SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.     

Campylobacter jejuni group III phage CP81 contains many T4-like genes without belonging to the T4-type phage group: implications for the evolution of T4 phages

CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.     

Genetic Insertions and Diversification of the PolB-Type DNA Polymerase (gp43) of T4-Related Phages

In Escherichia coli phage T4 and many of its phylogenetic relatives, gene 43 consists of a single cistron that encodes a PolB family (PolB-type) DNA polymerase. We describe the divergence of this phage gene and its protein product (gp43) (gene product 43) among 26 phylogenetic relatives of T4 and discuss our observations in the context of diversity among the widely distributed PolB enzymes in nature. In two T4 relatives that grow in Aeromonas salmonicida phages 44RR and 25, gene 43 is fragmented by different combinations of three distinct types of DNA insertion elements: (a) a short intercistronic untranslated sequence (IC-UTS) that splits the polymerase gene into two cistrons, 43A and 43B, corresponding to N-terminal (gp43A) and C-terminal (gp43B) protein products; (b) a freestanding homing endonuclease gene (HEG) inserted between the IC-UTS and the 43B cistron; and (c) a group I intron in the 43B cistron. Phage 25 has all three elements, whereas phage 44RR has only the IC-UTS. We present evidence that (a) the split gene of phage 44RR encodes a split DNA polymerase consisting of a complex between gp43A and gp43B subunits; (b) the putative HEG encodes a double-stranded DNA endonuclease that specifically cleaves intron-free homologues of the intron-bearing 43B site; and (c) the group I intron is a self-splicing RNA. Our results suggest that some freestanding HEGs can mediate the homing of introns that do not encode their own homing enzymes. The results also suggest that different insertion elements can converge on a polB gene and evolve into a single integrated system for lateral transfer of polB genetic material. We discuss the possible pathways for the importation of such insertion elements into the genomes of T4-related phages.     

Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli

Numerous T4-like Escherichia coli phages were isolated from human stool and environmental wastewater samples in Bangladesh and Switzerland. The sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages T4, RB69, RB49, and JS98. Thus, JS98 defines a new major subgroup of E. coli T4-like phages. We conducted an analysis of the 169-kb JS98 genome sequence. Overall, 198 of the 266 JS98 open reading frames (ORFs) shared amino acid sequence identity with the reference T4 phage, 41 shared identity with other T4-like phages, and 27 ORFs lacked any database matches. Genes on the plus strand encoded virion proteins, which showed moderate to high sequence identity with T4 proteins. The right genome half of JS98 showed a higher degree of sequence conservation with T4 and RB69, even for the nonstructural genes, than did the left genome half, containing exclusively nonstructural genes. Most of the JS98-specific genes were found in the left genome half. Two came as a hypervariability cluster, but most represented isolated genes, suggesting that they were acquired separately in multiple acquisition events. No evidence for DNA exchange between JS98 phage and the E. coli host genome or coliphages other than T4 was observed. No undesired genes which could compromise its medical use were detected in the JS98 genome sequence.     

Homing endonuclease I-TevIII: dimerization as a means to a double-strand break

Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H–N–H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3′ extensions. The enzyme has a distinct catalytic domain, which contains the H–N–H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H–N–H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H–N–H enzyme family.     

Plasticity of the gene functions for DNA replication in the T4-like phages

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.     

Group I intron homing in Bacillus phages SPO1 and SP82: a gene conversion event initiated by a nicking homing endonuclease

Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease.     

Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family

Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 03058-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ~60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the 'EDxHD' family, are distinct from previously characterized homing endonucleases.     

The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille

Here we describe the discovery of a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille. Although the intron insertion site is identical to that of the Bacillus subtilis phages SPO1 and SP82 introns, the Bastille intron differs from them substantially in primary and secondary structure. Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease of the H-N-H family, I-BasI, with a cleavage site identical to that of the SPO1-encoded enzyme I-HmuI. Unlike I-HmuI, which nicks both intron-minus and intron-plus DNA, I-BasI cleaves only intron-minus alleles, which is a characteristic of typical homing endonucleases. Interestingly, the C-terminal portions of these H-N-H phage endonucleases contain a conserved sequence motif, the intron-encoded endonuclease repeat motif (IENR1) that also has been found in endonucleases of the GIY-YIG family, and which likely comprises a small DNA-binding module with a globular ββααβ fold, suggestive of module shuffling between different homing endonuclease families.     

Genomic and phylogenetic traits of Staphylococcus phages S25-3 and S25-4 (family Myoviridae,genus Twort-like viruses)

The genomes of Staphylococcus phages S25-3 and S25-4 (family Myoviridae, genus Twort-like viruses) were sequenced and analyzed from an evolutionary perspective. The genome-based phylogeny and genome analyses of phages S25-3 and S25-4 showed that they had diverged evolutionarily from the majority of this viral genus based on the presence of mobile genetic elements, i.e., a putative transposase and the homing endonuclease I-MsaI. These results suggest that genetic elements such as transposases and homing endonucleases are likely to be involved with the evolution of some Twort-like phages, including phages S25-3 and S25-4.     

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.     

A highly sensitive selection method for directed evolution of homing endonucleases

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80–100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.     

Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages

About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges.     

Purification and characterization of the SegA protein of bacteriophage T4, an endonuclease related to proteins encoded by group I introns.

Although not encoded by an intron, the bacteriophage T4 SegA protein shares common amino acid motifs with a family of proteins found within mobile group I introns present in fungi and phage. Each of these intron-encoded proteins is thought to initiate the homing of its own intron by cleaving the intronless DNA at or near the site of insertion. Previously, we have found that SegA also cleaves DNA. In this report, we have purified the SegA protein and characterized this endonuclease activity extensively. SegA protein cleaved circular and linear plasmids, DNA containing unmodified cytosines, and wild-type T4 DNA containing hydroxymethylated, glucosylated cytosines. In all cases, certain sites on the DNA were highly preferred for cleavage, but with increasing protein concentration or time of incubation, cleavage occurred at many sites. SegA cleaving activity was stimulated by the presence of ATP or ATP gamma S. Sequence analysis of three highly preferred cleavage sites did not reveal a simple consensus sequence, suggesting that even among highly preferred sites, SegA tolerates many different sequences. A T4 segA amber mutant that we constructed had no phenotype, and PCR analyses indicated that several T-even-related phages lack the segA gene. Taken together, our results show that SegA is an endonuclease with a hierarchy of site specificity, and these results are consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely related T-even phages.     

Studies on the substrate specificity of the T 4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline-N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coliB_s−1 with UV-inactivated phage T4v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B_s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4v₁ is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.