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Filamentous bacteriophage predominantly infect Gram-negative bacteria and make an important contribution to host physiology, ecology, and virulence, including production of deadly toxins, such as cholera toxin. The unique filamentous structure, small genome size (4–12 kbp), replicative and/or integrative mode of inheritance, simple cultivation, and easy genomic manipulation sparked considerable attention to this class of bacteriophage for a number of applications, including cloning, sequencing, recombinant protein expression, phage display technology, and nanotechnology. This book covers a range of topics that can be grouped into two themes: impact of diverse filamentous phage on their host bacteria (five chapters) and applications of Escherichia coli Ff phage (eight chapters).

F-specific filamentous phage of Escherichia coli (Ff: f1, M13 or fd) are long thin filaments (860 nm x 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70 °C in 1 % SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative “dipstick” lateral flow diagnostic assay for human plasma fibronectin.

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.

In this study we describe BI-K0058, a new inhibitor of the transcription-termination factor Rho belonging to a different chemical class from bicyclomycin, the only known antibiotic acting on Rho. BI-K0058 inhibits the poly(C)-dependent ATPase activity of Rho with an IC(50) of 25 microM as well as in vitro transcription-termination of two natural substrates, the Salmonella enterica hisG cistron and the f1 phage intergenic region. BI-K0058 does not affect photolabeling of Rho by ATP. The results of gel mobility shift experiments with a natural RNA substrate demonstrate that BI-K0058 inhibits the formation of the ATP-independent high affinity Rho-RNA complex.

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.

The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the C-terminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV-'InvG43 protein becomes dependent on the co-expression of another gene, invH, for function in phage assembly. [3H]-palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV-'InvG43 and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.

Recombinant f1 phage particles containing cDNA of the human urokinase typeplasminogen activator (u-PA) under the control of the simian virus 40 (SV4O) early promoter were used for transfection of monkey COS-7 cells using a DEAE dextran method. The fibrinolytic activity of u-PA was detected in the culture medium of the 10 5 cells aansfected with the phage particles containing single-stranded (ss) DNA of more than 0.2 ng. This finding will lead us to develop a simple and efficient method for expression cloning using mammalian cells.

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.

It is a mystery how some viruses are able to replicate inside the host cell, producing progeny that escape without damaging the host's plasma membrane. In a Perspective, [Joshua Zimmerberg][1] describes new research ([ Marciano et al .][2]) demonstrating that the f1 phage (a virus that infects bacteria) is able to export new progeny out of the bacterial host using a phage-encoded pIV protein, which forms a gated channel in the bacterial outer membrane. Zimmerberg compares this mechanism of export to that of other viruses and discusses what these new findings teach us about protein translocation in eukaryotic cells. [1]: http://www.sciencemag.org/cgi/content/full/284/5419/1475 [2]: http://www.sciencemag.org/cgi/content/short/284/5419/1516

We describe the use of transcriptional fusions to the phage shock protein (psp) promoter. These fusions are expressed only when cells are infected by filamentous phage. In an application, the psp promoter was fused to the protein coding part of filamentous phage gene III (gIII). Protein III (pIII) is needed to complement mutant f1 phage containing a deletion of gIII, but its synthesis also renders cells resistant to infection. By inducing pIII production from psp–gIII only in the cells that are already infected with phage, it was possible to obtain plaques from phage in which gIII had been completely deleted. gIII was deleted from two helper phages: R408 and VCSM13, which were then propagated on cells containing the psp–gIII fusion. These two phages were tested for use in a phage display method that requires generation of noninfectious, phagemid-containing virion-like particles. Both helpers worked, but R408d3 was superior to VCSM13d3, because it generated about 1800-times fewer background infectious particles.

A major challenge in basic and applied biological research is the engineering of small proteins with pre-determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin-6. In previous work, the 'minibody', a 61 residue polypeptide consisting of a beta-pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin-6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokine's biological activity. The approach described here could in principle be applied to other molecular targets.

Bacteriophage f1-infected cells are relatively insensitive to a variety of colicins including E1, E2, and K; an f1 gene product is responsible for this insensitivity. A colicin-tolerant mutant cannot grow f1 phage; f1 DNA does not penetrate this mutant properly. One interpretation of these results is that an f1 gene product blocks the penetration of colicins which is necessary for their action, whereas the colicin-tolerant mutation blocks the penetration of colicins as well as phage f1 DNA.

The region of the bacteriophage f1 genome near the gene IV/intergenic region (IG) junction previously was shown to contain a sequence necessary for efficient packaging of single-stranded (SS) DNA into phage particles (the f1 “morphogenetic signal”) [ G. P. Dotto, V. Enea, and N. D. Zinder (1981) Virology114, 463–473]. The DNA content of f1 phage/pBR322 chimeric plasmid-transformed, phage-infected bacteria has been investigated. The chimeric plasmids, constructed by Dotto et al. (1981) , contained the f1 origins of DNA replication and, in some cases, also contained the “morphogenetic signal.” Chimeric plasmid SS DNA was detected in phage-infected bacteria harboring any one of these chimeric plasmids, and the majority of this SS DNA was found complexed to the phage gene V protein. Therefore, the “morphogenetic signal” is not required for the formation of the gene V protein/f1 SS DNA complex but instead must function at a later stage of filamentous phage morphogenesis.

We have analyzed the results of three gene II amber × gene II amber f1 phage crosses. Each was done in a non-restricting (K) host, and in a restricting (B) host. In each cross, only one parent was sensitive to B restriction. The other parent was protected from B restriction, either because of a combination of genetic mutation at one site governing sensitivity to B restriction, and B specific modification at the other, or because of genetic mutation at both sites. In all cases, B restriction resulted in the disruption of linkage relationships between the gene II region and the unselected sensitivity site markers. Previously we have shown that when such crosses involve a protected parent which is B modified at both sensitivity sites, linkage relationships remain the same under restricting and non-restricting conditions. Hence, the SB sites seem to manifest a striking marker effect in restricted crosses in which protection is conferred by mutation rather than modification. Taken together, these results imply that, in a restricted cross, the B host specificity system can distinguish a protected parent which is B modified from one which is a sensitivity site mutant. Since such a distinction could be made most easily on an intermediate structure containing hybrid DNA, we interpret these results in terms of a recombination mechanism mediated by an asymmetric heteroduplex.

K368, a male derivative of Escherichia coli strain CRT4634, is unable to replicate its DNA at 42°. It is a dnaA type mutant. It is also unable to support f1 phage growth at 42°, although a return to 33° is immediately followed by active phage production. Studies of the DNA found inside K368 cells infected and kept at 42° show the presence of material sedimenting like viral RF and SS. However, there is a defect in the single-stranded DNA molecules made under these conditions. Many of these molecules are linear instead of circular. We interpret these facts in terms of a ligaselike activity necessary for the completion of f1 viral single-strand synthesis.

The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively. The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified. [35S]cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage. This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA. We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins. The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.

The major coat protein of infecting f1 phage is incorporated into the inner membrane of the host cell, even in the absence of phage f1DNA penetration and replication. The major coat protein monomers are reutilized in the assembly of new phage. They are not conserved as a single unit but behave as independent units which are slowly incorporated into newly manufactured phage.

Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.

RNA transcription was investigated during the development of F1 phage, which is specific for the strict anaerobe Clostridium sporogenes. RNA species transcribed during F1 phage infection were characterized with respect to time of appearance and molecular weight by polyacrylamide gel electrophoresis. Ten mRNA species were characterized, of which five were produced early in infection and five were synthesized late in infection. All the above 10 species were transcribed from one strand of F1DNA, the heavy strand. Two additional mRNA species were transcribed from the light strand of F1 phage DNA later in infection. Throughout the F1 phage infective cycle, rRNA was continuously synthesized by cells of C. sporogenes.

We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication. Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage. However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria. A plasmid dependent on the f1 replicon fails to transform IHF mutants. By gel retardation analysis, we show that IHF specifically binds to the origin of replication. DNase I "footprinting" experiments demonstrate that IHF binds to multiple sites within the replication enhancer sequence, a cis-acting, A + T-rich sequence that potentiates f1 DNA replication. Moreover, the effect of IHF mutation on f1 growth is suppressed by initiator protein (f1 gene II) mutations that restore efficient replication from origins that lack a functional replication enhancer sequence. This genetic evidence supports the conclusion that the replication enhancer sequence is the site of action of IHF.

A strain was constructed which contains mutations in the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) such that filamentous phage f1 cannot grow. The complementation of either mutation with its wild-type allele permits phage growth. We used this strain to select f1 phage which contain a cloned trxB gene. The location of the gene on the cloned fragment was determined, and its protein product was identified. Plasmid subclones that contain this gene overproduce thioredoxin reductase.

A plasmid vector, pJBS633, that facilitates the construction of translational fusions of genes of interest to the coding region of the mature form of TEM beta-lactamase has been developed. Transformants containing in-frame fusions can be identified by their ability to grow when plated at high inocula on agar containing ampicillin (Ap). The cellular location of the beta-lactamase moiety of the fusion proteins can then be determined since only those that direct the translocation of the beta-lactamase across the cytoplasmic membrane to the periplasm result in the ability of individual cells of Escherichia coli to form isolated colonies in the presence of Ap. Conversely, those fusion proteins in which the beta-lactamase moiety remains cytoplasmic do not protect individual cells against Ap. Transformants expressing the latter class of fusion proteins can, however, be identified when plated at high inocula since, as cells start to lyse, the cytoplasmic beta-lactamase activity is released and provides Ap resistance to the surrounding cells. The vector contains the origin of replication of f1 phage so that single-stranded plasmid DNA can be obtained in the appropriate orientation to allow sequencing across the fusion junction using a universal primer complementary to the start of the coding region of mature TEM beta-lactamase. pJBS633 should be useful as a general vector for the construction of beta-lactamase fusions and, in particular, for the analysis of protein export signals and the determination of the organisation of proteins in the E. coli cytoplasmic membrane.

The effect of male-specific filamentous deoxyribonucleic acid (f1) and isometric ribonucleic acid (MS2) bacteriophages on the formation of mating pairs inEscherichia coliconjugation was examined directly in the Coulter counter. When a sufficient multiplicity of infection (MOI) was used, the f1 phage immediately and completely inhibited the formation of mating pairs. On the other hand, the MS2 phage at a relatively high MOI also inhibited the formation of mating pairs significantly although not completey. The inhibitory effect of MS2 phage was dependent on the time of addition and the MOI used. At relatively low MOI (400) MS2 phage disrupted the mating pairs already formed. Some preformed mating pairs were resistant to the high MOI of MS2 phages, however, and the “sensitive” (to high MOI) mating pairs seem to mature into “resistant” mating pairs as a function of time. We conclude that the tip of an F pilus is the specific attachment site for mating. The following process of mating pair formation has been formulated by deduction. (i) The sides of F pili weakly contact female cells, (ii) then the tips of F pili attach to the specific receptor sites to form initial mating pairs, and (iii) those pairs mature into mating pairs that are resistant to the high MOI of MS2 phages. The high MOI of MS2 prevents the first step, whereas f1 phages affect the second step—the binding between the tips of F pili and the receptor sites.