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• W2894927071 abstract "•Nutrient limitation upregulates the relative expression of the rDNA operon, rrnH•Ribosomes bearing the 16S rRNA, rrsH, can modulate the general stress response•rrsH-bearing ribosomes impact drug resistance, cell motility, and biofilm formation•Naturally occurring rRNA sequence variation encodes altered ribosome function Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. The ribosome is a two-subunit, multi-megadalton RNA protein complex that translates mRNA into protein through temporally coordinated transient interactions with cellular factors and tRNA. Although ribosomes are widely considered homogeneous assemblies that only passively contribute to gene expression, emerging evidence suggests that the composition of the actively translating ribosome may contribute to mRNA-specific changes in gene expression (Dinman, 2016Dinman J.D. Pathways to specialized ribosomes: the brussels lecture.J. Mol. Biol. 2016; 428: 2186-2194Crossref PubMed Scopus (46) Google Scholar, Genuth and Barna, 2018Genuth N.R. Barna M. The discovery of ribosome heterogeneity and its implications for gene regulation and organismal life.Mol. Cell. 2018; 71: 364-374Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Sauert et al., 2015Sauert M. Temmel H. Moll I. Heterogeneity of the translational machinery: Variations on a common theme.Biochimie. 2015; 114: 39-47Crossref PubMed Scopus (38) Google Scholar). In bacteria, stress-induced cleavage of the anti-Shine-Dalgarno sequence from rRNA and antibiotic-induced shedding of ribosomal proteins from assembled ribosomes have been suggested to enhance the translation of leaderless mRNAs (Sauert et al., 2015Sauert M. Temmel H. Moll I. Heterogeneity of the translational machinery: Variations on a common theme.Biochimie. 2015; 114: 39-47Crossref PubMed Scopus (38) Google Scholar). In eukaryotes, imbalances in the stoichiometry of specific ribosomal proteins within the assembled ribosome have been linked to alterations in gene expression (Ferretti et al., 2017Ferretti M.B. Ghalei H. Ward E.A. Potts E.L. Karbstein K. Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements.Nat. Struct. Mol. Biol. 2017; 24: 700-707Crossref PubMed Scopus (34) Google Scholar, Shi et al., 2017Shi Z. Fujii K. Kovary K.M. Genuth N.R. Röst H.L. Teruel M.N. Barna M. Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide.Mol. Cell. 2017; 67: 71-83.e7Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Varied levels of post-transcriptional rRNA modification have also been associated with changes in ligand binding and translational fidelity (Jack et al., 2011Jack K. Bellodi C. Landry D.M. Niederer R.O. Meskauskas A. Musalgaonkar S. Kopmar N. Krasnykh O. Dean A.M. Thompson S.R. et al.rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells.Mol. Cell. 2011; 44: 660-666Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The potential link between translation efficiency and ribosome concentration has, however, been raised as a confounding factor in the proposed mechanisms of gene-specific translational control (Culviner and Laub, 2018Culviner P.H. Laub M.T. Global analysis of the E. coli toxin MazF reveals widespread cleavage of mRNA and the inhibition of rRNA maturation and ribosome biogenesis.Mol. Cell. 2018; 70: 868-880.e10Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, Lodish, 1974Lodish H.F. Model for the regulation of mRNA translation applied to haemoglobin synthesis.Nature. 1974; 251: 385-388Crossref PubMed Scopus (248) Google Scholar, Mills and Green, 2017Mills E.W. Green R. Ribosomopathies: there’s strength in numbers.Science. 2017; 358: eaan2755Crossref PubMed Scopus (88) Google Scholar). Questions as to how changes in ribosome composition could be efficiently regulated have also been voiced (Briggs and Dinman, 2017Briggs J.W. Dinman J.D. Subtractional heterogeneity: a crucial step toward defining specialized ribosomes.Mol. Cell. 2017; 67: 3-4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, Leslie, 2017Leslie M. There are millions of protein factories in every cell. Surprise, they’re not all the same.Science. 2017; http://www.sciencemag.org/news/2017/06/there-are-millions-protein-factories-every-cell-surprise-they-re-not-all-sameGoogle Scholar). Although the roles of rRNA in diverse aspects of ribosome function are firmly established (Noller, 2005Noller H.F. RNA structure: reading the ribosome.Science. 2005; 309: 1508-1514Crossref PubMed Scopus (236) Google Scholar), the potential contributions of endogenously encoded rRNA sequence variation to gene expression regulation has received relatively little attention. The majority of organisms natively encode multiple highly homologous yet distinct genes for the rRNA components of the ribosome (Prokopowich et al., 2003Prokopowich C.D. Gregory T.R. Crease T.J. The correlation between rDNA copy number and genome size in eukaryotes.Genome. 2003; 46: 48-50Crossref PubMed Scopus (271) Google Scholar, Sun et al., 2013Sun D.-L. Jiang X. Wu Q.L. Zhou N.-Y. Intragenomic heterogeneity of 16S rRNA genes causes overestimation of prokaryotic diversity.Appl. Environ. Microbiol. 2013; 79: 5962-5969Crossref PubMed Scopus (134) Google Scholar). For instance, the E. coli K-12 MG1655 (K12) genome encodes seven rDNA operons (rrnA-E, G, and H), each of which contains genes for the core 16S, 23S, and 5S rRNA elements of the two-subunit ribosome (Figures 1A–1C; Blattner et al., 1997Blattner F.R. Plunkett 3rd, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. et al.The complete genome sequence of Escherichia coli K-12.Science. 1997; 277: 1453-1462Crossref PubMed Scopus (5544) Google Scholar). The rRNA gene products encoded by these operons are distinguished by sequence variation at 23 positions in the 16S rRNA, 35 positions in the 23S rRNA, and 4 positions in the 5S rRNA (Figures 1C, 1D, and S1A–S1E). As each of these operons is constitutively expressed (Condon et al., 1992Condon C. Philips J. Fu Z.Y. Squires C. Squires C.L. Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli.EMBO J. 1992; 11: 4175-4185Crossref PubMed Google Scholar), the translating ribosome pool in E. coli—as in most organisms—is intrinsically heterogeneous. rDNA copy number is typically associated with cellular growth and proliferation rates (Condon et al., 1995Condon C. Liveris D. Squires C. Schwartz I. Squires C.L. rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation.J. Bacteriol. 1995; 177: 4152-4156Crossref PubMed Scopus (151) Google Scholar, Gyorfy et al., 2015Gyorfy Z. Draskovits G. Vernyik V. Blattner F.F. Gaal T. Posfai G. Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number.Nucleic Acids Res. 2015; 43: 1783-1794Crossref PubMed Scopus (36) Google Scholar). However, E. coli rDNA operons possess functionally distinct promoters and are located non-contiguously within the genome (Figure 1B), suggesting that they may be differentially regulated (Condon et al., 1992Condon C. Philips J. Fu Z.Y. Squires C. Squires C.L. Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli.EMBO J. 1992; 11: 4175-4185Crossref PubMed Google Scholar, Hillebrand et al., 2005Hillebrand A. Wurm R. Menzel A. Wagner R. The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies.Biol. Chem. 2005; 386: 523-534Crossref PubMed Scopus (26) Google Scholar, Hirvonen et al., 2001Hirvonen C.A. Ross W. Wozniak C.E. Marasco E. Anthony J.R. Aiyar S.E. Newburn V.H. Gourse R.L. Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli.J. Bacteriol. 2001; 183: 6305-6314Crossref PubMed Scopus (80) Google Scholar). Context-dependent expression of specific rDNA operons has been documented in bacteria, parasites, zebrafish, and mammals (Condon et al., 1992Condon C. Philips J. Fu Z.Y. Squires C. Squires C.L. Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli.EMBO J. 1992; 11: 4175-4185Crossref PubMed Google Scholar, Gunderson et al., 1987Gunderson J.H. Sogin M.L. Wollett G. Hollingdale M. de la Cruz V.F. Waters A.P. McCutchan T.F. Structurally distinct, stage-specific ribosomes occur in Plasmodium.Science. 1987; 238: 933-937Crossref PubMed Scopus (243) Google Scholar, Kim et al., 2007Kim H.-L. Shin E.-K. Kim H.-M. Ryou S.-M. Kim S. Cha C.-J. Bae J. Lee K. Heterogeneous rRNAs are differentially expressed during the morphological development of Streptomyces coelicolor.FEMS Microbiol. Lett. 2007; 275: 146-152Crossref PubMed Scopus (13) Google Scholar, Locati et al., 2017Locati M.D. Pagano J.F.B. Girard G. Ensink W.A. van Olst M. van Leeuwen S. Nehrdich U. Spaink H.P. Rauwerda H. Jonker M.J. et al.Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development.RNA. 2017; 23: 1188-1199Crossref PubMed Scopus (25) Google Scholar, López-López et al., 2007López-López A. Benlloch S. Bonfá M. Rodríguez-Valera F. Mira A. Intragenomic 16S rDNA divergence in Haloarcula marismortui is an adaptation to different temperatures.J. Mol. Evol. 2007; 65: 687-696Crossref PubMed Scopus (55) Google Scholar, Parks et al., 2018Parks M.M. Kurylo C.M. Dass R.A. Bojmar L. Lyden D. Vincent C.T. Blanchard S.C. Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression.Sci. Adv. 2018; 4: eaao0665Crossref PubMed Scopus (45) Google Scholar). The physiological significance of these changes and the impacts of the encoded rRNA sequence variation are, however, not presently known. Here, we show that the E. coli rDNA operon, rrnH, becomes more highly expressed on a relative basis in response to nutrient limitation. The rrnH operon encodes the 16S rRNA gene, rrsH, which is conserved in enterobacteria and is distinguished by ten sequence variants in the small ribosomal subunit head domain. We find that rrsH-bearing ribosomes causally impact transcriptional and translational expression of coherent gene sets, including stress response genes regulated by the RpoS sigma factor, the master regulator of the general stress response (Battesti et al., 2011Battesti A. Majdalani N. Gottesman S. The RpoS-mediated general stress response in Escherichia coli.Annu. Rev. Microbiol. 2011; 65: 189-213Crossref PubMed Scopus (452) Google Scholar, Fredriksson et al., 2007Fredriksson A. Ballesteros M. Peterson C.N. Persson O. Silhavy T.J. Nyström T. Decline in ribosomal fidelity contributes to the accumulation and stabilization of the master stress response regulator sigmaS upon carbon starvation.Genes Dev. 2007; 21: 862-874Crossref PubMed Scopus (38) Google Scholar). Functional analyses reveal that the expression of rrsH-bearing ribosomes alters RpoS protein levels and mediates phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility. These effects are shown to involve the stress-related protein factors, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE, each of which interacts with the ribosome near the region of sequence variation within the small subunit head domain (Brown et al., 2016Brown A. Fernández I.S. Gordiyenko Y. Ramakrishnan V. Ribosome-dependent activation of stringent control.Nature. 2016; 534: 277-280Crossref PubMed Scopus (73) Google Scholar, Neubauer et al., 2009Neubauer C. Gao Y.-G. Andersen K.R. Dunham C.M. Kelley A.C. Hentschel J. Gerdes K. Ramakrishnan V. Brodersen D.E. The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE.Cell. 2009; 139: 1084-1095Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Shasmal et al., 2016Shasmal M. Dey S. Shaikh T.R. Bhakta S. Sengupta J. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.Sci. Rep. 2016; 6: 19936Crossref PubMed Scopus (8) Google Scholar). These findings establish that an endogenously encoded rDNA operon in bacteria with conserved sequence variation exhibits context-dependent expression and that ribosomes bearing this variant rRNA have the capacity to alter stress response gene expression and physiology. Accurate quantification of the relative intragenomic expression of rDNA alleles using high-throughput sequencing methods is challenged by read mapping uncertainty that arises from the high sequence homology of rRNA genes (Treangen and Salzberg, 2011Treangen T.J. Salzberg S.L. Repetitive DNA and next-generation sequencing: computational challenges and solutions.Nat. Rev. Genet. 2011; 13: 36-46Crossref PubMed Scopus (765) Google Scholar). Conventional protocols for RNA sequencing (RNA-seq) library preparation also include steps designed to deplete rRNA. Here, we employed RNA-seq without rRNA depletion, together with an expectation-maximization algorithm that accounts for read mapping uncertainty (Li et al., 2010Li B. Ruotti V. Stewart R.M. Thomson J.A. Dewey C.N. RNA-seq gene expression estimation with read mapping uncertainty.Bioinformatics. 2010; 26: 493-500Crossref PubMed Scopus (507) Google Scholar), to infer the relative expression of rRNA variants in E. coli. We validated this approach by sequencing mixtures of ribosome populations of known proportions isolated from E. coli exclusively expressing distinct 16S rRNAs from a multi-copy plasmid (Asai et al., 1999Asai T. Condon C. Voulgaris J. Zaporojets D. Shen B. Al-Omar M. Squires C. Squires C.L. Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons.J. Bacteriol. 1999; 181: 3803-3809Crossref PubMed Google Scholar, Brosius et al., 1981Brosius J. Ullrich A. Raker M.A. Gray A. Dull T.J. Gutell R.R. Noller H.F. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli.Plasmid. 1981; 6: 112-118Crossref PubMed Scopus (367) Google Scholar) to correctly recall the mixing proportions with high accuracy (Figure S1F; STAR Methods). The approach also reliably tracked the expression of heat-shock-induced, plasmid-borne, MS2-tagged ribosomes (Youngman and Green, 2005Youngman E.M. Green R. Affinity purification of in vivo-assembled ribosomes for in vitro biochemical analysis.Methods. 2005; 36: 305-312Crossref PubMed Scopus (50) Google Scholar) in wild-type E. coli cells and determined the extent of their purification from a mixed ribosome pool (Figure S1G). These controls demonstrate that RNA-seq coupled with expectation maximization can accurately infer rRNA expression levels even among genes that are >99% homologous. With a robust method for quantifying the relative expression levels of rRNA variants, we sought to determine whether nutrient limitation-induced stress alters the distribution of rDNA allele expression. In E. coli, growth in nutrient-limiting minimal media is a well-established, natural stress (Hengge-Aronis, 1993Hengge-Aronis R. Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in E. coli.Cell. 1993; 72: 165-168Abstract Full Text PDF PubMed Scopus (436) Google Scholar) that induces broad, adaptive changes in gene expression relative to growth in complex media via the general stress response (Tao et al., 1999Tao H. Bausch C. Richmond C. Blattner F.R. Conway T. Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media.J. Bacteriol. 1999; 181: 6425-6440Crossref PubMed Google Scholar). To examine rDNA operon expression, we grew E. coli in complex or minimal media and performed RNA-seq on total RNA, representing the entire expressed transcriptome, and on polysomal RNA, representing RNAs actively engaged in protein synthesis (STAR Methods). Consistent with a stress-induced reduction in global translation, the ribosome and polysome levels decreased for cells grown in minimal media (Figures 1E and 1F). As anticipated, we also observed widespread differences in gene expression, where 1,314 and 1,427 genes were found to be differentially expressed in the total and polysomal RNA pool, respectively (Figure S1H; STAR Methods). As expected, the genes observed to be differentially expressed significantly overlapped with those previously associated with nutrient limitation-induced stress, respecting direction of change (p < 1e−6; STAR Methods; Tao et al., 1999Tao H. Bausch C. Richmond C. Blattner F.R. Conway T. Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media.J. Bacteriol. 1999; 181: 6425-6440Crossref PubMed Google Scholar). Confirming that nutrient limitation activates canonical aspects of the general stress response, promoters targeted by RpoS (Gama-Castro et al., 2016Gama-Castro S. Salgado H. Santos-Zavaleta A. Ledezma-Tejeida D. Muñiz-Rascado L. García-Sotelo J.S. Alquicira-Hernández K. Martínez-Flores I. Pannier L. Castro-Mondragón J.A. et al.RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond.Nucleic Acids Res. 2016; 44: D133-D143Crossref PubMed Scopus (222) Google Scholar) were enriched among upregulated transcriptional units (TUs) (Cho et al., 2009Cho B.-K. Zengler K. Qiu Y. Park Y.S. Knight E.M. Barrett C.L. Gao Y. Palsson B.Ø. The transcription unit architecture of the Escherichia coli genome.Nat. Biotechnol. 2009; 27: 1043-1049Crossref PubMed Scopus (183) Google Scholar; p = 4.5e−5). Notably, RpoS is one of only two genes commonly upregulated among studies of global gene expression during acute and chronic nutrient stress (Figure S1I). In both minimal and complex media, all seven rDNA operons were expressed, and their relative expression levels were >99.6% correlated between total and polysomal fractions (Figures S1J and S1K). Thus, all expressed rRNAs are assembled into mature, translation-competent ribosomes with equivalent efficiency. Strikingly, six of the seven E. coli rDNA operons were differentially expressed between minimal and complex media (Figures 1G and S1L). These data unexpectedly reveal that modulation of the rRNA composition of the actively translating ribosome pool is a feature of nutrient limitation-induced stress. The rDNA operons located proximal to the origin of replication, oriC (rrnA, rrnB, and rrnE), with the exception of rrnC, were relatively downregulated in minimal media, and those located distally (rrnD, rrnG, and rrnH), particularly the rrnH operon located farthest from the origin of replication, were relatively upregulated (Figures 1B and 1G). Consequently, ribosomes assembled from the rRNA components of the rrnH operon become relatively enriched in the actively translating ribosome pool under nutrient limitation-induced stress. In E. coli MG1655, the rrnD, rrnG, and rrnH operons are distinguished by variants in distinct structural domains of the 16S rRNA component of the small subunit. rrsD and rrsG 16S rRNAs possess sequence variants within the “foot” domain comprising helices 6–11 (h6–h11). The rrsH 16S rRNA is distinguished by ten variant nucleotides within the “head” domain, nine of which cluster within the helix 33 (h33) “beak” region (Figures 1C, 1D, and S1A–S1C). The presence of identical h33 variants in the highly divergent E. coli MRE600 strain (Kurylo et al., 2016Kurylo C.M. Alexander N. Dass R.A. Parks M.M. Altman R.A. Vincent C.T. Mason C.E. Blanchard S.C. Genome sequence and analysis of Escherichia coli MRE600, a colicinogenic, nonmotile strain that lacks RNase I and the type I methyltransferase, EcoKI.Genome Biol. Evol. 2016; 8: 742-752Crossref PubMed Scopus (16) Google Scholar) prompted us to examine further the conservation of these variants. A broader phylogenetic analysis of the family Enterobacteriaceae, which includes E. coli and other enteric bacteria, revealed that 20.8% (1,773/8,511) of sequenced genomes encode at least one 16S rRNA with an h33 sequence identical to that of rrsH (Figure 2A). This includes Salmonella enterica, which diverged from E. coli over 120 million years ago (Ochman and Wilson, 1987Ochman H. Wilson A.C. Evolution in bacteria: evidence for a universal substitution rate in cellular genomes.J. Mol. Evol. 1987; 26: 74-86Crossref PubMed Scopus (503) Google Scholar). In contrast, only 3.6% (309/8,511) encode a 16S rRNA with a foot domain sequence identical to that of rrsG, the second most highly upregulated 16S rRNA. We also found that rDNA operons encoding the rrsH h33 sequence are more distally located from the origin of replication relative to other rDNA operons than expected by chance (p < 1e−4; STAR Methods). Given the inverse correlation between rRNA gene conversion and genomic distance (Hashimoto et al., 2003Hashimoto J.G. Stevenson B.S. Schmidt T.M. Rates and consequences of recombination between rRNA operons.J. Bacteriol. 2003; 185: 966-972Crossref PubMed Scopus (59) Google Scholar), we speculate that conservation of the rrnH operon sequence variation may relate to its tendency to occupy a relatively distal position within the bacterial genome. In light of the relative increase in rrnH expression observed to accompany nutrient limitation-induced stress and the evolutionary conservation of the rrsH h33 sequence, we hypothesized that rrsH-bearing ribosomes may contribute to the modulation of gene expression regulation. To test this hypothesis, we engineered two Δ7prrn E. coli strains that lack all endogenous rDNA operons and express plasmid-borne copies of either the rrnB operon or the rrnB operon containing the ten rrsH variants within the small-subunit head domain (STAR Methods). We refer to these plasmids as pKK3535-BBB and pKK3535-HBB, where the gene of origin for the 16S, 23S, and 5S rRNAs is listed in 5′ to 3′ orientation. The corresponding strains are referred to as Δ7prrn-BBB and Δ7prrn-HBB, respectively. This plasmid-based expression system bypasses complexities associated with chromosome position-dependent impacts on rDNA expression and controls for tRNA gene copy number (Figures 1A and 1B). As described below, this system also enabled the purification of pure ribosome populations for in vitro investigations and provides a consistent means of examining the impacts of rRNA sequence variation across distinct strains and genetic contexts. The Δ7prrn-BBB and Δ7prrn-HBB strains were confirmed by deep sequencing to be isogenic, except for the intended 16S rRNA variants, and exhibited indistinguishable doubling times in minimal media (STAR Methods). We therefore used the Δ7prrn-BBB and Δ7prrn-HBB strains as a controlled setting in which to investigate the impacts of endogenously encoded rRNA sequence variation on gene expression. To examine whether sequence variants in the ribosome’s head domain regulate transcriptional and translational aspects of gene expression, we performed RNA-seq on total and polysomal RNA, respectively, harvested from Δ7prrn-BBB and Δ7prrn-HBB strains, which were both grown in minimal media. Strikingly, this analysis revealed that 864 genes (19% of the annotated transcriptome) were differentially expressed at the level of transcription, with 440 genes exhibiting similar differences in the actively translating polysome pool (Figures 2B and S2A). The altered expression of a subset of these genes was verified by real-time qPCR (Figure S2B). These findings reveal that evolutionarily conserved, endogenously encoded sequence variation within the small subunit head domain of the ribosome has the capacity to substantially alter gene expression at the levels of both transcription and translation. Notably, the genes that were differentially expressed in Δ7prrn-HBB relative to Δ7prrn-BBB strains were overrepresented for those regulated during acute and chronic nutrient stress (p < 1e−6; STAR Methods; Durfee et al., 2008Durfee T. Hansen A.-M. Zhi H. Blattner F.R. Jin D.J. Transcription profiling of the stringent response in Escherichia coli.J. Bacteriol. 2008; 190: 1084-1096Crossref PubMed Scopus (232) Google Scholar, Tao et al., 1999Tao H. Bausch C. Richmond C. Blattner F.R. Conway T. Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media.J. Bacteriol. 1999; 181: 6425-6440Crossref PubMed Google Scholar, Traxler et al., 2008Traxler M.F. Summers S.M. Nguyen H.-T. Zacharia V.M. Hightower G.A. Smith J.T. Conway T. The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli.Mol. Microbiol. 2008; 68: 1128-1148Crossref PubMed Scopus (289) Google Scholar). They also significantly overlapped with those differentially expressed between wild-type cells grown in minimal versus complex media, respecting direction of change (p < 1e−6 for both polysomal and total RNA; Figure S2C; STAR Methods). Hence, two bacterial strains, whose only sequence distinctions are ten naturally encoded variant nucleotides within the small subunit head domain of the ribosome, exhibit substantial differences in the expression of gene sets associated with nutrient limitation-induced stress. The observed differences in stress response-related gene expression led us to hypothesize that rrsH-bearing ribosomes contribute to transcriptional aspects of the general stress response. RpoS and the (p)ppGpp alarmone mediate important transcriptional impacts on the stress response whose own regulation occurs, at least in part, during protein synthesis (Battesti et al., 2011Battesti A. Majdalani N. Gottesman S. The RpoS-mediated general stress response in Escherichia coli.Annu. Rev. Microbiol. 2011; 65: 189-213Crossref PubMed Scopus (452) Google Scholar, Hauryliuk et al., 2015Hauryliuk V. Atkinson G.C. Murakami K.S. Tenson T. Gerdes K. Recent functional insights into the role of (p)ppGpp in bacterial physiology.Nat. Rev. Microbiol. 2015; 13: 298-309Crossref PubMed Scopus (278) Google Scholar). In line with RpoS and (p)ppGpp contributions to the observed distinctions in gene expression, genes differentially expressed in the Δ7prrn-HBB strain significantly overlapped with RpoS- and (p)ppGpp-dependent gene sets identified in genetic knockout studies, respecting direction of change (p < 1e−6 for all tests; Figures 2C and 2D; STAR Methods; Dong and Schellhorn, 2009Dong T. Schellhorn H.E. Control of RpoS in global gene expression of Escherichia coli in minimal media.Mol. Genet. Genomics. 2009; 281: 19-33Crossref PubMed Scopus (89) Google Scholar, Traxler et al., 2008Traxler M.F. Summers S.M. Nguyen H.-T. Zacharia V.M. Hightower G.A. Smith J.T. Conway T. The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli.Mol. Microbiol. 2008; 68: 1128-1148Crossref PubMed Scopus (289) Google Scholar). TUs more highly expressed in Δ7prrn-HBB relative to Δ7prrn-BBB were also enriched for those whose promoters are regulated by RpoS (p = 2.9e−12; Gama-Castro et al., 2016Gama-Castro S. Salgado H. Santos-Zavaleta A. Ledezma-Tejeida D. Muñiz-Rascado L. García-Sotelo J.S. Alquicira-Hernández K. Martínez-Flores I. Pannier L. Castro-Mondragón J.A. et al.RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond.Nucleic Acids Res. 2016; 44: D133-D143Crossref PubMed Scopus (222) Google Scholar). As anticipated from these analyses, RpoS protein levels were found to be higher in the Δ7prrn-HBB strain (Figure 2D; STAR Methods), while significant changes in RpoS mRNA levels were not observed in either the total or polysomal RNA pools (Table S4). We therefore conclude that the ten-nucleotide variants within the head domain of rrsH-bearing ribosomes contribute to RpoS-mediated changes in the stress response gene expression program" @default.
• W2894927071 created "2018-10-12" @default.
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• W2894927071 date "2018-10-01" @default.
• W2894927071 modified "2023-10-01" @default.
• W2894927071 title "Endogenous rRNA Sequence Variation Can Regulate Stress Response Gene Expression and Phenotype" @default.
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