A P. aeruginosa relA spoT double mutant is avirulent in the fly feeding model of infection. (A, C, and E) Mortality of flies infected with P. aeruginosa PAO1 (▴), relA mutant (▪), and relA spoT (•) mutant, as well as the negative control of sterile sucrose (▾), was monitored for 14 days following infection. In panel A, the data points making up the survival curve and standard errors of the means (SEM) (error bars) were generated from three replicate experiments with 30 to 60 flies per strain. In panel C, flies were transferred to fresh bacterial filters on days 6 and 12 (indicated by the vertical broken lines); the data making up the survival curve and SEM were generated from one experiment with 40 flies per strain. In panel E, the vector control strain, the relA spoT mutant carrying pUCP28T (relA spoT pUCP28T) (▵), and the complemented strain, the relA spoT mutant carrying pSS6 (relA spoT pSS6) (□) were also examined; the data points making up the survival curve and SEM were from one experiment with 60 flies per strain. (B and D) Enumeration of viable bacteria remaining on fly feeding filters containing strain PAO1 (▴), relA mutant (▪), and relA spoT mutant (•) throughout the experiment. CFUs were determined in triplicate for each filter. Graphed data represent the average CFU/filter, with error bars (mainly too small to be visible) denoting SEM. In panel B, one filter per strain was removed every 2 days for the duration of the experiment. Note that the colony morphology of the relA spoT mutant changed throughout the experiment from the original large colonies (▾) to a small-colony variant; thus, both total counts and large-colony counts are shown for this strain. In panel D, flies were transferred to fresh bacterial filters on days 6 and 12. CFU counting was performed on the inoculum on days 0, 6, and 12, and also on the removed filters on days 6, 12, and 14 (end of experiment). In this experiment, no small colonies were observed for the relA spoT mutant.

A P. aeruginosa relA spoT double mutant is avirulent in the rat lung infection model. Quantitative bacteriology of rat lung agar bead infections with P. aeruginosa PAO1 and the relA and relA spoT mutants on day 7 of the infection. Data represent the mean of total bacterial counts ± SEM from the lungs of four animals for each strain. No significant differences were observed between the means of strain PAO1 and the relA mutant. There was a significant difference between the mean values for the colony counts of strain PAO1 and the relA spoT mutant (P < 0.0001).

Virulence factor synthesis is diminished in a P. aeruginosa relA spoT double mutant. All virulence factor assay data represent the means of three or four replicate experiments, performed in triplicate; the error bars indicate the standard deviations. The relA spoT mutant carrying pUCP28T and the relA spoT mutant carrying pSS6 are shown as pUCP28T and pSS6, respectively, in the figure. (A) Pyocyanin production was determined by extraction of culture supernatants with chloroform and hydrochloric acid; the absorbance at 520 nm of the resulting aqueous phase corresponds to the concentration of pyocyanin. (B) Elastase production was quantified as the size of zones of clearing produced by bacterial colonies on reverse-elastin agar. (C) Protease production was measured as the size of zones of clearing produced by bacterial colonies on dialyzed BHI-skim milk agar. (D) Siderophore production was assessed as the size of the orange halos produced by bacterial colonies on low-iron CAS blue agar.

A P. aeruginosa relA spoT double mutant is outcompeted by the wild-type strain PAO1 during prolonged coculture in diluted rich medium. Pure cultures of strain PAO1 and the relA spoT double mutant were grown overnight in BHI broth and then inoculated into diluted (0.1 strength) BHI either individually or mixed in a ratio of 10:1 for the relA spoT mutant to strain PAO1. These cultures were incubated at 37°C with aeration for 5 to 6 days without supplementation with fresh medium. (A) Results of viable cell counting for pure and mixed cultures, which was performed daily in triplicate for each culture. Error bars denote standard errors of the means. (B) Results of plating onto selective media to determine the proportion of cells in the mixed culture comprised by each strain. The mixed culture was plated daily on LB agar for single colonies, and 100 randomly selected colonies were subsequently restreaked onto media containing antibiotics selective for the relA spoT mutant. Data represent the means of three independent experiments; error bars denote standard deviations.

The virulence of the P. aeruginosa relA spoT double mutant is similar to that of the relA mutant, but both are less virulent than P. aeruginosa PAO1 in the Drosophila nicking infection model. Survival curves for D. melanogaster challenged with wild-type PAO1 or the stringent response mutants are shown. Flies were nicked in the abdomen with a needle dipped into a standardized culture of P. aeruginosa. Fly mortality was measured hourly on 3 sets of 30 flies. Significant differences were observed between the PAO1 survival curve and the survival curve of either the relA mutant (P = 0.0034) or the relA spoT mutant (P < 0.0001). There is not a significant difference between the survival curves for the relA and relA spoT mutants (P = 0.113).

Swarming and twitching motilities are reduced in the relA spoT mutant compared to the relA mutant and the parental PAO1 strain. The graphs show the means of motility from three replicate experiments performed in triplicate for strain PAO1, relA mutant, relA spoT mutant, the vector control strain, the relA spoT mutant carrying pUCP28T (“pUCP28T” in the figure), and the complemented strain, the relA spoT mutant carrying pSS6 (“pSS6” in the figure), overexpressing a plasmid-borne copy of spoT. Error bars denote the SEM. (A and B) The mean diameters of spread for swarming motility (A) and twitching motility (B) are shown.

Stringent response mutants of P. aeruginosa are more susceptible to abiotic stresses. The abilities of strain PAO1 (▴), relA mutant (▪), and relA spoT double mutant (•) to survive a variety of stresses were examined. All data are from three or four replicate experiments; error bars denote standard deviations. (A) Diluted cultures were heat shocked at 50°C for 60 min, with duplicate samples taken every 10 min to assess the number of viable cells remaining. Cell survival is expressed as a percentage of the viable cell count at time zero. (B) The sensitivity of strains to oxidative stress was assessed by measuring the size of the zones of clearing in the bacterial lawn surrounding discs soaked in 30% hydrogen peroxide. In this assay, the vector control strain, the relA spoT mutant carrying pUCP28T (“pUCP28T” in the figure), and the complemented strain, the relA spoT mutant carrying pSS6 (“pSS6” in the figure), were also tested. (C and D) Sensitivity to osmotic stress was examined by resuspending cultures in minimal medium containing a high concentration of solute (4 M sodium chloride in panel C and 2 M sucrose in panel D). Viable cell counting was performed after 2, 4, 6, and 24 h, with cell survival represented as the percentage of the viable cell count at time zero.