Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 12:8:839.
doi: 10.3389/fmicb.2017.00839. eCollection 2017.

RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin

Darija Viducic  1   2 , Keiji Murakami  1 , Takashi Amoh  1 , Tsuneko Ono  2 , Yoichiro Miyake  1
Affiliations

RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin

Darija Viducic et al. Front Microbiol. .

Abstract

Pseudomonas aeruginosa has developed diverse strategies to respond and adapt to antibiotic stress. Among the factors that modulate survival in the presence of antibiotics, alternative sigma factors play an important role. Here, we demonstrate that the alternative sigma factor RpoN (σ54) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase ΔrpoN mutant cells is suppressed by the loss of the alternative sigma factor RpoS. Transcriptional analysis indicated that RpoN positively regulates the expression of RsmA, an RNA-binding protein, in the P. aeruginosa stationary growth phase in a nutrient-rich medium. The loss of RpoS led to the upregulation of gacA expression in the nutrient-limited medium-grown stationary phase cells. Conversely, in the logarithmic growth phase, the ΔrpoS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase ΔrpoN mutant cells grown in nutrient-rich medium resulted in decreased expression of gacA, relA, and rpoS without altering the expression of rsmA relative to wild-type PAO1. The observed downregulation of gacA and relA in the ΔrpoN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the ΔrpoN mutant background. The tobramycin-tolerant phenotype of the ΔrpoNΔrpoS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase ΔrpoS and ΔrpoNΔrpoS mutant cells demonstrated increased expression of gacA in response to tobramycin. Together, these results suggest that a complex regulatory interaction between RpoN, RpoS, the Gac/Rsm pathway, and RelA modulates the P. aeruginosa response to tobramycin.

Keywords: Pseudomonas aeruginosa; RpoN; RpoS; antibiotic tolerance; tobramycin.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Time-dependent killing assay of stationary-phase (A) and logarithmic phase (B) wild-type PAO1 and the ΔrpoN, ΔrpoS, ΔrpoNΔrpoS, and ΔrpoN/rpoN+ mutants treated with 32 μg/ml tobramycin grown in LB. Wild-type PAO1 was grown without tobramycin and served as a growth control. Percent survival at the indicated time points was calculated by dividing the number of CFU/ml after antibiotic treatment by the CFU/ml before addition of tobramycin. The experiment was performed in triplicate. Error bars indicate SDs. P ≤ 0.05 (*), ≤0.01 (**), or ≤0.001(***) vs. wild type.
Figure 2
Figure 2
Time-dependent killing assay of stationary-phase (A) and logarithmic phase (B) wild-type PAO1 and the ΔrpoN, ΔrpoS, ΔrpoNΔrpoS, and ΔrpoN/rpoN+ mutants treated with 32 μg/ml tobramycin grown in a defined minimal (AB) medium supplemented with 0.2% glucose and 0.2% CAA. Wild-type PAO1 was grown without tobramycin and served as a growth control. Percentage survival at the indicated time points was calculated by dividing the number of CFU/ml after antibiotic treatment by the CFU/ml before addition of tobramycin. The experiment was performed in triplicate. Error bars indicate SDs. P ≤ 0.05 (*), ≤0.01 (**) vs. wild type.
Figure 3
Figure 3
Growth rate analysis for wild-type PAO1, ΔrpoN mutant, ΔrpoS mutant, ΔrpoNΔrpoS mutant, and ΔrpoN/rpoN+ mutant grown in the LB medium (A) and AB minimal medium supplemented with 0.2% glucose and 0.2% CAA (B) at 37°C. Growth curves showing the absorbance at 595 nm plotted over time. Error bars indicate SDs. P ≤ 0.05 (*), ≤0.01 (**), or ≤0.001(***) vs. wild type.
Figure 4
Figure 4
Expression of gacA, rsmA, relA, and rpoS genes in stationary phase wild-type PAO1 and the ΔrpoN, ΔrpoS, ΔrpoNΔrpoS, and ΔrpoN/rpoN+ mutants grown in the LB medium (A,C,E,G) and in AB minimal medium (B,D,F,H). The gacA, rsmA, relA, and rpoS transcript levels were measured by qRT-PCR, were normalized to omlA expression, and the levels are expressed relative to the wild-type PAO1 at time = 0 h. The time points at which the cells were sampled for transcriptional analysis were t = 0 h and t = 24 h after the addition of 32 μg/ml tobramycin, as indicated. All results are the average of at least three independent experiments, and the error bars represent SDs. P ≤ 0.05 (*), ≤0.01 (**).
Figure 5
Figure 5
Expression of gacA (A), rsmA (B), rpoS (C), and relA (D) in logarithmic phase wild-type PAO1 and the ΔrpoN, ΔrpoS, ΔrpoNΔrpoS, and ΔrpoN/rpoN+ mutants grown in the LB medium in the presence of 32 μg/ml tobramycin. The transcript levels were measured by qRT-PCR, were normalized to omlA expression, and are expressed relative to wild-type PAO1 at time = 0 h. The time points at which the cells were sampled for transcriptional analysis were t = 0 h and t = 3 h after the addition of tobramycin, as indicated. All results are the average of at least three independent experiments, and the error bars represent SDs. P ≤ 0.05 (*), ≤0.01 (**).
Figure 6
Figure 6
Proposed model for the involvement of RpoN in the regulation of tobramycin tolerance in the stationary growth (A) and logarithmic phase growth (B) in nutrient-rich medium. (A) RpoN stimulates the expression of the genes involved in the translation apparatus and positively affects the synthesis of ribosome components. Upon exposure to tobramycin, RpoN counteracts the negative effects of tobramycin on translation by increasing the expression of relA, which in turn produces the effector of the stringent response, ppGpp. ppGpp promotes survival in the presence of tobramycin by inducing translational inactivity and affecting the growth rate. The translational inactivity produced by ppGpp blocks tobramycin from exerting its effect on the ribosome and consequently leads to tobramycin tolerance. In addition, RpoN positively affects the expression of rpoS in the presence of tobramycin and employs RsmA to overcome the effect of tobramycin, likely by increasing the mRNA stability of a gene potentially involved in tobramycin tolerance. RpoN increases the expression of gacA in response to tobramycin, which probably occurs through a ppGpp-dependent pathway. (B) During the logarithmic phase of growth, RpoN employs the RpoS-dependent pathway, which in turn activates additional stress-response genes promoting the survival to tobramycin. RpoN profoundly downregulates the expression of rsmA in response to tobramycin. Furthermore, RpoS negatively affects the expression of gacA in the presence of tobramycin. The mutant deficient in rpoN and rpoS demonstrates (i) the loss of tobramycin-mediated inhibition of relA expression, (ii) a consequent increase in ppGpp production, and (iii) suggests ppGpp-dependent upregulation of gacA, which together lead to tobramycin tolerance. The detailed regulation network is described in the Discussion. OM, outer membrane; PS, periplasmic space; IM, inner membrane; TOB, tobramycin; → represents positive control; ⊥ represents negative control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amato S. M., Fazen C. H., Henry T. C., Mok W. W., Orman M. A., Sandvik E. L., et al. . (2014). The role of metabolism in bacterial persistence. Front. Microbiol. 5:70. 10.3389/fmicb.2014.00070 - DOI - PMC - PubMed
    1. Baharoglu Z., Krin E., Mazel D. (2013). RpoS plays a central role in the SOS induction by sub-lethal aminoglycoside concentrations in Vibrio cholerae. PLoS Genet. 9:e1003421. 10.1371/journal.pgen.1003421 - DOI - PMC - PubMed
    1. Baker C. S., Morozov I., Suzuki K., Romeo T., Babitzke P. (2002). CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli. Mol. Microbiol. 44, 1599–1610. 10.1046/j.1365-2958.2002.02982.x - DOI - PubMed
    1. Battesti A., Majdalani N., Gottesman S. (2011). The RpoS-mediated general stress response in Escherichia coli. Annu. Rev. Microbiol. 65, 189–213. 10.1146/annurev-micro-090110-102946 - DOI - PMC - PubMed
    1. Bjarnsholt T., Jensen P. Ø., Burmølle M., Hentzer M., Haagensen J. A., Hougen H. P., et al. . (2005). Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 151, 373–383. 10.1099/mic.0.27463-0 - DOI - PubMed

LinkOut - more resources