phnE frameshift reversion frequency: beyond density effects to genomic prevalence.

UNLABELLED: Phosphonates are alternative phosphorus sources widely present in environmental settings and in commercially relevant compounds such as pesticides and antibiotics. In Escherichia coli, uptake and assimilation of phosphonates are mediated by the 14-gene phnCDEFGHIJKLMNOP operon. In the laboratory, strain K-12 and its derivatives, phnE, encoding the phosphonate permease, carry an 8 bp insertion that causes a frameshift and premature termination. Early studies reported unusually high frequencies of phn revertants selected on phosphonates as the sole phosphorus source. Separately, high cell density has been proposed to suppress mutation emergence in multiple systems. Here, we reexamined the frequency of phn revertants in E. coli K-12. When phosphate impurities were rigorously eliminated from the medium, estimated revertant frequencies decreased by orders of magnitude, contradicting earlier reports. Revertant frequency was not significantly affected by bacterial density on selective plates. Furthermore, under phosphate-limiting conditions, the presence of a functional phnE allele did not confer a measurable fitness advantage. Finally, an in silico survey revealed that approximately 15% of E. coli strains lack functional phn genes, suggesting that loss of phosphonate utilization may be favored in specific ecological or physiological contexts.

IMPORTANCE: Phosphonates are an important phosphorus reservoir in many environments, and their utilization by Escherichia coli depends on the phn operon. This study reexamines the unusually high frequencies of phnE revertants reported for laboratory K-12 strains. By controlling for phosphate contamination in selective media, we show that reversion frequencies are substantially lower than previously estimated and largely independent of cell density. We further find that restoration of phnE function does not confer a detectable fitness advantage under phosphate limitation. Consistent with this observation, genomic analyses reveal that loss of phn function is common among natural E. coli isolates. Together, these findings suggest that loss of phn function may represent an adaptive strategy in a substantial fraction of natural populations.