Activation of the Cryptic PhnE Permease Promotes Rapid Adaptive Evolution in a Population of Escherichia coli K-12 Starved for Phosphate

Escherichia coli K-12 suffers acetic acid stress during prolonged incubation in glucose minimal medium containing a limiting concentration of inorganic phosphate (0.1 mM P-i), which decreases the number of viable cells from 6 x 10(8) to <= 10 CFU/ml between days 6 and 14 of incubation. Here we show that following two serial transfers into P-i-limiting medium, evolved mutants survived prolonged incubation (approximate to 10(7) CFU/ml on day 14 of incubation). The evolved strains that overtook the populations were generally PhnE(+), whereas the ancestral K-12 strain carries an inactive phnE allele, which prevents the transport of phosphonates. The switching in phnE occurred with a high frequency as a result of the deletion of an 8-bp repeated sequence. In a mixed culture starved for P-i that contained the K-12 ancestral strain in majority, evolved strains grew through PhnE-dependent scavenging of probably organic phosphate esters (not phosphonates or P-i) released by E. coli K-12 between days 1 and 3, before acetic acid excreted by E. coli K-12 reached toxic levels. The growth yield of phnE(+) strains in mixed culture was dramatically enhanced by mutations that affect glucose metabolism, such as an rpoS mutation inactivating the alternative sigma factor RpoS. The long-term viability of evolved populations was generally higher when the ancestral strain carried an inactive rather than an active phnE allele, which indicates that cross-feeding of phosphorylated products as a result of the phnE polymorphism may be essential for the spread of mutants which eventually help populations to survive under P-i starvation conditions.