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

Abstract

The bacteriophage P1 hot gene product is a homolog of the θ subunit of E. coli DNA polymerase III. Previous studies with hot cloned on a plasmid have shown that Hot protein can substitute for θ, as evidenced by its stabilizing effect on certain dnaQ mutator mutants carrying an unstable pol III proofreading subunit (ε subunit). These results are consistent with Hot, like θ, being a replication protein involved in stabilizing the intrinsically unstable ε 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.

Evidence for Moonlighting Functions of the Subunit of Escherichia coli DNA Polymerase III

“…This is the case for the genome of the phage P1. Strikingly, expression of the P1 hot gene is not strictly related to the replication cycle of the phage but is also related to the lysogenic growth stage (16). These data were interpreted as HOT benefiting phage replication, but a significant difference in phage yield upon the thermal induction of hot ϩ and hot-minus phages was not found (16).…”

“…Strikingly, expression of the P1 hot gene is not strictly related to the replication cycle of the phage but is also related to the lysogenic growth stage (16). These data were interpreted as HOT benefiting phage replication, but a significant difference in phage yield upon the thermal induction of hot ϩ and hot-minus phages was not found (16). If the unique function of HolE is to act as a component of the Pol III core, the gene products of the corresponding plasmid-carried holE genes must alter the composition of Pol III core in those enterobacteria that randomly incorporate them by acquiring the corresponding plasmid.…”

“…When expressed from the holE promoter, hot is able to complement a ⌬holE defect (15). Interestingly, hot is expressed not only during the late stage of phage development but during the lysogenic state and early stages of a lytic induction (16). This complex expression pattern was interpreted as HOT affecting the replication machinery to benefit phage replication.…”

Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

“…It is important to remark that these observations were obtained with strains cultured in LB medium at 37 °C, and containing a wild-type dnaQ allele. Interestingly, when holE was deleted in genetic backgrounds featuring a defective dnaQ allele, mutation frequencies increased dramatically [29,43–45]. In particular, it is interesting to consider the experiments performed in the presence of the dnaQ49 allele, which is thought to code for a temperaturesensitive proofreading subunit [43–45].…”

“…Interestingly, when holE was deleted in genetic backgrounds featuring a defective dnaQ allele, mutation frequencies increased dramatically [29,43–45]. In particular, it is interesting to consider the experiments performed in the presence of the dnaQ49 allele, which is thought to code for a temperaturesensitive proofreading subunit [43–45]. When holE was deleted in a dnaQ49 background, the mutation frequencies increased dramatically even at the lower temperatures of 25 and 30 °C compared to the control dnaQ49 strain, suggesting that at these temperatures the absence of θ further increased the intrinsic instability of DnaQ49 [43].…”