Coping with Hard Times: Death as an Option

by Elio

First, some musings about life and death, two matters that don't seem to occupy the same space in our brain. We tend to celebrate the former and resent the latter, and we often see the world through this perspective. Only on reflection do we realize how wrong this is, especially with regard to biology. The cycle of life depends fundamentally on living things dying. But this is not always the prevailing way we approach biological questions. Think of how long it took for the concept of apoptosis (in itself an old realization) to take hold. In microbiology, most of the early modern studies on bacterial physiology dealt with growth and seldom with how bacteria deal with adverse conditions, such as starvation, and death. But all this is changing, as we realize that organisms have evolved to cope as much with danger as with good times. And their coping is often due to a collaborative effort, which is why the adaptation to hard times is a primary concern of the emergent science of Microbial Sociology.

Among the big-time adjustments in our views of the microbial world has been the recognition in the last few decades that bacteria do not particularly behave as individuals. That was the preferred way of thinking about them, not the least because it made life simpler. Remember that modern biology (the post-World War II variety) was greatly influenced by physicists, who brought along a predilection for simple units as model systems (e.g., a single atom, ergo a single T4 coliphage particle or one E. coli cell). As is now obvious, this is an incomplete way of thinking because bacteria interact in important and diverse ways with members of their same or other species. So intricate are these interactions that bacteria are beginning to vie for sophistication in communication with the social insects. Remember, both speak a chemical language.

High in the list of signposts of bacterial sociability are biofilm formation, quorum sensing, and, newer on the scene, cannibalism (click here for a recent paper). Some time ago, another, perhaps even less expected facet of microbial sociology was introduced, namely the notion of programmed cell death. It was found that plasmids make proteins that can kill the bacteria that carry them. Lucky for the bugs, such "toxins," as they were named, are neutralized by "antitoxins." Antitoxins are proteins from the same operon that negate the effect of the toxins by binding to them. All is good as long as the cell carries the plasmid and both proteins are expressed. However, when a cell is rid of the plasmid, the antitoxin is degraded faster than the toxin, ergo the cell dies. This system, aptly named the "addiction module" by Michael Yarmolinsky, had the obvious virtue of making full sense, at least from the point of view of the plasmid.

Control Pathways for the MazE/MazF system. The MazE antitoxin (blue oval) sequesters the MazF toxin (red oval). MazF is liberated under some conditions (e.g., starvation with formation of the alarmone ppGpp, the presence of antibiotics or reactive oxygen species). MazF can also be degraded by the protease ClpAP (magenta diamond). The EDF peptide (green star) is a fragment of G6PDH (green sphere), perhaps formed by ClpAP action. Source

Enter a similarly acting pair of proteins, encoded not by plasmids but by the E. coli chromosome. Discovered in 1996 by Hanna Engelberg-Kulka and collaborators at the Hebrew University, such TA (toxin-antitoxin) modules were soon found in many other bacterial species, sometimes in great abundance. (The tubercle bacillus has 88 putative such TA modules!) Killing by the toxins is manifested only under conditions of stress, such as starvation, the presence of reactive oxygen species, or antibiotic action. As to why this happens, one thought is that the death of some cells helps the rest of the population to survive, possibly by the survivors feasting — cannibal-like — on their dead siblings. But, given the great variety of TA modules and their lethal mechanisms, it seems likely that bacterial programmed cell death has evolved repeatedly and for different purposes.

The story of the chromosomal toxin-antitoxin systems is best illustrated by the first module to be discovered, the duo of a toxin called MazF and its antitoxin, MazE. MazF is a sequence-specific ribonuclease that cleaves mRNAs at ACA sites, rendering the mRNA unavailable for the translation of vital proteins. MazE binds avidly to MazF, which is why cells in propitious environments can go on their merry way. However, stresses such as the ones mentioned above, lead to the dissociation of the two proteins, allowing the toxin to become active. But there is more to it. E. coli secretes a pentapeptide that, by binding to MazF, enhances its toxic ribonucleolytic activity and, in the process, keeps MazE from binding to MazF. Called EDF for "extracellular death factor," this is not your ordinary peptide. It has an unusual-looking sequence (asn-asn-try-asn-asn, or NNWNN) and is derived from proteolysis of a perfectly respectable household enzyme, glucose-6-phosphate-dehydrogenase (G6PDH). Just why this protein carries the right peptide and thus has been chosen as its source is a topic of speculation. Note again that quite a number of other mechanisms for programmed bacterial death are known. For example, a recent report proposes that death can also result from inhibition of peptidoglycan synthesis.

© Structural similariities of an EDF-MazF model and the solved structure of MazE-MazF. MazF is the white surface, EDF is blue, and the corresponding portion of MazE is magenta. Conserved hydrogen bonds to MazF are shown arrows). (D) The central tryptophan of EDF overlaps perfectly with MazE Trp73. Source

How does EDF work? It mimics a portion of the MazE sequence important for its binding to MazF, especially with regard to the central tryptophan that corresponds to a critical tryptophan in MazE. The parallel interaction of EDF-MazF and MazE MazF awaits structural analysis of the EDF-MazF complex.But matters are more intricate, as seen by the presence of two binding sites on MazF for mRNA. This paper explains much of it, plus it introduces another toxin, ChpBK, that has equally tantalizing properties.

Added to the magic of EDF is the fact that it acts as a quorum sensing signal, meaning that it comes into play at high bacterial densities where interactions between cells become especially relevant . But the MazF protein also has unusual properties: It regulates the synthesis of both "death proteins and "survival proteins," thus playing the role of a master regulator. This leads not only to the death of some members of the population but to the survival of the others. Neat or what?

Fortunately, much remains to be explained about the whole phenomenon of self-induced bacterial cell death. I say "fortunately" because it seems like an easy prophecy to say that more magic will be uncovered, both on the level of the biochemical mechanisms involved and on their grander biological significance. For the latter, we may have to wait for more in naturam experiments, which is a far more demanding proposition than what can be done in the laboratory. But then, I always thought that doing biochemistry is easier than doing ecology.

Reference

Belitsky M, Avshalom H, Erental A, Yelin I, Kumar S, London N, Sperber M, Schueler-Furman O, Engelberg-Kulka H. (2011). The Escherichia coli Extracellular Death Factor EDF Induces the Endoribonucleolytic Activities of the Toxins MazF and ChpBK. Molecular cell, 41 (6), 625–635. PMID 21419338