Absence of Ribosome Modulation Factor Alters Growth and Competitive Fitness of Escherichia coli

Abstract

During stationary phase in Escherichia coli, the expression of the ribosome modulation factor (RMF) protein participates in the dimerization of two 70S ribosomes, ultimately creating a 100S particle. 100S ribosomes are commonly thought to function to preserve ribosomes as growth ceases and cells begin to catabolize intracellular components, including proteins, during their transition into stationary phase. Here, we show that the rates of stationary-phase ribosomal degradation are increased in an rmf mutant strain that cannot produce 100S ribosomes, resulting in deficiencies in outgrowth upon reinoculation into fresh medium. Upon coinoculation in LB medium, the mutant exhibits a delay in entry into log phase, differences in growth rates, and an overall reduction in relative fitness during competition. Unexpectedly, the rmf mutant exhibited shorter generation times than wild-type cells during log phase, both in monoculture and during competition. These doubling times of ∼13 min suggest that failure to maintain ribosomal balance affects the control of cell division. Though the timing of entry into and exit from log phase is altered, 100S ribosomes are not essential for long-term viability of the rmf mutant when grown in monoculture. IMPORTANCE Ribosomes are the sole source in any cell for new protein synthesis that is vital to maintain life. While ribosomes are frequently consumed as sources of nutrients under low-nutrient conditions, some ribosomes appear to be preserved for later use. The failure to maintain the availability of these ribosomes can lead to a dire consequence upon the influx of new nutrients, as cells are unable to efficiently replenish their metabolic machinery. It is important to study the repercussions, consequences, and mechanisms of survival in cells that cannot properly maintain the availability of their ribosomes in order to better understand their mechanisms of survival during competition under nutrient-depleted conditions.

Keywords: 100S ribosome; competitive fitness; long-term survival; ribosome modulating factor.

FIG 1

Growth of the rmf mutant is impaired in the presence of wild-type cells. Monoculture (A) and coculture (B) growth of wild type (open circles, black lines) and mutant (open squares, gray lines) is shown. Lines represent average viable cell counts (CFU/mL). Error bars represent the standard deviations; n = 3.

FIG 2

Altered log phase exhibited by the rmf mutant in both monoculture and coculture. Monoculture (A) and coculture (B) growth of wild type (open circles, black lines) and mutant (open squares, gray lines) during the first 320 min of outgrowth is shown. Lines represent average viable cell counts (CFU/mL). Error bars represent the standard deviations; n = 3.

FIG 3

Comparison of calculated numbers of ribosomes per cell between growth phases in the wild-type and rmf mutant strains. Averages of the estimated numbers of ribosomes are shown, with error bars representing the standard deviations. Two-sample t test was performed to determine significant differences between the growth phases for each strain (*, P < 0.05; **, P < 0.01).

FIG 4

Reinoculation of mutant cells during monoculture growth restores the wild-type phenotype. From monocultures of wild type (black) and mutant (gray), 1:1,000 dilutions were made at the following times: 120 min (A), 150 min (B), 200 min (C), and 320 min (D). Thin black lines and gray lines represent wild-type growth and mutant growth, respectively. Bold lines with open circles (wild type) and open squares (rmf mutant) represent the average CFU/mL of the replicates; n = 3.

FIG 5

Reinoculation of coculture does not restore wild-type phenotype to the mutant. From competition cultures of wild type (black) and mutant (gray), 1:1,000 dilutions were made at the following times: 120 min (A), 150 min (B), 200 min (C), and 320 min (D). Thin black lines and gray lines represent wild-type growth and mutant growth, respectively. Bold lines with open circles (wild type) and open squares (rmf mutant) represent the average CFU/mL of the replicates; n = 3.