Mechanisms involved in the formation of plaque-forming derivatives from over-sized hybrid phages between bacteriophage P1 and the R plasmid NR1

Three case histories document how subsequent events of genomic rearrangements and selection interplay in the evolution of infectious bacteriophage genomes carrying acquired genes. Two of the phages studied were plaque-forming P1CmTc recombinants derived from P1Cm1 and P1Tc1, both of which are hybrids between phage P1 and the R plasmid NR1. In the formation of the P1CmTc4 genome a postulated intermediate underwent IS1-mediated deletion formation. From the same intermediate P1CmTc1 must have evolved by IS1-mediated inversion followed by homologous recombination with a parental phage DNA. The third case documents formation of the P1Cm2 genome by “illegitimate” intramolecular recombination in the genome of P1-r-det, a hybrid between P1 and NR1.     

Genomic characterization of mycobacteriophage Giles: evidence for phage acquisition of host DNA by illegitimate recombination

A characteristic feature of bacteriophage genomes is that they are architecturally mosaic, with each individual genome representing a unique assemblage of individual exchangeable modules. Plausible mechanisms for generating mosaicism include homologous recombination at shared boundary sequences of module junctions, illegitimate recombination in a non-sequence-directed process, and site-specific recombination. Analysis of the novel mycobacteriophage Giles genome not only extends our current perspective on bacteriophage genetic diversity, with more than 60% of the genes unrelated to other mycobacteriophages, but offers novel insights into how mosaic genomes are created. In one example, the integration/excision cassette is atypically situated within the structural gene operon and could have moved there either by illegitimate recombination or more plausibly via integrase-mediated site-specific recombination. In a second example, a DNA segment has been recently acquired from the host bacterial chromosome by illegitimate recombination, providing further evidence that phage genomic mosaicism is generated by nontargeted recombination processes.     

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.     

Mobile Antibiotic Resistance Encoding Elements Promote Their Own Diversity

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.     

Involvement of transposable elements in the formation of hybrid phages between bacteriophage P1 and the R plasmid NR1

Homologous recombination between IS1 elements present on both replicons, P1 and NR1, resulted in P1-NR1 cointegrates and P1-RTF and P1-r-det phages. Cointegration between P1 and NR1-B, and NR1 derivative with multiple DNA rearrangements including insertion of the transposable element γδ, was also mediated by reciprocal recombination in IS1 sequences. However, all 4 hybrids studied carried deletions promoted by γδ residing on NR1-B. Further IS1-mediated deletions on the hybrid genomes resulted in plaque-forming P1Cm phages.     

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.     

Acquisition and rearrangement of sequence motifs in the evolution of bacteriophage tail fibres

Molecular analysis reveals a surprising sharing of short gene segments among a variety of large double-stranded DNA bacteriophages of enteric bacteria. Ancestral genomes from otherwise unrelated phages, including λ Mu, P1, P2 and T4, must have exchanged parts of their tail-fibre genes, Individual genes appear as mosaics with parts derived from a common gene pool. Therefore, horizontal gene transfer emerges as a major factor in the evolution of a specific part of phage genomes. Current concepts of homologous recombination cannot account for the formation of such chimeric genes and the recombinational mechanisms responsible are not known. However, recombination sites for DNA invertases and recombination site-like sequences are present at the boundaries of gene segments conferring the specificity for the host receptor. This, together with the properties of the DNA inversion mechanism, suggests that these site-specific recombination enzymes could be responsible for the exchange of host-range determinants.     

Multiple Pathways of Duplication Formation with and Without Recombination (RecA) in Salmonella enterica

Duplications are often attributed to “unequal recombination” between separated, directly repeated sequence elements (>100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10⁻³–10⁻⁵/cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F′₁₂₈ plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10⁻⁴/cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by single-strand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0–36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication.     

DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, ϕw39 and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.     

Characterization of the Holliday Junction Resolving Enzyme Encoded by the Bacillus subtilis Bacteriophage SPP1

Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (oriL), and when replication progresses, the fork might stall by the binding of the origin binding protein G38P to the late replication origin (oriR). Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from P _E2 and P _E3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the P _E2 operon (genes 44 and 45) showed that they are in fact a single gene, re-annotated here as gene 44, that encodes a single polypeptide, named gene 44 product (G44P, 27.5 kDa). G44P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G44P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G44P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in B. subtilis cells. These in vitro and in vivo data suggest a role for G44P in replication restart during the transition to concatemeric viral replication.     

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1

Summary The genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

Evolution of the long range structure of satellite DNAs in the genus Apodemus.

The temperate bacteriophage Mu causes mutations by inserting its DNA randomly into the genes of its host bacterium Escherichia coli. It is shown here that Mu DNA can be precisely excised from the different integration sites and that as a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lac⁺ revertants from six strains, containing heat-inducible prophage Mu cts62 at different locations in the Z gene of the lac operon, were readily obtained by first introducing the X mutation into Mu cts62. The lac⁺ revertants produced wild-type β-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu cts62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. V. ilv+ Deletion mutants of F14

The structure of a number of F′ilv episomes derived from F14 by bacteriophage P1-mediated transduction have been determined by the electron microscope heteroduplex method. F16, F25, F310 and F312 are all simple deletion mutants of F14. F316 is essentially the same but contains a small insertion (0.8 kilobase) of DNA of unknown origin within the F sequences at 78.6 F. The length of these plasmids are all about the same as that of phage P1 DNA itself. The sequences of F and the sequences of bacterial DNA that are present on the episomes are contiguous on the parental F14. Thus, their structures are consistent with the usual model for the mechanism of P1 transduction. The physical order of ilv genes is also consistent with previous genetic mapping. From this order one can determine the polarity of the Escherichia coli K12 chromosomal sequences on F14 and its F′ilv derivatives relative to the F sequences. This order is consistent with the known counterclockwise transfer order of the parental Hfr AB313. F′ilv episomes carry only one copy of the 2.8 to 8.5 F sequence, which is present as a direct duplication on F14. The F′ilv episomes are genetically stable, whereas F14 is unstable because of reciprocal recombination between the two duplicate sequences. The strain F316/AB2070 is different in several respects. All of the bacteria carry P1 phage DNA. As noted above, F316 itself carries a small insertion. Two transfer-defective deletion mutants, F316Δ(65.4-78.6) and F316Δ-(78.6-0.6) are also present in the population of F316/AB2070 cells. In each case, the deletion borders on one of the junctions of inserted DNA and F14 DNA in F316. Thus, these junctions appear to be hot spots for deletion formation.     

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli.

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec⁺ function is not required. In Rec⁺ strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.     

Cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives suggest molecular mechanisms for P1-mediated transduction of small plasmids

Summary We characterized cointegrates formed in an Escherichia coli rec ^– strain between bacteriophage P1 genomes and small plasmids related to pBR322. The partners were, on the one hand, either phage P1 DNA, which carries one copy of IS1, or phage P1-15 DNA, a derivative which lacks the IS1, and, on the other hand, plasmids containing either a split IS1 or no IS1. In the presence of IS1 sequences on both partners, cointegrates were usually formed by reciprocal recombination between IS1 sequences. Cointegrates between P1 and a plasmid carrying no IS1 sequence were formed by transpositional cointegration mediated by IS1 of P1. Cointegrates between P1-15 and small plasmids containing a split IS1 were formed by one of three ways: (a) acquisition of an IS1 by P1-15 followed by reciprocal recombination between IS1 sequences, (b) transpositional cointegration mediated by the split IS1 element, Tn2657, or (c) involvement of the invertible segment carried on P1-15 DNA. Most cointegrates segregated into the small plasmids and phage P1 derivatives. A comparison of the phenomena studied and of their frequencies allowed us to conclude that cointegrate formation is a molecular mechanism involved in the transduction of plasmids smaller than those packageable into P1 virions, although it does not seem to be the only process used.     

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host''s chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.     

The DNA Replication Factor RFC1 Is Required for Interference-Sensitive Meiotic Crossovers in Arabidopsis thaliana

During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways.     

The invertible P-DNA segment in the chromosome of Escherichia coli.

In the chromosome of many strains of Escherichia coli K12 the excisable element e14 is found, which contains an invertible DNA region. This invertible P region, and the gene responsible for the inversion (pin) were cloned, together with other e14 sequences. The element e14 contains a gene which kills the host cell. This can be repressed by a function also coded by e14. The kil and repressor genes as well as the attachment site of the element were mapped in different regions of the element. The invertible segment and pin gene were sequenced. The invertible segment is 1794 bp long, and contains one large internal open reading frame of 879 bp and reading frames which overlap the end pont of the invertible segment. Although pin highly homologous to gin of phage Mu, neither the genetic organization of the P segment nor the sequence of the putative proteins resemble the invertible G segment of phage Mu (which codes for genes involved in tail fiber assembly). The complete DNA sequences of both invertible segments were screened for homology. No resemblance was found. The P segment is flanked by inverted repeat sequences of 16 bp. Comparison of these with related inversion systems points out that the recombination site maps probably within a 2-bp region. This cross-over site is contained within a short palindromic sequence (AAACC AA GGTTT) which is more or less conserved in the recombination sites of all related DNA invertases.