Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. Although many methods for protease activity and specificity profiling exist, none of these combine the advantages of combinatorial synthetic libraries, i.e., high diversity, equimolar concentration, custom design regarding peptide length, and randomization, with the sensitivity and detection power of mass spectrometry. Here, we developed such a method and applied it to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e., Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) that has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a subpopulation of cells arises that hyperproduces metabolically costly antibiotics, resulting in a division of labor that increases colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is similar to altruistic worker castes in social insects or somatic cells in multicellular organisms. To understand these mutant cells’ reproductive and genomic fate after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that in contrast to wild-type cells, putatively altruistic mutant cells continue to decline in fitness during transfer while they lose more fragments from their chromosome ends. In addition, the base-substitution rate in mutants increases roughly 10-fold, possibly due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with DNA damage due to point mutations and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest the cells arising in the S. coelicolor division of labor are analogous to altruistic reproductively sterile castes of social insects.

Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery.

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a sub-population of cells arise that hyper-produce metabolically costly antibiotics, resulting in a division of labor that maximizes colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is altruistic, much like worker castes in eusocial insects. To understand the reproductive and genomic fate of these mutant cells after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that, in contrast to wild-type cells, altruistic mutant cells continue to significantly decline in fitness during transfer while they delete larger and larger fragments from their chromosome ends. In addition, altruistic mutants acquire a roughly 10-fold increase in their base-substitution rates due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with irreversible DNA damage due to point mutation and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest that the altruistic cells arising in this division of labor are equivalent to reproductively sterile castes of social insects.

In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a sub-population of cells arise that hyper-produce metabolically costly antibiotics, resulting in a division of labor that maximizes colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is altruistic, much like worker castes in eusocial insects. To understand the reproductive and genomic fate of these mutant cells after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that, in contrast to wild-type cells, altruistic mutant cells continue to significantly decline in fitness during transfer while they delete larger and larger fragments from their chromosome ends. In addition, altruistic mutants acquire a roughly 10-fold increase in their base-substitution rates due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with irreversible DNA damage due to point mutation and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest that the altruistic cells arising in this division of labor are equivalent to reproductively sterile castes of social insects.