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The tail fiber adhesins are the primary determinants of host range in the T4-type bacteriophages. Among the indispensable virion components, the sequences of the long tail fiber genes and their associated adhesins are among the most variable. The predominant form of the adhesin in the T4-type phages is not even the version of the gene encoded by T4, the archetype of the superfamily, but rather a small unrelated protein (gp38) encoded by closely related phages such as T2 and T6. This gp38 adhesin has a modular design: its N-terminal attachment domain binds at the tip of the tail fiber, whereas the C-terminal specificity domain determines its host receptor affinity. This specificity domain has a series of four hypervariable segments (HVSs) that are separated by a set of highly conserved glycine-rich motifs (GRMs) that apparently form the domain's conserved structural core. The role of gp38's various components was examined by a comparative analysis of a large series of gp38 adhesins from T-even superfamily phages with differing host specificities. A deletion analysis revealed that the individual HVSs and GRMs are essential to the T6 adhesin's function and suggests that these different components all act in synergy to mediate adsorption. The evolutionary advantages of the modular design of the adhesin involving both conserved structural elements and multiple independent and easily interchanged specificity determinants are discussed.

Approximately 170 bacteriophages with morphologies similar to T4 have been identified (1). These T4-like phages have been isolated on a wide range of bacterial hosts that grow in diverse environments (1, 2, 72). T4, the type phage of this family, is probably the best-understood virulent phage. Its genome has been entirely sequenced, and its life cycle is extremely well understood; however, until recently little was known about all the other T4-type phages. Genomic hybridization and PCR analysis revealed that the T4-type phages vary considerably in their distance from T4 (54, 62, 72). Based on such data and limited sequence analysis, we can distinguish subgroups of the T4 type (54, 62, 72). The T-even subgroup shares considerable nucleic acid sequence homology with T4; for example, quantitative hybridization between the genomes of T2, T4, and T6 indicates that more than 90% of their sequences are nearly identical (18). As a result of their close relationship to T4, the T-even genomes can usually be analyzed and sequenced using PCR primers based on the T4 sequence (62, 71, 72). Although such comparisons confirmed that most of the T4-type phages were very close to T4, a few of them, such as RB69 (81) and SV14 (54), were clearly chimeras. The genomes of these phages have blocks of sequence that diverge significantly from T4. The origin of these sequences became obvious with the characterization of the pseudo-T-even phages (54). The members of this subgroup of the T4-type phages (e.g., RB49 and 44rr2.8t) are more diverse in their host range than the T-even phages, and their genomes are phylogenetically distant from T4 (54, 62). Only a few genes of phage RB49, for example, still retain sufficient homology to hybridize with T4 DNA under stringent conditions (54). The sequencing of the most conserved segments of the RB49 genome revealed that they encoded the structural proteins of the head (gp23 and gp24), the collar (gp20), and the contractile tail (gp18 and gp19) (54). Homologues of most of the structural components of the T4 virion were thought to be present in the pseudo-T-even phages (54). A small number of plasmids containing randomly cloned DNA of RB49 have been previously analyzed (54), and these had two sorts of sequences. A minority contained sequences that lacked homology to any entry in the NCBI database. The majority contained distant homologues to both nonstructural and structural genes of T4. Aside from their significant divergence in nucleotide sequence, the T-even and the pseudo-T-even phages also differ in the modifications of their DNA. The T-even genomes contain hydroxyl-methylcytosine in place of cytosine, and these residues are generally glucosylated, which provides additional protection against host restriction systems (12, 29). The DNA of the pseudo-T-even phages does not appear to have these nucleotide modifications (54). Consequently, the pseudo-T-even phages must have evolved a transcription and replication apparatus that was adapted to this difference in the DNA template they use. Furthermore, Southern analysis of several different pseudo-T-even phages revealed that they are as distant from each other as they are from T4 (54). Their phylogenetic distance from T4, and the genome plasticity that this implies, motivated us to further investigate the pseudo-T-even subgroup of phages. In this communication we have used an efficient partial sequencing strategy to compare the genomes of the T-even phage T4 and the pseudo-T-even phage RB49. Our snapshot of the RB49 genome indicates that it has diverged substantially and relatively uniformly from T4, and thus it is the first phage of this type to be analyzed. In addition to extensive random genomic sequencing, this study involved the targeted sequencing and characterization of two regions that contain the DNA replication genes. Although the genomes of T4 and RB49 share many features, they have important differences. A functional analysis of the RB49 promoters revealed a fundamental difference in the regulation of the gene expression in T4 and RB49. RB49 employs only two classes of temporally regulated promoters, rather than the three in T4. The evolutionary processes capable of generating such diversity within a phage family are discussed.

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3′ poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the β and γ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca , following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3′ polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.

THE Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E1,2. Both enzymes are important in RNA processing and messenger RNA degradation3–14. Here we report that enolase and RhlB are two other major components of the degradosome. Enolase is a glycolytic enzyme with an unknown role in RNA metabolism. RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes15–26. We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase. Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation. These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover.

The adsorption specificity of T4 is determined by the tip of the gene 37 tail fibers which bind to receptors on the bacterial surface. T4 infects only Escherichia coli and closely related Shigella species, but rare host range mutants can be isolated that infect Yersinia pseudotuberculosis I, an evolutionally distant bacterium. Some of these mutations result in amino acid residue substitutions in the C-terminal portion of gene 37, but others involve unequal exchanges between a series of sequence motifs (His boxes) in the same region. The duplication or mutational alteration of this segment apparently suffices for phage adsorption to a Yersinia receptor. It is suggested that recombination between the His box sequences can generate diversity in phage host range by shuffling receptor recognition domains.

We have constructed a strain that overexpresses E coli poly(A) polymerase (PAP I). The recombinant protein was soluble, and a partially purified extract had high levels of poly(A) polymerising activity. An antiserum raised against the overexpressed PAP I has permitted two types of analysis: the identification of other E coli proteins that may interact with PAP I, and the search for PAP I-like proteins in other bacteria. Immunoprecipitation experiments suggest that PAP I is associated with a 48-kDa protein. This protein remains to be identified. Western blotting using the antiserum against E coli PAP I revealed related proteins in a variety of Gram-negative bacteria and in B subtilis. A comparison of the E coli protein with putative poly(A) polymerases recently identified in H influenza and B subtilis showed highly conserved sequences in the amino terminal and central portions of the proteins that may be important for enzyme activity.

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the ∼50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.

The ratio of the FtsZ to FtsA proteins determines the correct initiation of cell division in Escherichia coli. The genes for these proteins are contiguous on the chromosome. Although both genes are transcribed from common promoters, the presence of ftsZ-specific promoters, along with differences in the efficiency of translation of their respective mRNAs, contribute to the increased relative expression of ftsZ. We report here that the polycistronic ftsA-ftsZ transcripts are cleaved by RNase E and that this cleavage affects the decay of ftsA and ftsZ mRNA. As a consequence of the cleavage, RNase E also contributes to the differential expression of the two genes.

Immediately after T4 bacteriophage infection, the Escherichia coli nucleoid undergoes rapid delocalization. The ndd gene of T4 is responsible for this nuclear disruption phenomenon. We have cloned two alleles of this gene and studied the effects of their expression on E. coli cells. We have shown that the Ndd protein (i) is able to reproduce the disruption of the nucleoid characteristic of T4 infection, (ii) is highly toxic and results in a logarithmic decrease in cell viability, and (iii) inhibits genomic DNA replication by blocking progression of replication forks. Induction of Ndd does not result in degradation of genomic DNA and does not significantly alter the general processes of transcription and translation during the entire period of exponential cell death. These results support the notion that the target of Ndd is some aspect of the nucleoid architecture.

Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.

Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7 kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease identified as having a general role in the chemical decay of E. coli mRNA.

Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage PhiMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with beta-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.

We have examined the mechanism of the self-regulation of bacteriophage T4D genes 32 and 43. It has been demonstrated that the rate of P32 synthesis can increase substantially in situations in which new RNA synthesis is blocked either by the drug rifampicin or by inactivation of a temperature-sensitive host RNA polymerase. In addition, RNAs have been extracted from various infections which have large differences in the in vivo rate of P32 synthesis. Determination of the in vitro capacity of these RNAs to direct the translation of P32 indicates only small differences in the relative amounts of gene 32 mRNA in such infections. These results are interpreted as suggesting gene 32 self-regulation operates by regulation of the translation of gene 32 mRNA. In contrast, experiments with gene 43 mutants show that overproduction of P43 is blocked when further RNA synthesis is prevented. Thus, the self-regulation of gene 43 appears to operate by control of transcription.

Two new expression vectors have been constructed to take advantage of several useful properties of bacteriophage T4-infected Escherichia coli. These plasmids, pRDB8 and pRDB9, contain the promoter region and start codon of T4 gene 32, a contiguous multiple cloning site (MCS), and translation and transcription termination signals. DNA fragments inserted into the MCS are transcribed and translated at a high level in both uninfected and phage T4-infected cells. Furthermore, the extreme stability of the hybrid mRNA after infection permits the specific biosynthetic labeling of the protein encoded by the cloned gene. In addition, the cloned gene product is stabilized, since the host-mediated degradation of foreign proteins is inhibited by phage infection. The properties of this expression system were demonstrated with the constant region of a rabbit immunoglobulin lambda light chain (C lambda) gene. Although proteolytic degradation of the C lambda fusion protein was rapid in uninfected cells, degradation was blocked in phage-infected cells and the protein accumulated in greater amounts.

The construction of a plasmid vector which facilitates the cloning and recovery of blunt-ended DNA fragments is described.This plasmid,called pHP34, differs from pBR322 by a 10-bp insertion which introduces a unique SmaI site immediately flanked by two Eco Rl sites.Blunt-ended DNA fragments cloned in the SmaI site can be recovered by digestion with Eco VI.Small cloned fragments can be chemically sequenced using a strategy which does not require their purification.The use of a plasmid related to pHP34 for in vitro mutagenesis by the insertion of a DNA linker fragment conferring an antibiotic resistance is also discussed.

Deoxyribonucleic acid replication and genetic recombination were investigated after infection of Escherichia coli with ligase-deficient r II bacteriophage T4D. The major observations are: (i) deoxyribonucleic acid synthesis is discontinuous, (ii) the discontinuities are more slowly repaired than in wild-type infection, (iii) host ligase is required for viability, and (iv) genetic recombination is increased.

Following infection of E. coli B with ligase-deficient rII bacteriophage T4D recombination between linked markers is increased 4.2 fold and heterozygote frequency increased 2.3 fold. In such infection recombination occurs at a rapid rate for an extended period. This is in contrast to the time course of recombination observed in wild-type, lysis-inhibited, or lysis-defective (gene t defective) infection. In all of these cases recombination under standard cross conditions occurs early in the vegetative cycle. The increased recombination in ligase-deficient rII infection is reduced in a bacterial strain which produces greater than normal levels of host ligase. These results indicate that ligase has a crucial role not only in the replication of DNA but also in recombination. The level of ligase may determine whether DNA replication occurs with or without concomitant recombination.

In T4-infected cells, the gene 32 monocistronic mRNA is very stable. To study the molecular basis for this stability, we have constructed chimeric plasmids containing the monocistronic promoter and the gene 32 translation initiation sequence fused to either most of the E. coli lac operon or only a segment of the lacZ gene, followed by the gene 32 transcription terminator. The resulting hybrid transcripts are unstable in uninfected cells. In phage-infected cells, however, the hybrid mRNAs are at least as stable as gene 32 mRNA itself. Analysis of other plasmid constructs indicates that the sequences on the gene 32 mRNA from its 5' end to slightly beyond the initiation codon suffice to stabilize these hybrids. Studies with a series of deletions of the gene 32 leader sequence suggest that an RNA sequence near the gene 32 initiation codon is involved. Various models to explain this mRNA stabilization are discussed.

A new method for in vitro insertional mutagenesis of genes cloned in Escherichia coli is presented. This simple procedure combines the advantages of in vitro DNA linker mutagenesis with those of in vivo transposition mutagenesis. It makes use of the omega fragment, a 2.0-kb DNA segment consisting of an antibiotic resistance gene (the Smr/Spcr gene of the R100.1 plasmid) flanked by short inverted repeats carrying transcription and translation termination signals and synthetic polylinkers. The omega fragment is inserted into a linearized plasmid by in vitro ligation, and the recombinant DNA molecules are selected by their resistance to streptomycin and spectinomycin. The omega fragment terminates RNA and protein synthesis prematurely, thus allowing the definition and mapping of both transcription and translation units. Because of the symmetrical structure of omega, the same effect is obtained with insertions in either orientation. The antibiotic resistance gene can be subsequently excised from the mutated molecules, leaving behind its flanking restriction site(s).

This paper describes the construction and characterization of a chimeric plasmid that encodes the single-stranded DNA-binding protein of bacteriophage T4D (the product of gene 32). The plasmid contains a 2·6 × 10 3 base Hin dIII segment of T4 DNA that includes genes 59 and 32 as well as a portion of gene 33. Isolation of bacteria carrying the recombinant plasmid became possible when the segment of phage DNA contained an amber mutation in gene 32. This suggests that a functional gene 32 is deleterious to the cell. Using antibody to gene 32 protein, we have been able to demonstrate expression of the plasmid-borne gene 32 in uninfected bacteria. Deletion variants of the gene 32 plasmid have been constructed in vitro . These have been used to align the genetic map of the region with the restriction map and to study phage gene expression from the plasmid in both infected and uninfected cells. In phage-infected cells the level of functional gene 32 product regulates the efficiency of translation of its own messenger RNA. We also observe such self-regulation for gene 32 present on the plasmid.

The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6 X 10(6) Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.

Gene 32 from bacteriophage T4 is transcribed as precursor transcripts which are processed to a stable product. This processing of the gene 32 mRNA was observed in RNase III or P-deficient strains of Escherichia coli. However, after infection of an RNase E-deficient strain, the amount of processed transcript was significantly reduced while the levels of the precursor transcripts remained high. RNase E therefore appears to have an essential role in the processing of the gene 32 mRNA. We have mapped the exact 5' end of the processed transcript by primer extension. The cleavage occurs near a stem-loop structure at a site which shows some similarity to other known RNase E cleavage sites. The effects of the processing on the differential stability of the upstream and downstream sequences, and on gene expression, are discussed.

We have constructed a derivative of the broad-host-range vector RSF1010. This plasmid, p alpha omega, contains an expression cassette derived from bacteriophage T4 gene 32, into which we have inserted the coding sequence for the xylE enzyme (C2,3O) of the TOL plasmid pWWO. The composite plasmid, p alpha xylE omega, was transferred by conjugal mobilisation into a variety of Gram-negative bacteria (Agrobacter, Paracoccus, Erwinia, Pseudomonas, Rhizobium and Xanthomonas). High levels of C2,3O activity were found in almost all of the extracts. Polyacrylamide gel electrophoresis of these extracts revealed a prominent protein band at 35 kDa whose identity as the C2,3O gene product was confirmed by immunoblotting. We have mapped the 5' ends of the gene 32/xylE hybrid transcripts. In all of the Gram-negative bacteria, the proximal P2 promoter is the most efficient promoter in the cassette. In most of the strains a weaker and more distal promoter activity (Pl) was also detected. In both uninfected and phage-infected Escherichia coli cells, the transcript produced from this promoter is processed at a specific site upstream from the gene 32 start codon. The same processing occurred in all the bacterial species examined. The decay of the hybrid xylE transcript has been analyzed in E. coli and Erwinia, and in both strains this mRNA was among the most stable.

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli , overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.