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YoeB toxin is activated during thermal stress.

Janssen BD, Garza-Sánchez F, Hayes CS - Microbiologyopen (2015)

Bottom Line: Moreover, heat-activated YoeB does not induce growth arrest nor does it suppress global protein synthesis.In fact, E. coli cells proliferate more rapidly at elevated temperatures and instantaneously accelerate their growth rate in response to acute heat shock.We propose that heat-activated YoeB may serve a quality control function, facilitating the recycling of stalled translation complexes through ribosome rescue pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California.

No MeSH data available.


Related in: MedlinePlus

Overexpression of lon and rpoH induces A-site mRNA cleavage. (A) Overexpression of lon induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in ssrA−Δrnb backgrounds at 37°C. Where indicated, the lon or lon(S679A) genes were overexpressed from a plasmid-borne arabinose-inducible promoter. (B) Lon immunoblot analysis. Urea-soluble protein was isolated from cells of the indicated genotype that had been cultures at 30°C, 37°C or 42°C for 2.5 h. The bottom panel shows Lon levels in ssrA−Δrnb cells that had been cultured at 30°C for 1.5 h, then shifted to 42°C for the indicated number of minutes. (C) Overexpression of rpoH induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in the indicated genetic backgrounds at 37°C. Where indicated (+), rpoH was overexpressed from a plasmid-borne arabinose-inducible promoter. In (A and C), the migration position of flag-(m)ybeL-PP transcript that is truncated at the stop codon is indicated, and horizontal arrows indicate an additional yoeB-dependent transcript. (D) Immunoblot analysis of Lon. Urea-soluble protein was isolated from cells of the indicated genotype that had been grown at 37°C. Where indicated (+), the σ32 heat-shock transcription factor (rpoH) was overexpressed. Samples were analyzed by immunoblot using polyclonal antisera to Lon protease.
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fig04: Overexpression of lon and rpoH induces A-site mRNA cleavage. (A) Overexpression of lon induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in ssrA−Δrnb backgrounds at 37°C. Where indicated, the lon or lon(S679A) genes were overexpressed from a plasmid-borne arabinose-inducible promoter. (B) Lon immunoblot analysis. Urea-soluble protein was isolated from cells of the indicated genotype that had been cultures at 30°C, 37°C or 42°C for 2.5 h. The bottom panel shows Lon levels in ssrA−Δrnb cells that had been cultured at 30°C for 1.5 h, then shifted to 42°C for the indicated number of minutes. (C) Overexpression of rpoH induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in the indicated genetic backgrounds at 37°C. Where indicated (+), rpoH was overexpressed from a plasmid-borne arabinose-inducible promoter. In (A and C), the migration position of flag-(m)ybeL-PP transcript that is truncated at the stop codon is indicated, and horizontal arrows indicate an additional yoeB-dependent transcript. (D) Immunoblot analysis of Lon. Urea-soluble protein was isolated from cells of the indicated genotype that had been grown at 37°C. Where indicated (+), the σ32 heat-shock transcription factor (rpoH) was overexpressed. Samples were analyzed by immunoblot using polyclonal antisera to Lon protease.

Mentions: Christensen et al. 2004 have shown that overproduced Lon inhibits cell growth largely by activating YoeB. These findings indicate that YefM is particularly susceptible to proteolysis and suggest that thermal activation may be due to increased Lon levels. To address this possibility, we first confirmed that truncated flag-(m)ybeL-PP transcripts accumulate in cells that overexpress lon at 37°C (Fig.4A, lane 2). This mRNA processing was not observed when lon was induced in ΔyefM-yoeB cells (Fig.4A, lane 4). We also determined that protease activity is required for this effect, because truncated messages were not detected when catalytically inactive Lon(Ser679Ala) was overproduced (Fig.4A, lane 3) (Botos et al. 2004). Having established that increased Lon is sufficient to activate YoeB in our system, we then examined endogenous Lon levels by immunoblot. Given that Lon is considered to be a heat-shock protein (Phillips et al. 1984), we were surprised to find that Lon antigen levels were remarkably constant in cells cultured for 2.5 h at 30°C, 37°C, and 42°C (Fig.4B). Lon levels were also unchanged regardless of ssrA or rnb genetic background (Fig.4B). We further examined whether Lon levels increase transiently in response to temperature up-shift, but observed no significant increase over the first few minutes of heat stress (Fig.4B, lower blot). These data show that Lon overproduction can activate YoeB, but this mechanism does not account for activation at elevated temperature.


YoeB toxin is activated during thermal stress.

Janssen BD, Garza-Sánchez F, Hayes CS - Microbiologyopen (2015)

Overexpression of lon and rpoH induces A-site mRNA cleavage. (A) Overexpression of lon induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in ssrA−Δrnb backgrounds at 37°C. Where indicated, the lon or lon(S679A) genes were overexpressed from a plasmid-borne arabinose-inducible promoter. (B) Lon immunoblot analysis. Urea-soluble protein was isolated from cells of the indicated genotype that had been cultures at 30°C, 37°C or 42°C for 2.5 h. The bottom panel shows Lon levels in ssrA−Δrnb cells that had been cultured at 30°C for 1.5 h, then shifted to 42°C for the indicated number of minutes. (C) Overexpression of rpoH induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in the indicated genetic backgrounds at 37°C. Where indicated (+), rpoH was overexpressed from a plasmid-borne arabinose-inducible promoter. In (A and C), the migration position of flag-(m)ybeL-PP transcript that is truncated at the stop codon is indicated, and horizontal arrows indicate an additional yoeB-dependent transcript. (D) Immunoblot analysis of Lon. Urea-soluble protein was isolated from cells of the indicated genotype that had been grown at 37°C. Where indicated (+), the σ32 heat-shock transcription factor (rpoH) was overexpressed. Samples were analyzed by immunoblot using polyclonal antisera to Lon protease.
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Related In: Results  -  Collection

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fig04: Overexpression of lon and rpoH induces A-site mRNA cleavage. (A) Overexpression of lon induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in ssrA−Δrnb backgrounds at 37°C. Where indicated, the lon or lon(S679A) genes were overexpressed from a plasmid-borne arabinose-inducible promoter. (B) Lon immunoblot analysis. Urea-soluble protein was isolated from cells of the indicated genotype that had been cultures at 30°C, 37°C or 42°C for 2.5 h. The bottom panel shows Lon levels in ssrA−Δrnb cells that had been cultured at 30°C for 1.5 h, then shifted to 42°C for the indicated number of minutes. (C) Overexpression of rpoH induces A-site mRNA cleavage. flag-(m)ybeL-PP transcripts were expressed in the indicated genetic backgrounds at 37°C. Where indicated (+), rpoH was overexpressed from a plasmid-borne arabinose-inducible promoter. In (A and C), the migration position of flag-(m)ybeL-PP transcript that is truncated at the stop codon is indicated, and horizontal arrows indicate an additional yoeB-dependent transcript. (D) Immunoblot analysis of Lon. Urea-soluble protein was isolated from cells of the indicated genotype that had been grown at 37°C. Where indicated (+), the σ32 heat-shock transcription factor (rpoH) was overexpressed. Samples were analyzed by immunoblot using polyclonal antisera to Lon protease.
Mentions: Christensen et al. 2004 have shown that overproduced Lon inhibits cell growth largely by activating YoeB. These findings indicate that YefM is particularly susceptible to proteolysis and suggest that thermal activation may be due to increased Lon levels. To address this possibility, we first confirmed that truncated flag-(m)ybeL-PP transcripts accumulate in cells that overexpress lon at 37°C (Fig.4A, lane 2). This mRNA processing was not observed when lon was induced in ΔyefM-yoeB cells (Fig.4A, lane 4). We also determined that protease activity is required for this effect, because truncated messages were not detected when catalytically inactive Lon(Ser679Ala) was overproduced (Fig.4A, lane 3) (Botos et al. 2004). Having established that increased Lon is sufficient to activate YoeB in our system, we then examined endogenous Lon levels by immunoblot. Given that Lon is considered to be a heat-shock protein (Phillips et al. 1984), we were surprised to find that Lon antigen levels were remarkably constant in cells cultured for 2.5 h at 30°C, 37°C, and 42°C (Fig.4B). Lon levels were also unchanged regardless of ssrA or rnb genetic background (Fig.4B). We further examined whether Lon levels increase transiently in response to temperature up-shift, but observed no significant increase over the first few minutes of heat stress (Fig.4B, lower blot). These data show that Lon overproduction can activate YoeB, but this mechanism does not account for activation at elevated temperature.

Bottom Line: Moreover, heat-activated YoeB does not induce growth arrest nor does it suppress global protein synthesis.In fact, E. coli cells proliferate more rapidly at elevated temperatures and instantaneously accelerate their growth rate in response to acute heat shock.We propose that heat-activated YoeB may serve a quality control function, facilitating the recycling of stalled translation complexes through ribosome rescue pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California.

No MeSH data available.


Related in: MedlinePlus