O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, a likely cause for how it lost its O antigen

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies reported the presence of independent mutations that abolish OAg biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for loss of OAg during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS mostly contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-carried OAg intermediates in the bacterial periplasm interfere with peptidoglycan synthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for. Hence our work provides a likely explanation for the long-held mystery of how E. coli K-12 lost its OAg production and opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria. Significance statement Escherichia coli K-12 is the most studied microorganism, widely used in laboratories for studying bacteriology and as a tool for molecular biology. The reason why it is devoid of O antigen remains a long-standing question. Our work has uncovered a previously unknown selection pressure of bile salts on bacterial O antigen biogenesis, which provides a plausible scenario for how the early propagation of E. coli K-12 strains in bile salt containing media could have led to loss of O antigen in K-12. Our results also suggest that the accumulation of O antigen intermediates in the bacterial periplasm may interfere with bacterial cell wall synthesis, which paves a new research direction into the interplay of different cell envelope component synthesis pathways. ### Competing Interest Statement The authors have declared no competing interest.