Stepwise accumulation of SecA mutations enables high-stringency azide-based selection in Escherichia coli.

The global spread of multidrug-resistant bacteria has reduced the effectiveness of many conventional antibiotics and complicated genetic analyses that rely on antibiotic resistance genes as selection markers. Genetic selection systems that do not rely on antibiotic-resistance genes are therefore increasingly important, particularly for multidrug-resistant strains. Sodium azide (Az) inhibits the essential ATPase SecA, but previously reported secA mutations confer only limited resistance and have not been developed into practical plasmid-based markers; plasmid-borne secA (F598L) confers only marginal resistance in an Az-sensitive background. Here, we show that this limited resistance results from coexistence of plasmid-derived mutant SecA with chromosomal wild-type SecA, which attenuates the resistant phenotype. The combination of F598L with a newly identified G516C mutation markedly enhanced Az resistance. Chromosomal replacement with secA (G516C/F598L) confirmed that wild-type SecA suppresses the resistant phenotype when coexpressed. Using error-prone PCR, we further generated cumulative SecA mutants and identified a variant harboring five amino acid substitutions (G516C, F598L, L279P, S678I, and A867G) that supported substantially improved, though progressively reduced, growth in liquid culture at Az concentrations up to 200 µg ml