The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment

Abstract

Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent.

FIG. 1.

The rpoS819 allele confers a competitive advantage in 10-day-old heat-killed, conditioned medium. Wild-type (solid lines) and rpoS819 (dashed lines) cells from 1-day-old cultures were inoculated at 1:1,000 into unbuffered (A) or pH 7 buffered heat-killed conditioned medium (B). Four representative trials of each competition experiment are shown. The symbols (▴, ▵, ○, and □) indicate competition pairs. Asterisks indicate that viable counts were below the level of detection (<10³ CFU/ml).

FIG. 2.

Competitive fitness of wild-type and rpoS819 cells in LB batch culture is dependent on pH. Wild-type (solid lines) and rpoS819 (dashed lines) cells from 1-day-old cultures were inoculated at 1:1,000 into LB buffered at pH 9 (A), pH 7 (B), or pH 5 (C). Competitions where markers were switched between competing strains gave similar results. Three representative trials of each experiment are shown. The symbols (▵, ○, and □) indicate competition pairs. Asterisks indicate that viable counts were below the level of detection (<10³ CFU/ml).

FIG. 3.

pH determines the strength of the GASP phenotype. Wild-type strain ZK1143 was incubated for 10 days in LB buffered at pH 9 (A and D), pH 7 (B and E), or pH 5 (C and F). Aliquots of these cells were then inoculated as a minority (dashed lines) into 1-day-old cultures of ZK1142 (solid lines) grown in unbuffered LB (A to C) or in LB buffered at pH 9 (D), pH 7 (E), or pH 5 (F). Competitions in which markers were switched between competing strains gave similar results. Three representative trials of each experiment are shown. The symbols (▵, ○, and □) indicate competition pairs. Asterisks indicate that viable counts were below the level of detection (<10³ CFU/ml).

FIG. 4.

Competitive fitness of wild-type and rpoS819 cells incubated in minimal medium supplemented with Casamino Acids is dependent on pH. Wild-type (solid lines) and rpoS819 (dashed lines) cells from 1-day-old cultures were inoculated at 1:1,000 into minimal medium supplemented with Casamino Acids at pH 9 (A), pH 7 (B), or pH 5 (C). Competitions where markers were switched between competing strains gave similar results. Three representative trials of each experiment are shown. The symbols (▵, ○, and □) indicate competition pairs. Asterisks indicate that viable counts were below the level of detection (<10³ CFU/ml).

FIG. 5.

Competitive fitness of wild-type and rpoS819 cells in glucose minimal medium is dependent on pH. Wild-type (solid lines) and rpoS819 (dashed lines) cells from 1-day-old cultures were inoculated at 1:1,000 into minimal medium supplemented with glucose at pH 9 (A), pH 7 (B), or pH 5 (C). Competitions where markers were switched between competing strains gave similar results. Three representative trials of each experiment are shown. The symbols (▵, ○, and □) indicate competition pairs. Asterisks indicate that viable counts were below the level of detection (<10³ CFU/ml).