Phage Summits

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 200455513001314Meeting ReportThe 2004 Phage SummitS. Adhya et al.

Molecular Microbiology (2005) 55(5), 1300–1314 doi:10.1111/j.1365-2958.2005.04509.x

MicroMeeting

2004 ASM Conference on the New Phage Biology: the ‘Phage Summit’

Sankar Adhya,1 Lindsay Black,2 David Friedman,3 model systems of unequalled power and facility for Graham Hatfull,4 Kenneth Kreuzer,5 Carl Merril,6 studying fundamental biological issues. In addition, Amos Oppenheim,7 Forest Rohwer8 and Ry Young9* the New Phage Biology is also populated by basic and 1Laboratory of Molecular Biology, Center for Cancer applied scientists focused on ecology, evolution, Research, National Cancer Institute, 37 Convent Dr., Rm nanotechnology, bacterial pathogenesis and phage- 5138, Bethesda, MD 20892-4264, USA. based immunologics, therapeutics and diagnostics, 2Department of Biochemistry and Molecular Biology, resulting in a heightened interest in bacteriophages University of Maryland Medical School, 108 N. Greene per se, rather than as a model system. Besides con- Street, Baltimore, MD 21201-1503, USA. stituting another landmark in the long history of a 3Department of Microbiology and Immunology, University field begun by d’Herelle and Twort during the early of Michigan, 5641 Medical Science Building II, Ann Arbor, 20th century, the Summit provided a unique venue for MI 48109-0620, USA. establishment of new interactive networks for collab- 4Pittsburgh Bacteriophage Institute, 4249 5th Avenue, orative efforts between scientists of many different University of Pittsburgh, Pittsburgh, PA 15260, USA. backgrounds, interests and expertise. 5Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, NC 27710, USA. Introduction 6Section on Biochemical Genetics, National Institute of Mental Health, NIH, Bethesda, MD 20892, USA. The ASM Conference on the New Phage Biology was held 7Department of Molecular Genetics and Biotechnology, on 1–5 August in Key Biscayne, Florida. This meeting, The Hebrew University – Hadassah Medical School, PO known as the ‘Phage Summit’, was the first major interna- Box 12272, Ein Karem, Jerusalem, Israel 91120. tional meeting dedicated to the biology of bacteriophage 8Department of Biology, San Diego State University, 5500 in decades. There were more than 350 registrants from Campanile Dr., San Diego, CA 92182-4614, USA. 24 countries and 188 universities, research institutions, 9Department of Biochemistry and Biophysics, Texas A&M companies and government agencies. The strong University, 2128 TAMU, College Station, TX 77843-2128, response to the announcement of the Summit reflects a USA. vigorous and very broad-based re-emergence of interest in phage biology, beyond its historical role as the facile model system used to establish the fundamentals of Summary molecular genetics. As was made evident at the Confer- In August, more than 350 conferees from 24 countries ence, the classical phage systems like l, T4 and T7, with attended the ASM Conference on the New Phage Biol- their unparalleled genetic power, have themselves been ogy, in Key Biscayne, Florida. This meeting, also energized by the admixture of new biophysical, spectro- called the Phage Summit, was the first major interna- scopic and fluorescence technologies and by the tional gathering in decades devoted exclusively to enhanced context provided by the explosion of sequence phage biology. What emerged from the 5 days of the and structural information. In addition, the Summit Summit was a clear perspective on the explosive revealed that the ‘new phage biology’ spans a number of resurgence of interest in all aspects of bacteriophage other areas, including ecology, evolution, structural biol- biology. The classic phage systems like l and T4, ogy and molecular pathogenesis. Moreover, applications reinvigorated by structural biology, bioinformatics of our burgeoning knowledge of bacteriophage are fea- and new molecular and cell biology tools, remain tured in nanotechnology, vaccine design and phage- based therapeutics, diagnostics and prophylactics. Accepted 1 December, 2004. *For correspondence. E-mail The Conference, co-chaired by S. Adhya (National Can- [email protected]; Tel. (+1) 979 845 2087; Fax (+1) 979 862 4718. cer Institute, Bethesda) and R. Young (Texas A&M), was

The 2004 Phage Summit 1301 constituted as a series of keynote addresses and platform pairs could be measured, a fact exploited by Benzer in his sessions structured on the traditional Phage Meetings at paradigm work in constructing the first fine-structure map Cold Spring Harbor, with short talks followed by some- of a gene. Summers’ analysis showed that, although the times contentious question periods. In addition, there conceptualization of the system was later chronicled to be were more than 250 posters, which remained displayed a sudden flash of inspiration, in reality the rII system’s throughout the entire conference. Platform speakers were utility became apparent over months of indirect and often asked to provide posters with supporting information, a tangential experimentation. By demystifying this legend- device which helped the conferees from so many distinct ary achievement and relating it to the realities of disciplines negotiate the widely divergent terminologies laboratory science, Summers’ presentation provided a and diverse experimental systems. Because of the short perfect historical backdrop to the proceedings, connecting talk format and an audience with such heterogeneous everyone present to the golden era of classical phage expertise, the presentations were distilled to their most genetics. arresting scientific content, leading to highlight after highlight and vibrant question periods after each talk. Several Regulation, development and phage–host conferees noted how many times during the sessions interactions there were audible gasps and even spontaneous out- breaks of applause. Moreover, the frequent nucleation of Regulation, development and phage–host interactions discussion groups around posters during breaks in the continue to be dynamic areas of intensive study in phage platform talk schedule lent an air of ferment and excite- biology, as was evident from the 73 abstracts, three key- ment to the entire proceedings. note talks and the first session of oral presentations, Adding to this stimulating, almost revivalist atmosphere, presided over by A. Oppenheim (Hebrew University, the first keynote talk, by W.C. Summers (Yale University), Jerusalem). A number of talks centred on the l lysis- reminded everyone that the origins of modern biological lysogeny paradigm, which, although after more than science are grounded in phage biology, showing vintage 50 years is easily the most intensively studied molecular pictures from a ‘phage summit’ of 1952 with Nobelists developmental decision in biology, continues to reward its Delbrück, Hershey and Luria, among others, in atten- investigators. The keynote speaker in the session, J. Little dance (Fig. 1). Summers’ fascinating talk examined in (University of Arizona), took a systems biology approach detail the laboratory notebooks of Seymour Benzer from relating to the gene regulation circuitry of phage l and its the period when the famous T4 rII studies were concep- evolution. He discussed two sets of findings. First, his tualized and begun. Benzer discovered that T4 rII mutants group constructed phages in which a crucial regulatory would not plate on Escherichia coli K-12 carrying the l protein, Cro, is replaced with the Lac repressor; several prophage. The non-permissive phenotype was so tight of these phages behaved like the wild type in their ability that recombination frequencies between individual base to support the lysis-lysogeny decision and in their UV

Fig. 1. A previous ‘Phage Summit’ at Royau- mont, France in 1952. Left to right: Alfred Her- shey, Jacques Monod, Elie Wollman, Gunther Stent, Max Delbrück. (From W. Summers’ talk on the history of the rII genetic system.) Photo- graph used with permission from S. Benzer.

1302 S. Adhya et al. dose–response for prophage induction (Atsumi and Little, required for N-dependent anti-termination in phage l; 2004). Second, his group individually removed features of King et al., 1996) and that the modification persists as the circuitry previously thought crucial to its proper oper- long as the put RNA is tethered to RNAP. Cleaving an ation: differential affinity of CI and Cro for the OR operators RNA loop that tethers put RNA to the active centre of (Little et al., 1999), positive autoregulation of cI gene RNAP eliminates anti-termination in vitro (Sen et al., expression and cooperative DNA binding by CI. In the 2001). Terminators located as far as 10 kb from put are latter two cases, suppressors were needed to restore efficiently anti-terminated in vivo, suggesting that the near-wild-type behaviour. Little put these findings in the tether functions by increasing the local concentrations of context of a two-stage model for evolution of a complex the interacting partners. circuit. In the first step, simple modules recombine to Temperate coliphage 186, a member of the P2 family, create a more complex wiring diagram. The ability to is unrelated in sequence to phage l but the two phages replace Cro with Lac repressor supports a modular orga- are remarkably similar in lifestyles. By comparing the nization for the existing circuit. In the second stage, refine- genetic switches of the two phages, K. Shearwin (Univer- ments (such as autoregulation) are added to a simpler sity of Adelaide, Australia) reported how different solutions functional circuit, yielding the more elaborate circuitry can evolve to solve a single biological problem. In contrast found in modern phages. The ability to remove certain to the divergent lytic and lysogenic promoters of the l refinements without destroying the circuitry supports this switch, the corresponding promoters of 186 are arranged aspect of the model. A. Oppenheim described constructs face-to-face, giving rise to convergent transcription. Such using various fluorescence protein fusions that allow the an arrangement leads to transcriptional interference, with continuous kinetic monitoring of phage regulatory func- the strong lytic pR promoter inhibiting the weak lysogenic tions. The results beautifully showed that when lysogeny promoter pL. The interference is critical for the lysogenic is favoured, the transcription activator protein CII plays a switch. In vivo results showed that the interference needs key role in activating repressor synthesis and in inhibiting the passage of the converging RNAP over the weak pro- late gene expression. This experimental tool should allow moter. Shearwin suggested a ‘sitting duck’ model (Callen better mathematical description of the lysis–lysogeny et al., 2004) in which open complexes at the weak pro- decision process. Another approach in studying the sys- moter are sensitive to head-on collisions with elongating tems behaviour of phage was presented by L. Chan (D. RNAP (Fig. 2). Endy group, Massachusetts Institute of Technology), who Although the historic focus on the classic coliphages reported the use of experimentally determined parame- was critical for the vertical development and unparalleled ters in making a mathematical model for phage T7 devel- rigor of phage molecular genetics, workers in new phage opment (Endy et al., 1997). The Endy group is making systems are now finding diverse and equally compelling progress in engineering a re-factored T7 by separating regulatory stories. E. Semenova (K. Severinov group, genes and regulatory elements, making a new phage that Waksman Institute, Piscataway, NJ) described an unusual is simpler to study and model (Endy et al., 2000). Xanthomonas phage, Xp10, with a genome organized Lambdoid phages usually encode two anti-terminators, similarly to lambdoid phages. Surprisingly, Xp10 relies on the N and Q proteins, which act by modifying host RNA both host RNAP and a phage-encoded T7-like single- polymerase (RNAP) in the early and late operons, respec- subunit RNAP for expression of its late genes (Semenova tively, and work on these systems continues to be exciting. et al., 2004). Such conjoint action by different RNAPs in A. Das (University of Connecticut, Farmington) reported a single regulatory phase is unprecedented for a bacte- that his group has identified the target of N in RNAP to riophage. Rapid progress in the detailed molecular char- be in the beta subunit flap, a flexible domain that consti- acterization of Gram-positive hosts was reported. For tutes part of the RNA exit channel. He also presented example, M. Pedersen (K. Hammer group, Technical Uni- evidence that N binds a RNA:DNA hybrid and showed that versity of Denmark, Lyngby) reported that Alt, the late the hybrid-binding domain in N is distinct from those that gene activator of lactococcal phage TP901-1, has a C- bind nut site RNA and the RNAP flap (Das et al., 2003). terminal motif that causes binding to four direct repeats These results lead to a new concept of anti-termination in upstream of the late promoter. In the Lactobacillus phage which N is proposed to form a stable sliding clamp on the A2, the genes for both the major capsid and major tail RNA:DNA hybrid that impedes RNA release by either Rho proteins as well as the gene that encodes the lysogenic helicase or the terminator hairpins. A contrasting story repressor Cro are expressed as two products, by virtue was told by R. Weisberg (National Institute of Child Health of efficient -1 frame-shifting mediated by ‘slippery and Human Development, Bethesda), who related that in sequences’ and RNA stem-loop structures, as reported the lambdoid phage HK022 RNAP is modified by tran- by I. Rodriguez (J.E. Suarez group, Universidad de scription through anti-termination sequence called a put Oviedo, Oviedo) (Garcia et al., 2004). Finally, M. Clokie site (for polymerase utilization, analogous to the nut site (N. Mann group, University of Warwick, UK) reported that

The 2004 Phage Summit 1303

Fig. 2. ‘Sitting duck’ model for the regulation of the lysogenic promoter of phage 186. Three potential mechanisms of transcriptional interference by the convergent promoters of bacteriophage 186 are shown. These are: (i) collisions between elongating polymerases, (ii) occlusion of the weak promoter (pL) by passage across it of RNAP originating from the opposing strong promoter (pR) and (iii) the ‘sitting duck’ mechanism, where initiation intermediates waiting to fire at pL are removed by collision with polymerase coming from pR. At the wild-type interpromoter distance of 62 bp, the ‘sitting duck’ mechanism makes by far the major contribution to the observed 5.6-fold interference. From the presentation of K. Shearwin (University of Adelaide). more than half of cyanobacterial phages examined con- Repair, chaired by K. Kreuzer (Duke University), began tained photosynthetic genes that are expressed through- with a keynote talk about the phage T4 replication fork by out the phage infection cycle. These phages may have N. Nossal (National Institutes of Health, Bethesda). Using important implications for the communal photosynthetic chain-terminating nucleotides and a special 450 bp circu- physiology of the picophytoplankton and consequently on lar substrate, her group showed that inhibition of the lag- biogeochemical cycles. Surprisingly, sequence compari- ging-strand polymerase does not block leading strand sons indicated that the phage-encoded photosynthetic synthesis, arguing against a tight coupling of the leading genes are closely related to but distinct from those of their and lagging-strand enzymes. She showed electron cyanobacterial hosts (Millard et al., 2004). microscopy studies performed in collaboration with J. Grif- In a keynote address, L. Rothman-Denes (University of fith (University of North Carolina, Chapel Hill) in which the Chicago) described the virion-associated RNAP (vRNAP) entire replication complex was visualized and analysed of coliphage N4 (Kamtekar et al., 2002). Unlike all other (Chastain et al., 2003). Nossal’s group is pioneering a double-stranded DNA (dsDNA) phages characterized to novel technique involving ‘biopointers’, in which proteins date, N4 does not use the host RNAP to transcribe its are tagged with biotinylation epitopes and then visualized early genes. Instead, vRNAP is injected into the host at by adding a 0.2 kb DNA molecule covalently linked to the onset of infection and is required for injection of the streptavidin (Fig. 3). As an example of the power of this early half of the genome. Limited proteolysis revealed technique, Nossal showed electron micrographs revealing three domains in the vRNAP polypeptide: the N-domain, that the helicase loading protein, gp59, stays with the required for the early DNA injection; the C-terminal travelling replication fork. domain required for vRNAP encapsidation; and the cen- Other intriguing aspects of T4 DNA metabolism were tral 1100 aa domain (mini-vRNAP) that has all the tran- also highlighted. D. Shub (State University of New York, scriptional properties of full length vRNAP. Rothman- Albany) discussed the arrangement of the topoisomerase Denes described the crystal structure of mini-vRNAP, genes 39 and 60, separated by about 1000 bp in T4, and determined by her collaborator, K. Murakami (Pennsylva- in related phages, where the two reading frames are nia State, PA), showing that the alpha-carbon backbone fused. He presented evidence that the intervening DNA in at the active site is completely superimposable with T7 T4 encodes a special endonuclease, MobA, which directs RNAP. vRNAP interactions with its single-stranded, hair- DNA mobility reactions. Most intron mobility systems pin-containing DNA promoter were mapped to the same involve an endonuclease that makes double-stranded domain structures used by T7 RNAP to interact with its breaks, but MobA makes site-specific single-stranded dsDNA promoter. She also reported results showing that breaks, which apparently initiate the recombination reac- the role of host single-strand DNA binding protein is to tion involved in DNA mobility (also see Landthaler et al., help template recycling by binding to the RNA product 2004). Kreuzer described experiments that utilize the (Davydova and Rothman-Denes, 2003). ‘awesome power of T4 rII genetics’ demonstrating that in double-stranded break repair, the two ends both interact with the same homologous chromosome (see Shcherba- Phage replication, recombination and repair kov et al., 2002; Stohr and Kreuzer, 2002). Interestingly, The session on Phage Replication, Recombination and mutations that greatly reduce the amount of gp46, which

1304 S. Adhya et al. primed DNAPs is part of a loop that covers the DNA- binding cleft. Indeed, a mutant polymerase lacking the region is impaired in both processivity and strand displacement. The short talks in the remainder of the session had many highlights worthy of mention. Two talks were con- cerned with the application of new technologies. In an overview of his revolutionary ‘recombineering’ technology (Court et al., 2002), D. Court (National Cancer Institute, Frederick, MD) detailed how genes can be directly mutated at high efficiency (>30%) by electroporation of ª70 nt single-stranded oligonucleotides targeted to the lagging-strand template in cells deficient for mismatch repair (Costantino and Court, 2003). A. Segall (San Diego State) summarized interesting work on the isolation of oligopeptide inhibitors that bind the Holliday junction and thereby inhibit enzymes such as l Int and the E. coli proteins RecG and RuvABC (Boldt et al., 2004), including one class isolated from peptide libraries and found to inhibit bacterial growth and to generate DNA breaks (Cas- sell et al., 2000; Cassell and Segall, 2003; Boldt et al., 2004) (K.V. Kepple, J.L. Boldt and A.M. Segall, submitted). Unusual DNA transactions were the focus of talks by W.-M. Huang (University of Utah) and D. Manna (P. Hig- gins laboratory, University of Alabama at Birmingham). Huang reported on chromosomes with hairpin ends, such Fig. 3. Electron micrograph of a replication fork with bacteriophage that the entire chromosome is a continuous single- T4 proteins. The locations of the biotin-tagged leading and lagging- stranded DNA (ssDNA) folded back on itself. These DNAs, strand DNA polymerases are shown by biopointers composed of which are found in bacteria (e.g. Borrelia spp. and Agro- streptavidin attached to 179 bp biotinylated DNA. Rolling circle replication of M13 DNA by T4 DNA polymerase, clamp, clamp-loader, bacterium tumefaciens), phages (e.g. N15) and plasmids, helicase, helicase loader, primase, RNaseH, ligase and 32 ssDNA- require protelomerase (Huang et al., 2004), an enzyme in binding protein gives a long linear product. The nascent lagging- the type IB topoisomerase/tyrosine-recombinase family of strand fragment is folded into a duplex DNA loop at the fork. The dense complex at the fork (enclosed in the dashed circle) contains proteins. Huang’s group has been able to clone the duplex the replication proteins, as well as the protein-covered single- version of the end sequence into a plasmid and to dem- stranded regions of the lagging-strand that are folded into a compact onstrate in vitro conversion of the plasmid into a hairpin structure (Chastain et al., 2003). There is a single biotin tag at the N- terminus of each T4 DNA polymerase. Unbound replication proteins molecule via the protelomerase. Manna described were removed by gel filtration before addition of the streptavidin DNA genomic analyses of phage Mu transposition sites, which pointers. (Unpublished experiments of N.G. Nossal, P. Chastain, A.M. have hot and cold spots that could be correlated with high- Makhov and J.D. Griffith; from the presentation by N.G. Nossal.) affinity binding sites for the Mu B protein and with areas of intense transcription respectively (Manna et al., 2004). is the T4 homologue of eukaryotic Rad50 protein, appear DNA partitioning and entry into the cell was also a to debilitate this end co-ordination. The eukaryotic Rad50 focus. R. Edgar (M. Yarmolinsky group, National Institutes protein and its partner Mre11 (T4 homologue is gp47) are of Health, Bethesda) presented in vivo studies on the likewise implicated in end co-ordination in eukaryotic cells. partitioning of the plasmid prophage form of phage P1. In The highlight of another keynote presented by M. Salas DNA gyrase-inhibited cells, the observed blockage by (Universidad Autonoma, Madrid) was new insight on the ParB of rotational diffusion of DNA in dimeric plasmids structure–function relationships in the DNAP of Bacillus carrying two centromeres was taken as evidence for ParB- subtilis phage f29. This remarkably processive enzyme mediated centromere pairing (Edgar et al., 2001). The has protein priming, DNA polymerization, proof reading blockage of rotational diffusion of DNA in monomeric plas- 3¢-5¢ exonuclease and strand displacement activities mids carrying a single centromere to which ParB binds (Blanco and Salas, 1996). Its crystal structure, determined was found to depend on the presence of ParA, suggesting in collaboration with the T. Steitz group (S. Kamtekar, that ParA enhances pairing or, more likely, tethers a ParA– et al., submitted) shows that one of the two domains that ParB–DNA complex to an unidentified cellular component are conserved in both eukaryotic and prokaryotic protein- (Edgar et al., 2001). W. Robins (I. Molineux group, Univer-

The 2004 Phage Summit 1305 sity of Texas at Austin) showed convincing experiments 1 kb in either direction before cleavage. Phylogenetic that transcription is the driving force for the translocation analysis of these and the other known terminases indi- of T7 DNA into the host, namely that transcriptional ter- cated that the different types of terminases largely fall into minators and anti-terminators modulate both the rate and separable and robust groups, suggesting that terminase efficiency of entry of phage T7 DNA into the cell. sequences can be used to predict the type of ends virion Taken together, the talks in this area clearly showed that DNAs will have. Black reported that T4 DNA, packaged phage continue to provide some of the best model sys- into capsids at ª500 mg ml-1, can have a number of tems for investigating the fundamental mechanisms of unusual twists, such as more than 1000 embedded but DNA metabolism. Many connections of these talks to DNA invisible proteins, and offered data suggesting that the transactions in eukaryotes, including aspects relevant to popular rotary portal mechanism of packaging is far from human disease, were obvious throughout the session. established. The session was also marked by incisive questions and Talks by B. Fane (University of Arizona) and A. Kuhn comments from a highly participatory audience. (University of Hohenheim, Germany) focused on protein factors that are required for capsid assembly. Fane discussed the role of fX174 internal scaffolding protein, D, Structural biology and morphogenesis in facilitating several steps during morphogenesis without A number of provocative findings were reported at the being absolutely required for any of them. Selections for Morphogenesis and Assembly session, chaired by L. assembly-competent mutants that can use successively Black (University of Maryland, Baltimore). Among these smaller fragments of D have identified several functions was a report by D. Bamford (University of Helsinki) of the of the protein. Kuhn described the role of the host mem- structure of the tectivirus PRD1, which has an external brane protein YidC in insertion of the major procoat of protein coat surrounding a biological membrane enclos- filamentous phage M13 into the bilayer. YidC protein, ing a linear dsDNA genome. The high-resolution structure which has homologues in mitochondria and chloroplasts, of this unusual phage supports the interesting proposal was reconstituted into proteoliposomes where, in the that only a relatively low number of protein folds are capa- presence of a transmembrane electrochemical potential, ble of assembling an icosahedral virus coat (Abrescia it supported efficient membrane insertion of microgram et al., 2004; Cockburn et al., 2004). Accordingly, it is pro- quantities of procoat in a topologically correct orientation. posed that viruses fall into distinct groups, each having a F. Arisaka (Tokyo Institute of Technology), R. Duda (R. common ancestor and sharing similar structural and Hendrix laboratory, University of Pittsburgh) and E. Gold- assembly principles, bringing phylogeny to virus classifi- berg (Tufts School of Medicine, Boston) reported struc- cation. Seen in this light, one can question whether the ture–function studies of some of the sophisticated extraordinarily large number of phylogenetically disputa- proteins involved in virion function, from lysozymes to tious phage genomes are an illusion in the larger scheme proteases to nano-worthy adsorption fibre materials. of things. Arisaka described newly determined features of the pre- Reports on DNA packaging in tailed dsDNA phages viously published structure of the T4 tail lysozyme, which showed how complex this terminal DNA transaction can undergoes proteolytic processing at a specific site. By become. M. Feiss (University of Iowa) presented results replacing the residue at the cleavage site and thus block- showing that the baroque interaction of l terminase with ing the cleavage, a structure that, after cleavage, had the cos packaging control sequences and host factors can been too flexible to be visible in the crystal structure was be replaced in a near relative with a much simpler revealed. Surprisingly, although acquiring a cold-sensitive sequence recognition process that is independent of the phenotype, the phage with the uncleavable lysozyme Integration Host Factor. This rigorous analysis of packag- remained infection competent. Duda has found that a C- ing is consistent with other findings that increasing com- terminal peptide from the HK97 maturation protease is plexity can be layered onto, and perhaps is subsequently necessary and sufficient to direct the protease or a heter- removed from, a basic working evolutionary plan. S. ologous protein into the interior of the HK97 prohead. Casjens (University of Utah) described the head-full pack- Except for this peptide, the protease appears to be related aging strategy of the Salmonella generalized transducing to capsid proteases from viruses as distant as the Herp- phage ES18. This phage is like P22 and other phages in esviruses. This encapsidation signal might be used to recognition of a specific pac site to initiate DNA packaging generate a capsid-like protein coat around enzymes or but is unlike them in that it then makes packaging initiation other biomolecules for storage, protection and targeted cleavages in the concatemeric substrate DNA anywhere delivery. Goldberg described initial experiments aimed at within a region of about 2 kb centred on the putative pac exploiting the basic structure of the T4 tail fibre as struts site. Casjens’ model for this is that, after recognition, the for nanodevices. The length of these struts can be ES18 terminase can slide around on the DNA for up to adjusted and they can also be decorated with peptide

Fig. 4. The T4 baseplate structure in the hexagonal (left) and star (right) conformations. Colours identify different proteins: gp7 (red), gp8 (blue), gp9 (green), gp10 (yellow), gp11 (cyan) and gp12 (magenta). For clarity, the full- length short-tail ﬁbres are shown in the star- shaped baseplate. The model of the disordered receptor-binding domain is based on the crystal structure and the corresponding density from the hexagonal baseplate map. For consistency, the long-tail ﬁbres are shown as rods in both conformations. From the presentation by P. Leiman.

display ligands (Hyman et al., 2002). In a recent develop- genes are shared among phage genomes and suggested ment, a heat-stable coiled-coil motif has been engineered recombination mechanisms that could give rise to these into the strut, eliminating the need for the gp38 chaperone mosaic relationships. These processes include assort- that is required for the gp37 trimerization in T4 long tail ment of modules by homologous recombination between fibre maturation (Qu et al., 2004) shared modules, and the generation of new mosaic P. Leiman (M. Rossman laboratory, Purdue) presented boundaries by illegitimate recombination and selection for a truly remarkable study, using a combination of X-ray function (Hendrix, 2002). Hendrix also described the iden- crystallography and electron cryo-microscopy (cryoEM), tification of ‘morons’, segments of DNA, typically contain- revealing the structural changes that occur in bacterioph- ing an open reading frame (ORF) flanked by a promoter age T4 on attachment to the host cell (Leiman et al., and a terminator, that are present in one genome but 2004). The cryoEM reconstructions of the T4 baseplate absent in a close relative. Many morons appear to encode were obtained in the pre- and post-attachment conforma- functions that may be beneficial to the host, suggesting tions and interpreted in terms of its component proteins, that these are acquired by phages as one means of ‘pay- integrating atomic structures of baseplate proteins, genet- ing their rent’ when housed as prophages in the chromo- ics and biochemical data (Fig. 4). This analysis suggests somes of their hosts. Hatfull reviewed the structure of 30 that re-orientation of the long-tail fibres following specific mycobacteriophage genomes, typically ª70 kb and highly interactions of the tips with the cell surface up to 1450 Å diverse at the nucleotide and protein levels, with a perva- away changes the network of protein interactions in the sive mosaicism in which single genes are frequently mod- baseplate, resulting in the hexagon to star transition. The ules (Pedulla et al., 2003). The predicted ORFs can be fibres, acting through their baseplate attachment proteins, grouped into approximately 1500 protein families, but act as levers that move the baseplate towards the cell fewer than 15% of these are homologous to proteins surface. During these transitions, most baseplate proteins described outside this mycobacteriophage group. The rotate as rigid bodies with little change in their overall seven complete genome sequences of Listeria phages structure. The presentation culminated in a movie simula- described by M. Loessner (Swiss Federal Institute of tion that, while almost frighteningly realistic, was a high- Technology, Zurich) are clearly also built as mosaics, and light of the meeting. exhibit a number of unusual features. For example, phage A118 integrates into the gene encoding a homologue of the B. subtilis ComK competence regulator, and phage Phage genomics and evolution PSA, like phage A2 mentioned above, utilizes unusual Mosaicism of phage genomes was a major theme of this frame-shifting schemes to express its structural proteins session chaired by G. Hatfull (University of Pittsburgh). (Zimmer et al., 2003). The keynote speaker, R. Hendrix (University of Pitts- A contrast to the diversity of these Listeria and Myco- burgh), described how modules containing one or more bacteria phages was presented by A. Nilsson (Stockholm

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1300–1314 The 2004 Phage Summit 1307 University) and his collaborator and keynoter, E. Haggård- site of random replacements in the expression locus. Ljungquist (Stockholm University), who described the P2 Interestingly, related systems appear to exist in a wide phage family, which has prophage representatives in a variety of other phages and bacteria, although it remains wide spectrum of proteobacteria, including almost 30% of unclear as to whether the bacterial systems are in fact the strains in the ECOR library (Ochman and Selander, prophage-associated. Clark discussed an intriguing plas- 1984; Nilsson et al., 2004). Bacteriophage P2 belongs to mid prophage of the Sodalis glossinidius, the secondary a family of related phages that can be found in different endosymbiont of the tsetse fly. The plasmid appears to be proteobacteria. An analysis of the late genes of P2-like a ‘heterodimeric’ genome related to ancestral phages coliphages has revealed such a high degree of identity resembling the lambdoid phage HK620 and the Salmo- that they may be regarded as slight variants of the same nella phage epsilon-15. It has two sets of virion structural phage (Nilsson and Haggård-Ljungquist, 2001). Phyloge- genes, one set complete and presumably active, the other netic analyses of P2 relatives from different host species set reduced and consisting of pseudogenes. Clark spec- show that they are a conservative group of phages without ulated that endosymbionts are relatively isolated from modular evolution. The take-home-lesson here is that phage infection and selective pressure on resident phage these phages may have evolved clonally, as they follow is reduced, allowing the persistence of evolutionary inter- the evolution of their respective hosts. Moreover, dramatic mediate forms which would be eliminated more rapidly in new findings illustrated how P2-like phages evolve with free-living bacteria exposed to frequent infection. their E. coli host and compete with each other. At one M. Sullivan (S. Chisholm group, Massachusetts Institute specific DNA site, 10 out of 11 analysed prophages in the of Technology) described the genomes of three Prochlo- ECOR library contain different horizontally transferred rococcus phages representing a T7-like podovirus, P- gene cassettes, probably encoding proteins benefiting the SSP7, and two T4-like myophages, P-SSM2 and P-SSM4 host and integrated by a site-specific recombination event (Sullivan et al., 2003). All three of these phages contain (Nilsson et al., 2004). In addition, the P2-like coliphages the gene encoding the D1 protein of the photosystem II can be divided into at least five immunity classes which reaction centre. While the role of this phage gene is not contain different developmental switches. clear, it is speculated that it might compensate for the host K. Stedman (Portland State University, OR) and J. D1 function, which is rapidly turned over, thus maintaining Karam (Tulane Medical School, New Orleans) further photosynthesis during phage infection. This and other emphasized the diversity emerging from the current wave observations (integrase gene in the T7-like phage; phos- of phage genomics. Karam reported a comparative phate-response genes and carbon metabolic genes in the genomic analysis of T4-like phages, for which six com- T4-like phages) suggest that these phage genomes plete genome sequences are available (see http:// adapted to infect open ocean photosynthetic hosts by co- phage.bioc.tulane.edu/), revealing interesting diversity of opting genes from their hosts. genome size and organization, the functions for genome Both the R. Young and Hatfull laboratories emphasized modification and DNA metabolism, and content of mobile the utility of phage discovery and genomics as educa- elements (Bebenek et al., 2002). Stedman described tional tools. E. Summers (R. Young group, Texas A&M) viruses of extreme archaeal thermophiles that, by electron reported on a programme of genomic sequencing of microscopy, were found to have many bizarre and unusual Burkholderia phages, in which the bulk sequencing and morphologies. The complete sequences of Sulfolobus sol- bioinformatic analyses are performed by teams of under- fataricus phages SSV1, SSV2 and SSV3 have been com- graduates. A new branch of the Mu family is represented pared, providing insights into their essential and non- by the phage BcepMu, which is a prophage in a Burkhold- essential functions (Wiedenheft et al., 2004). Interestingly eria lineage that infects human cystic fibrosis patients closely related viruses appear to be abundant and some (Summer et al., 2004). The BcepMu genome carries have been characterized from both Yellowstone and potential pathogenesis determinants. Besides providing Kamchatka. serious scientific advances, these reports illustrate the Work by A.J. Clark (University of Arizona), in collabora- suitability of phage genomics as educational facilitators tion with C. Dale (University of Utah), and A. Hodes (J. that may play an important role in generating the next Miller laboratory, University of California at Los Angeles generation of phage biologists. School of Medicine) dealt with specific mechanisms by which diversity is generated. Hodes described a variation- Phage in pathogens and phage–bacteria interactions generating process in the Bordetella phage BPP-1 and its relatives, in which a phage-encoded reverse transcriptase The keynote speaker in this session, chaired by D. Fried- copies information from an invariant template cassette man (University of Michigan), was M. Waldor (Tufts School into the mtd tail gene (Doulatov et al., 2004). In the proof Medicine, Boston) who led off with a provocative report cess, nearly every adenine residue in the template is a on the nature of the immunity (repressor/operator) system

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1300–1314 1308 S. Adhya et al. of Vibrio cholerae phage CTXf. Unusual in being a tem- Rex also appears to play a role in optimizing the develop- perate filamentous phage, CTXf carries the gene encod- ment of the induced l prophage. ing cholera toxin, the major virulence factor of this The co-opting of host proteins by B. subtilis phage f29 important pathogen (Waldor and Mekalanos, 1996; Kim- to implement its strategy for adapting to a changing phys- sey and Waldor, 2004). Waldor and colleagues found that iological bacterial environment was the subject of a talk like the l family of phages, the CTX prophage is induced by W. Meijer (Universidad Autonoma, Madrid). In collabo- by the SOS response. In that response, RecA, activated ration with M. Salas, Meijer found that the bacterial by ssDNA produced by DNA damage and subsequent machinery involved in segregation of the bacterial repair activity, stimulates the auto-cleavage of the LexA genome into the prespore is also responsible for spore- protein, the master regulator that controls expression of a entrapment of the phage genome. In addition, the bacte- large number of enzymes involved in DNA repair. l CI rial Spo0A protein, the master regulator for entry in sporu- repressor is the only protein involved in repression of the lation, silences the early phage promoters. The end result l prophage and its auto-cleavage is also stimulated by is phage DNA in the spore awaiting germination to begin activated RecA. The Waldor group found that CTXf differs its life cycle. from l in that expression from the prophage is repressed The consequences of the interminable struggle for by both a prophage-encoded repressor, RstR, and by the dominance between the phage and its bacterial host were host LexA protein. Thus, unlike l, expression of the CTXf brought home by the talk of G. Kaufmann (Tel Aviv Uni- prophage is, in part, directly controlled by a bacterial pro- versity). Phage T4 infection results in the inactivation of tein that serves as a sensor of the physiological state of the bacterial restriction modification enzyme EcoPrrI and the bacterium. that, in turn, activates an associated tRNALys-specific anti- Friedman presented studies on the physiology of codon nuclease (ACNase) termed PrrC (Amitsur et al., lysogens with prophages that carry the genes encoding 2003). Cleavage of tRNALys by this ACNase occurs 5¢ to shiga toxin (Stx). His group had shown that the toxin is the wobble base. Unless thwarted, this activity would stop produced and released from a subpopulation of sponta- the progression of the progression of the phage, but the neously induced cells (Wagner et al., 2001). Using a phage-encoded RNA repair enzymes polynucleotide reporter system designed to quantify the fraction of spon- kinase and RNA ligase normally offset the damage. Struc- taneously induced lysogens (Livny and Friedman, 2004), tural studies reveal ACNase to be a hexamer of PrrC. An it was found that Stx-encoding lambdoid phages induce NTPase domain that interacts with EcoPrrI occupies the more readily than do non-Stx-encoding lambdoid phages, ª260 aa amino-proximal portion of PrrC while the ACNase suggesting that the former may have evolved a more activity resides in the remaining ª130 aa. Known PrrC sensitive trigger for Stx production and release. The next homologues are sporadically distributed among distantly application of this reporter system is to determine whether related bacterial groups. All of them feature the catalytic there is increased induction in situ leading to high levels ACNase residues but anti-codon recognition is found only of Stx in the animal gut. in a subset. Thus, the various PrrC restriction RNases Dean Scholl (National Institutes of Health, Bethesda) must differ in substrate specificity. discussed a newly isolated T7-like coliphage, M59-2, that Another talk by M. Smith (University of Aberdeen, Scot- recognizes and degrades the extracellular polysaccharide land) dramatized the intricate interactions possible in the colanic acid. Cells that produce this structure are resistant battles for survival between phages and bacteria. Protec- to many LPS-specific phages such as T7 but are suscep- tion from infection by phage f31 is afforded Streptomyces tible to M59-2. Like many other polysaccharide-specific coelicolor by the Pgl system (Sumby and Smith, 2002). phages, M59-2 encodes a tail enzyme that is probably Unlike classical restriction systems, the Pgl+ bacterium responsible for this activity. As colanic acid is important supports one round of growth of f31, but the progeny of for biofilm formation in E. coli, this phage and/or the tail this infection is unable to go through any further rounds enzyme could be useful in biofilm control. of growth in S. coelicolor. Thus, initially infected bacteria L. Thomason (National Cancer Institute, Frederick, MD) form a defensive line that blocks further infection of the reported on studies re-examining a legacy phenomenon, developing S. coelicolor hyphae. Although one of the the physiological role of the l rex gene products. In a genes involved in this exclusionary process encodes a series of studies originating in the work by S. Benzer putative DNA adenine methyltransferase, suggesting noted above, Rex proteins have been shown to be respon- modification of the f31 DNA during replication in S. coeli- sible for the exclusion of T4 rII mutants by the l prophage. color, evidence for such a process has as yet not been Thomason found that overexpression of the rex genes obtained. Moreover, the obvious genetic approach has not affects the physiology of the bacterium, resulting in over- been successful, in that it has not been possible to select production of NADH when the bacteria are grown in the mutants of f31 that escape the exclusionary process. It presence of certain sugars such as mannitol and fructose. seems likely that multiple sites on the f31 DNA are

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1300–1314 The 2004 Phage Summit 1309 involved, requiring multiple mutations for the DNA to described another T4-like phage genome, SPM2, which escape this mysterious ‘bait and switch’ strategy of the carries the rate-limiting genes for cyanobacterial photo- bacterium. synthesis, providing a context for the earlier presentation by Clokie, in which physiological data support the notion that these photosynthesis genes are important for phage Phage ecology and taxonomy replication. This story is also similar to that presented by The overarching theme of this session, chaired by F. M. Sullivan in the Phage Genomics and Evolution session Rohwer (San Diego State), was repeated reminders about (i.e. other cyanophage genomes carry photosynthesis just how much is left to learn about phage. C. Suttle genes). (University of British Columbia), R. Sandaa (University of In fact, lateral gene transfer by phages was a recurring Bergen) and Rohwer started the session with talks show- theme within the session and throughout the meeting. ing that the vast majority of phage diversity, as assessed Suttle and Rohwer, for example, presented data showing at the sequence level, remains uncharacterized. Sandaa’s that phage genes are moving between environments and work in solar salterns showed that phage diversity over large geographical distances. A critical new concept appears to be highest at the intermediate salinities. This with health implications is that phage-encoded genes is consistent with patterns found in macroorganisms and involved in pathogenesis also appear to be moving highlights the emergence of classical ecological theory through the environment, possibly independent of their with microbial studies. The saltern work also directly con- microbial hosts. nected to a major theme of the section, namely viruses of Significant progress is being made in our understanding extremophiles. For example, A. Kropinski (Queen’s Uni- of phage ecology. E. Wommack (University of Delaware) versity, Kingston, Ontario) presented the first genome and and R. Sandaa showed how viral populations change over genetic characterization of an alkaliphilic phage, BCJA1. environmental and seasonal gradients (Wommack and In his presentation, truly a high-point in the conference, Colwell, 2000). These data sets are essential for the mod- D. Prangishvili (Institut Pasteur, Paris, France) literally elling efforts of investigators, like A. Rabinovitch (Ben- stunned the audience with his description of the excep- Gurion University of the Negev, Israel), a physicist who, in tional novel morphotypes and diversity of phages of Cre- collaboration with colleagues A. Zaritsky and I. Aviram, narchaeota growing geo-thermally heated areas of high has proposed that bacteria in a culture might be protected volcanic activity (i.e. acidic environments above 80∞C) by the bodies of their dead compatriots (Rabinovitch et al., (Prangishvili, 2003; Prangishvili and Garrett, 2004). Seven 2003). Mathematical modelling and real-time detection of novel virus families have been established based on phage infection using green fluorescence protein is being unique features of morphotype and genome organization used by I. Calvo-Bado (L. Wellington laboratory, University of these viruses. About 95% of genes of the sequenced of Warwick, UK) to study phage ecology in soil environ- genomes show no similarity to genes in public databases, ments. These system biology approaches to phage biol- suggesting unusual solutions to biological problems. Most ogy are likely to bring dramatic changes in the field of remarkably, some of these phages have capsids that phage ecology in the near future. change morphology over a period of days after being released from their hosts. Phage in therapeutics and biotechnology The addition of all these new phage morpho- and gen- otypes highlighted the need of the phage community to With 59 abstracts, this popularity of this area rivalled even update our taxonomy systems. R. Edwards (University of that of the classical topics of gene regulation and phage– Tennessee, Memphis, TN) presented work suggesting host interactions. C. Merril (National Institutes of Health, that phage taxonomy systems based on free-phage prop- Bethesda) chaired the platform session and reviewed the erties versus genomic information are essentially compat- potential of phage as an anti-bacterial agent for clinical ible. This presentation was followed by a lively discussion infections. He stressed the need for quantitative data con- in which the difficulties of the various options for phage cerning interactions between phage, pathogenic bacteria taxonomy were thoroughly examined. and the infected animal, as therapeutic phage concentra- Members of well-known phage groups are also excep- tions need to be sufficient to lower titres of pathogens tionally diverse and contain significant amounts of novel before the latter reach a lethal concentration for the mam- sequence. H. Krisch (Centre National de la Recherche malian host. Merril reviewed experiments showing that l Scientifique, Toulouse, France) showed that two T4-like phage administered to animals can avoid being cleared phages, RB49 and Aeh1, both have large segments in by the mammalian host defence system by single mis- their genomes with sequences that are unrelated to the sense changes in the capsid protein (Merril et al., 1996). T4 sequence, to each other, and to anything else in This provides a tool for exploring the molecular and cel- the databases. N. Mann (University of Warwick, UK) lular basis of this clearance, which would be a critical

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1300–1314 1310 S. Adhya et al. consideration in any systemic application of phage thera- Phage and their components can also be used as vac- peutics. Merril also emphasized that for the realization of cines and to enhance vaccine systems. R. Calendar (Uni- clinical phage therapy applications in the USA there is a versity of California, Berkeley) and colleagues used need for enhanced methods of identification of infecting phage-based integration vectors (Lauer et al., 2002) to bacterial strains, availability of useful therapeutic phage enhance a Listeria monocytogenes vaccine system. The strains, appropriate methods of phage purification and the capacity of L. monocytogenes to grow as a facultative development of protocols for phage administration. Infor- intracellular bacterium in mammalian cells provides its mation on phage genetics and physiology, which is known proteins direct access to the T cell-mediated immunity in detail for only a few phage strains, needs to be extended system. By using phage-based integration vectors, anti- to phage strains of therapeutic interest for helping in the genic target genes can be introduced into two different elimination of any phage-encoded toxin genes and neutral sites on the L. monocytogenes chromosome. The enhancing therapeutic properties of phage strains (Merril use of this system to express tumour antigens resulted in et al., 2003). significant tumour regression and long-term survival in The keynote speaker for the session, V. Fischetti (Rock- tumour-bearing mice (Brockstedt et al., 2004). Calendar efeller University), demonstrated the effectiveness of also modified the system to make it safer for human appli- phage endolysins in destroying bacteria in vitro and pre- cations by replacing a drug-resistance marker with the L. dicted that these ‘enzybiotics’ will provide new anti-bacte- monocytogenes alanine racemase (dal) gene. rial agents for the treatment of antibiotic-resistant Research on the use of whole-phage particles as a bacteria. Fischetti noted that phage endolysins are in gen- delivery vehicle for a DNA vaccine against Yersinia pestis eral specific for their host species and genera, offering a was presented by J.R. Clark (J. March group, Moredun potential for avoiding damage to commensal organisms. Research Institute, Penicuik, UK). The gene for the V- In vivo mouse experiments demonstrated that a single antigen, which has been shown to give protection against dose of specific endolysin was sufficient to clear either Y. pestis infection, was cloned into plasmid and bacte- streptococcal or pneumococcal bacterial strains colonized riophage vectors under the control of a eukaryotic expres- in the nasal or oral cavities of the mice. In addition, a sion cassette. The V-antigen DNA vaccine which was single intravenous dose of enzyme given to mice made delivered using the bacteriophage vector gave IgG2a septicemic with Streptococcus pneumoniae reduced the responses significantly higher than that from the plasmid- bacterial titres in the blood by two orders of magnitude. borne vaccine, following intramuscular delivery. Interest- Fischetti reported that phage enzymes specific for Strep- ingly, while phages delivered orally (by gavage needle) tococcus pyogenes, S. pneumoniae and Bacillus anthra- were not as efficacious as phages given by intramuscular cis are available. D. Donovan (United States Department inoculation, the orally administered phage preparation still of Agriculture, Beltsville, MD) discussed his efforts to matched the performance of the intramuscular plasmid express phage endolysins in the mammary gland of trans- vaccine. Similarly, l and plasmid vectors containing the genic dairy cows. The goal is to control and prevent mas- gene for the hepatitis B surface antigen (HBSAg) under titis, a disease that costs the dairy industry approximately the control of the eukaryotic cytomegalovirus promoter $2 billion annually. His group has developed an expres- were used for intradermal vaccination in cannulated sion cassette in which the gene for the endolysin of a sheep. In the case of phage administrations, effective anti- Group B Streptococcus phage endolysin is inserted phage titres were found in the draining lymph after the between the 5¢ and 3¢ flanking regions of the ovine b- second inoculation, along with a significant IgM and IgG lactoglobulin gene and is testing its expression in cultured anti-HBSAg response. The authors suggested that the cells. The phage endolysin is highly specific for strepto- virus-like properties of the phage particles result in them coccal species and thus would be expected to have no being taken up by professional antigen presenting cells effect on the enteric bacteria of humans. (such as dendritic cells) where efficient expression of the Development of a phage endolysin for staphylococcal vaccine genes can occur (Clark and March, 2004). infections was presented by S. O’Flaherty (P. Ross group, The capacity of phage to deliver genes in mammalian Teagasc, Cork, Ireland). The Ross laboratory has suc- hosts was graphically demonstrated by C. Gorman- cessfully expressed an intron-containing endolysin gene Zanghi (S. Dewhurst group, University of Rochester, NY) of phage K, a lytic phage with broad host range among with images of light emission from mice inoculated intra- Staphylococcus aureus strains, in both E. coli and Lacto- dermally with l phage carrying a luciferase gene. A sim- coccus lactis. In both cases zymograms with dead staphy- ilar delivery system is being used with a l construct in lococcal cells indicated that active recombinant LysK which there is a C-terminal fusions between the gpD product was produced, so it may be possible to use these external virion protein and the IgG-binding domains of bacteria for endolysin production for both medical and staphylococcal protein A and streptococcal protein G. veterinary applications. Purified l phage with both fusion types are being used

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1300–1314 The 2004 Phage Summit 1311 in conjunction with antibodies specific for common den- in sugar-beet (I. Kolonyuk, Kyiv National University, dritic cell receptors to target human and murine dendritic Ukraine). Throughout the duration of the conference, cells in vitro. Successful gene transductions are meetings with discussions and interactions at these and evaluated by luciferase and green fluorescent protein other posters were especially energized by the evident expression. momentum in biotechnological and therapeutic applica- By using two ‘non-essential proteins’ displayed at high tions of phage biology. density on the outer capsid surface of the phage T4, T. Sathaliyawala (V. Rao laboratory, Catholic University of The view from the Summit America, Washington, DC) has adapted this phage as a vaccine vector. He presented evidence that the capsid The 2004 ASM Conference on the New Phage Biology ‘decoration’ proteins Hoc (155 copies per capsid), a highly was a unique opportunity for scientists from many different immunogenic 39 kDa molecule, and Soc (810 copies per areas of specialization but with a common interest in bac- capsid), a 9.7 kDa molecule, can be used for display teriophage biology to come together in one place and gain fusions. Several full-length foreign proteins fused to these a sense of the new, vital currents in the field. Many new proteins have been displayed on the surface of the T4 networks of contact and collaboration were established as capsid, demonstrating the potential of using such phage- attendees from these diverse areas met and mingled with based nanoparticles as customized multicomponent vac- each other and with scientists from the traditional phage cines. The idea was further pursued with a phage display- systems. Everone benefited from the energetic participa- ing the HIV capsid protein P24 fused to either the N- tion of G. Bertani (Fig. 5), who in 1951 published the first terminus or the C-terminus of Hoc. Humoral and cellular paper on temperate phages (Bertani, 1951). The phages responses showed high P24-specific IgG antibody that P1 and P2 were both carried in the Lisbonne strain of E. lasted for 6 weeks after the last booster dose. In addition, coli and became two of the most fertile systems for phage P24-specific INFg and IL4 secreting cells were detected molecular genetics (Bertani, 2004). With S. Luria, Bertani in the spleen and lymph nodes. also furnished the acronym LB to the most widely used In a novel application of phage components, P. Guo growth medium in bacteriology. (Purdue University, West Lafayette, IN) proposed the use of the self-assembling motor pRNA of bacteriophage f29 as a targeting and delivery vector for therapeutic RNAs. The natural function of pRNA is to form a hexameric ring as part of a motor complex which facilitates f29 DNA packaging. As hexamer formation occurs by interaction of pRNA interlocking loops, it was possible to make pRNAs that self-assemble in vitro to form dimers or trimers efficiently by making complementary mutations in these loops. Engineering a CD4 receptor-binding RNA aptamer into the structure helped delivery of siRNA or a ribozyme moeity to a CD4 overexpressing T cell line by receptor- mediated endocytosis. Guo noted that the use of 30– 40 nm complexes reduces the problem of short half-life that confronts experimenters wishing to use the siRNAs in mammalian systems. Because of the limited time available for oral presentations in this area, many compelling stories were presented as posters. For example, there were posters on the alter- ation of restriction/modifications systems in lactococcal starter cultures to protect them from phage interference for cheese production (O. McAuliffe, Teagasc, Cork, Ire- land), an extensive review of the clinical application of phage therapy in Poland (A. Gorski, Institute of Immunol- ogy and Experimental Therapy, Warsaw), the use of phage to treat respiratory and systemic colibacillosis in poultry (W. Huff, United States Department of Agriculture/ Agricultural Research Service, Fayetteville, AR), and the Fig. 5. G. Bertani, more than 50 years after the first paper on isolation of phage for the control of bacterial diseases lysogenic phage (P1 and P2) (Bertani, 1951; 2004).

Highlights of the Conference will be displayed on the cific modulation of fidelity by polymerase accessory pro- Phage Summit web pages on the Bacteriophage Ecology teins. Genetics 162: 1003–1018. Group website. To help cement the networking experi- Bertani, G. (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol ence, a Phage People database assembled for promul- 62: 293–300. gating the Conference will also be available and can be Bertani, G. (2004) Lysogeny at mid-twentieth century: P1, obtained in various formats by downloading from the site P2, and other experimental systems. J Bacteriol 186: 595– or by emailing the author (R.Y.). The database contains 600. not only contact information but also a summary of Blanco, L., and Salas, M. (1996) Relating structure to research expertise and interests for many scientists inter- function in f29 DNA polymerase. J Biol Chem 271: 8509– ested in phage. 8512. Boldt, J.L., Pinilla, C., and Segall, A.M. (2004) Reversible In view of the overwhelming response to the announce- inhibitors of lambda integrase-mediated recombination effi- ment of the meeting and the widespread enthusiasm with ciently trap Holliday junction intermediates and form the which every keynote talk, platform session and poster basis of a novel assay for junction resolution. J Biol Chem session was received, there is no doubt that the Phage 279: 3472–3483. Summit should be repeated. Plans are now under active Brockstedt, D.G., Giedlin, M.A., Leong, M.L., Bahjat, K.S., discussion, and suggestions for leadership, venue and Gao, Y., Luckett, W., et al. (2004) Listeria-based cancer infrastructure are earnestly solicited (phagesummit@ vaccines that segregate immunogenicity from toxicity. Proc Natl Acad Sci USA 101: 13832–13837. tamu.edu). Callen, B.P., Shearwin, K.E., and Egan, J.B. (2004) Tran- scriptional interference between convergent promoters Acknowledgements caused by elongation over the promoter. Mol Cell 14: 647– 656. The authors wish to thank Daisy Wilbert for clerical assis- Cassell, G.D., and Segall, A.M. (2003) Mechanism of inhibi- tance in the preparation of the manuscript, and the Con- tion of site-specific recombination by the Holliday junction- ference office of the American Society for Microbiology for trapping peptide WKHYNY: insights into phage lambda their tireless efforts in helping to organize the meeting. integrase-mediated strand exchange. J Mol Biol 327: 413– Essential support for the Conference was provided by a 429. grant from NIAID and funds from NICHD; we thank Milton Cassell, G., Klemm, M., Pinilla, C., and Segall, A. (2000) Dissection of bacteriophage lambda site-specific recombi- Hernandez of NIAID and Robert A. Weisberg of NICHD nation using synthetic peptide combinatorial libraries. J Mol for their invaluable assistance in securing these Biol 299: 1193–1202. resources. We are very grateful to colleagues from the Chastain, P.D., Makhov, A.M., Nossal, N.G., and Griffith, J. private sector who support phage biology with their enthu- (2003) Architecture of the replication complex and DNA siasm and encouragement and who arranged for funding loops at the fork generated by the bacteriophage T4 pro- for this conference: J. Ramachandran, GangaGen (Palo teins. J Biol Chem 278: 21276–21285. Alto); David Martin, Avidbiotics (San Francisco); Tony Clark, J.R., and March, J.B. (2004) Bacterial viruses as human vaccines? Expert Rev Vaccines 3: 463–476. Smithyman, Special Phage Services (Brookvale, New Cockburn, J.J., Abrescia, N.G., Grimes, J.M., Sutton, G.C., South Wales), Gregory Bogosian, Monsanto (St. Louis) Diprose, J.M., Benevides, J.M., et al. 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