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What Is the Link between Stringent Response, Endoribonuclease Encoding Type II Toxin – Antitoxin Systems and Persistence?

View Article: PubMed Central - PubMed

ABSTRACT

Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is toxin–antitoxin systems (TASs) which are also implicated in a wide range of stress-related phenomena. Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140–1150 reported an interesting link between ppGpp mediated stringent response, TAS, and persistence. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for transcriptional activation of yefM/yoeB TAS, which is an indirect indication of Lon-dependent degradation of YefM antitoxin. The Δ10 strain, an Escherichia coli MG1655 derivative in which the 10 TAS are deleted, is more sensitive to ciprofloxacin compared to wild type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the persistence related implications based on Δ10 strain are void. We conclude that the transcriptional regulation and endoribonuclease activity of YefM/YoeB TAS is independent of ppGpp and inorganic polyphosphate. Therefore, we urge for thorough inspection and debate on the link between chromosomal endoribonuclease TAS and persistence.

No MeSH data available.


Related in: MedlinePlus

(A) Growth curve of MG1655 and Δ10 strains. 2 μL of the diluted cultures were inoculated into 200 μL of LB in microtitre plate wells in triplicates. The microtitre plates were incubated at 37°C with 170 rpm shaking. Optical density at 595 nm was measured in a microtitre plate reader (BioradTM). The closed triangles and closed squares represent the OD of MG1655 and the Δ10 strains respectively. The open triangles and open squares represent the growth rates (change in OD per hour) of MG1655 and the Δ10 strain respectively. (B) Percent CFU in optimal conditions in 12 h. Overnight cultures were incoculated into tubes containing 3 ml LB broth and grown at 37°C with 170 rpm shaking for 12 h. 10 μL of the culture was diluted appropriately and plated on LB plates and incubated overnight. (C) Biofilm assay for prolonged duration. 2 μL of inoculum was added into 200 μL of LB broth in 96-well microtitre plate. The plates were incubated at 37°C for 16, 24, 48, and 72 h. The plates were washed and stained with 1% crystal violet. Then washed thrice and the bound crystal violet was redissolved using ethanol and OD was measured at 595 nm. Experiments were carried out independently thrice in quadruplicates. Error bars indicate standard error. (Two tailed ∗∗P < 0.001, ∗P < 0.01).
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Figure 4: (A) Growth curve of MG1655 and Δ10 strains. 2 μL of the diluted cultures were inoculated into 200 μL of LB in microtitre plate wells in triplicates. The microtitre plates were incubated at 37°C with 170 rpm shaking. Optical density at 595 nm was measured in a microtitre plate reader (BioradTM). The closed triangles and closed squares represent the OD of MG1655 and the Δ10 strains respectively. The open triangles and open squares represent the growth rates (change in OD per hour) of MG1655 and the Δ10 strain respectively. (B) Percent CFU in optimal conditions in 12 h. Overnight cultures were incoculated into tubes containing 3 ml LB broth and grown at 37°C with 170 rpm shaking for 12 h. 10 μL of the culture was diluted appropriately and plated on LB plates and incubated overnight. (C) Biofilm assay for prolonged duration. 2 μL of inoculum was added into 200 μL of LB broth in 96-well microtitre plate. The plates were incubated at 37°C for 16, 24, 48, and 72 h. The plates were washed and stained with 1% crystal violet. Then washed thrice and the bound crystal violet was redissolved using ethanol and OD was measured at 595 nm. Experiments were carried out independently thrice in quadruplicates. Error bars indicate standard error. (Two tailed ∗∗P < 0.001, ∗P < 0.01).

Mentions: Recently we have shown that TAS are horizontally transferring genes and are integrated within the intergenic regions between important ‘core’ genomic regions (Ramisetty and Santhosh, 2016). Maisonneuve et al. (2011) reported that Δ10 strain formed lesser persisters compared to wild type strain when challenged with ciprofloxacin and Ampicillin. Hence, we speculated that deletion of 10 TAS could compromise the expression of flanking genes due to polar effects resulting in decreased fitness of the Δ10 strain. We analyzed the differences in between E. coli MG1655 and Δ10 strains (Maisonneuve et al., 2011) fitness by growth curve, maximal CFU per ml in stationary phase and biofilm formation. We observed that the maximum growth rate (change in OD/hour) of MG1655 was 0.35 while that of Δ10 strain was 0.27 (Figure 4A). During the 8 h growth curve study in 96 well microtitre plates, the maximum absorbance at 595 nm was 1.05 for MG1655 while it was 0.95 for Δ10 strain (Figure 4A). We also noticed that the optical density of the overnight cultures of Δ10 strain grown in tubes was consistently lower than that of MG1655. These observations indicated that the Δ10 strain may have metabolic deficiencies. To confirm this further we determined the CFU/ml of both the strains after 12 h of growth in tube containing LB medium at 37°C with 170 rpm. We observed that MG1655 yielded 7.99 × 1012 CFU/ml while the Δ10 strain yielded 4.92 × 1012 CFU/ml which is ≈40% lesser than the CFU/ml of wild type (Figure 4B). In a given set of conditions, the difference in the CFU/ml of two strains of a species is an indication of difference in their respective fitness.


What Is the Link between Stringent Response, Endoribonuclease Encoding Type II Toxin – Antitoxin Systems and Persistence?
(A) Growth curve of MG1655 and Δ10 strains. 2 μL of the diluted cultures were inoculated into 200 μL of LB in microtitre plate wells in triplicates. The microtitre plates were incubated at 37°C with 170 rpm shaking. Optical density at 595 nm was measured in a microtitre plate reader (BioradTM). The closed triangles and closed squares represent the OD of MG1655 and the Δ10 strains respectively. The open triangles and open squares represent the growth rates (change in OD per hour) of MG1655 and the Δ10 strain respectively. (B) Percent CFU in optimal conditions in 12 h. Overnight cultures were incoculated into tubes containing 3 ml LB broth and grown at 37°C with 170 rpm shaking for 12 h. 10 μL of the culture was diluted appropriately and plated on LB plates and incubated overnight. (C) Biofilm assay for prolonged duration. 2 μL of inoculum was added into 200 μL of LB broth in 96-well microtitre plate. The plates were incubated at 37°C for 16, 24, 48, and 72 h. The plates were washed and stained with 1% crystal violet. Then washed thrice and the bound crystal violet was redissolved using ethanol and OD was measured at 595 nm. Experiments were carried out independently thrice in quadruplicates. Error bars indicate standard error. (Two tailed ∗∗P < 0.001, ∗P < 0.01).
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Figure 4: (A) Growth curve of MG1655 and Δ10 strains. 2 μL of the diluted cultures were inoculated into 200 μL of LB in microtitre plate wells in triplicates. The microtitre plates were incubated at 37°C with 170 rpm shaking. Optical density at 595 nm was measured in a microtitre plate reader (BioradTM). The closed triangles and closed squares represent the OD of MG1655 and the Δ10 strains respectively. The open triangles and open squares represent the growth rates (change in OD per hour) of MG1655 and the Δ10 strain respectively. (B) Percent CFU in optimal conditions in 12 h. Overnight cultures were incoculated into tubes containing 3 ml LB broth and grown at 37°C with 170 rpm shaking for 12 h. 10 μL of the culture was diluted appropriately and plated on LB plates and incubated overnight. (C) Biofilm assay for prolonged duration. 2 μL of inoculum was added into 200 μL of LB broth in 96-well microtitre plate. The plates were incubated at 37°C for 16, 24, 48, and 72 h. The plates were washed and stained with 1% crystal violet. Then washed thrice and the bound crystal violet was redissolved using ethanol and OD was measured at 595 nm. Experiments were carried out independently thrice in quadruplicates. Error bars indicate standard error. (Two tailed ∗∗P < 0.001, ∗P < 0.01).
Mentions: Recently we have shown that TAS are horizontally transferring genes and are integrated within the intergenic regions between important ‘core’ genomic regions (Ramisetty and Santhosh, 2016). Maisonneuve et al. (2011) reported that Δ10 strain formed lesser persisters compared to wild type strain when challenged with ciprofloxacin and Ampicillin. Hence, we speculated that deletion of 10 TAS could compromise the expression of flanking genes due to polar effects resulting in decreased fitness of the Δ10 strain. We analyzed the differences in between E. coli MG1655 and Δ10 strains (Maisonneuve et al., 2011) fitness by growth curve, maximal CFU per ml in stationary phase and biofilm formation. We observed that the maximum growth rate (change in OD/hour) of MG1655 was 0.35 while that of Δ10 strain was 0.27 (Figure 4A). During the 8 h growth curve study in 96 well microtitre plates, the maximum absorbance at 595 nm was 1.05 for MG1655 while it was 0.95 for Δ10 strain (Figure 4A). We also noticed that the optical density of the overnight cultures of Δ10 strain grown in tubes was consistently lower than that of MG1655. These observations indicated that the Δ10 strain may have metabolic deficiencies. To confirm this further we determined the CFU/ml of both the strains after 12 h of growth in tube containing LB medium at 37°C with 170 rpm. We observed that MG1655 yielded 7.99 × 1012 CFU/ml while the Δ10 strain yielded 4.92 × 1012 CFU/ml which is ≈40% lesser than the CFU/ml of wild type (Figure 4B). In a given set of conditions, the difference in the CFU/ml of two strains of a species is an indication of difference in their respective fitness.

View Article: PubMed Central - PubMed

ABSTRACT

Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is toxin&ndash;antitoxin systems (TASs) which are also implicated in a wide range of stress-related phenomena. Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140&ndash;1150 reported an interesting link between ppGpp mediated stringent response, TAS, and persistence. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for transcriptional activation of yefM/yoeB TAS, which is an indirect indication of Lon-dependent degradation of YefM antitoxin. The &Delta;10 strain, an Escherichia coli MG1655 derivative in which the 10 TAS are deleted, is more sensitive to ciprofloxacin compared to wild type MG1655. Furthermore, we show that the &Delta;10 strain has relatively lower fitness compared to the wild type and hence, we argue that the persistence related implications based on &Delta;10 strain are void. We conclude that the transcriptional regulation and endoribonuclease activity of YefM/YoeB TAS is independent of ppGpp and inorganic polyphosphate. Therefore, we urge for thorough inspection and debate on the link between chromosomal endoribonuclease TAS and persistence.

No MeSH data available.


Related in: MedlinePlus