Abstract
Rapid bacterial growth depends on the speed at which ribosomes can translate mRNA into proteins. mRNAs that encode successive stretches of proline can cause ribosomes to stall, substantially reducing translation speed. Such stalling is especially detrimental for species that must grow and divide rapidly. Here, we focus on di-prolyl motifs (XXPPX) and ask whether their prevalence varies with growth rate. To find out we conducted a broad survey of such motifs in >3000 bacterial genomes across 35 phyla. Indeed, fast-growing species encode fewer motifs than slow-growing species, especially in highly expressed proteins. We also found many di-prolyl motifs within thermophiles, where prolines can help maintain proteome stability. Moreover, bacteria with complex, multicellular lifecycles also encode many di-prolyl motifs. This is especially evident in the slow-growing phylum Myxococcota. Bacteria in this phylum encode many serine-threonine kinases, and many di-prolyl motifs at potential phosphorylation sites within these kinases. Serine-threonine kinases are involved in cell signaling and help regulate developmental processes linked to multicellularity in the Myxococcota. Altogether, our observations suggest that weakened selection on translational rate, whether due to slow or thermophilic growth, may allow di-prolyl motifs to take on new roles in biological processes that are unrelated to translational rate.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Data availability
All genomes used in this study are publicly available from JGI’s IMG database [35]. Taxon IDs corresponding to every genome are listed in Supplementary Dataset 1, along with the genomic characteristics calculated for this study. Results from analyses of individual proteins are presented in Supplementary Dataset 2 and results of all PGLS models are listed in Supplementary Table 1. R scripts and all files needed to reproduce these analyses are available at https://github.com/tessbrewer/proline_project.
References
Russell JB, Cook GM. Energetics of bacterial growth: balance of anabolic and catabolic reactions. Microbiol Rev. 1995;59:48–62.
Klumpp S, Scott M, Pedersen S, Hwa T. Molecular crowding limits translation and cell growth. PNAS. 2013;110:16754–9.
Pedersen S. Escherichia coli ribosomes translate in vivo with variable rate. EMBO J. 1984;3:2895–8.
Ran W, Higgs PG. Contributions of speed and accuracy to translational selection in bacteria. PLoS One. 2012;7:e51652.
Vieira-Silva S, Rocha E. The systemic imprint of growth and its uses in ecological (meta)genomics. PLoS Genet. 2009;6:1–15.
Roller BRK, Stoddard SF, Schmidt TM. Exploiting rRNA operon copy number to investigate bacterial reproductive strategies. Nat Microbiol. 2016;1:1–7.
Buskirk AR, Green R. Ribosome pausing, arrest and rescue in bacteria and eukaryotes. Philos Trans R Soc B. 2017;372:20160183–11.
Wohlgemuth I, Brenner S, Beringer M, Rodnina MV. Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates. J Biol Chem. 2008;283:32229–35.
Pavlov MY, Watts RE, Tan Z, Cornish VW, Ehrenberg M, Forster AC. Slow peptide bond formation by proline and other N-alkylamino acids in translation. PNAS. 2009;106:50–54.
Mandal A, Mandal S, Park MH. Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution. PLoS One. 2014;9:e111800–13.
Adzhubei AA, Sternberg MJE, Makarov AA. Polyproline-II helix in proteins: structure and function. J Mol Biol. 2013;425:2100–32.
Elam WA, Schrank TP, Campagnolo AJ, Hilser VJ. Evolutionary conservation of the polyproline II conformation surrounding intrinsically disordered phosphorylation sites. Protein Sci. 2013;22:405–17.
Ball LJ, Kühne R, Schneider-Mergener J, Oschkinat H. Recognition of proline-rich motifs by protein-protein-interaction domains. Angew Chem Int Ed Engl. 2005;44:2852–69.
Starosta AL, Lassak J, Peil L, Atkinson GC, Virumäe K, Tenson T, et al. Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site. Nucleic Acids Res. 2014;42:10711–9.
Woolstenhulme CJ, Guydosh NR, Green R, Buskirk AR. High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep. 2015;11:13–21.
Hersch SJ, Elgamal S, Katz A, Ibba M, Navarre WW. Translation initiation rate determines the impact of ribosome stalling on bacterial protein synthesis. J Biol Chem. 2014;289:28160–71.
Lassak J, Wilson DN, Jung K. Stall no more at polyproline stretches with the translation elongation factors EF‐P and IF‐5A. Mol Microbiol. 2016;99:219–35.
Yanagisawa T, Sumida T, Ishii R, Takemoto C, Yokoyama S. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nature. 2010;17:1136–43.
Park J-H, Johansson HE, Aoki H, Huang BX, Kim H-Y, Ganoza MC, et al. Post-translational modification by beta-lysylation is required for activity of Escherichia coli elongation factor P (EF-P). J Biol Chem. 2012;287:2579–90.
Lassak J, Keilhauer E, Fürst M, Wuichet K, Gödeke J, Starosta AL, et al. Arginine-rhamnosylation as new strategy to activate translation elongation factor P. Nat Chem Biol. 2015;11:266–70.
Tollerson R, Witzky A, Ibba M. Elongation factor P is required to maintain proteome homeostasis at high growth rate. PNAS. 2018;115:1–6.
Peng WT, Banta LM, Charles TC, Nester EW. The chvH locus of Agrobacterium encodes a homologue of an elongation factor involved in protein synthesis. J Bacteriol. 2001;183:36–45.
Rajkovic A, Hummels KR, Witzky A, Erickson S, Gafken PR, Whitelegge JP, et al. Translation control of swarming proficiency in Bacillus subtilis by 5-amino-pentanolylated elongation factor P. J Biol Chem. 2016;291:10976–85.
Navarre WW, Zou SB, Roy H, Xie JL, Savchenko A, Singer A, et al. PoxA, YjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. Mol Cell. 2010;39:209–21.
Hummels KR, Kearns DB. Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P. PLoS Genet. 2019;15:e1008179–27.
Rajkovic A, Erickson S, Witzky A, Branson OE, Seo J, Gafken PR, et al. Cyclic rhamnosylated elongation factor P establishes antibiotic resistance in Pseudomonas aeruginosa. MBio. 2015;6:1–9.
Yanagisawa T, Takahashi H, Suzuki T, Masuda A, Dohmae N, Yokoyama S. Neisseria meningitidis translation elongation factor P and its active-site arginine residue are essential for cell viability. PLoS One. 2016;11:e0147907–27.
Krafczyk R, Qi F, Sieber A, Mehler J, Jung K, Frishman D, et al. Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria. Commun Biol. 2021;4:1–11.
Qi F, Motz M, Jung K, Lassak J, Frishman D. Evolutionary analysis of polyproline motifs in Escherichia coli reveals their regulatory role in translation. PLoS Comput Biol. 2018;14:e1005987–19.
Karlin S, Mrázek J, Campbell A, Kaiser D. Characterizations of highly expressed genes of four fast-growing bacteria. J Bacteriol. 2001;183:5025–40.
Dethlefsen L, Schmidt TM. Performance of the translational apparatus varies with the ecological strategies of bacteria. J Bacteriol. 2007;189:3237–45.
Weissman JL, Hou S, Fuhrman JA. Estimating maximal microbial growth rates from cultures, metagenomes, and single cells via codon usage patterns. PNAS. 2021;118:1–10.
Hersch SJ, Wang M, Zou SB, Moon K-M, Foster LJ, Ibba M, et al. Divergent protein motifs direct elongation factor P-mediated translational regulation in Salmonella enterica and Escherichia coli. MBio. 2013;4:1–10.
Pinheiro B, Scheidler CM, Kielkowski P, Schmid M, Forné I, Ye S, et al. Structure and function of an elongation factor P subfamily in Actinobacteria. Cell Rep. 2020;30:4332–42. e5.
Chen I-MA, Markowitz VM, Chu K, Palaniappan K, Szeto E, Pillay M, et al. IMG/M: integrated genome and metagenome comparative data analysis system. Nucleic Acids Res. 2017;45:D507–D516.
Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25:1043–55.
Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics. 2020;36:1925–7.
Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol. 2011;7:e1002195–16.
Madin JS, Nielsen DA, Brbic M, Corkrey R, Danko D, Edwards K, et al. A synthesis of bacterial and archaeal phenotypic trait data. Sci Data. 2020;7:1–8.
Novembre JA. Accounting for background nucleotide composition when measuring codon usage bias. Mol Biol Evol. 2002;19:1390–4.
Erdos G, Dosztányi Z. Analyzing protein disorder with IUPred2A. Curr Protoc Bioinforma. 2020;70:1–15.
Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7.
Price MN, Dehal PS, Arkin AP. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009;26:1641–50.
Pagel M. Inferring the historical patterns of biological evolution. Nature. 1999;401:877–84.
Revell LJ. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol. 2011;3:217–23.
Orme D, Freckleton R, Thomas G, Petzoldt T, Fritz S, Isaac N, et al. caper: comparative analyses of phylogenetics and evolution in R. 2018; https://CRAN.R-project.org/package=caper.
Wickham H. ggplot2: elegant graphics for data analysis. 2016. Springer-Verlag New York.
Symonds MRE, Blomberg SP. A primer on phylogenetic generalized least squares. In: Garamszegi L (eds). Modern phylogenetic comparative methods and their application in evolutionary biology. (Springer, Berlin, Heidelberg, 2014) pp. 105–30.
Watanabe K, Suzuki Y. Protein thermostabilization by proline substitutions. J Mol Catal B Enzym. 1998;4:167–80.
Sabath N, Ferrada E, Barve A, Wagner A. Growth temperature and genome size in bacteria are negatively correlated, suggesting genomic streamlining during thermal adaptation. Genome Biol Evol. 2013;5:966–77.
Goldman BS, Nierman WC, Kaiser D, Slater SC, Durkin AS, Eisen JA, et al. Evolution of sensory complexity recorded in a myxobacterial genome. PNAS. 2006;103:15200–5.
Long AM, Hou S, Ignacio-Espinoza JC, Fuhrman JA. Benchmarking microbial growth rate predictions from metagenomes. ISME J. 2020;15:1–13.
Rocha EPC. Codon usage bias from tRNA’s point of view: redundancy, specialization, and efficient decoding for translation optimization. Genome Res. 2004;14:2279–86.
Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol. 2000;66:1328–33.
Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012;40:D109–D114.
Perez J, Castaneda-García A, Jenke-Kodama H, Muller R, Munoz-Dorado J. Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome. PNAS. 2008;105:15950–5.
Shi L, Pigeonneau N, Ravikumar V, Dobrinic P, Macek B, Franjevic D, et al. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues. Front Microbiol. 2014;5:1–13.
Jakob U, Kriwacki R, Uversky VN. Conditionally and transiently disordered proteins: awakening cryptic disorder to regulate protein function. Chem Rev. 2014;114:6779–805.
Starosta AL, Lassak J, Peil L, Atkinson GC, Woolstenhulme CJ, Virumäe K, et al. A conserved proline triplet in Val-tRNA synthetase and the origin of elongation factor P. Cell Rep. 2014;9:476–83.
Nariya H, Inouye S. A protein Ser/Thr kinase cascade negatively regulates the DNA-binding activity of MrpC, a smaller form of which may be necessary for the Myxococcus xanthus development. Mol Microbiol. 2006;60:1205–17.
Stein EA, Cho K, Higgs PI, Zusman DR. Two Ser/Thr protein kinases essential for efficient aggregation and spore morphogenesis in Myxococcus xanthus. Mol Microbiol. 2006;60:1414–31.
Iakoucheva LM, Radivojac P, Brown CJ, OConnor TR, Sikes JG, Obradovic Z, et al. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 2004;32:1037–49.
Elsen S, Swem LR, Swem DL, Bauer CE. RegB/RegA, a highly conserved redox-responding global two-component regulatory system. Microbiol Mol Biol R. 2004;68:263–79.
Tawa P, Stewart RC. Kinetics of CheA autophosphorylation and dephosphorylation reactions. Biochemistry. 1994;33:7917–24.
Yoshida T, jian CaiS, Inouye M. Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator. Mol Microbiol. 2002;46:1283–94.
Cho M-H, Wrabl JO, Taylor J, Hilser VJ. Hidden dynamic signatures drive substrate selectivity in the disordered phosphoproteome. PNAS. 2020;117:1–11.
Acknowledgements
We acknowledge funding from the European Research Council under Grant Agreement No. 739874, as well as from Swiss National Science Foundation grant 31003A_172887, and from the University Priority Research Program in Evolutionary Biology at the University of Zurich. We thank members of the Wagner lab for helpful discussions, particularly Andrei Papkou and Pouria Dasmeh, and Michael Engel for figure design input.
Author information
Authors and Affiliations
Contributions
TEB and AW conceived and designed the project. TEB performed all computational analyses. TEB and AW wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
About this article
Cite this article
Brewer, T.E., Wagner, A. Translation stalling proline motifs are enriched in slow-growing, thermophilic, and multicellular bacteria. ISME J 16, 1065–1073 (2022). https://doi.org/10.1038/s41396-021-01154-y
Received:
Revised:
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41396-021-01154-y
This article is cited by
-
Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor
BMC Biology (2026)
-
Enhancing crop resilience towards drought: by integrating nanotechnology, microbiomes, and growth-promoting rhizobacteria
Discover Agriculture (2024)
