DNA sequence heterogeneity in the genes of T-even type Escherichia coli phages encoding the receptor recognizing protein of the long tail fibers

Summary Genes (g) 36 and 37 code for the proteins of the distal half of the long tail fibers of phage T4, gene product (gp) 35 links the distal half to the proximal half of this fiber. The receptor, lipopolysaccharide, most likely is recognized by gp37. Using as probe a restriction fragment consisting of most of g36 and g37 of phage T4 the genes corresponding to g35, g36, and g37 of phages T2 and K3 (using the E. coli outer membrane proteins OmpF and OmpA, respectively, as receptors) have been cloned into plasmid pUC8. Partial DNA sequences of g37 of phage K3 have been determined. One area, corresponding to residues 157 to 210 of the 1026 residue gp37 of phage T4, codes for an identical sequence in phage K3. Another area corresponds to residues 767 to 832 of the phage T4 sequence. Amino acid residues 767 to 832 of the phage T4 sequence are almost identical in both phage proteins while the remainder is rather different. DNAs of T2, T4, T6, another T-even type phage using protein Tsx as a receptor, and 10 different T-even type phages using the OmpA protein as a receptor have been hybridized with restriction fragments covering various parts of the g37 area of phage K3. With probably only one exception all of the 13 phages tested possess unique genes 37 and within the majority of these, sequences highly homologous to parts of g37 of K3 are present in a mosaic type fashion. Other regions of these genes 37 did not show any homology with the K3 probes; in case of the OmpA specific phage M1 absence of homology was evident in most of its g37 even including the area that should serve for recognition of the cellular receptor. In sharp contrast to this situation it was found that a major part of the gene (g23) coding for the major capsid protein is rather highly conserved in all phages studied. The extreme variability in sequences existing in genes 37 might be a consequence of phages during evolution being able to more or less drastically change their receptor specifities.     

Stoichiometry and Domainal Organization of the Long Tail-fiber of Bacteriophage T4: A Hinged Viral Adhesin

The long-tail fibers (LTFs) form part of bacteriophage T4's apparatus for host cell recognition and infection, being responsible for its initial attachment to susceptible bacteria. The LTF has two parts, each ∼70 to 75 nm long; gp34 (140 kDa) forms the proximal half-fiber, while the distal half-fiber is composed of gp37 (109 kDa), gp36 (23 kDa) and gp35 (30 kDa). LTFs have long been thought to be dimers of gp34, gp37 and gp36, with one copy of gp35. We have used mass mapping by scanning transmission electron microscopy (STEM), quantitative SDS-PAGE, and computational sequence analysis to study the structures of purified LTFs and half-fibers of both kinds. These data establish that the LTF is, in fact, trimeric, with a stoichiometry of gp34: gp37: gp36: gp35=3:3:3:1. Averaged images of stained and unstained molecules resolve the LTF into a linear stack of 17 domains. At the proximal end is a globular domain of ∼145 kDa that becomes incorporated into the baseplate. It is followed by a rod-like shaft (33 × 4 nm; 151 kDa) which correlates with a cluster of seven quasi repeats, each 34 to 39 residues long. The proximal half-fiber terminates in three globular domains. The distal half-fiber consists of ten globular domains of variable size and spacing, preceding a needle-like end domain (15 × 2.5 nm; 31 kDa). The LTF is rigid apart from hinges between the two most proximal domains, and between the proximal and distal half-fibers. The latter hinge occurs at a site of local non-equivalence (the “kneecap”) at which density, correlated with the presence of gp35, bulges asymmetrically out on one side. Several observations indicate that gp34 participates in the sharing of conserved structural modules among coliphage tail-fiber genes to which gp37 was previously noted to subscribe. Two adjacent globular domains in the proximal half-fiber match a pair of domains in the distal half-fiber, and the rod domain in the proximal half-fiber resembles a similar domain in the T4 short tail-fiber (gp12). Finally, possible structures are considered; combining our data with earlier observations, the most likely conformation for most of the LTF is a three-stranded β-helix.     

Organization and evolution of bacterial and bacteriophage primase-helicase systems

Summary Amino acid sequences of primases and associated helicases involved in the DNA replication of eubacteria and bacteriophages T7, T3, T4, P4, and P22 were compared by computer-assisted methods. There are two types of such systems, the first one represented by distinct helicase and primase proteins (e.g., DnaB and DnaG proteins of Escherichia coli), and the second one by single polypeptides comprising both activities (gp4 of bacteriophages T7 and T3, and alpha protein of bacteriophage P4). Pronounced sequence similarity was revealed between approximately 250 amino acid residue N-terminal domains of stand-alone primases and the primase-helicase proteins of T7(T3) and P4. All these domains contain, close to their N-termini, a conserved Zn-finger pattern that may be implicated in template DNA recognition by the primases. In addition, they encompass five other conserved motifs some of which may be involved in substrate (NTP) binding. Significant similarity was also observed between the primase-associated helicases (DnaB, gp12 of P22 and gp41 of T4) and the C-terminal domain of T7(T3) gp4. On the other hand the C-terminal domain of P-alpha of P4 is related to another group of DNA and RNA helicases. Tentative phylogenetic trees generated for the primases and the associated helicases showed no grouping of the phage proteins, with the exception of the primase domains of bacteriophages T4 and P4. This may indicate a common origin for one-component primase-helicase systems. Two scenarios for the evolution of primase-helicase systems are discussed. The first one involves fusion of the primase and helicase components (T7 and T3) or fusion of the primase component with a different type of helicase domain (P4). The second possibility is the duplication of an ancestral gene encoding a gp4-like bifunctional protein followed by divergence of the copies, one of which retains the primase and the other the helicase domain.     

Characterization of a novel intramolecular chaperone domain conserved in endosialidases and other bacteriophage tail spike and fiber proteins

Folding and assembly of endosialidases, the trimeric tail spike proteins of Escherichia coli K1-specific bacteriophages, crucially depend on their C-terminal domain (CTD). Homologous CTDs were identified in phage proteins belonging to three different protein families: neck appendage proteins of several Bacillus phages, L-shaped tail fibers of coliphage T5, and K5 lyases, the tail spike proteins of phages infecting E. coli K5. By analyzing a representative of each family, we show that in all cases, the CTD is cleaved off after a strictly conserved serine residue and alanine substitution prevented cleavage. Further structural and functional analyses revealed that (i) CTDs are autonomous domains with a high alpha-helical content; (ii) proteolytically released CTDs assemble into hexamers, which are most likely dimers of trimers; (iii) highly conserved amino acids within the CTD are indispensable for CTD-mediated folding and complex formation; (iv) CTDs can be exchanged between proteins of different families; and (v) proteolytic cleavage is essential to stabilize the native protein complex. Data obtained for full-length and proteolytically processed endosialidase variants suggest that release of the CTD increases the unfolding barrier, trapping the mature trimer in a kinetically stable conformation. In summary, we characterize the CTD as a novel C-terminal chaperone domain, which assists folding and assembly of unrelated phage proteins.     

Receptor-recognizing proteins of T-even type bacteriophages. The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb

Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers. Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose. The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced. Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous. The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved. Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area. The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature. In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area. It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands.     

DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3

The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4. The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor. The CO2H termini of the polypeptides are located at the free ends of the fibers. Morphologically, the three phages are essentially identical, but they use different receptors. The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively. DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic. The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures. About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36. This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology. The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins. Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine. It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4. Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not. Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues. This area is missing in the T4 protein. This region may serve as a binding site for polypeptides 38 of phages K3 and T2. The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA.     

Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.     

Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin

The folding of beta-structured, fibrous proteins is a largely unexplored area. A class of such proteins is used by viruses as adhesins, and recent studies revealed novel beta-structured motifs for them. We have been studying the folding and assembly of adenovirus fibers that consist of a globular C-terminal domain, a central fibrous shaft, and an N-terminal part that attaches to the viral capsid. The globular C-terminal, or "head" domain, has been postulated to be necessary for the trimerization of the fiber and might act as a registration signal that directs its correct folding and assembly. In this work, we replaced the head of the fiber by the trimerization domain of the bacteriophage T4 fibritin, termed "foldon." Two chimeric proteins, comprising the foldon domain connected at the C-terminal end of four fiber shaft repeats with or without the use of a natural linker sequence, fold into highly stable, SDS-resistant trimers. The structural signatures of the chimeric proteins as seen by CD and infrared spectroscopy are reported. The results suggest that the foldon domain can successfully replace the fiber head domain in ensuring correct trimerization of the shaft sequences. Biological implications and implications for engineering highly stable, beta-structured nanorods are discussed.     

ORF334 in Vibrio phage KVP40 plays the role of gp27 in T4 phage to form a heterohexameric complex

KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate.     

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.     

Recombinant expression and purification of T4 phage Hoc, Soc, gp23, gp24 proteins in native conformations with stability studies

Understanding the biological activity of bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.     

The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein

The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.     

Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins.

We show, using dot matrix comparisons and statistical analysis of sequence alignments, that seven sequenced sigma factors, E. coli sigma-70 and sigma-32, B. subtilis sigma-43 and sigma-29, phage SP01 gene products 28 and 34, and phage T4 gene product 55, comprise a homologous family of proteins. Sigma-70, sigma-32, and sigma-43 each have two copies of a sequence similar to the helix-turn-helix DNA binding motif seen in CRP, and lambda repressor and cro proteins. B. subtilis sigma-29, SP01 gp28, and SP01 gp34 have at least one copy similar to this sequence. We propose that a second sequence, conserved in all seven proteins is the core RNA polymerase binding site. A third region, present only in sigma-70 and sigma-43, may also be involved in interaction with core. Available mutational evidence supports our model for sigma factor structure.     

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.     

The wac gene product of bacteriophage T4 contains coiled-coil structural patterns

The bacteriophage T4 late gene wac (whisker antigen control) encodes the protein which forms the fibrous structure on the neck of the virion called whiskers. Amino acid sequence analysis of wac gene product, as deduced from the nucleotide sequence, indicate ten alpha-helical domains (19-40 residues long) with coiled-coil structural patterns. These regions comprise about 70% of the entire 486 amino acid sequence. The alpha-helices are separated by short stretches of polypeptide chain which are similar to the loop regions of the globular protein sequences. We propose a structural model for the dimer of wac gene product molecule, that we call fibritin in which two polypeptide chains associate in a parallel fashion and form a segmented alpha-helical coiled-coil rod similar to epidermal keratins.     

T4-Like genome organization of the Escherichia coli O157:H7 lytic phage AR1

We report the genome organization and analysis of the first completely sequenced T4-like phage, AR1, of Escherichia coli O157:H7. Unlike most of the other sequenced phages of O157:H7, which belong to the temperate Podoviridae and Siphoviridae families, AR1 is a T4-like phage known to efficiently infect this pathogenic bacterial strain. The 167,435-bp AR1 genome is currently the largest among all the sequenced E. coli O157:H7 phages. It carries a total of 281 potential open reading frames (ORFs) and 10 putative tRNA genes. Of these, 126 predicted proteins could be classified into six viral orthologous group categories, with at least 18 proteins of the structural protein category having been detected by tandem mass spectrometry. Comparative genomic analysis of AR1 and four other completely sequenced T4-like genomes (RB32, RB69, T4, and JS98) indicated that they share a well-organized and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. The major diverse features between these phages include the modules of distal tail fibers and the types and numbers of internal proteins, tRNA genes, and mobile elements. Codon usage analysis suggested that the presence of AR1-encoded tRNAs may be relevant to the codon usage of structural proteins. Furthermore, protein sequence analysis of AR1 gp37, a potential receptor binding protein, indicated that eight residues in the C terminus are unique to O157:H7 T4-like phages AR1 and PP01. These residues are known to be located in the T4 receptor recognition domain, and they may contribute to specificity for adsorption to the O157:H7 strain.