Xxxiv

A Functional organization of the PRD1 genome including the promoters (P) and potential transcription terminators (open lollipop, forward direction filled lollipop, reverse direction). The genome is organized into five operons. The early operons (OE1 and OE2) are transcribed inward from the genome ends. The three late operons (OL1-OL3) in the middle of the genome are transcribed in the same direction as OE1. B The genes are identified by Roman numerals and ORFs by...

Pm CroJB

Figure 8-3 Molecular basis for bistability of l circuitry. A The OR region is repeated from Figure 8-2D. B Occupancy pattern of OR operators at moderate CI levels. CI binds as a dimer, and dimers at OR1 and OR2 make cooperative interactions. Contrary to this cartoon, structural evidence suggests that both subunits of a dimer touch the other dimer evidence with LexA suggests that the hinge region connecting the dimerization and DNA-binding domains is shorter than implied here. C Occupancy...

P9

Figure 16-2 A simplified diagram of cystovirus structure. double-stranded RNA is contained inside a core of P1, P2, P4, and P7. This structure is within a shell of P8 (51) in all members except f 8, where P8 is a membrane component. The lipid-containing membrane surrounds the P8 shell. Attachment proteins P6 and P3 are in the membrane along with P9 (the major membrane protein) and P10. See thebacteriophages.org frames_0160.htm for a color version of this figure. protein and is present in 120...

I

Figure 15-1 Genetic maps of four representative single-stranded RNA phages. Phages with a read-through protein are classified as alloleviviruses. ORF1 in AP 205 is thought to encode a lysis function. The map of AP205 shown here is a correction of that published in (43). The difference is due to an additional C-residue found lately at position 460. strains. Species I has a longer and more elaborate 3' UTR (2) and a small insertion in the replicase gene. The alloleviviruses are also composed of...

RNA Processing and Degradation

Host RNases, especially the RNase E complex (69, 144, 298, 410) now called the degradosome (see (391) for review) and Rnase III (75, 88), contribute to processing of T4-encoded tRNAs (352) and to degradation of T4 mRNAs (69, 281, 291). In at least one case RNase III cleavage activates translation from an internal ribosome binding site to produce a truncated terminase protein, gp17' (119). Selective degradation of early T4 transcripts at late times is directed by at least two mechanisms. A T4...

Gene Expression

Since Microviridae contain single-stranded genomes of positive polarity, the negative strand must be synthesized before any genes can be transcribed and proteins translated. Unlike larger double-stranded DNA bacteriophages, Microviridae do not utilize trans-acting mechanisms to ensure temporal gene expression. Therefore, the timing and relative production of viral proteins is entirely dependent on cis-acting regulation signals promoters, transcription terminators, mRNA stability sequences, and...

Evolution

As mentioned earlier, the essentialgenes of the most closely related T-even phages are arranged in the same order, and their sequences are similar, but the genomes of different members of the family have different nonessential genes interspersed between these essential genes. These differences first became evident as insertion or substitution loops in electron micrographs of heteroduplex DNA prepared in vitro by annealing single strands of T2, T4, and T6 DNA (201) (figure 18-2), and have been...

M

Figure 16-7 Agarose gel electrophoresis of double-stranded RNA isolated from virions. Lane V shows the distribution of normal segments L, M, and S. Lane a shows double-stranded RNA from bacteriophage f2007, which has a deletion in segment M. Lane b shows RNA from f2064, which has normal L, an MS chimera picked up from pLM1114, and a normal segment S. Lane c shows RNA from f2323, which contains normal L, the MS chimera shown in b, and a deleted segment S that contains no genes and is only 798...

Genome Capsid Interactions and the Final Collapse

Unlike large dsDNA bacteriophages (38), the f X174 genome does not exist as a dense core in the capsid. Instead, the DNA binding protein and basic capsid amino acid residues tether it to the capsid's inner surface. There are 60 copies of protein J per virion, one associated with each coat protein. In the atomic model, the protein forms an S-shaped polypeptide chain devoid of secondary structure. The C-terminus of the protein is tightly associated with a cleft, located near the center of the...

Specialized Transduction

Transduction is the transfer of genes from one host cell to another by a virus. There are two types generalized (which results from errors in packaging) and specialized (which results from errors in excision from the chromosome of a lysogenic bacterium). Generalized transduction is not directly related to lysogeny. It is most easily observed with temperate phages, but only because the potential transduc-tants formed by a virulent phage are generally destroyed by lysis. This lysis is not caused...

Dennis H Bamford Jaana K H Bamford

Bacteriophage PM2 is the only described member of the Corticoviridae family (1). It was isolated from sea-water collected from a polluted bay off the coast of Chile (13). The original host was a common marine bacterium Pseudoalteromonas espejiana BAL-31 (originally Pseudomonas BAL-31 14, 18), which is the source of the DNA exonu-clease BAL-31. Alternatively, Pseudoalteromonas sp. ER72M2 obtained from the East River, New York, by Leonard Mindich (26) can be used as a host for PM2. PM2 is the...

Host Factors

Mutations in nusA, nusB, rpsJ (nusE), and rho (nusD) that result in a failure of the host E. coli to support N-mediated antitermination identified genes whose products were likely to be involved in N action (54). A direct role for the Nus factors in N action was demonstrated in two ways. First, this was shown using an in vitro system with S30 extracts derived from nus mutants and controls with plasmids expressing the wild-type allele (34). Extracts from E. coli derivatives with nus mutations...

Paul J Jardine Dwight L Anderson

Imagine trying to stuff a string more than 6 mm long into a sphere that is fifty nanometers in diameter. The hole in the sphere that the string must enter is only twice as wide as the string itself. The string is stiff, with a persistence length on the order of 50 nm. It is also negatively charged and self-repulsive. The string must be organized such that it can be pulled out easily, so no knots or tangles are permitted. When the sphere is full, the string will have a near crystalline density....

Info

The data are summarized from the comprehensive table deposited at the website associated with this book, which was adapted from a similar website-deposited table, with permission (112). For this table, holin gene families (columns B and D) are defined as orthologous groups with sufficient sequence similarity to permit manual alignment. If at least one holin in the family is associated with an endolysin which has been shown to support sec-mediated externalization or has an apparent TMD or...

Why Did Holins Evolve

In principle, lysis requires only that the cell wall be at least partly degraded, or that its synthesis be corrupted during growth. Thus, to achieve lysis a phage needs only to produce a muralytic enzyme with a secretory signal sequence or to generate a polypeptide that blocks murein biosynthesis. However, it appears that all phages with sufficient genomic space have evolved a more sophisticated lysis system, involving at least one other protein, the holin, which imposes a strict temporal...

The Other Paradigm secMediated Lysis

Although the first comprehensive review of phage lysis did not appear until 1992, the clarity and elegance of the l paradigm rapidly imposed itself as a kind of dogma. The complementary phenotypes of two S missense alleles, SA52G and SA52V, with unconditional plaque-formation defects illustrate this point. The former causes lysis too early, before the first virion is assembled, whereas the latter is lysis-defective, indistinguishable from the null Sam7 in terms of the unabated accumulation of...

The N Antitermination Protein

The N gene (15) located immediately downstream of the early pL promoter encodes a 12.2 kDa protein (46, 72) that, as discussed, acts at NUT sites to modify RNA Pol to a termination-resistant form. To a first approximation N proteins have specificity for their cognate NUT sequence, although when overexpressed in vivo, N proteins can act at noncog-nate NUT sites (47,150). Genetic studies suggested an interaction between N and RNA Pol. E. coli carrying a mutation (ron) in the rpoB gene (encoding...

The HK022 PUT Sequence

Phage HK022 has PUT sequences downstream of the pL and pR promoters in place of the NUT sequences found in other lambdoid phages. Although other phages with PUT sites have recently been identified (R. Farooque and R. King, personal communication), all the work to date on this anti-termination system has focused on HK022. Unlike NUT, PUT appears to modify RNA Pol to a termination-resistant form without the aid of either phage- or host-encoded proteins (22, 92, 124). The put sites (figure 9-2B)...

Echos in Coliphage Secreted Endolysins

Sequence comprisons also reveal that a number of endo-lysins from phages of Gram-negative bacteria also have N-terminal sequences that could engage the sec system. There is a hydrophobic N-terminal extension on the predicted endolysins, all orthologs of T4 E, from coliphage P1 and from the lambdoid phages 21, P22, PS119 and PS3 (figure 10-6). The length of the hydrophobic sequence and the distribution of flanking charged residues are, however, indicative of a N-terminal TMD, a signal anchor or...

T4Evoked Modifications of Ribosomes

Translation of host messages ceases immediately after T4 infection. Mysteriously, it is inhibited by infection with DNA-free particles, so-called ghosts (100). It is thought that some cells can recover from this immediate turn off, but in successfully infected cells, ribosomes have already been programmed to translate T4 messages exclusively. A few host membrane proteins are the notable exception. There is extensive, controversial literature, reviewed with great fairness (428), concerning to...

Phage Population Growth

Lysis involves a tradeoff between maximizing per-infection phage productivity and minimizing the phage generation While phage organismal ecology emphasizes virion production and survival (above) and phage community ecology has an emphasis on host population dynamics (below), the emphasis of phage population ecology (table 5-1) is on phage population growth and intraspecific competition, either within bacterial cultures (5) or within individual infected bacteria (28). Like any organism living...

Overall Organization

The PRD1 virion consists of a linear double-stranded DNA genome surrounded by an envelope containing host lipids and viral proteins. The major coat protein forms a protective shell over this membrane punctuated only by minor vertex and cementing proteins. Cryo-electron microscopy and image reconstruction of the virion (13, 57) revealed an icosahedral particle with average dimensions vertex to vertex of 69.8 nm, edge to edge of 65.5 nm, and facet to facet of 63.7 nm (figure 13-2A, B, C). There...

Phage Plaque Growth

Phage can grow within soft agar overlays a simple, spatially structured environment as plaques punctuating otherwise opaque bacterial lawns. Phage growth in plaques may be considered to occur in four stages (47) (i) initial host adsorption of seeded phage, (ii) an initial round of bacterial infection, (iii) an enlargement phase'' that involves multiple rounds of adsorption, infection, and release, and (iv) the end of the enlargement phase, which typically is associated with physiological...

And Assembly

The six long tail fibers of T4 are oriented for assembly onto the baseplate by the fibritin (wac) whiskers (436). Some other structure must serve this function in T2, since it lacks whiskers. Fibers about 3.0-4.0 nm thick and 150 nm long are made by the joining of two half-fibers at a kinked angle of about 150 . The half-fiber bound to the baseplate is constructed from three molecules of gp34 (71), and much of the fiber is likely composed of a three-stranded, b-helical structure. The half-fiber...

Transcription Patterns

This discussion is based on the information contained in figure 9-1. Similar transcription patterns have been observed with all lambdoid phages that have been examined. However, the small differences between l and other members of the lambdoid family have provided important insights into the mechanism of regulatory processes that act during transcription elongation. These processes, transcription termination and antitermination, play important roles in the temporal regulation of transcription...

References

Casadaban. 1987. In vivo DNA cloning with mini-Mu replicon cosmid and a helper lambda phage. Gene 51 77-84. 2. Groisman, E. A., B. A. Castilho. and M. J. Casadaban. 1984. In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc. Natl. Acad. Sci. USA 81 1480-1483. 3. Hayashi,T., K. Makino, M. Ohnishi, K. Kurokawa, K. Ishii, K. Yokoyama, C.-G. Han, E. Ohtsubo, K. Nakayama,T. Murata, M. Tanaka,T. Tobe,T. Iida, H....

P22 Psu9

Figure 10-6 Signal-anchor-release (SAR) domains in endolysins of Gramnegative bacteria. The N-terminal sequence of the endolysins of the lambdoid phages 21 (22), and PS119 (GenBank accession CAA09710), P22 (30), and the non-lambdoid phages P1 (30), T4 (81), and HP1 (7) are shown. The N-terminal SAR domain is underlined in the P1, HP1, and R21 endolysins (118b). The catalytic triad correspnding to residues E11, D20, and T26 of T4 lysozme are boxed (87). The dashed brackets arrow shows the...

Structure

Early studies on the structure of PRD1 have focused on elucidating its architecture by analyzing the virion by dissociation, the effects of mutations, and the characterization of recombinant proteins (reviewed in 5). These approaches continue to be very useful (9,17,19, 27-29, 34, 51, 53, 54, 64) and have now been complemented by X-ray crystallography (7,11,12, 69, 70), Raman spectroscopy (3, 65, 66), mass spec-trometry (33), small-angle X-ray scattering (63), antibody labeling (26) and...

Peqwnaigvlv0iihavmtyltnlyfkirednrrsrsrdepnve

Figure 10-4 Alignment of representatives of the S21 class II holin family. Sequences are compared with SPA2. Changes within either TMD resulting in increased or decreased side-chain bulk is indicated by shading or boxes, respectively. Dlp12 and Qin are cryptic lambdoid prophages of E. coli K-12 (90). PA2 is the lambdoid prophage of a porcine strain of E. coli (16). 933W is the lambdoid prophage carrying the Shiga-like toxin gene in the hemorrhagic E. coli strain 0157 H7 (86). to make a holin is...

Prophage Induction The Switch from the Lysogenic State to the Lytic State

Although the lysogenic state is extremely stable, lysogens can switch to the lytic state. They do so by taking advantage of a host regulatory system termed the SOS response (11,21). In response to conditions that damage DNA or inhibit DNA replication, perhaps 40 SOS genes are expressed at elevated levels. The products of these genes help the cell cope with the DNA damage in various ways. The SOS response involves the interplay of two regulatory proteins RecA and LexA. During normal growth, LexA...

The Q Transcription Antiterminator

The Q gene of l, located 5.8 kb beyond the pR promoter and distal to at least four transcription terminators, requires N antitermination for expression. Q function, in turn, is required for full expression of the phage late genes (39, 87) and serves as a second antitermination factor (figure 9-1) (43, 139). Q, like N, can antiterminate Rho-dependent and Rho-independent terminators (58,186). As we will see the requirements for and the mechanics of modification of the RNA Pol complex are very...

A

Figure 15-11 Acceptance or rejection of various RNA structure elements introduced in a noncoding region of MS2. At the left we show the wild-type structure, containing the stop codon (UAG italics) of the maturation gene. Mutant A has been given a big unstructured loop that is almost fully deleted during passaging (double arrows). Mutant C, containing a nearly perfect hairpin extension, is genetically stable for many generations. Mutant R with the perfect hairpin extension is an RNase III...

Repressor Control During Lysogenization

Infection by a temperate phage has an indeterminate outcome. Some cells in the culture become lysogenic, whereas others are channeled into lytic development. If 100 of the cells gave a lytic response, the phage would be virulent. If 100 gave a lysogenic response, the phage would not form plaques and would not be readily recognizable as a phage. How these dual potentialities are achieved is best understood in l. Chapter 8 covers present understanding of how the lysis lysogeny decision is made in...

Evolution and Evolutionary Studies

In the past decade, two approaches have been taken to investigate Microviridae evolution. In one approach, pioneered by the Drs. J. J. Bull and H. A. Wichman and colleagues, viruses are placed under selective conditions, either high temperature or host variations, and grown for numerous generations in a chemostat (23,24,31,69,137). At various time intervals, individual genomes are sequenced. Therefore the appearance and disappearance of beneficial mutations can be monitored. Of course, host...

Stephen T Abedon

Phage ecology is the study of the interactions between phage and their environments. These interactions are consequential, particularly to the extent that they affect bacteria. During traditional molecular phage characterization, however, only minimal consideration of ecology is made. Contrasting this tendency, here I consider phage organismal, population, community, and ecosystem ecology (Table 5-1). For additional approaches to the review of phage ecology, as well as the related field of...

Allan Campbell

Cell lines harboring latent viruses are common both in eukaryotes and in prokaryotes. Prokaryotes harboring latent phages are lysogenic, and the latent form of the phage is called a prophage. Phages that can enter such a latent state are called temperate, although some authors erroneously refer to them as lysogenic. Lysogeny was discovered because some bacterial isolates spontaneously produce small amounts of infectious phage. It was later shown that lysogenic bacteria could arise during...

Rz and Rz1

The Rz (153 codons) and Rzl (60 codons) genes are unique in biology one segment of DNA occupied by two genes in alternate reading frames but responsible for the same phenotype (56, 123). In the presence of millimolar concentrations of divalent cations, null alleles of Rz or Rzl, or their related P22 equivalents, cause lysis to terminate in metastable spherical cell forms (30, 122, 123). Rzl has a signal peptidase II cleavage site and is predicted to be a 40-residue, proline-rich lipoprotein...

Outlook

As suggested throughout this chapter, many complexities and redundancies of T4 are perhaps best understood in terms of its evolutionary history. Exchange of genes and acquisition of genes from other genomes (by lateral transfer) has been the raw material for natural selection under different environmental conditions. It is already well established that many genes are shared by rather different phages growing in different hosts and environments (155). Moreover, phages with contractile tail...

Post Transcriptional Controls

As with other phages, post-transcriptional mechanisms modulate expression of T4 genes in a variety of ways (i) by modulating translation initiation via potential secondary RNA structures and or by coupling translation of two or more genes, (ii) by translational repression, (iii) by proteins affecting ribosome structure and function, (iv) by processing of primary transcripts, and (v) by the rate of degradation. In addition, expression of at least four T4 genes requires splicing, or in a...

Prophage Insertion by Transposition

The most familiar example of insertion by transposition is coliphage Mu-1. There are fundamental differences between the life cycles of Mu-1 and l. In Mu-1, there is no circular form of the viral DNA. Also, viral DNA is always inserted into host DNA, even in the virion. Both replication and insertion result from transposition of viral DNA from one host site to another. Transposition is diagrammed in figure 7-5. Transposition has two possible outcomes, replicative and nonreplicative, of which...

FX174 et al the Microviridae

BRENTLINGER, APRIL D. BURCH, MIN CHEN, SUSAN HAFENSTEIN, ERICA MOORE, CHRISTOPHER R. NOVAK, AND ASAKO UCHIYAMA Sometime in the 1920s, a plaque was placed in a test tube labeled 174. It must have had a certain luster. Since then that phage has challenged genetic, biochemical, and biophysical dogmas. From the very beginning, it was unique. It was unusually tiny, readily passing through the smallest of ultrafilters. Sertic and Bulgakov (111) used its size to define a race...

Evolution of Phage Latent Period

From an ecological perspective we can divide the members of phage populations into two groups prereproductive and reproductive. Prereproductive phage are those engaged in either adsorption (including the extracellular search for susceptible bacteria) or the eclipse during these periods the phage is not generating mature phage progeny. Reproductive phage, by contrast, are those infecting bacteria during the period of phage-progeny maturation. For phage that must lyse their host bacteria to...

Phage Assembly

Filamentous phage assembly is a secretory process. Assembly occurs in the cytoplasmic membrane, and nascent phages are secreted from the cell as they assemble (figure 12-5). All eight of the phage-encoded proteins that are directly involved in assembly are integral membrane proteins. This includes three nonvirion proteins pI and its restart partner, pXI, in the cytoplasmic membrane (69) and pIV in the outer membrane (12) and the five viral coat proteins, which reside in the cytoplasmic membrane...

Head Structure and Assembly

The head assembly pathway is summarized in figure 18-6. The completed T4 head is composed primarily of the major cleaved capsid protein, gp23*, arranged in an icosahedral surface lattice of T 13, elongated along a 5-fold axis (267). The length of the T4 head is defined by a number, Q, related to the icosahedral T number. An illustration of Q numbers is shown in figure 18-7 together with models of someT4 head lengths. The 5-fold vertices, except for one, are occupied by the cleaved vertex...

Plasmaviridae Shape

AParentheses indicate approximate numbers of poorly characterized isolates that may or may not represent independent species. bArchaea. aParentheses indicate approximate numbers of poorly characterized isolates that may or may not represent independent species. bArchaea. known leviviruses are plasmid-specific coliphages that adsorb to F or sex pili. They have been divided, by serology and other criteria, into two genera. Several as yet unclassified leviviruses are specific for other plasmid...

Structure of the Phage Particle

The image of a filamentous phage obtained by atomic force microscopy is shown in figure 12-2. They are about 6.5 nm in diameter, with a length determined by the size of the genome, normally 6-7 kb of single-stranded DNA. The 6400-nucleotide Ff genome is encapsidated in a 930 nm particle a 221-nucleotide microphage variant is only 50 nm long (152), and the presence of cloned DNA in the phage genome makes the particle proportionately longer. Although there is no theoretical limit to the amount of...

The HK022 Nun Protein

Although not identified as a regulatory protein for its own genome, the action of the HK022 nun gene product is so tied to the action of l N that it warrants discussion in this review. The HK022 nun gene is located in the same relative position on the HK022 genome as the various N genes are located on the genomes of other lambdoid phages. However, the Nun protein does not act as an antitermination protein for HK022 but, instead, acts by terminating transcription on the l genome at sites not...

Prophages Inserted by Reciprocal Site Specific Recombination

L is one of the many phages that insert their DNA into the host chromosome by reciprocal recombination between the chromosome and a circular form of the phage DNA. The overall reaction comprises cointegration of the two circles. The recombination takes place between a unique site on the phage DNA and a unique site on the E. coli Table 7-1 Modes of Prophage Maintenance Insertion By site-specific recombination Coliphage l Salmonella phage P22 Mycobacterial phage L5 Vibrio phage CTX Phi...

DNA Replication

Positive polarity single-stranded DNA replication strategies are complex, occurring in three separate stages. These processes will be briefly summarized a more complete discussion can be found in the previous edition of this book (62). Stage I DNA replication involves the conversion of the single-stranded genome into a covalently closed, double-stranded, circular molecule, called RF I DNA (replicative form one DNA). Purified single-stranded Microviridae DNA produces progeny in transfection...

Lysis Without Lysozyme

It appears that all phages with double-stranded nucleic acid genomes use the holin-endolysin system for lysis. This must reflect a powerful evolutionary pressure for optimization of the lytic event, both in terms of efficiency of release of the progeny virions and, probably more so, in terms of terminating the vegetative cycle at a time appropriate for the particular host and environment. However, no lysozyme is encoded or produced by three classes of very simple lytic phages with small,...

Lysis Lysogeny Decision

As described above, CI must be present to stimulate expression of PRM in the lysogenic state. In a cell that is following the lysogenic pathway, the initial burst of CI is provided by expression of another promoter, PRE (9, 16). Regulation of this promoter involves physiological events that are not fully understood. The direct positive regulator of PRE is the phage protein CII, which binds at PRE (Figure 8-2C) and activates its expression. The level of CII in the cell is in turn under complex...

Harald Brussow And Frank Desiere

Many phage researchers believe that phages are as old as their bacterial hosts. If this hypothesis is true, then we have to postulate elements of vertical evolution for phages. In view of the postulated antiquity of these relationships we might not expect sequence similarities between more distantly related phages. Comparative phage genomics can reveal DNA sequence, protein sequence, or gene map similarities in the absence of sequence similarities between increasingly diverged phages. For even...

Coda

Transcription antitermination can serve two different roles useful to the phage. First, it allows the phage to acquire new genes even if the genes have associated transcription terminators. If an acquired gene locates within an operon, the antitermination mechanism enables transcription to reach downstream genes even if the acquired gene has an associated downstream terminator. For example, in phages carrying genes encoding Shiga toxin, even though the stx genes have an associated promoter,...

Structure of Packaged DNA

Whatever the mechanism of DNA translocation, it must overcome the energetic barrier of compacting the DNA and deliver the DNA into the prohead to confer the proper structure and organization within the head. DNA packaging in dsDNA phages is endothermic. Analogies are often made between DNA packaging and the collapse of DNA into a torus, or DNA toroid, in the presence of polyamines such as spermidine (29) or hexamine cobalt (98). While the final structures share structural similarities, such as...

Perspectives and Directions

In recent years remarkable strides have been made in our understanding of the molecular basis of phage lysis and its regulation. The progress has raised many new questions. That there are at least two general strategies for lysis is clear. All complex phages seem to use holin-endolysin lysis, whereas two of the prototype small ssDNA and ssRNA phages encode proteins which act as inhibitors of cell wall synthesis. The diversity of holins has always been a stunning, and somewhat daunting feature...

Lysis Systems At Least Two Strategies

Most bacteria have a murein cell wall that constitutes the principal barrier to host lysis. Compromising the cell wall is thus the fundamental goal for lytic processes. Two general strategies to accomplish lysis have been described (121). Phages with double-stranded nucleic acid genomes use the holin-endolysin strategy. In this scheme, the phage elaborates a murein-degrading enzyme, an endolysin (also known as lysozyme), specifically dedicated to lysis, and a second, membrane-embedded protein,...

Jan Van Duin Nina Tsareva

The single-stranded RNA coliphages were discovered by Tim Loeb and Norton Zinder in 1961 as the result of a search for phages whose infection cycle depends on E. coli F-pili, normally used for bacterial conjugation. Loeb and Zinder plated filtered samples of raw New York City sewage on E. coli strains and screened for phages that would produce plaques on male (F+) but not female (F ) bacteria. The first isolate, named f1, turned out to be a filamentous phage with a single-stranded DNA genome...

PRD1 Dissecting the Genome Structure and Entry

BUTCHER, JAANA K. H. BAMFORD, AND DENNIS H. BAMFORD There are six extremely similar phage isolates, from different parts of the world, that all infect Gramnegative bacteria harboring a conjugative plasmid. One of these viruses is PRD1. Viruses with this morphology (protein capsid surrounding a membrane vesicle containing the linear double-stranded DNA genome) also infect Grampositive hosts. These viruses have been classified together forming the Tectiviridae family...

Myoviridae

The same pattern of overall DNA sequence alignment and graded relatedness was also observed in phages with a contractile tail (Myoviridae). E. coli phage Mu (representative of the Mu-like genus in the Myoviridae family) and E. coli O157 Sakai prophage Sp18 could be aligned over about half of the genome length at the DNA level in a mosaic fashion (figure 4-4F). Mu shared with the Shewanella prophage MuSo2 protein sequence similarity, but only overall genome organization in the absence of...

The NUT Site

Information gained from genetic and structural studies as well as genome comparisons has extended our understanding of the nature and function of interactions at NUT RNA. The nut sites have been divided into three components boxA, a spacer, and boxB (figure 9-2A). Although the interactions at NUT involve a complex formed between all of the components, the following discussion will focus on each of the component sites individually. A comparison of boxA sites yielded a consensus sequence,...

Contributors

Abedon, Department of Microbiology, Ohio State University, Mansfield, OH 44906, USA Hans-W. Ackermann, Department of Medical Biology, Faculty of Medicine, Laval University, Qc G1K7p4, Quebec, Canada Sankar Adhya, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Juan C. Alonso, Departmento de Biotechnologia Microbiana, Centro Nacional de Biotecnologia-Consejo Superior de Investigaciones...

Reduced Phage Productivity

Host density impacts on community stability by affecting the peak phage densities that follow community-wide host lysis. With more phage than hosts within a batch-culture system, eventually all sensitive bacteria may become adsorbed and lysed (10, 29). However, in continuous culture there will be decay in free-phage densities due to outflow from the growth vessel. Since the rate at which host cells are found by free phage is a function of free-phage density (1), there is a race between the...

Antiholin Diversity and Raison dEtre

As stated above, many holin genes have dual-start motifs, with two potential start codons separated by at least one codon for a basic amino acid. In the few other cases tested (P22 13, 21 S, and f 29 14), eliminating codon 1 accelerates the onset of lysis, which is presumptive evidence that the dual start functions analogously to that of S (6, 77, 102). At least for S21, the holin gene of lambdoid phage 21, the longer product has been shown to have antiholin character, based on the N-proximal...

Receptor Recognition

PRD1 belongs to the class of broad-host-range, donor-specific phages, which infect cells only when IncP-, IncN-, or IncW-type multiple drug resistance conjugative plasmids are present. Among the hosts are several opportunistic human pathogens such as E. coli, Salmonella enterica, and Pseudomonas aeruginosa. Plasmid functions in the phage life cycle are dispensable after the genome has entered the cell (23, 43). These plasmids, the best studied of which is the IncP plasmid RP4 (50), encode the...

Contribution of Early Adsorbers to Phage Population Growth

Phage adsorption occurs essentially as an exponential decay in free-phage density (figure 5-2A). For any phage cohort released at a given moment into a population of potential hosts, phage adsorption occurs such that some constant fraction of remaining free phage will adsorb over any given interval. As a consequence, more phage from a given cohort will adsorb during an earlier interval compared with a later interval. Furthermore, if by chance a phage adsorbs a host earlier rather than later,...

Regulation in a Lysogen

In a lysogenic bacterium, the genes of the phage lytic cycle must be turned off, or the cell would die. Turnoff is caused by a phage-coded repressor (usually a protein, but in some cases, as with phage P4, an antisense RNA). The repressor prevents transcriptional initiation from key phage promoters. This turns off almost all phage transcription. Repressor acts directly on only a few promoters (in l, there are two). These are the promoters from which lytic development is initiated. Phage genes...

Hansw Ackermann

Bacteriophages or phages were discovered twice in a short time. The British pathologist Frederick William Twort described in 1915 the glassy transformation of Micrococcus colonies by an unknown agent. Felix Hubert d'Heer-elle, a French Canadian then working at the Pasteur Institute of Paris, observed the destruction of Shigella bacteria in broth (5). Contrary to Twort, he clearly recognized the viral nature of this phenomenon and devoted the rest of his scientific career to it. He coined the...

How Do Holins Work

The foregoing has promoted the idea that holins are remarkable proteins, honed by powerful evolutionary forces to be the simplest and most adjustable of, if not clocks, biological timers. Moreover, it also seems clear that holins are sensitive to the energy state of the membrane and thus, perhaps, constitute the simplest of gated membrane assemblies. The reader has no doubt noticed, however, that there is very little mechanistic insight into how holins work, however elegant and amusing the...

FX174 E

The fX174 genome has only 10 genes, and the lysis gene, E, spans only 91 codons in an alternate reading frame embedded in an essential gene D encoding a capsid scaffolding protein (figure 10-9). E is a membrane protein its first 35 residues define the lytic function and the membrane insertion domain. Although its topology is uncertain, several lines of evidence indicate the C-terminus is cytoplasmic. Replacement of the entire C-terminal cytoplasmic domain by b-galactosidase, GFP, or any folded...

Modification and Restriction of T4 DNA

In the following discussion the term restriction is used in its broadest meaning and is not limited to type II restriction enzymes. The complex modifications and restrictions of T4 DNA and of other DNA by T4 can be best rationalized as a result of ongoing evolution that is accelerated by strong selective forces and exchanges between the genomes of T4 and its host, including plasmid and prophage genomes that reside in the host. As mentioned above, T4 DNA contains HMC instead of C residues. This...

Filamentous Phage Display

Phage display is an extraordinarily powerful method of linking phenotype and genotype in the selection of proteins or peptides (see also chapter 44). The principle is very simple a DNA sequence is inserted into a gene encoding a phage coat protein so as to make a fusion protein in which the coat protein now displays the protein product of the inserted sequence. If a ligand is bound to a solid support, those phages expressing a protein or peptide that binds to the ligand will be immobilized, the...

Gisela Mosig Fred Eiserling

Bacteriophage Diagram

BacteriophagesT2,T4, andT6 were among seven Escherichia coli phages selected by Max Delbr ck to study fundamentals of viral replication in a limited number of model viruses. These studies led to the first formulation of many concepts that are now accepted foundations of molecular biology the fundamental differences between growth of viruses and cells figure 18-1 109 the demonstration that nucleic acids of virus particles suffice to establish infection and to direct synthesis of complete virions...

Prophage Genomics

At the time of this writing, the GenBank phage database comprises 200 complete phage genome sequences. An equivalent number of phage sequences were passively acquired as prophages in bacterial genome sequencing projects. The scientific value of these prophage sequences was only recently recognized 13, 14 . It goes beyond their potential to double the content of the current phage database and to correct a bias of the database towards selected phage systems coliphages, dairy phages,...

Tls

Figure 17-3 Consensus sequence logos of the T1 and TLS 21 nucleotide direct repeats. Produced using WebLogo Figure 17-3 Consensus sequence logos of the T1 and TLS 21 nucleotide direct repeats. Produced using WebLogo of rho-independent terminators. In coliphage T4 these sites frequently contained a UUCG or GNRA loop sequence 70 that none of the putative T1 TLS terminators possess. The presence of transcriptional terminators led us to speculate that either a transcriptional read-through system...

R1

Figure 15-12 Comparison of RNA secondary structures in the 3' UTR in four representative RNA phages. SP is a species IV allolevivirus. PP7 and AP205 resemble SP in their simple 3' UTR structure. MS2 has a more elaborate folding with many extra elements shaded . The stopcodon of replicase is boxed R stop . Also boxed is a conserved sequence in the top of U1 UGCUU . In Qb and SP these nucleotides form a pseudoknot with their complement 1200 nucleotides upstream figure 15-6 . For MS2, PP7, and...

Gregory J German Rajeev Misra Andrew M Kropinski

Haemophilus Phage Hp2

Although T1 got its initial fame from its use, in 1943, by Salvador Luria and Max Delbr ck in their landmark fluctuation test 64 , an entirely different reason has resulted in its notoriety since then. This phage's resistance to desiccation results in its persistence for weeks in an aerosol which in turn created a real nightmare for laboratories engaged in research employing E. coli K-12 strains. It was not uncommon to hear cries from laboratory workers that their overnight bacterial cultures...