The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III theta subunit, is expressed during both lysogenic and lytic growth stages

Abstract

The bacteriophage P1 hot gene product is a homolog of the theta subunit of E. coli DNA polymerase III. Previous studies with hot cloned on a plasmid have shown that Hot protein can substitute for theta, as evidenced by its stabilizing effect on certain dnaQ mutator mutants carrying an unstable pol III proofreading subunit (epsilon subunit). These results are consistent with Hot, like theta, being a replication protein involved in stabilizing the intrinsically unstable epsilon proofreading function. However, the function of hot for the viral life cycle is less clear. In the present study, we show that the hot gene is not essential. Based on its promoter structure, hot has been previously classified as a "late" phage gene, a property that is not easily reconciled with a presumed replication function. Here, we clarify this issue by demonstrating that P1 hot is actively expressed both during the lysogenic state and in the early stages of a lytic induction, in addition to its expression in the late stage of phage development. The results indicate that P1 hot has a complex expression pattern, compatible with a model in which Hot may affect the host replication machinery to benefit overall phage replication.

Fig. 1

Phage yields (PFU) after thermal induction of P1 lysogens. (A) Phages carrying a kanamycin-resistance cassette (kan) in three different P1 loci (Tn9 (cat), hot, or humD) as detailed in the text. ‘wt’ represents the parental P1 c1–100 Tn9 lysogen. Induction and titering were performed as described in Section 2. (B) As in (A) but after removal of the kan insert from hot or humD, indicated as Δhot or ΔhumD. The results represent the mean value (±S.E.M.) for 20 independent cultures for each indicated strain as calculated by “Prism” software.

Fig. 2

Mutator or antimutator effect of Hot protein produced by P1 lysogens, as monitored by the frequency of rifampicin-resistant (Rif^R) mutants. (A) Control experiment for dnaQ923 or dnaQ930 strains containing ΔholE, holE⁺, or P1 hot on the E. coli chromosome. The strains were NR16320 (dnaQ923, ΔholE), NR16319 (dnaQ923), NR16321 (dnaQ923, ΔholE::hot), NR16329 (dnaQ930, ΔholE), NR16328 (dnaQ930), and NR16330 (dnaQ930, ΔholE::hot) as described previously [13]. For comparison, typical Rif^R mutant frequencies for non-mutator (dnaQ⁺) strains are (0.02–0.05) × 10⁶. (B) Effect of Hot protein on dnaQ923 and dnaQ930 strains when the hot gene is expressed from the P1 prophage. Strains NR16320 (dnaQ923 ΔholE) and NR16329 (dnaQ930 ΔholE) [13] were infected by bacteriophage P1 c1–100 Tn9 and its Δhot derivative to create lysogens. Fifteen independent lysogens for each strain were used to measure the frequency of rifampicin-resistant mutants. Cultures grown overnight at 30 °C in LB + chloramphenicol. The graph shows median values and the interquartile ranges for the frequency of rifampicin-resistant mutants as calculated by the statistical analysis software Prism (GraphPad). (C) As in (B) above, but here the bacterial hosts are holE⁺ strains NR17116 (dnaQ49) and NR16328 (dnaQ930) [13].

Fig. 3

Expression of hot during a P1 lytic cycle. QPCR methods were used to assay expression of the P1 hot gene upon thermal induction of P1 c1–100 Tn9 in strain MG1655 along with that of the known late gene 16 and the chromosomal holE gene. RNA and cDNA samples were prepared as described in Section 2. RNA was quantified using RiboGreen reagent (Invitrogen) and, for each sample, an identical amount of RNA was carried into the cDNA preparation step. Standard QCPR curves were obtained by serial dilutions of purified DNA samples, establishing that, under the conditions used, one cycle of PCR reflected a doubling of the DNA amounts. The C_t (threshold cycle) values obtained from the real-time PCR analysis were converted into the fold increases or decreases displayed on the Y-axis as follows. At each time point, the C_t value was subtracted by the starting value C_t0, yielding (C_t − C_t0) = n, representing the number of cycles by which the PCR product reached the threshold “earlier”. n was then converted to a corresponding cDNA increase by 1/2ⁿ, which is represented on the Y-axis. A melting curve experiment was also included as part of each run to verify the correctness of the amplified DNA product (Stratagene). The resulting numbers were analyzed by “Prism” software (GraphPad), and each point on the graph represents the mean value (±S.E.M.). In the indicated (representative) experiment, each data point represents the average of six samples (two independent cultures analyzed in triplicate). The average C_t0 values were 23.2 for holE, 27.3 for hot, and 33.7 for gene 16. Note that due to the large effects for gene 16 expression (panel B), the data for this gene are shown on a logarithmic scale.