Energy-dependent degradation of lambda O protein in Escherichia coli

Abstract

Protein 0 of bacteriophage A is a short-lived protein which has a key role in the replication of the phage DNA in Escherichia coli. Here we present evidence that XO degradation is energy dependent: it is impaired by cyanide and a-methylglucoside, both of which inhibit cellular energy metabolism. Removal of these inhibitors restored the degradation of XO. Our experiments suggest that limited Amounts of cellular energy are sufficient to support XO degradation. In addition, degradation of XO protein is prevented by a mutation in the E. coli clpP gene, but not by a mutation in the clpA gene. These results suggest that the ClpP protease is involved in the energy-dependent degradation of the XO protein.Most proteins in Escherichia coli are relatively stable, with half-lives that are greater than the doubling time of the cell (9). In contrast, a number of proteins in the E. coli cell have been found to be highly unstable. These proteins are subjected to rapid degradation by various proteases, some of which are energy dependent (12). Among the proteins which are subjected to rapid degradation in E. coli are several proteins of bacteriophage X which regulate the life cycle of the phage (6,13,14,19,25,26). For example, the antitermination protein N of bacteriophage X is degraded by the product of the E. coli lon gene, which is an ATP-dependent protease (22). On the other hand, XcII protein, which has a key role in the decision between the phage's lytic or lysogenic cycles, is subjected to degradation by the Hfl system, which is energy independent (2, 12). Several other X proteins are also subjected to proteolysis through as yet unidentified degradation pathways. These include the short-lived XO protein, which has a key role in the phage X DNA replication (1,5,7,27,28). Degradation of XO protein is prevented by the E. coli rexB gene product (24). Here, we present evidence that the rapid degradation of XO in E. coli is energy dependent. In addition, our results support the findings that the degradation of XO is not prevented in an E. coli strain carrying a mutation in the lon gene (11). However, XO degradation is inhibited in E. coli cells carrying a mutation in the clpP gene. Thus, our results suggest that the product of the clpP gene, which has a proteolytic activity, is involved in the energy-dependent degradation of the XO protein.Effect of energy metabolism inhibitors on XO degradation. XO protein is rapidly degraded in E. coli cells (20,21). We have previously studied XO degradation by using plasmid pRS4, which carries the XO gene under the control of the APL promoter (24). Here, using the same experimental system, we examined whether metabolic energy is required for the degradation of XO. For this purpose, we studied the fate of the XO protein in E. coli cells under conditions of impaired cellular energy production. XO protein was labelled for 2 min in E. coli CSR603(XcI857Sam7) carrying plasmid pRS4 (Table 1) grown in the presence of glucose, as described previously (24). The cells were washed and suspended in a glu...

Recognition, Targeting, and Hydrolysis of the λ O Replication Protein by the ClpP/ClpX Protease

“…Later reports demonstrated that the O protein has a chemical half-life of just 1.5 min in -infected cells (48 -50) and that an ATP-dependent protease mediated this rapid turnover (50,51). In light of the identification of the responsible protease as the ClpP/ClpX protease (6, 7) and of recent findings that O protein bound to the replication origin is, both in vivo (52) and in vitro (53), stabilized against proteolytic attack, it is likely that the studies reported here will provide useful insights into the O turnover process in vivo.…”

Global Transcriptional Analysis of Virus-Host Interactions between Phage ϕ29 and Bacillus subtilis

“…On the other hand, several bacterial energydependent proteases have been shown to degrade regulatory proteins of bacteriophages. For example, E. coli ClpP degrades protein O of the phage, which has a main role in the replication of viral DNA (30). Similarly, it is possible that ClpP degrades some 29 proteins required for viral development.…”

Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli.

“…The turnover rate of Phd is slow relative to a number of rates of energy-dependent proteolyses reported in the literature (8,24). This slow turnover (a half-life of almost two bacterial generations) minimizes the risk that variations in protease activity might lead to premature activation of Doc.…”

“…The few substrates identified so far for the recently discovered ClpXP protease of E. coli (9) are all encoded by bacteriophages: the 0 replication protein of bacteriophage A (298 amino acid residues) (10,24), mutant forms of the bacteriophage Mu repressor protein (196 amino acid residues) (32), and the antidote protein Phd (73 amino acid residues) of bacteriophage P1. Phd, being the smallest of the three, would appear to be a promising substrate for studies of protease target specificity.…”