Evolution of DNA Polymerase Families: Evidences for Multiple Gene Exchange Between Cellular and Viral Proteins

A phylogenetic analysis of the five major families of DNA polymerase is presented. Viral and plasmid sequences are included in this compilation along with cellular enzymes. The classification by Ito and Braithwaite (Ito and Braithwaite 1991) of the A, B, C, D, and X families has been extended to accommodate the ``Y family' of DNA polymerases that are related to the eukaryotic RAD30 and the bacterial UmuC gene products. After analysis, our data suggest that no DNA polymerase family was universally conserved among the three biological domains and no simple evolutionary scenario could explain that observation. Furthermore, viruses and plasmids carry a remarkably diverse set of DNA polymerase genes, suggesting that lateral gene transfer is frequent and includes non-orthologous gene displacements between cells and viruses. The relationships between viral and host genes appear very complex. We propose that the gamma DNA polymerase of the mitochondrion replication apparatus is of phage origin and that this gene replaced the one in the bacterial ancestor. Often there was no obvious relation between the viral and the host DNA polymerase, but an interesting exception concerned the family B enzymes: in which ancient gene exchange can be detected between the viruses and their hosts. Additional evidence for horizontal gene transfers between cells and viruses comes from an analysis of the small damage-inducible DNA polymerases. Taken together, these findings suggest a complex evolutionary history of the DNA replication apparatus that involved significant exchanges between viruses, plasmids, and their hosts.     

Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.     

Phylogenetic and complementation analysis of a single-stranded DNA binding protein family from lactococcal phages indicates a non-bacterial origin

Background

The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14_bIL67-like proteins) have been identified and characterized structurally and biochemically.

Methodology/Principal Findings

This study focused on the determination of phylogenetic relationships between Orf14_bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14_bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14_bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli.

Conclusions/Significance

Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages.     

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

Bacteriophages engage in complex dynamic interactions with their bacterial hosts and with each other. Bacteria have numerous mechanisms to resist phage infection, and phages must co‐evolve by overcoming bacterial resistance or by choosing an alternative host. Phages also compete with each other, both during lysogeny by prophage‐mediated defense against viral attack and by superinfection exclusion during lytic replication. Phages are enormously diverse genetically and are replete with small genes of unknown function, many of which are not required for lytic growth, but which may modulate these bacteria–phage and phage–phage dynamics. Using cellular toxicity of phage gene overexpression as an assay, we identified the 93‐residue protein gp52 encoded by Cluster F mycobacteriophage Fruitloop. The toxicity of Fruitloop gp52 overexpression results from interaction with and inactivation of Wag31 (DivIVA), an essential Mycobacterium smegmatis protein organizing cell wall biosynthesis at the growing cellular poles. Fruitloop gene 52 is expressed early in lytic growth and is not required for normal Fruitloop lytic replication but interferes with Subcluster B2 phages such as Hedgerow and Rosebush. We conclude that Hedgerow and Rosebush are Wag31‐dependent phages and that Fruitloop gp52 confers heterotypic superinfection exclusion by inactivating Wag31.     

Genetic analysis of bacterial plasmid promiscuity

The genetic basis of the promiscuous behaviour of bacterial plasmids has been investigated by study of the incompatibility P-1 group of conjugative plasmids of gram-negative bacteria. Both transposon mutagenesis and the construction of minireplicons linking varying combinations of the plasmid genome have shown that specific genomic regions control the conjugational transfer and vegetative replication of the plasmid in specific bacterial hosts. These include the plasmid DNA primase gene, the origin of plasmid transfer, a region near the origin of transfer, the origin of plasmid vegetative replication, thetrans- acting gene essential for the initiation of plasmid replication and a region involved in its regulation. DNA sequence analysis has identified the requirement of specific direct repeats within the origin of replication for plasmid replication in some but not in other hosts. The cloning of some of the trans-acting genes onto multicopy cloning vectors and complementation tests have shown that the requirements of these gene products vary in different hosts and that the plasmid has evolved genetic strategies for their optimal expression.     

Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep₂₀₀₉ and orf17. One of these genes, rep₂₀₀₉, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori₂₀₀₉), which was identical for all four phages. DNA fragments representing the ori₂₀₀₉ sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori₂₀₀₉ sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep₂₀₀₉ and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.     

The existence conditions for bacterial plasmids: Theory and reality

Bacteria abound with conjugative and nonconjugative plasmids that often carry genes determining a number of environmental adaptations. Plasmids may also encode genes that enable them to transmit themselves infectiously to new host cells, by conjugation or mobilization. The question of whether plasmids can be maintained in a bacterial community as parasitic DNA, that is, while conferring a selective disadvantage to their host, serves as a basic hypothesis in theoretical studies of the population biology of plasmids. The conditions necessary for the establishment and maintenance of plasmids have been determined analytically for the simplest possible models. Based on these a priori conditions, on some reconsiderations and extensions of these models, and on recent estimates of transfer rates of liquid and surface bacterial populations, it will be argued that within a bacterial population, a parasitic lifestyle is unlikely for most naturally occurring plasmids. This result raises anew the problem of how cryptic plasmids are maintained and why plasmids encode costly and elaborate genes for horizontal transfer.     

Diversity, biology and evolution of IncQ-family plasmids

Plasmids of IncQ-family are distinguished by having a unique strand-displacement mechanism of replication that is capable of functioning in a wide variety of bacterial hosts. In addition, these plasmids are highly mobilizable and therefore very promiscuous. Common features of the replicons have been used to identify IncQ-family plasmids in DNA sequence databases and in this way several unstudied plasmids have been compared to more well-studied IncQ plasmids. We propose that IncQ plasmids can be divided into four subgroups based on a number of mutually supportive criteria. The most important of these are the amino acid sequences of their three essential replication proteins and the observation that the replicon of each subgroup has become fused to four different lineages of mobilization genes. This review of IncQ-family plasmid diversity has highlighted several events in the evolution of these plasmids and raised several questions for further research.     

The global distribution and evolutionary history of the pT26-2 archaeal plasmid family

Although plasmids play an important role in biological evolution, the number of plasmid families well-characterized in terms of geographical distribution and evolution remains limited, especially in archaea. Here, we describe the first systematic study of an archaeal plasmid family, the pT26-2 plasmid family. The in-depth analysis of the distribution, biogeography and host–plasmid co-evolution patterns of 26 integrated and 3 extrachromosomal plasmids of this plasmid family shows that they are widespread in Thermococcales and Methanococcales isolated from around the globe but are restricted to these two orders. All members of the family share seven core genes but employ different integration and replication strategies. Phylogenetic analysis of the core genes and CRISPR spacer distribution suggests that plasmids of the pT26-2 family evolved with their hosts independently in Thermococcales and Methanococcales, despite these hosts exhibiting similar geographic distribution. Remarkably, core genes are conserved even in integrated plasmids that have lost replication genes and/or replication origins suggesting that they may be beneficial for their hosts. We hypothesize that the core proteins encode for a novel type of DNA/protein transfer mechanism, explaining the widespread oceanic distribution of the pT26-2 plasmid family.     

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.     

Conflicting selection alters the trajectory of molecular evolution in a tripartite bacteria–plasmid–phage interaction

Bacteria engage in a complex network of ecological interactions, which includes mobile genetic elements (MGEs) such as phages and plasmids. These elements play a key role in microbial communities as vectors of horizontal gene transfer but can also be important sources of selection for their bacterial hosts. In natural communities, bacteria are likely to encounter multiple MGEs simultaneously and conflicting selection among MGEs could alter the bacterial evolutionary response to each MGE. Here, we test the effect of interactions with multiple MGEs on bacterial molecular evolution in the tripartite interaction between the bacterium, Pseudomonas fluorescens, the lytic bacteriophage, SBW25φ2, and conjugative plasmid, pQBR103, using genome sequencing of experimentally evolved bacteria. We show that individually, both plasmids and phages impose selection leading to bacterial evolutionary responses that are distinct from bacterial populations evolving without MGEs, but that together, plasmids and phages impose conflicting selection on bacteria, constraining the evolutionary responses observed in pairwise interactions. Our findings highlight the likely difficulties of predicting evolutionary responses to multiple selective pressures from the observed evolutionary responses to each selective pressure alone. Understanding evolution in complex microbial communities comprising many species and MGEs will require that we go beyond studies of pairwise interactions.     

Genomes and characterization of phages Bcep22 and BcepIL02, founders of a novel phage type in Burkholderia cenocepacia

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.     

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.     

A hypothesis for DNA viruses as the origin of eukaryotic replication proteins

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli.

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.     

'Cells' and 'organisms' as a habitat for DNA.

Although the bulk of the hereditary information in bacteria is organized as a single chromosome, it has been known for some years now that bacteria may also carry pieces of self-replicating extrachromosomal DNA. These units are known as plasmids. Sometimes such plasmids carry the information necessary to give rise to mature bacterial viruses under appropriate conditions, but in other cases they specify the production of enzymes and other proteins which alter the bacterial phenotype. Plasmids are often inessential for survival of bacteria, although they may widen the range of environmental conditions under which they flourish. Thus plasmids may be thought of as adventitious additions to the genetic content of bacterial cells. Recently it has become clear that furthur organizational units of DNA are to be found in bacterial cells. These units are called insertion sequences and transposons. Unlike plasmids and the chromosome, however, these DNA units do not carry enough genetic information to specify their own independent replication: they must rely on plasmids or the chromosome for that purpose. Nevertheless they behave in many respects as independent functional units. Although it is possible to think of the chromosome, plasmids and transposons/insertion sequences as three distinct hierarchies of bacterial DNA, genes may move from one hierarchy to another; and such transitions have important implications for the evolution of bacterial populations. Moreover, their study in bacteria may throw much light on the type of DNA interactions occurring in higher cells.