In Escherichia coli, programmed cell death is mediated through ''addiction modules'' consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of rexB, one of the few genes expressed in the lysogenic state of bacteriophage , prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3,5-bispyrophosphate (ppGpp) and thus by amino acid starvation. RexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of RexB through its action on the ClpP proteolytic subunit. We also propose that the rex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the ''survival operon'' of phage .
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...
Penicillinase (beta-lactamase I, EC 3.5.2.6) secreted by Bacillus cereus, strain 569/H, was covalently attached to aminoethyl cellulose via glutaraldehyde. The immobilized derivative shows increased thermostability and decreased susceptibility to conformational changes induced by certain substrates of penicillinase. The decline in the rate of hydrolysis of such substrates was consequently suppressed by immobilization. A marked increase in Km was observed with all substrates except for the unsubstituted 6-aminopenicillanic acid. The altered properties of the new derivative are attributed to the constraint imposed by immobilization on the conformational flexibility of the enzyme molecule. Thus, apart from obvious technological interest, immobilized penicillinase provides a useful model for the study of the role of flexibility in the function of an enzyme.
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