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LAMBDA: A Prophage Detection Benchmark for Genomic Language Models.

Transformer-based genomic sequence models represent an emerging frontier in computational biology. Yet, their embeddings have not yet shown the same level of predictive power as natural and protein language models, indicating a gap between current implementations and theoretical promise. Existing benchmarks for DNA language models primarily focus on classifying regulatory elements in eukaryotic genomes, leaving open the fundamental question of whether these models learn sequence-level features across whole genomes. We introduce LAMBDA, a benchmark designed to rigorously evaluate genome language model embeddings through phage-bacteria sequence discrimination across four categories of increasing complexity: probing tasks, fine-tuning assessments, diagnostic tests, and genome-wide prophage detection. Our comprehensive analysis of current genomic language models provides novel insights into the importance of training data quality relative to model size, the need for domain-specific training, and the application of genomic language models for detecting prophage sequences. This benchmark represents a challenging genomic annotation task in the bacterial domain and addresses a key computational problem with direct relevance to microbiology and medicine.

DNA language model

Replication of prophage P1 during the cell cycle of Escherichia coli.

We have followed, by DNA-DNA hybridization, the variation in the number of copies of prophage P1 relative to two chromosomal markers when the doubling time of the host cells is modified by a change in carbon source. The ratio of P1/chromosome terminus undergoes a twofold decrease when the cell doubling time increases from 24 to 215 min, whereas the ratio of P1/chromosome origin increases 1.4 fold; both ratios tend towards unity at slow growth rates. This suggests that the replication of prophage P1 is not simultaneous with chromosome initiation or chromosome termination. The chromosome replication time is unaffected by the presence of P1, and remains constant over the range of doubling times studied, with a value of about 4o min. Following amino acid starvation, the P1/chromosome origin ratio increases from 0.7 to 0.9, suggesting that P1 retains the ability to replicate after chromosome initiation has stopped and in the absence of essential amino acids. The results are discussed with reference to similar studies done on F and R1.

Cell Division

Mutation of lambda during prophage induction by nitrosamides.

Approximately 6% of Escherichia coli K12 (lambda wild-type) cells whose prophage was induced by treatment with N-methyl-N-nitrosourea initiated plaques on E. coli K12S which contained wholly or mostly clear plaque-forming mutants (lambdac). "Fuzzy" plaque-forming mutants (lambdaf) were also recognised, at lesser frequencies. Less marked mutation occurred during prophage induction by N-ethyl-N-nitrosourea, and no apparent mutation occurred during induction by methyl and iso-propyl methanesulphonates, or by a non-inducing treatment of the lysogen with ethyl methanesulphonate. Mutagenic effects of treatment of susceptible host cells or of phage alone, prior to infection, seem not to account for the phenomenon described.

Coliphages

Induction of lambda prophage and of mutations to streptomycin resistance in separate small fractions of a lysogenic derivative of Escherichia coli B/r by very low doses of ultraviolet light.

The number of induced mutations to streptomycin resistance is compared at doses of ultraviolet (UV) light between 0.2 and 6.4 J/m2 in a Uvr- (excision-deficient) derivative of E. coli B/r, strain WU, and in its lambda lysogen, strain WU(lambda). At UV doses up to about 1 J/m2, which converts about 5% of the lysogenic population into enfective centers, no difference is observed in the number of mutations to streptomycin resistance produced by the two strains. It is concluded that the capacity to produce UV-induced mutations is not coupled with lysis due to the induction of lambda prophage at low doses of UV radiation. At UV doses above 1 J/m2, the number of mutatants detected in the lysogenic strain decreases appreciably compared to the number detected in the nonlysogen, and is only about 10% as high at UV doses of 3 J/m2 and higher, doses which cause maximal induction of prophage. The results are compatible with the operation of a common "all-or-none" induction signal resulting in expression of UV-inducible functions at high UV doses, but not at low doses.

Coliphages

Insertion sequence IS2 near the gene for prophage lambda excision.

In this study we characterize a variant of the lambdacI857S7 prophage, designated lambdabi2cI857S7, which carries a DNA insertion. The insertion sequence is IS2, and it resides in the antipolar orientation II just upstream from the gene for prophage excision (xis) at 61.6%lambda. This bi2 insertion mutant could prove valuable for studies on possible recombination functions of IS2 DNA and of its effect on the lambda integration and excision functions.

Chromosome Mapping

Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn.

Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC. The transducing phages had been isolated from a strain of E. coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster [Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1978) Proc. Natl. Acad. Sci. USA 75, 5099--5103]. When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells. In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC. The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC. In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria. These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides. Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes.

Adenosine Triphosphatases

Deletions induced by heat treatment of E. coli K12 lysogenic for lambda prophages.

We have investigated the production of prophage deletions in heat-induced lambda lysogens of E. coli K12. Our results are indicative of a direct action of the heat-induced prophage in producing deletions. The temperature of 40 degrees used for the experiments may be critical to prove this effect. The phage function involved in deletion formation is not known.

Chromosome Aberrations

Chromosome replication in an Escherichia coli dnaA mutant integratively suppressed by prophage P2.

Escherichia coli CRT4624-P2sig5 is a dnaA mutant in which integration of the prophage P2sig5 has occurred at the attP2II site (min 85). This strain was integratively suppressed, and when cells were shifted to 42 degrees C replication was initiated at a site in or near the P2 prophage. Initially, this replication occurred primarily in the direction that corresponds to the clockwise direction on the genetic map. Replication also occurred in the counterclockwise direction, but the initiation of replication in this direction occurred approximately 40 min later than the initiation of replication in the other direction. Because of this delay, the replication forks that traveled in the clockwise direction were the first to arrive in the region of the replication terminus. These replication forks ceased replication near the aroD locus (min 37), and it is proposed that the replication terminus is between the aroD and rac loci (min 31). A model is proposed for the cycle of chromosome replication in this strain at 42 degrees C.

Chromosomes, Bacterial

Viral R plasmid Rphi6P: properties of the penicillinase plasmid prophage and the supercoiled, circular encapsidated genome.

Properties of the viral R plasmid Rphi6P are described. As a temperate bacteriophage, it plaques on the facultative phototroph Rhodopseudomonas sphaeroides. Under aerobic conditions the phage had a latent period of 180 min, a burst time of 200 min, and a burst size of 15 to 20 particles per infective center. The encapsidated viral genome occurred as a supercoiled, circular DNA duplex with a mean contour length of 16.5 +/- 10 micron. Percent guanine plus cytosine, as calculated from thermal denaturation profiles, was 63.5. Mitomycin C-induced loss of the prophage suggested an extrachromosomal location in the host cell. Use of this curing agent enabled the isolation of a plasmid-free strain of R. sphaeroides. Biophysical analysis of the plasmid-free strain lysogenized with Rphi6P confirmed that the prophage occurred as a plasmid in the host cell.

Adsorption

Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.

Antigen-Antibody Complex

Orientation of the tox gene in the prophage of corynebacteriophage beta.

The orientation of the gene for diphtheria toxin, tox, in the prophage of converting corynebacteriophage beta has been determined. The orientation of tox in prophage and that reported simultaneously by Holmes (1976) for vegetative phage are compatible with the hypothesis that beta phage is inserted into the chromosome of its bacterial host by means of a mechanism similar to that described for lambda phage, and that the phage attachment site lies between the tox and imm genes. The position of three tox mutations that are phenotypically CRM- has also been determined. Relative to the tox-45 mutation, they are located more proximally to the end of the tox structural gene that corresponds to the amino terminal of diphtheria toxin.

Bacteriophages

The role of temperate bacteriophage SP beta in prophage-mediated interference in Bacillus subtilis.

Virulent bacteriophage phi 1 grows on a variety of Bacillus subtilis strains, mutants of this virus which abortively infect the transformable bacillus. B. subtilis 168, while retaining the ability to productively infect related bacteria have been found. In the present study, we demonstrate that the inability of one such variant, phi 1m, to develop normally in strain 168 is mediated by cryptic prophage SP beta. The latter is a temperate bacteriophage which is carried by B. subtilis 168 and most strains derived from this bacterium. Phi 1 m infection of SP beta lysogens begins with apparently normal adsorption, penetration, and inititaion of virus-directed syntheses. At about the 20th min of the latent period, however, there is an abrupt cessation of nucleic acid synthesis and cellular respiration, accompanied by a change in cell permeability. This course of events can be altered to a permissive infection by mutation in the mpi gene of SP beta, by mutation in the spoOA gene of the host, or by growing SP beta lysogens at high temperature. In addition, we found a second class of phi 1 mutants which abortively infect B. subtilis 168 derivatives even in the absence of the SP beta prophage.

Bacillus subtilis

[Restriction and modification in Staphylococcus aureus: properties of resistance plasmids and prophages].

Experiments on elimination and transfer of resistance-plasmids in S. aureus (controlling resistance to penicillin, chloramphenicol and oxytetracycline) show that these plasmids have no restricting influence on phages used for typing of staphylococci. Prophages in lysogenic strains control a mechanism of restriction and modification which is active on phages and on chromosomal markers. The resistance-plasmids used in these experiments are insensitive to prophage controlled restriction.

Chloramphenicol

[Induction of prophage gamma by means of bleomycin treatment].

The kinetics of induction of lambda prophage with bleomycin A2-CP3, mitomycin C and a combination of these two antibiotics have been studied. Bleomycin has been found to cause a rapid induction of prophage in exponentially growing cultures of E. coli K12 (lambda), and the maximal values of including activity are obtained after treatment for 16 minutes with a concentration of 10 mug/ml. The per cent of induction, calculated from the total number of surviving cells, including the phage producing cells, better expresses the dynamics of the phenomenon. The number of inducing events in the population of exposed cells is proportional to the length of bleomycin treatment. These values are greater than those observed after treatment with mitomycin, at the same concentration. Inducing activity is enhanced by combination of the two antibiotics and this effect is not additive, but synergic. After the 16 minutes of treatment with bleomycin the burst sizes are lower than those which are obtained with mitomycin or with the combination of the two antibiotics. The results are interpreted as a consequence and a confirmation of the different mechanisms of action that distinguishes bleomycin from mitomycin.

Bleomycin

Studies on megacinogeny in Bacillus cereus. II. Bacillus cereus isolates characterized by prophage-controlled production of megacin A (phospholipase A).

Five out of a number of Bacillus cereus strains isolated from soil produced high titre specific bacteriocin (megacin A) in mitomycin C-induced cultures. In the course of cultivation with ethidium bromide, the strains gave off segregants not producing bacteriocin (cin-). The lysate of two wild strains formed plaques on the corresponding cin- bacteria. The two phages (wx23 and wx26) were identical in antigenic structure with phage wx was present in the lysate of B. cereus strain W, and converted cin- derivatives into cultures producing megacin A (phospholipase A). The phages produced plaques at 26 degrees C but not at 37 degrees C. In the lysates of the remaining three strains phages were not detected with biological and morphological methods; these cultures have been assumed to carry defective prophage genome. As the corresponding prophages are responsible for the determination of inducible phospholipase A production, phages named wx seem to form a separate group of B. cereus phages.

Bacillus cereus

The value of a prophage-borne defense system in phage-phage competition.

Temperate phages that incorporate into their bacterial hosts' genomes often encode defense systems that protect their hosts from superinfection by unrelated phages. Yet the evolutionary value of such defenses to the phage remains unclear. We present a minimal theoretical framework to quantify the selective advantage of a prophage-borne defense system in competition between temperate phages infecting the same bacterial host. The model reveals regimes in which a "defensive phage" can invade and persist despite growth costs, regimes of bistability, and others in which all phage types coexist due to a rock-paper-scissors-like dynamic between defensive, non-defensive, and defense-loss variants. Because defense systems can be non-transitive, true rock-paper-scissors relations can lead to persistent oscillations. These results identify simple conditions under which phage-encoded defense systems are evolutionarily stable, providing testable predictions for the prevalence and maintenance of these systems in natural microbial communities.

Prophages