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^-1. Collectively, these results demonstrate that cumulative SecA mutations can overcome Az-mediated inhibition and provide a plasmid-borne marker that does not itself encode antibiotic resistance. In the present prototype, stable recovery of transformants still required co-selection with tetracycline, and selection by Az alone remains to be achieved. The system nonetheless represents a proof of concept in E. coli that, given the conserved essentiality of SecA, is conceptually extendable to other bacteria, including multidrug-resistant strains.