No abstract

In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its molecular mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions. MazF is a stable toxin, and MazE is an unstable antitoxin. Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P 2 is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.

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 .

The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine. This UGA decoding process requires a cis -acting mRNA element called 'selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure. In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon. Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli . Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac' Z fusion. We found that the whole stem-loop RNA structure of the E.coli fdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo . Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon. Based on these observations, we present a new model for the minimal E.coli SECIS.

"Addiction modules" consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The Escherichia coli chromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E. coli mazEF system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited. Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEF systems.In Escherichia coli cultures, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of the second gene is long lived and toxic, whereas the product of the first is short lived and antagonizes the lethal action of the toxin. Until recently, such genetic systems of bacterial programmed cell death have been found mainly in a number of E. coli extrachromosomal elements (for reviews, see references 5, 7, 8, and 15), where they are responsible for what is called the postsegregational killing effect; they are responsible for the death of plasmid-free cells. When bacteria lose the extrachromosomal elements, the cured cells are selectively killed because the unstable antitoxin is degraded faster than is the more stable toxin. One of the best-studied systems belonging to this category is the phd-doc module of plasmid prophage P1 (9, 10). This module consists of an operon the organization of which is similar to that of the operons of other addiction modules: the antitoxic gene, phd (prevents host death), precedes the toxic gene, doc (death on curing). Doc acts as a cell toxin to which the short-lived Phd protein is an antidote. Phd is degraded by the serine protease ClpPX (10). Like that of other previously described addiction modules, the expression of phd-doc is also subjected to an autoregulatory circuit (11,12). The cellular target of Doc is not yet known. However, based on recent studies it is assumed to be a step in protein synthesis (7; M. Yarmolinsky, personal communication).Members of our group have reported on the E. coli mazEF system, the first known regulatable prokaryotic chromosomal addiction module (1). It consists of two genes, mazE and mazF, located downstream from the relA gene in the rel operon (13). Through our work, we have found that mazEF has all the properties required for an addiction module. MazF is toxic and long lived, while MazE is antitoxic and short lived and is degraded by the ClpPA serine protease. MazE and MazF are coexpressed, and they interact. In addition, the mazEF system has a unique property: its expression is regulated by guanosine-3Ј5Ј-bispyrophosphate (ppGpp), which is synthesized by the RelA protei...