New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.

To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.     

Novel replicative properties of a capsid mutant of bacteriophage phi chi 174.

A capsid mutant of bacteriophage phi chi 174 demonstrates altered requirements for the conversion of viral single-stranded DNA to double-stranded replicative form DNA. In the presence of puromycin at 42 C, wild-type phi chi 174 is unable to complete this replicative event, whereas phi chi ahb is able to do so. Furthermore, in contrast to wild-type phi chi 174, formation of phi chi ahb parental replicative form DNA is sensitive to rifampin under certain experimental conditions. These data suggest that the mutant capsid proteins of phi chi ahb influence the biosynthesis of phi chi ahb complementary strand DNA.     

Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis.

By use of the Bacillus subtilis bacteriophage cloning vehicle phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages phi 105Rec phi 1 (3.85-kilobase insert) and phi 105Rec phi 4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE+ strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage phi 105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either phi 105Rec phi 1 or phi 105Rec phi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages phi 105Rec phi 1 and phi 105Rec phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA+ gene product antibodies. Collectively, these data demonstrate that the recE+ gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination.     

Co-infection weakens selection against epistatic mutations in RNA viruses

Co-infection may be beneficial in large populations of viruses because it permits sexual exchange between viruses that is useful in combating the mutational load. This advantage of sex should be especially substantial when mutations interact through negative epistasis. In contrast, co-infection may be detrimental because it allows virus complementation, where inferior genotypes profit from superior virus products available within the cell. The RNA bacteriophage phi6 features a genome divided into three segments. Co-infection by multiple phi6 genotypes produces hybrids containing reassorted mixtures of the parental segments. We imposed a mutational load on phi6 populations by mixing the wild-type virus with three single mutants, each harboring a deleterious mutation on a different one of the three virus segments. We then contrasted the speed at which these epistatic mutations were removed from virus populations in the presence and absence of co-infection. If sex is a stronger force, we predicted that the load should be purged faster in the presence of co-infection. In contrast, if complementation is more important we hypothesized that mutations would be eliminated faster in the absence of co-infection. We found that the load was purged faster in the absence of co-infection, which suggests that the disadvantages of complementation can outweigh the benefits of sex, even in the presence of negative epistasis. We discuss our results in light of virus disease management and the evolutionary advantage of haploidy in biological populations.     

Genes and regulatory sequences of bacteriophage phi X174

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.     

Pervasive genomic recombination of HIV-1 in vivo

Recombinants of preexisting human immunodeficiency virus type 1 (HIV-1) strains are now circulating globally. To increase our understanding of the importance of these recombinants, we assessed recombination within an individual infected from a single source by studying the linkage patterns of the auxiliary genes of HIV-1 subtype B. Maximum-likelihood phylogenetic techniques revealed evidence for recombination from topological incongruence among adjacent genes. Coalescent methods were then used to estimate the in vivo recombination rate. The estimated mean rate of 1.38 x 10(-4) recombination events/adjacent sites/generation is approximately 5.5-fold greater than the reported point mutation rate of 2.5 x 10(-5)/site/generation. Recombination was found to be frequent enough to mask evidence for purifying selection by Tajima's D test. Thus, recombination is a major evolutionary force affecting genetic variation within an HIV-1-infected individual, of the same order of magnitude as point mutational change.     

Circular and linear simian virus 40 DNAs differ in recombination.

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.     

Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme

RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCD(K177Q) enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot chi and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB(K29Q)CD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecB(K29Q)CD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize chi and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to chi, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.     

A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi

In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.     

Modelling bacterial speciation

A central problem in understanding bacterial speciation is how clusters of closely related strains emerge and persist in the face of recombination. We use a neutral Fisher-Wright model in which genotypes, defined by the alleles at 140 house-keeping loci, change in each generation by mutation or recombination, and examine conditions in which an initially uniform population gives rise to resolved clusters. Where recombination occurs at equal frequency between all members of the population, we observe a transition between clonal structure and sexual structure as the rate of recombination increases. In the clonal situation, clearly resolved clusters are regularly formed, break up or go extinct. In the sexual situation, the formation of distinct clusters is prevented by the cohesive force of recombination. Where the rate of recombination is a declining log-linear function of the genetic distance between the donor and recipient strain, distinct clusters emerge even with high rates of recombination. These clusters arise in the absence of selection, and have many of the properties of species, with high recombination rates and thus sexual cohesion within clusters and low rates between clusters. Distance-scaled recombination can thus lead to a population splitting into distinct genotypic clusters, a process that mimics sympatric speciation. However, empirical estimates of the relationship between sequence divergence and recombination rate indicate that the decline in recombination is an insufficiently steep function of genetic distance to generate species in nature under neutral drift, and thus that other mechanisms should be invoked to explain speciation in the presence of recombination.     

Estimating the Relative Roles of Recombination and Point Mutation in the Generation of Single Locus Variants in Campylobacter jejuni and Campylobacter coli

Single locus variants (SLVs) are bacterial sequence types that differ at only one of the seven canonical multilocus sequence typing (MLST) loci. Estimating the relative roles of recombination and point mutation in the generation of new alleles that lead to SLVs is helpful in understanding how organisms evolve. The relative rates of recombination and mutation for Campylobacter jejuni and Campylobacter coli were estimated at seven different housekeeping loci from publically available MLST data. The probability of recombination generating a new allele that leads to an SLV is estimated to be roughly seven times more than that of mutation for C. jejuni, but for C. coli recombination and mutation were estimated to have a similar contribution to the generation of SLVs. The majority of nucleotide differences (98?% for C. jejuni and 85?% for C. coli) between strains that make up an SLV are attributable to recombination. These estimates are much larger than estimates of the relative rate of recombination to mutation calculated from more distantly related isolates using MLST data. One explanation for this is that purifying selection plays an important role in the evolution of Campylobacter. A simulation study was performed to test the performance of our method under a range of biologically realistic parameters. We found that our method performed well when the recombination tract length was longer than 3?kb. For situations in which recombination may occur with shorter tract lengths, our estimates are likely to be an underestimate of the ratio of recombination to mutation, and of the importance of recombination for creating diversity in closely related isolates. A parametric bootstrap method was applied to calculate the uncertainty of these estimates.     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

The 1.5-Mb region spanning the myotonic dystrophy locus shows uniform recombination frequency.

The myotonic dystrophy (DM) mutation has been identified as a heritable unstable CTG trinucleotide repeat sequence. The intergenerational amplification of this sequence is an example of a new class of dynamic mutations responsible for human genetic diseases. To ascertain whether recombination activity influences, or is affected by, the presence of this unique sequence, a comprehensive study of the physical and genetic mapping data for the 1.5-Mb region of human chromosome 19q13.3, which contains the DM locus, was conducted. The recombination rate for this region was examined by correlating genetic distance to physical distance for six selected marker loci. The following markers span the DM region: 19qCEN-p alpha 1.4 (D19S37)-APOC2-CKM-pE0.8 (D19S115)-pGB2.6 (DM)-p134c (D19S51)-19qTER. Initial linear regression analysis of these two parameters failed to reveal a significant linear correlation (coefficient of determination, r2 = .19), suggesting nonuniform rates of recombination. However, the presence of a recombination hot spot was believed to be unlikely, as the marker-to-marker pairs that showed the greatest deviation in recombination frequency were not restricted to a specific region of the 1.5 Mb studied and had relatively broad confidence intervals, as reflected by low LOD values. A second linear regression analysis using only marker intervals with high LOD scores (Zmax > 22) showed linear correlation (r2 = .68) for the entire 1.5-Mb region. This analysis indicated a relatively uniform recombination frequency in the 1.5-Mb region spanning the DM locus. Furthermore, the recombinations observed were neither under- nor overrepresented on DM chromosomes. Consequently, recombination activity is unlikely to influence, or be affected by, the presence of the DM mutation.     

A weak effect of background selection on trinucleotide microsatellites in maize

Artificial selection during the domestication of maize is thought to have been predominantly positive and to have had little effect on the surrounding neutral diversity because linkage disequilibrium breaks down rapidly when physical distance increases. However, the degree to which indirect selection has shaped neutral diversity in the maize genome during domestication remains unclear. In this study, we investigate the relationship between local recombination rate and neutral polymorphism in maize and in teosinte using both sequence and microsatellite data. To quantify diversity, we estimate 3 parameters expected to differentially reflect the effects of indirect selection and mutation. We find no general correlation between diversity and recombination, indicating that indirect selection has had no genome-wide impact on maize diversity. However, we detect a weak correlation between heterozygosity and recombination for trinucleotide microsatellites deviating from the stepwise mutation model and located within genes (rho = 0.32, P < 0.03). This result can be explained by a background selection hypothesis. The fact that the same correlation is not confirmed for nucleotide diversity suggests that the strength of purifying selection at or near this class of microsatellites is higher than for nucleotide mutations.     

Analysis of intragenic recombination at wx in rice: correlation between the molecular and genetic maps within the locus.

In plant genomes as well as other eukaryotic genomes, meiotic recombination does not occur uniformly. At the level of the gene, high recombination frequencies are often observed within genetic loci in maize, but this feature of intragenic recombination is not seen at the csr1 locus in Arabidopsis. These observations suggest that meiotic recombination in plant genomes varies considerably among species. In the present study we investigated meiotic recombination at the wx locus in rice. The mutation sites of wx mutants induced by ethyl methanesulfonate (EMS) treatment or gamma-ray irradiation and a spontaneous wx mutant were physically characterized, and the genetic distances between those wx mutation sites were estimated by pollen analysis. Based on these results, the recombination frequency at the wx locus in rice was estimated as 27.3 kb/cM, which was about 10 times higher than the average for the genome, suggesting that there was a radically different rate of meiotic recombination for intra- and intergenic regions in the rice genome.     

Kick-starting the ratchet: the fate of mutators in an asexual population

Muller's ratchet operates in asexual populations without intergenomic recombination. In this case, deleterious mutations will accumulate and population fitness will decline over time, possibly endangering the survival of the species. Mutator mutations, i.e., mutations that lead to an increased mutation rate, will play a special role for the behavior of the ratchet. First, they are part of the ratchet and can come to dominance through accumulation in the ratchet. Second, the fitness-loss rate of the ratchet is very sensitive to changes in the mutation rate and even a modest increase can easily set the ratchet in motion. In this article we simulate the interplay between fitness loss from Muller's ratchet and the evolution of the mutation rate from the fixation of mutator mutations. As long as the mutation rate is increased in sufficiently small steps, an accelerating ratchet and eventual extinction are inevitable. If this can be countered by antimutators, i.e., mutations that reduce the mutation rate, an equilibrium can be established for the mutation rate at some level that may allow survival. However, the presence of the ratchet amplifies fluctuations in the mutation rate and, even at equilibrium, these fluctuations can lead to dangerous bursts in the ratchet. We investigate the timescales of these processes and discuss the results with reference to the genome degradation of the aphid endosymbiont Buchnera aphidicola.     

Bond-optimized ring closure for proline: comparison of conformations and semiempirical energies with small molecule X-ray structures

A method is described for generating proline ring structures by successive addition of atoms, wherein ring closure is achieved by optimizing the fit to known ring bond-angles and one closing bond-length ("bond-optimized ring closure"). Two ring torsion angles are fixed independently within broad, allowed ranges, and the remaining torsion angles are determined uniquely in most cases. The independent torsion angles are chosen as phi and chi 2, and ring closure is achieved without prohibitive strain through most of the ranges -130 degrees less than phi less than -20 degrees and -60 degrees less than chi 2 less than 60 degrees. Comparisons of predicted ring structures to 191 X-ray diffraction structures from the literature, starting with the known values of phi and chi 2, yielded root-mean-square deviations of 4.8 degrees in chi 1, 4.7 degrees in chi 3, 8.3 degrees in chi 4, and 0.3-2% in the ring bond angles and the N-C delta distance. Semiempirical energies were calculated for the optimized structures using three sets of energy parameters from the literature. The energy surfaces show broad minima coinciding with the torsion angle regions in which the highest concentrations of observed structures are found. Two of the sets of energy parameters produce double minima corresponding to the "up" and "down" puckered conformations.     

Recombination estimation under complex evolutionary models with the coalescent composite-likelihood method

The composite-likelihood estimator (CLE) of the population recombination rate considers only sites with exactly two alleles under a finite-sites mutation model (McVean, G. A. T., P. Awadalla, and P. Fearnhead. 2002. A coalescent-based method for detecting and estimating recombination from gene sequences. Genetics 160:1231-1241). While in such a model the identity of alleles is not considered, the CLE has been shown to be robust to minor misspecification of the underlying mutational model. However, there are many situations where the putative mutation and demographic history can be quite complex. One good example is rapidly evolving pathogens, like HIV-1. First we evaluated the performance of the CLE and the likelihood permutation test (LPT) under more complex, realistic models, including a general time reversible (GTR) substitution model, rate heterogeneity among sites (Gamma), positive selection, population growth, population structure, and noncontemporaneous sampling. Second, we relaxed some of the assumptions of the CLE allowing for a four-allele, GTR + Gamma model in an attempt to use the data more efficiently. Through simulations and the analysis of real data, we concluded that the CLE is robust to severe misspecifications of the substitution model, but underestimates the recombination rate in the presence of exponential growth, population mixture, selection, or noncontemporaneous sampling. In such cases, the use of more complex models slightly increases performance in some occasions, especially in the case of the LPT. Thus, our results provide for a more robust application of the estimation of recombination rates.     

Massively parallel resequencing of the isogenic Drosophila melanogaster strain w1118; iso-2; iso-3 identifies hotspots for mutations in sensory perception genes

We used the Illumina reversible-short sequencing technology to obtain 17-fold average depth (s.d.~8) of ~94% of the euchromatic genome and ~1-5% of the heterochromatin sequence of the Drosophila melogaster isogenic strain w1118; iso-2; iso-3. We show that this strain has a ~9 kb deletion that uncovers the first exon of the white (w) gene, ~4 kb of downstream promoter sequences, and most of the first intron, thus demonstrating that whole-genome sequencing can be used for mutation characterization. We chose this strain because there are thousands of transposon insertion lines and hundreds of isogenic deficiency lines available with this genetic background, such as the Exelixis, Inc., and the DrosDEL collections. We compared our sequence to Release 5 of the finished reference genome sequence which was made from the isogenic strain y1; cn1 bw1 sp1 and identified ~356,614 candidate SNPs in the ~117 Mb unique sequence genome, which represents a substitution rate of ~1/305 nucleotides (~0.30%). The distribution of SNPs is not uniform, but rather there is a ~2-fold increase in SNPs on the autosome arms compared with the X chromosome and a ~7-fold increase when compared to the small 4th chromosome. This is consistent with previous analyses that demonstrated a correlation between recombination frequency and SNP frequency. An unexpected finding was a SNP hotpot in a ~20Mb central region of the 4th chromosome, which might indicate higher than expected recombination frequency in this region of this chromosome. Interestingly, genes involved in sensory perception are enriched in SNP hotspots and genes encoding developmental genes are enriched in SNP coldspots, which suggests that recombination frequencies might be proportional to the evolutionary selection coefficient. There are currently 12 Drosophila species sequenced, and this represents one of many isogenic Drosophila melanogaster genome sequences that are in progress. Because of the dramatic increase in power in using isogenic lines rather than outbred individuals, the SNP information should be valuable as a test bed for understanding genotype-by-environment interactions in human population studies.     

Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.