Molecular Basis and Ecological Relevance of Caulobacter Cell Filamentation in Freshwater Habitats.

All living cells are characterized by certain cell shapes and sizes. Many bacteria can change these properties depending on the growth conditions. The underlying mechanisms and the ecological relevance of changing cell shape and size remain unclear in most cases. One bacterium that undergoes extensive shape-shifting in response to changing growth conditions is the freshwater bacterium Caulobacter crescentus When incubated for an extended time in stationary phase, a subpopulation of C. crescentus forms viable filamentous cells with a helical shape. Here, we demonstrated that this stationary-phase-induced filamentation results from downregulation of most critical cell cycle regulators and a consequent block of DNA replication and cell division while cell growth and metabolism continue. Our data indicate that this response is triggered by a combination of three stresses caused by prolonged growth in complex medium, namely, the depletion of phosphate, alkaline pH, and an excess of ammonium. We found that these conditions are experienced in the summer months during algal blooms near the surface in freshwater lakes, a natural habitat of C. crescentus, suggesting that filamentous growth is a common response of C. crescentus to its environment. Finally, we demonstrate that when grown in a biofilm, the filamentous cells can reach beyond the surface of the biofilm and potentially access nutrients or release progeny. Altogether, our work highlights the ability of bacteria to alter their morphology and suggests how this behavior might enable adaptation to changing environments.IMPORTANCE Many bacteria drastically change their cell size and morphology in response to changing environmental conditions. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus and related species transform into filamentous cells in response to conditions that commonly occur in their natural habitat as a result of algal blooms during the warm summer months. These filamentous cells may be better able to scavenge nutrients when they grow in biofilms and to escape from protist predation during planktonic growth. Our findings suggest that seasonal changes and variations in the microbial composition of the natural habitat can have profound impact on the cell biology of individual organisms. Furthermore, our work highlights that bacteria exist in morphological and physiological states in nature that can strongly differ from those commonly studied in the laboratory.

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.