Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons

Reviewer #1 (Public Review):

Summary:

In this manuscript by Beardslee and Schmitz, the authors undertook a screen for potential degrons - short peptide sequences at the C-terminus that would target the toxin VapC for degradation. The authors randomly mutagenized 5 amino acids appended to the C-terminus of VapC and transformed this library into E. coli to look for surviving cells when the VapC gene was expressed. The authors found an enrichment for tags ending Ala-Ala, and found that this enrichment was dependent on the presence of the ClpXP protease, since this sequence was not similarly enriched in a mutant lacking this protease. Moreover, the authors identify the sequence FKLVA as the tag with the highest fold enrichment in the screen and confirm that GFP tagged with this sequence is degraded by ClpXP with similar kinetics to GFP tagged with the ssrA-derived tag.

Strengths:

This study has two major implications for understanding the nature of degrons in E. coli. First, peptides ending Ala-Ala, and especially degrons resembling the ssrA degron are likely the most degradation-promoting sequences in E. coli. Second, these findings suggest that ClpXP is the most central protease, at least for this particular protein with a randomized C-terminus under the particular conditions of this screen. It is also notable that the ribosome quality control protein RqcH tags truncated proteins with an alanine tag in a template-free manner when the large ribosomal subunit is obstructed. Although E. coli doesn't encode RqcH, the utility of alanine-tagging for protein degradation likely extends to other organisms.

Weaknesses:

The authors remark and show that mutations that inactivate the VapC protein are enriched potentially more than the proteolysis tags. This is a limitation of the study and the authors have done well to describe this as it will inform future screens. Perhaps using a protein with more intermediate toxicity in future screens would help to prioritize C-terminal mutations instead of toxin-inactivating mutations.

For clarity, the authors should explain why the NNK structure of the random codons was used. Why is it important that the codon end with a G or T?

Authors state on page 7 that by determining enrichment of individual tags they can rank the relative Km for proteolysis of the individual tags. This statement is not accurate since the tag could variously impact its association with any of the proteases in the cell. Since Km is specific to each particular protease, these can't be ranked in vivo when all proteases are present.

Reviewer #2 (Public Review):

Summary:

The authors studied the sequence determinants of C-terminal tags that govern protein degradation in bacteria. They introduce a new strategy to determine degron sequences: Detox (Degron Enrichment by toxin). This unbiased approach links degron efficiency to cell growth as degrons are C-terminally fused to the toxin VapC, which inhibits protein translation. Selecting for bacterial growth and thus toxin degradation enabled the identification of potent degron derived from a randomized library of pentapeptides. Remarkably, most degrons show sequence similarity to the SsrA-tag, which is fused to incomplete polypeptides at stalled ribosomes by the tmRNA-tagging system. These findings underline the extraordinary efficiency of the SsrA-tag and the ClpXP protease in removing incomplete polypeptides and demonstrate that most proteins are spared from degradation by harboring different C-termini. The introduced method will be highly useful to determine degron sequences in other positions and other bacterial species.

Strengths:

The work introduces an innovative and powerful strategy to identify degron sequences in bacteria. The study is well-controlled and results have been thoroughly analyzed. It will now become important to broaden the technology, making it also accessible for more complex degrons.

Weaknesses:

The approach is efficient in identifying strong degron sequences that are predominantly recognized by the ClpXP protease. The sequence specificity of other proteolytic systems, however, is not efficiently addressed, pointing to a potential limitation of this technology. The GS-rich linker sequence connecting the degron and the toxin might also impact proteolysis and thus outcome.

Reviewer #3 (Public Review):

Summary:

This manuscript by Beardslee and Schmitz reports discoveries made from a genetic screen to identify C-terminal degrons that cause the efficient depletion of a potent toxin, which allowed for a deep assessment of amino acid patterns that promote protein turnover.

Strengths:

The key findings are that SsrA-like C termini are a dominant class of efficient degrons and that ClpP (X/A) mediates the turnover in most cases. Moreover, the data provides insight into the importance of residues situated farther into the degron and reveals aspects of the ClpX engagement and processivity process. The manuscript is clearly written and there is ample supporting data for the conclusions drawn. The figures are also informative.

Weaknesses:

There are only a few minor suggestions on data interpretation.

(1) Page 6: It is stated that "We plated cells on media containing 0 - 1% arabinose inducer, and observed that stronger induction of untagged VapC indeed correlates with smaller colony size; ... We conclude that VapC levels have a titratable effect on growth rate."

In E. coli with intact arabinose import/response systems, sub-saturating levels of arabinose do not generally lower the induction level of the PBAD promoter in each cell; rather, a sub-population of cells becomes induced [PMID: 9223333]. The bulk observation is a reduced expression level, and, in this case, slowed growth, but it seems more likely that the slow growth observed is from the induced cohort dying off as the cultures and colonies develop.

(2) Page 8: "At 6-hours post-induction,..."

Because these experiments were enrichments from initial pools of clones, the number of cell divisions is more informative than the hours of outgrowth or culture densities at harvest. It would be helpful if the authors could indicate, or at least estimate, the number of cell divisions. this could then be included in the results or methods section.

(3) Page 12: "It is possible that these sequences compromise VapC folding and solubility, or mimic inhibitory interactions made by hydrophobic segments of the VapB antitoxin that block VapC activity (43, 59)."

Later in the manuscript, Lon is presented as a minor player in the overall story, but Lon prefers hydrophobic degrons. Could that hydrophobic class be Lon substrates? (Possibly presented as an additional mechanism here or in the discussion of this class of tags.)

(4) Page 13: "Arg in the 2nd position was also associated with proteolysis, yet Arg is virtually absent from proteobacterial ssrA sequences."

The nucleic acid changes required for evolutionary drift from the predominant amino acid codons at this position in proteobacteria to Arg may require moving through several codons that notably impair the performance of the degron. Such a constraint may also be responsible, in part, for the observed conservation.