A fraction of a genetically homogeneous microbial population may survive exposure to stress such as antibiotic treatment. Unlike resistant mutants, cells regrown from such persistent bacteria remain sensitive to the antibiotic. We investigated the persistence of single cells of Escherichia coli with the use of microfluidic devices. Persistence was linked to preexisting heterogeneity in bacterial populations because phenotypic switching occurred between normally growing cells and persister cells having reduced growth rates. Quantitative measurements led to a simple mathematical description of the persistence switch. Inherent heterogeneity of bacterial populations may be important in adaptation to fluctuating environments and in the persistence of bacterial infections.

Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanism. Most recently, genome sequencing and “omics” profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.

An integrated approach is described that allows the domain-specific incorporation of optical probes into large recombinant proteins. The strategy is the combination of two existing techniques, expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues. The Src homology 3 (SH3) domain from the c-Crk-I adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using Escherichia coli Trp auxotrophs. Structural, biochemical, and thermodynamic studies show that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of the 7AW-labeled SH3 domain to the c-Crk-I Src homology 2 (SH2) domain, via EPL, generated the multidomain protein, c-Crk-I, with a domain-specific label. Studies of this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multidomain protein. The technology described here is likely to be a useful tool in enhancing our understanding of the behavior of modular domains in their natural context, within multidomain proteins.