Abstract
In a λimm434 lysogen, two proteins are expressed from the integrated prophage. Both are encoded by the same mRNA whose transcription initiates at the PRM promoter. One protein is the 434 repressor, needed for the establishment and maintenance of lysogeny. The other is Hex which is translated from an open reading frame that apparently partially overlaps the 434 repressor coding region. In the wild type host, disruption of the gene encoding Hex destabilizes λimm434 lysogens. However, the hex mutation has no effect on lysogen stability in a recA− host. These observations suggest that Hex functions by modulating the ability of RecA to stimulate 434 repressor autocleavage. We tested this hypothesis by identifying and purifying Hex to determine if this protein inhibited RecA‑stimulated autocleavage of 434 repressor in vitro. Our results show that in vitro a fragment of Hex prevents RecA-stimulated autocleavage of 434 repressor, as well as the repressors of the closely related phage P22. Surprisingly, Hex does not prevent RecA‑stimulated autocleavage of phage lambda repressor, nor the E. coli LexA repressor.
Highlights
Upon infection of a host cell, the lambdoid phages choose between two developmental fates, opting either to replicate and lyse the cell, or to enter the latent, or lysogenic phase in which the phages’chromosome is integrated into that of the host and is replicated along with it [1,2]
Computer analysis of the three reading frames in the mRNA initiated from promoter for repressor maintenance (PRM) of the bacteriophage
To ensure that the transcript initiated at PRM extends through the putative ORF downstream from the repressor gene, we isolated total bacterial RNA from MG1655(λimm434) lysogens, reverse transcribed this RNA using primer 1 (Figure 1) and the resulting cDNA was analyzed by PCR using a series of primers complementary to this region (Figure 1)
Summary
Upon infection of a host cell, the lambdoid phages choose between two developmental fates, opting either to replicate and lyse the cell, or to enter the latent, or lysogenic phase in which the phages’. In addition to the repressor gene many lambdoid bacteriophages encode additional proteins on the transcript initiated from PRM. Since these genes are not essential for lysogenic or lytic growth, they are considered accessory [8]. Bacteriophage 434 expresses, in addition to cI repressor, a second protein Hex, which is encoded on the mRNA transcript initiated from PRM. The results of genetic and biochemical experiments suggest that, when present, Hex increases the stability of bacteriophage 434 by interfering with RecA-mediated autocleavage of the phage 434 repressor. Consistent with this finding, we found that purified Hex blocks RecA-mediated stimulation of 434 repressor autocleavage. This finding suggests that Hex does not block RecA activity by directly interacting with it
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