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.     

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.     

Diversity and rearrangement of the human T cell rearranging gamma genes: nine germ-line variable genes belonging to two subgroups

We describe nine T cell gamma variable (V) gene segments isolated from human DNA. These genes, which fall into two subgroups, are mapped in two DNA regions covering 54 kb and probably represent the majority of human V gamma genes. One subgroup (V gamma I) contains eight genes, consisting of four active genes and four pseudogenes. The single V gamma II gene is potentially active. Sequence analysis of the V gamma I genes shows variation clustered in hypervariable regions, but somatic variability is restricted to N-region diversity. Studies on rearrangement in T cell lines and in thymic DNA show that major rearrangements can be observed that are attributable to the five active V gamma genes. In addition, human cells with the phenotype of helper T cells can undergo productive V gamma-J gamma joining.     

Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry

Much of the gene regulatory circuitry of phage lambda centers on a complex region called the O(R) region. This approximately 100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a lambda cloning vector that allowed us to clone fragments spanning the O(R) region. By using this vector, we carried out intensive mutagenesis of the P(RM) promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the -35 and -10 regions were randomized and cloned this pool into the vector, making a pool of P(RM) variant phage. About 10% of the P(RM) variants were able to lysogenize, suggesting that the lambda regulatory circuitry is compatible with a wide range of P(RM) sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.     

Grooming and group cohesion in primates: implications for the evolution of language

It is well established that allogrooming, which evolved for a hygienic function, has acquired an important derived social function in many primates. In particular, it has been postulated that grooming may play an essential role in group cohesion and that human language, as verbal grooming or gossip, evolved to maintain group cohesion in the hominin lineage with its unusually large group sizes. Here, we examine this group cohesion hypothesis and test it against the alternative grooming-need hypothesis which posits that rates of grooming are higher in species where grooming need (i.e. the motivation to groom for hygiene and its associated psychological reward) is more pronounced. This alternative predicts that the derived social function of grooming evolved mostly in those lineages that had the highest exposure to ectoparasites and dirt, i.e. terrestrial species. A detailed comparative analysis of 74 species of wild primates, controlling for phylogenetic non-independence, showed that terrestriality was a highly significant predictor of allogrooming time, consistent with the prediction. The predictions of the group cohesion hypothesis were not supported, however. Group size did not predict grooming time across primates, nor did it do so in separate intra-population analyses in 17 species. Thus, there is no comparative support for the group-cohesion function of allogrooming, which questions the role of grooming in the evolution of human language.     

Multiple independent horizontal transfers of informational genes from bacteria to plasmids and phages: implications for the origin of bacterial replication machinery

In contrast to the universality of other central genetic mechanisms, the replication machinery of Bacteria is clearly different from those of Archaea and Eukaryotes. A large number of bacterial genes involved in DNA replication can also be found in plasmids and phages. Based on this, it has been recently proposed that the ancestral bacterial genes were displaced by non-orthologous replication genes from plasmids and phages, which would explain the profound difference between Bacteria and the other domains of life. The alternative hypothesis is that these DNA replication genes have been frequently transferred from bacterial hosts to the genomes of their plasmids and phages. The phylogenetic analysis of the bacterial DNA replication proteins most abundant in databases (replicative helicase DnaB, single-strand binding protein Ssb and topoisomerase TopB) presented here supports the latter hypothesis. Each protein tree shows that sequences from plasmids and phages branch close to their bacterial-specific hosts, suggesting multiple independent horizontal transfers. Therefore, there is no evidence so far for non-orthologous gene displacement of these genes.     

Sequence specificity of the P6 pairing for splicing of the group I td intron of phage T4.

Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5' element (P6[5']) of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5'] and P6[3'] compensatory mutations. Second, all P6[5'] and P6[3'] mutants are defective in the first step of splicing, guanosine-dependent 5' splice site cleavage, whereas their activity at the 3' splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5']:[3'] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.     

A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydroxyalkanoic acids (HA _mcl ) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HA _scl ), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB^–4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HA _mcl and was expressed in GPp104 but not in PHB^–4. Correspondence to: A. Steinbüchel     

Isolation of Hox genes from the scyphozoan Cassiopeia xamachana: implications for the early evolution of Hox genes.

The isolation of Hox genes from two cnidarian groups, the Hydrozoa and Anthozoa, has sparked hypotheses on the early evolution of Hox genes and a conserved role for these genes for defining a main body axis in all metazoan animals. We have isolated the first five Hox genes, Scox-1 to Scox-5, from the third cnidarian class, the Scyphozoa. For all but one gene, we report full-length homeobox plus flanking sequences. Four of the five genes show close relationship to previously reported Cnox-1 genes from Hydrozoa and Anthozoa. One gene, Scox-2, is an unambiguous homologue of Cnox-2 genes known from Hydrozoa, Anthozoa, and also Placozoa. Based on sequence similarity and phylogenetic analyses of the homeobox and homeodomain sequences of known Hox genes from cnidarians, we suggest the presence of at least five distinct Hox gene families in this phylum, and conclude that the last common ancestor of the Recent cnidarian classes likely possessed a set of Hox genes representing three different families, the Cnox-1, Cnox-2, and Cnox-5 families. The data presented are consistent with the idea that multiple duplication events of genes have occurred within one family at the expense of conservation of the original set of genes, which represent the three ancestral Hox gene families.     

Bacteriophage T4 transfer RNA : III. Clustering of the genes for the T4 transfer RNA's

Escherichia coli cells infected with phage strains carrying extensive deletions encompassing the gene for the phage Ser-tRNA are missing the phage tRNA's normally present in wild-type infected cells. By DNA-RNA hybridization we have demonstrated that the DNA complementary to the missing tRNA's is also absent in such deletion mutants. Thus the genes for these tRNA's must be clustered in the same region of the genome as the Ser-tRNA gene. Physical mapping of several deletions of the Ser-tRNA and lysozyme genes, by examination of heteroduplex DNA in the electron microscope, has enabled us to locate the cluster, to define its maximum size, and to order a few of the tRNA genes within it. That such deletions can be isolated indicates that the phage-specific tRNA's from this cluster are dispensable.     

Gene duplication and the evolution of group II chaperonins: implications for structure and function

Chaperonins are multisubunit protein-folding assemblies. They are composed of two distinct structural classes, which also have a characteristic phylogenetic distribution. Group I chaperonins (called GroEL/cpn60/hsp60) are present in Bacteria and eukaryotic organelles while group II chaperonins are found in Archaea (called the thermosome or TF55) and the cytoplasm of eukaryotes (called CCT or TriC). Gene duplication has been an important force in the evolution of group II chaperonins: Archaea possess one, two, or three homologous chaperonin subunit-encoding genes, and eight distinct CCT gene families (paralogs) have been described in eukaryotes. Phylogenetic analyses indicate that while the duplications in archaeal chaperonin genes have occurred numerous times independently in a lineage-specific fashion, the eight different CCT subunits found in eukaryotes are the products of duplications that occurred early and very likely only once in the evolution of the eukaryotic nuclear genome. Analyses of CCT sequences from diverse eukaryotic species reveal that each of the CCT subunits possesses a suite of invariant subunit-specific amino acid residues ("signatures"). When mapped onto the crystal structure of the archaeal chaperonin from Thermoplasma acidophilum, these signatures are located in the apical, intermediate, and equatorial domains. Regions that were found to be variable in length and/or amino acid sequence were localized primarily to the exterior of the molecule and, significantly, to the extreme tip of the apical domain (the "helical protrusion"). In light of recent biochemical and electron microscopic data describing specific CCT-substrate interactions, our results have implications for the evolution of subunit-specific functions in CCT.     

Mhc-DRB genes of the pigtail macaque (Macaca nemestrina): implications for the evolution of human DRB genes.

The DRB family of human class II major histocompatibility complex (Mhc) loci is unusual in that individuals differ in the number and combination of genes (haplotypes) they carry. Indications are that both the allelic and haplotype polymorphisms of the DRB loci predate speciation. Searching for the evolutionary origins of these polymorphisms, we have sequenced five DRB clones isolated from a cDNA library of a pigtail macaque (Macaca nemestrina) B lymphocyte line. The clones represent five different genes which we designate Mane-DRB*01-Mane-DRB*05. The genes appears to be approximately equidistant from each other, so that allelic relationships between them cannot be established on the basis of the sequence data alone. If positions coding for the peptide-binding region of the class II beta chains are eliminated from sequence comparisons, the Mane-DRB genes appear to be most closely related to the human (HLA) DRB1 genes of the DRw52 group. We interpret this finding to indicate that the ancestral gene of the DRw52 group of human DRB1 alleles separated from the rest of the HLA-DRB1 alleles before the separation of the Old World monkeys (Cercopithecoidea) from the apes (Hominoidea) in the early Oligocene. After this separation, the ancestral DRB1 gene of the DRw52 group duplicated in the Old World monkey lineage to give rise to genes at three loci at least, while in the ape lineage this gene may have remained single and diverged into a number of alleles instead. These findings suggest that some of the polymorphism currently present at the DRB1 locus is greater than 35 Myr old.     

Morphology heterogeneity within a Campylobacter jejuni helical population: the use of calcofluor white to generate rod‐shaped C. jejuni 81‐176 clones and the genetic determinants responsible for differences in morphology within 11168 strains

Campylobacter jejuni helical shape is important for colonization and host interactions with straight mutants having altered biological properties. Passage on calcofluor white (CFW) resulted in C. jejuni 81‐176 isolates with morphology changes: either a straight morphology from frameshift mutations and single nucleotide polymorphisms in peptidoglycan hydrolase genes pgp1 or pgp2 or a reduction in curvature due a frameshift mutation in cjj81176_1105, a putative peptidoglycan endopeptidase. Shape defects were restored by complementation. Whole genome sequencing of CFW‐passaged strains showed no specific changes correlating to CFW exposure. The cjj81176_1279 (recR; recombinational DNA repair) and cjj81176_1449 (unknown function) genes were highly variable in all 81‐176 strains sequenced. A frameshift mutation in pgp1 of our laboratory isolate of the straight genome sequenced variant of 11168 (11168‐GS) was also identified. The PG muropeptide profile of 11168‐GS was identical to that of Δpgp1 in the original minimally passaged 11168 strain (11168‐O). Introduction of wild type pgp1 into 11168‐GS did not restore helical morphology. The recR gene was also highly variable in 11168 strains. Microbial cell‐to‐cell heterogeneity is proposed as a mechanism of ensuring bacterial survival in sub‐optimal conditions. In certain environments, changes in C. jejuni morphology due to genetic heterogeneity may promote C. jejuni survival.     

Directed evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code

To better understand how amino acid sequences specify unique tertiary folds, we have used random mutagenesis and phage display selection to evolve proteins with a high degree of sequence identity but different tertiary structures (homologous heteromorphs). The starting proteins in this evolutionary process were the IgG binding domains of streptococcal protein G (G(B)) and staphylococcal protein A (A(B)). These nonhomologous domains are similar in size and function but have different folds. G(B) has an alpha/beta fold, and A(B) is a three-helix bundle (3-alpha). IgG binding function is used to select for mutant proteins which retain the correct tertiary structure as the level of sequence identity is increased. A detailed thermodynamic analysis of the folding reactions and binding reactions for a pair of homologous heteromorphs (59% identical) is presented. High-resolution NMR structures of the pair are presented by He et al. [(2005) Biochemistry 44, 14055-14061]. Because the homologous but heteromorphic proteins are identical at most positions in their sequence, their essential folding signals must reside in the positions of nonidentity. Further, the thermodynamic linkage between folding and binding is used to assess the propensity of one sequence to adopt two unique folds.     

Molecular evolution of bacteriophages: Discrete patterns of codon usage in T4 genes are related to the time of gene expression

Summary Patterns of codon usage in certain coliphages are adapted to expression inEscherichia coli. Bacteriophage T4 may be an exception to test the rule, as it produces eight tRNAs with specificities that are otherwise rare inE. coli. A database of all known T4 DNA sequences has been compiled, comprising 174 genes and a total of 115 kb (approximately 70% of the T4 genome). Codon usage has been examined in all T4 genes; some of these are known to be expressed before, and some after, the production of phage tRNAs. The results show two different patterns of codon usage: by comparison with the early genes, the late genes exhibit a shift in preference toward those codons recognized by the phage-encoded tRNAs. The T4 tRNAs translate A-ending codons, and it is possible that the phage acquired the tRNA genes because the mutation bias of the T4 DNA polymerase forces the T4 genome toward A+T-richness.Presented at the NATO Advanced Workshop on Genome Organization and Evolution, held in Spetses, Greece, September 1990     

gpwac of the T4-type bacteriophages: structure, function, and evolution of a segmented coiled-coil protein that controls viral infectivity

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 approximately 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.     

Regulation of autologous immunity to the mouse 5T4 oncofoetal antigen: implications for immunotherapy

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.