CRISPR/Cas9-mediated knockout of a polyester synthase-like gene delays flowering time in alfalfa.

KEY MESSAGE: T-DNA and CRISPR/Cas9-mediated knockout of polyester synthase-like genes delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Thus, we here present the first report of edited alfalfa with delayed flowering.

Exploring the Role of the NO-Detoxifying Enzyme HmpA in the Evolution of Domesticated Alfalfa Rhizobia.

We have previously shown the extensive loss of genes during the domestication of alfalfa rhizobia and the high nitrous oxide emission associated with the extreme genomic instability of commercial inoculants. In the present note, we describe the molecular mechanism involved in the evolution of alfalfa rhizobia. Genomic analysis showed that most of the gene losses in inoculants are due to large genomic deletions rather than to small deletions or point mutations, a fact consistent with recurrent DNA double-strand breaks (DSBs) at numerous locations throughout the microbial genome. Genetic analysis showed that the loss of the NO-detoxifying enzyme HmpA in inoculants results in growth inhibition and high DSB levels under nitrosative stress, and large genomic deletions in planta but not in the soil. Therefore, besides its known function in the effective establishment of the symbiosis, HmpA can play a critical role in the preservation of the genomic integrity of alfalfa rhizobia under host-derived nitrosative stress.

Elimination of GlnKAmtB affects serine biosynthesis and improves growth and stress tolerance of Escherichia coli under nutrient-rich conditions.

Nitrogen is a most important nutrient resource for Escherichia coli and other bacteria that harbor the glnKamtB operon, a high-affinity ammonium uptake system highly interconnected with cellular metabolism. Although this system confers an advantage to bacteria when growing under nitrogen-limiting conditions, little is known about the impact of these genes on microbial fitness under nutrient-rich conditions. Here, the genetically tractable E. coli BW25113 strain and its glnKamtB-null mutant (JW0441) were used to analyze the impact of GlnK-AmtB on growth rates and oxidative stress tolerance. Strain JW0441 showed a shorter initial lag phase, higher growth rate, higher citrate synthase activity, higher oxidative stress tolerance and lower expression of serA than strain BW25113 under nutrient-rich conditions, suggesting a fitness cost to increase metabolic plasticity associated with serine metabolism. The overexpression of serA in strain JW0441 resulted in a decreased growth rate and stress tolerance in nutrient-rich conditions similar to that of strain BW25113, suggesting that the negative influence on bacterial fitness imposed by GlnK-AmtB can be traced to the control of serine biosynthesis. Finally, we discuss the potential applications of glnKamtB mutants in bioproduction processes.