Summary Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution.

SUMMARY Toxin-antitoxin (TA) systems are ubiquitous on bacterial chromosomes, yet the mechanisms regulating their activity, and the molecular targets of toxins, remain incompletely understood. Here, we identify SocAB, a new TA system in Caulobacter crescentus. Unlike canonical TA systems, the toxin SocB is unstable and constitutively degraded by the protease ClpXP; this degradation requires the antitoxin, SocA, as a proteolytic adaptor. We find that the toxin, SocB, blocks replication elongation through an interaction with the sliding clamp, driving replication fork collapse. Mutations that suppress SocB toxicity map to either the hydrophobic cleft on the clamp that binds DNA polymerase III or a clamp-binding motif in SocB. Our findings suggest that SocB disrupts replication by outcompeting other clamp-binding proteins. Collectively, our results expand the diversity of mechanisms employed by TA systems to regulate toxin activity and inhibit bacterial growth, and they suggest that inhibiting clamp function may be a generalizable antibacterial strategy.

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

A molecular evolutionary explanation of natural genetic variation requires analysis of specific variants' evolutionary dynamics. To pursue this for phosphoglucose isomerase (PGI) of Colias butterflies, whose polymorphism is maintained by strong natural selection, we assembled a large data set of wild haplotypes, highly variable at the amino acid and DNA levels. The most common electrophoretic, i.e., charge macrostate, allele class, 3, is conserved in its pattern of charged amino acid residues. The next most common macrostate, 4, has multiple patterns of charge, i.e., microstates, while less common (1, 2, 5, 6) macrostates are very diverse. Macrostate 4 shows significant linkage disequilibrium (LD) among its variants, especially for two groups of five haplotypes each. We find extensive intragenic recombination among all haplotypes except the two high-LD groups of macrostate 4, which display none. Phyletic relations among haplotypes are largely reticulate, again except for the high-LD groups of macrostate 4, which form clades with strong bootstrap support. Charge-changing and linked charge-neutral amino acid variants occur in diverse parts of PGI's sequence. Homology-based modeling of PGI's structure shows that these regions are related spatially in ways suggesting functional interaction. The high-LD groups of macrostate 4 display parallel amino acid variation in these regions. This pattern of haplotype clades with high LD among multiple varying sites, emerging from chaotically recombining variation, may be a "signature" of refinement of complex adaptive sequences by recombination and selection. It should be tested further in this study system and others as a possibly general feature of the evolution of living complexity.

Summary Heterogeneity within a clonal population of cells can increase survival in the face of environmental stress. In a recent issue of Science, Aldridge et al. (2012) demonstrate that cell division in mycobacteria is asymmetric, producing daughter cells that differ in size, growth rate, and susceptibility to antibiotics.