 85:5439-5443 The late operon of bacteriophage lambda contains the genes encoding the morphogenetic proteins of the phage. These genes are transcribed equally from the single late promoter. Although the functional half-lives of the mRNA for the various genes of this operon vary less than 2-fold, their relative rates of expression have been shown to vary by nearly 1000-fold. This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare. To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid lambda-beta-galactosidase proteins. The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes.
Murphy, K.C., L. Casey, N. Yannoutsos, A.R. Poteete, and R.W. Hendrix (1987) Localization of a DNA-binding determinant in the bacteriophage P22 Erf protein. J. Mol. Biol. 194:105-117 Four amber fragments of the recombination-promoting P22 Erf protein were characterized. The intact Erf monomer contains 204 amino acids. The amber mutations produce fragments of 190, 149, 130 and 95 amino acid residues, all of which are inactive in vivo. The 190 residue fragment is more susceptible to proteolysis in cell extracts than is intact Erf. It breaks down to a stable remnant that is slightly larger than the 149 residue fragment. The 149 and 130 residue fragments are stable; electron microscopy of the purified fragments reveals that they have similar morphologies, retaining the ring-like oligomeric structure, but lacking the tooth-like protruding portions of intact Erf. Intact Erf and the 149 residue fragment have similar affinities for single-stranded DNA; the affinity of the 130 residue fragment is 40-fold lower in low salt at pH 6.0. The 95 residue fragment is unstable in vivo. These observations, combined with previous observations, are interpreted as suggesting that the boundary of the amino-terminal domain of the protein lies between residues 96 and 130, that certain residues between 131 and 149 form part of an interdomain DNA-binding segment of the protein, that the boundary of the carboxy-terminal domain lies to the C-terminal side of residue 149, and that the carboxy-terminal domain is not necessary for assembly of the ring oligomer, although it is essential for Erf activity in vivo. Chandrasekhar, G.N., K. Tilly, C. Woolford, R.W. Hendrix, and C.P. Georgopolos (1986) Purification and properties of the groES morphogenetic protein of Escherichia coli. J. Biol. Chem. 261:12414-12419 The morphogenesis of lambda proheads is governed by the products of at least four bacteriophage-coded genes (B, C, E and Nu3) and two host-coded genes (groES (mopB) and groEL (mopA)). Earlier genetic experiments indicated that the phenotypes of some of the groES- mutations could be suppressed by mutations in the groEL gene, suggesting an interaction between the two groE proteins in vivo (Tilly, K., and Georgopoulos, C. P. (1982) J. Bacteriol. 149, 1082-1088). The Mr 15,000 groES protein was overproduced and purified to homogeneity by monitoring its presence after polyacrylamide gel electrophoresis. Both gel filtration on an AcA34 sizing column and glycerol gradient centrifugation indicate that the groES protein possesses an oligomeric structure of Mr 80,000. In agreement, electron microscopic pictures of the purified groES protein show that it possesses a symmetrical ring-like structure. The sequence of the first five amino acids and the overall composition of the purified protein match those predicted by the nucleotide sequence of the groES gene. The following results implicate a physical association between the groES and groEL proteins in vitro. The groES protein inhibits the weak ATPase activity of the groEL protein, with a maximal effect seen at a 1:1 molar ratio; the two proteins cosediment during glycerol gradient centrifugation in the presence of ATP and Mg2+; and the groES protein binds specifically to a groEL-affinity column. These results help explain why mutations in either of the groE genes exhibit similar phenotypes with respect to both lambda and bacterial growth.
Hendrix, R.W. (1985) Shape determination in virus assembly. Pp in Virus Structure and Assembly, Casjens, S., Ed. Jones and Bartlett, Cambridge, MA Cowing, D.W., J.C. Bardwell, E.A. Craig, C. Woolford, R.W. Hendrix, and C.A. Gross (1985) Consensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci., USA 82:2679-2683 We have identified promoters for the Escherichia coli heat shock operons dnaK and groE and the gene encoding heat shock protein C62.5. Transcription from each promoter is heat-inducible in vivo, and each is recognized in vitro by RNA polymerase containing sigma 32, the sigma factor encoded by rpoH (htpR) but not by RNA polymerase containing sigma 70. We compared the sequences of the heat shock promoters and propose a consensus promoter sequence, having T-N-t-C-N-C-c-C-T-T-G-A-A in the -35 region and C-C-C-C-A-T-t-T-a in the -10 region. These sequences differ from the consensus sequence recognized by holoenzyme containing sigma 70, the major sigma in E. coli. We suggest that the accumulated consensus sequences of promoters recognized by alternate forms of holoenzyme are compatible with a model in which sigma recognizes only the -10 region of the promoter.
Katsura, I., and R.W. Hendrix (1984) Length determination in bacteriophage lambda tails. Cell 39:691-698 We have isolated viable mutants of bacteriophage lambda that have in-frame deletions in gene H, which codes for a minor tail protein. They produce correspondingly smaller but active gene H protein products and assemble shorter-tailed phage particles. The deficiency in tail length for each mutant corresponds to the calculated shortening of the gene H protein caused by the deletion. These results show that the H protein determines tail length and argue strongly for a scheme in which the H protein is a ruler or template that measures length during tail assembly.
Tsui, L.C., and R.W. Hendrix (1983) Proteolytic processing of phage lambda tail protein gpH: timing of the cleavage. Virology 125:257-264 We describe a method for the rapid partial purification of intermediate structures of phage lambda tail assembly, using formaldehyde-fixed Escherichia coli cells to precipitate tail-related structures. The purification depends on the specific interaction between the E. coli lambda receptor protein and lambda tail protein gpJ. Protein compositions of tail assembly intermediates were analyzed to determine when in the assembly sequence the minor tail protein gpH is cleaved. gpH joins the tail precursor structure early in the pathway, during assembly of the initiator (a structure that becomes the tail tip). However, gpH is not cleaved until after initiator assembly is complete and after the tail shaft has polymerized onto the initiator. These results suggest that each gpH molecule is extended along the length of the tail. Our results also appear to eliminate an ambiguity in the tail assembly pathway determined by earlier experiments: we argue that gene G acts between genes H and M. Tsui, L.C., and R.W. Hendrix (1983) Role of gene T in phage lambda tail assembly. Virology 125:265-273 We have studied the phenotype of a heat-sensitive mutation that defines lambda tail gene T. Induction and growth at the nonpermissive temperature of a lambda Tts40 prophage results in production of morphologically normal but biologically inactive tails, which can be activated in vitro by lysates supplying the products of genes T, U, and Z. Thus, gene T acts between genes V and U in the tail assembly pathway or immediately after the completion of tail shaft polymerization. lambda Tts40 lysates also contain a substantial number of morphologically normal phage particles which are deficient in cleavage of the minor tail protein gpH. This result is consistent with the fact that gpH cleavage normally occurs later in the assembly pathway than the time found for gene T action. Purified lambda Tts40 virions that were produced at the permissive temperature are normal with respect to gpH cleavage. However, they are more sensitive to heat inactivation than are wild-type virions, suggesting that the T gene product is in the virion. At the permissive temperature, the mutant protein made by lambda Tts40 is active in vivo even if it has been synthesized at the nonpermissive temperature. Poteete, A.R., R.T. Sauer, and R.W. Hendrix (1983) Domain structure and quaternary organization of the bacteriophage P22 Erf protein. J. Mol. Biol. 171:401-418 The structure and activities of the recombination-promoting P22 Erf protein were examined in vitro. Treatment of the protein with elastase produces a stable amino-terminal fragment, consisting of amino acid residues 1 to (approximately) 136. We have purified this fragment, designated fragment B, to apparent homogeneity by gel filtration chromatography. Fragment B retains the oligomeric structure and single-stranded DNA binding specificity of intact Erf. It differs, however, in lacking the ability of intact Erf to bind single-stranded DNA into large aggregates following mild heat treatment of the protein. In addition, its binding to DNA may be weaker than that of intact Erf. Intact Erf sediments through a sucrose gradient as a discrete species with an apparent S20,w of approximately 11 X 7 S. Its sedimentation behavior is affected little, if at all, by concentration. Fragment B also sediments as a discrete species at approximately 10 X 4 S. In the electron microscope, intact Erf appears as rings, with 10 to 14 small projecting structures resembling the teeth of a gear. Fragment B is similar, except that it appears to lack the peripheral structures. From these observations, we conclude that Erf consists of at least two structurally and functionally distinct domains, and that it has a discrete ring-like oligomeric structure. Georgopoulos, C., K. Tilly, D. Drahos, and R. Hendrix (1982) The B66.0 protein of Escherichia coli is the product of the dnaK+ gene. J. Bacteriol. 149:1175-1177 Drahos, D.J., and R.W. Hendrix (1982) Effect of bacteriophage lambda infection on synthesis of GroE protein and other Escherichia coli proteins. J. Bacteriol. 149:1050-1063 We used two-dimensional gel electrophoresis to quantitate the changes in rates of synthesis that follow phage lambda infection for 21 Escherichia coli proteins, including groE and dnaK proteins. Although total protein synthesis and the rates of synthesis of most individual E. coli proteins decreased after infection, some proteins, including groE protein, dnaK protein, and stringent starvation protein, showed increases to rates substantially above their preinfection rates. Infection by lambda Q- affected host synthesis in the same way as infection by gamma+, whereas infection by lambda N- showed no detectable effect on host synthesis. Deletion of the early genes between att and N abolished the effect, and shorter deletions in this region gave intermediate effects. By this sort of deletion mapping, we show that a large part, though not all, of the effect of lambda infection on host protein synthesis can be ascribed to the early region that contains phage genes Ea10 and ral. We compared the changes in protein synthesis after infection with the changes that occur in uninfected cells upon heat shock or amino acid starvation. The spectrum of changes that occurred on infection was very different from that seen after heat shock but quite similar to that seen during amino acid starvation. Despite this similarity of the effects of lambda infection and starvation, we did not detect any increase in the level of guanosine tetraphosphate during infection. We show that the groE protein is the same protein as B56.5 of Lemaux et al. (Cell 13:427-434, 1978) and A protein of Subramanian et al. (Eur. J. Biochem. 67:591-601, 1976).
Tsui, L., and R.W. Hendrix (1980) Head-tail connector of bacteriophage lambda. J. Mol. Biol. 142:419-438 Hendrix, R.W. (1979) Bacteriophage assembly. Nature 277:172-173 Hendrix, R.W. (1979) Purification and properties of groE, a host protein involved in bacteriophage assembly. J. Mol. Biol. 129:375-392 Earnshaw, W.C., R.W. Hendrix, and J. King (1979) Structural studies of bacteriophage lambda heads and proheads by small angle X-ray diffraction. J. Mol. Biol. 134:575-594 Hendrix, R.W., and L. Tsui (1978) Role of the host in virus assembly: cloning of the Escherichia coli groE gene and identification of its protein product. Proc. Natl. Acad. Sci., USA 75:136-139 Correct assembly of the heads of bacteriophages lambda and T4 requires the function of the groE gene of the Escherichia coli host. We have isolated a transducing derivative of lambda, called lambda gt-Ec.groE, that carries a functional copy of the groE gene. Unlike wild-type lambda, this phage is able to form plaques on hosts with a mutant groE gene. We have isolated an amber mutation in the groE gene carried by the phage, and this has made it possible to identify the groE product as a protein of molecular weight 65,000. In the phage, the groE gene is under the control of an early phage promoter.
Hendrix, R.W. (1978) Symmetry mismatch and DNA packaging in large bacteriophages. Proc. Natl. Acad. Sci., USA 75:4779-4783 A model is presented for the mechanism of packaging double-stranded DNA into phage heads. The model is based on, and rationalizes, the mismatch in symmetry between the heads and tails of large bacteriophages. DNA movement is postulated to be mediated by a rotating protein structure at the tail-proximal vertex of the head.
Hendrix, R.W., and S.R. Casjens (1975) Assembly of bacteriophage lambda heads: protein processing and its genetic control in petit lambda assembly. J. Mol. Biol. 91:187-199 Casjens, S.R., and R.W. Hendrix (1974) Comments on the arrangement of the morphogenetic genes of bacteriophage lambda. J. Mol. Biol. 90:20-25 Hendrix, R.W., and S.R. Casjens (1974) Protein fusion during the assembly of phage lambda heads. J. Supramol. Struct. 2:329-336
Hendrix, R.W., and S.R. Casjens (1974) Protein fusion: a novel reaction in bacteriophage lambda head assembly. Proc. Natl. Acad. Sci., USA 71:1451-1455 Hendrix, R.W., and S.R. Casjens (1974) Protein cleavage in bacteriophage lambda tail assembly. Virology 61:156-159 |
Download the