E. coli-{varphi}X174 genotype to phenotype map reveals flexibility and diversity in LPS structures

Phage therapy is a promising method for the treatment of multi-drug-resistant bacterial infections. However, its long-term efficacy depends on understanding the evolutionary effects of the treatment. Current knowledge of such evolutionary effects is lacking, even in well-studied systems. We used the bacterium Escherichia coli C and its bacteriophage {Phi}X174, which infects cells using host lipopolysaccharide (LPS) molecules. We first generated 31 bacterial mutants resistant to {Phi}X174 infection. Based on the genes disrupted by these mutations, we predicted that these E. coli C mutants collectively produce eight unique LPS structures. We then developed a series of evolution experiments to select for {Phi}X174 mutants capable of infecting the resistant strains. During phage adaptation, we distinguished two types of phage resistance: one that was easily overcome by {Phi}X174 with few mutational steps ("easy" resistance), and one that was more difficult to overcome ("hard" resistance). We found that increasing the diversity of the host and phage populations could accelerate the adaptation of phage {Phi}X174 to overcome the hard resistance phenotype. From these experiments, we isolated 16 {Phi}X174 mutants that, together, can infect all 31 initially resistant E. coli C mutants. Upon determining the infectivity profiles of these 16 evolved phages, we uncovered 14 distinct profiles. Given that only eight profiles are anticipated if the LPS predictions are correct, our findings highlight that the current understanding of LPS biology is insufficient to accurately forecast the evolutionary outcomes of bacterial populations infected by phage.