Perspective Life, Death, Differentiation, and the Multicellularity of Bacteria Susan M. Rosenberg* Departments of Molecular and Human Genetics, Biochemistry and Molecular Biology, Molecular Virology and Microbiology, and the Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, United States of America In recent years, bacterial geneticists and microbiologists have begun moving away from the view that the clonal cell popula- tions they study in the lab are homoge- neous lots of identical, autonomous indi- viduals and toward one that was suggested decades ago [1], in which social and even multicellular attributes of bacteria are recognized. Bacterial clones display differ- entiation, development, cell–cell commu- nication, aging, and even apparent apo- ptosis, and not just the species with visually appreciable phase variations of surface proteins, spore formation, or variation between swimming and sessile cell types. These features appear to be ubiquitous, applying even to Escherichia coli, which has been long regarded as a laboratory model for producing homogeneous cell clones. Cellular behaviors seem ‘‘multicellular’’ when they appear to confer a group benefit. For example, in many circum- stances, bacterial cell–cell communication prevents isolated cells from running cellu- lar programs that work only in groups, like production of light, or attacking a host with toxin proteins [2]. Similarly, many (perhaps all) bacteria differentiate subpop- ulations that take risks, while the remain- ing cells stay aloof, hedging the clone’s bets—a process called bistability [3,4]. For example, stress responses instigate the turning up of mutation rate in a small subpopulation of starving E. coli cells (reviewed in [5]). This is clearly a danger- ous game for most of the cells whose genomes are mutated, but it is one that may provide a shot at producing a rare better-adapted mutant from a clone that is maladapted to its environment, i.e., one that is stressed. Similarly, small cell subpopulations of starving bacteria of many species take up foreign DNA, thus altering their genomes. In Bacillus subtilis, the same stress response activates compe- tence for transformation in the subpopu- lation as increased mutation rate under stress [6], perhaps offsetting the dangerous mutagenic pathway with the ability to regain favorable alleles from (albeit dead) neighbors. Similarly, many bacteria con- tinuously differentiate small subpopula- tions of temporarily growth-impaired ‘‘persister’’ cells that will lose in a race to colonize new territory rapidly, but can survive a transient blast of antibiotics that will kill their rapidly growing siblings, ensuring some survivors in a clone [3,4]. Perhaps the most surprising of multicel- lular-sounding bacterial behaviors is the differentiation of a cell subpopulation slated for programmed cell death. In developing vertebrates, apoptosis kills a layer of eyelid cells so that eyelids may open [7]. In bacteria, many death pro- grams are known but few are understood at this level of ‘‘organismal’’ function (see [8] for an example understood in the program of bacterial sporulation). In this issue of PLoS Genetics, Amitai and colleagues probe the mechanism of pro- grammed cell death caused by the MazF gene in E. coli [9]. Many bacteria have death genes as part of toxin/antitoxin (TA) gene modules. These are gene pairs usually co-transcribed in operons. The toxin is a stable, deadly protein but is bound and inactivated by the more labile antitoxin. Cells are safe until some cir- cumstance reduces expression of the operon. This shifts the balance in favor of the stable toxin, causing cell stasis or death. Stressors that induce this shift include various antibiotics, heat shock, starvation, DNA damage, possibly phage infection, and others. Several bactericidal antibiotics appear to kill E. coli, because they activate the MazEF system. Why do cells have TA systems? First discovered in plasmids, TA systems kill cells that lose the plasmid, causing ‘‘plas- mid addiction’’. However, TA pairs are abundant in bacterial chromosomes; E. coli has at least five pairs, and Mycobacterium tuberculosis may have 60 or more [10]. What are they doing in chromosomes? TA pairs might be selfish genetic elements, apoptosis genes, genome-stabilizing mod- ules that effectively prevent deletion of a chromosomal region [11], genes used for resisting plasmid addiction (by protecting against a plasmid-borne toxin with a chromosomal antitoxin [12]), or inducers of subpopulations of cells in stasis that transiently resist antibiotics (persister cells). The article by Amitai and colleagues offers surprising new details about the mecha- nism of MazF-mediated killing, and in doing so illuminates what this TA system might be doing for E. coli. Toxins can kill cells by several routes. Many are RNases, including MazF, which cleaves mRNAs containing the ACA sequence [13]. MazF expression results in a dramatic decrease in cellular protein levels, which was thought to be the cause of MazF-mediated cell death. Amitai et al. revisited the effect of MazF on total cellular protein levels and report the surprising discovery that although the levels of most proteins decrease, levels of some proteins actually increase after MazF overproduction (Figure 1). Amitai et al. displayed the proteomes of the MazF- treated cells on 2D gels and saw that cellular levels of most large proteins (over 20 kDa) decreased while many smaller than 20 kDa increased. They recovered 13 of the up-regulated proteins, identified them with mass spectrometry, then deleted the genes encoding each and tested their effects on MazF-dependent cell death after antibiotic treatment. Surprisingly, all of these proteins contained ACA sequences Citation: Rosenberg SM (2009) Life, Death, Differentiation, and the Multicellularity of Bacteria. PLoS Genet 5(3): e1000418. doi:10.1371/journal.pgen.1000418 Editor: Harmit S. Malik, Fred Hutchinson Cancer Research Center, United States of America Published March 13, 2009 Copyright: 2009 Susan M. Rosenberg. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grant R01 GM53158. Competing Interests: The author has declared that no competing interests exist. * E-mail: smr@bcm.edu PLoS Genetics | www.plosgenetics.org 1 March 2009 | Volume 5 | Issue 3 | e1000418