Targeted Knockout of CYP79A1 Reduces Cyanogenic Potential in Grain Sorghum.
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Journal Article↗
Biomedical subjects
David F Savage
Publications and source records attributed to David F Savage.
Polyphosphate acts as an architectural regulator of carbon fixation and nucleoid structure in cyanobacteria.
Polyphosphate (polyP) is a conserved inorganic polymer traditionally viewed as a stress-induced phosphate and energy reserve. In cyanobacteria, however, polyP granules are constitutively present and are frequently observed in close proximity to carboxysomes, the bacterial microcompartments that mediate CO2 fixation. Here, we show that polyP functions as a spatially organized regulator of the photosynthetic cytoplasm in Synechococcus elongatus. PolyP granules localize to the nucleoid and are periodically arranged along the cell axis, independent of the McdAB carboxysome positioning system. Despite this independence, polyP and carboxysomes associate non-randomly, and this association is enhanced when active carboxysome positioning by the McdAB system is disrupted. Loss of polyP synthesis leads to nucleoid expansion, an increased number of smaller carboxysomes with high mobility, and severe defects in growth under ambient CO2. Perturbation of polyP turnover further reveals structural connections to both carboxysomes and thylakoid membranes. Together, these findings identify polyP as an architectural integrator that couples chromosome organization, metabolic compartmentalization, and photosynthetic fitness.IMPORTANCEPolyphosphate (polyP) is a ubiquitous storage polymer found across all three domains of life. In bacteria, polyP is involved in virulence, energy metabolism, DNA structure, and stress. Here, we investigate the physical and functional relationships between polyP and the carboxysome-a carbon-fixing organelle that encapsulates the most abundant enzyme on Earth and is responsible for a significant amount of atmospheric CO2 sequestration. We discover that polyP is important for proper carboxysome assembly and organization in the cell and is also required for robust cell growth under ambient CO2 conditions. As carboxysomes are one class of the broader group of bacterial microcompartments, these findings have broader implications for polyP in the function and organization of the bacterial cytoplasm.
Polyphosphates↗
Stepwise DNA-unwinding gates TnpB genome-editing activity.
TnpB is a compact RNA-guided endonuclease and an evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited, and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived, partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, H4W-L304F-V305R (Ymu1-WFR), stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.
Gene Editing↗
Polyphosphate acts as an architectural regulator of carbon fixation and nucleoid structure in cyanobacteria.
Polyphosphate (polyP) is a conserved inorganic polymer traditionally viewed as a stress-induced phosphate and energy reserve. In cyanobacteria, however, polyP granules are constitutively present and frequently observed in proximity to carboxysomes, the bacterial microcompartments that mediate CO2 fixation. Here we show that polyP functions as a spatially organized regulator of the photosynthetic cytoplasm in Synechococcus elongatus. PolyP granules localize to the nucleoid and are periodically arranged along the cell axis, independently of the McdAB carboxysome positioning system. Despite this independence, polyP and carboxysomes associate non-randomly, and this association is enhanced when active carboxysome positioning by the McdAB system is disrupted. Loss of polyP synthesis leads to nucleoid expansion, an increased number of smaller carboxysomes with high mobility, and severe defects in growth under ambient CO2. Perturbation of polyP turnover further reveals structural connections to both carboxysomes and thylakoid membranes. Together, these findings identify polyP as an architectural integrator that couples chromosome organization, metabolic compartmentalization, and photosynthetic fitness.
Journal Article↗
Mutational scanning of TnpB reveals latent activity for genome editing.
TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.
Journal Article↗