[en] Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that appear to silence gene expression. One nucleoid-associated silencing factor is the conserved protein Hfq. Although seemingly nonspecific in its DNA binding properties, Hfq is strongly enriched at AT-rich DNA regions, characteristic of prophages and mobile genetic elements. Here, we demonstrate that polyphosphate (polyP), an ancient and highly conserved polyanion, is essential for the site-specific DNA binding properties of Hfq in bacteria. Absence of polyP markedly alters the DNA binding profile of Hfq, causes unsolicited prophage and transposon mobilization, and increases mutagenesis rates and DNA damage–induced cell death. In vitro reconstitution of the system revealed that Hfq and polyP interact with AT-rich DNA sequences and form phase-separated condensates, a process that is mediated by the intrinsically disordered C-terminal extensions of Hfq. We propose that polyP serves as a newly identified driver of heterochromatin formation in bacteria.
Beaufay, François ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'Ingénierie des Protéines (CIP) ; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
Amemiya, Haley M; Cellular and Molecular Biology Program, Michigan Medicine, University of Michigan, Ann Arbor, MI, USA ; Department of Computational medicine and Bioinformatics, Michigan Medicine, University of Michigan, Ann Arbor, MI, USA
Guan, Jian; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
Basalla, Joseph; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
Meinen, Ben A ; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA ; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA
Chen, Ziyuan ; Biophysics Program, University of Michigan, Ann Arbor, MI, USA
Mitra, Rishav ; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
Bardwell, James C A; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA ; Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA
Biteen, Julie S ; Biophysics Program, University of Michigan, Ann Arbor, MI, USA ; Department of Chemistry, University of Michigan, Ann Arbor, MI, USA
Vecchiarelli, Anthony G ; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
Freddolino, Lydia ; Department of Biological Chemistry, Michigan Medicine, University of Michigan, Ann Arbor, MI, USA
Jakob, Ursula ; Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA ; Department of Biological Chemistry, Michigan Medicine, University of Michigan, Ann Arbor, MI, USA
This work was supported by the National Institutes of Health grant R21-GM128022 (to J.S.B.), National Institutes of Health grant GM128637 (to P.L.F.), National Institute of Health grant GM122506 (to U.J.), National Institutes of Health Training grant T-32-GM007315 (to H.M.A.), National Science Foundation CAREER award no. 1941966 (to A.G.V.), and EMBO long-term fellowship ALTF 601-2016 (to F.B.). J.C.A.B. is funded by the Howard Hughes Medical Institute.
M. R. Brown, A. Kornberg, Inorganic polyphosphate in the origin and survival of species. Proc. Natl. Acad. Sci. U.S.A. 101, 16085-16087 (2004).
L. Xie, A. Rajpurkar, E. Quarles, N. Taube, A. S. Rai, J. Erba, B. Sliwinski, M. Markowitz, U. Jakob, D. Knoefler, Accumulation of nucleolar inorganic polyphosphate is a cellular response to cisplatin-induced apoptosis. Front. Oncol. 9, 1410 (2019).
M. J. Gray, W. Y. Wholey, N. O. Wagner, C. M. Cremers, A. Mueller-Schickert, N. T. Hock, A. G. Krieger, E. M. Smith, R. A. Bender, J. C. A. Bardwell, U. Jakob, Polyphosphate is a primordial chaperone. Mol. Cell 53, 689-699 (2014).
F. Beaufay, E. Quarles, A. Franz, O. Katamanin, W.-Y. Wholey, U. Jakob, Polyphosphate functions in vivo as an iron chelator and fenton reaction inhibitor. MBio 11, 4 (2020).
A. Du Toit, Phage induction in different contexts. Nat. Rev. Microbiol. 17, 126-127 (2019).
G. Posfai, G. Plunkett III, T. Feher, D. Frisch, G. M. Keil, K. Umenhoffer, V. Kolisnychenko, B. Stahl, S. S. Sharma, M. de Arruda, V. Burland, S. W. Harcum, F. R. Blattner, Emergent properties of reduced-genome Escherichia coli. Science 312, 1044-1046 (2006).
R. P. Rastogi, Richa, A. Kumar, M. B. Tyagi, R. P. Sinha, Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair. J. Nucleic Acids 2010, 592980 (2010).
T. Feher, B. Cseh, K. Umenhoffer, I. Karcagi, G. Posfai, Characterization of cycA mutants of Escherichia coli. An assay for measuring in vivo mutation rates. Mutat. Res. 595, 184-190 (2006).
D. J. Jin, C. A. Gross, Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J. Mol. Biol. 202, 45-58 (1988).
N. N. Rao, M. R. Gomez-Garcia, A. Kornberg, Inorganic polyphosphate: Essential for growth and survival. Annu. Rev. Biochem. 78, 605-647 (2009).
K. Murata, S. Hagiwara, Y. Kimori, Y. Kaneko, Ultrastructure of compacted DNA in cyanobacteria by high-voltage cryo-electron tomography. Sci. Rep. 6, 34934 (2016).
R. T. Dame, The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin. Mol. Microbiol. 56, 858-870 (2005).
J. McQuail, A. Switzer, L. Burchell, S. Wigneshweraraj, The RNA-binding protein Hfq assembles into foci-like structures in nitrogen starved Escherichia coli. J. Biol. Chem. 295, 12355-12367 (2020).
I. Moll, D. Leitsch, T. Steinhauser, U. Blasi, RNA chaperone activity of the Sm-like Hfq protein. EMBO Rep. 4, 284-289 (2003).
K. M. Wassarman, F. Repoila, C. Rosenow, G. Storz, S. Gottesman, Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev. 15, 1637-1651 (2001).
J. Orans, A. R. Kovach, K. E. Hoff, N. M. Horstmann, R. G. Brennan, Crystal structure of an Escherichia coli Hfq Core (residues 2-69)-DNA complex reveals multifunctional nucleic acid binding sites. Nucleic Acids Res. 48, 3987-3997 (2020).
A. Takada, M. Wachi, A. Kaidow, M. Takamura, K. Nagai, DNA binding properties of the hfq gene product of Escherichia coli. Biochem. Biophys. Res. Commun. 236, 576-579 (1997).
M. Kajitani, A. Kato, A. Wada, Y. Inokuchi, A. Ishihama, Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta. J. Bacteriol. 176, 531-534 (1994).
G. W. Li, D. Burkhardt, C. Gross, J. S. Weissman, Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624-635 (2014).
A. Zhang, D. J. Schu, B. C. Tjaden, G. Storz, S. Gottesman, Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets. J. Mol. Biol. 425, 3678-3697 (2013).
T. Vora, A. K. Hottes, S. Tavazoie, Protein occupancy landscape of a bacterial genome. Mol. Cell 35, 247-253 (2009).
P. L. Freddolino, H. M. Amemiya, T. J. Goss, S. Tavazoie, Dynamic landscape of protein occupancy across the Escherichia coli chromosome. PLoS Biol. 19, e3001306 (2021).
H. M. Amemiya, T. J. Goss, T. M. Nye, R. Hurto, L. A. Simmons, P. L. Freddolino, Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria. EMBO J., in press.
C. Sauter, J. Basquin, D. Suck, Sm-like proteins in Eubacteria: The crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res. 31, 4091-4098 (2003).
S. Melamed, A. Peer, R. Faigenbaum-Romm, Y. E. Gatt, N. Reiss, A. Bar, Y. Altuvia, L. Argaman, H. Margalit, Global mapping of small RNA-target interactions in bacteria. Mol. Cell 63, 884-897 (2016).
A. E. Posey, A. S. Holehouse, R. V. Pappu, Phase separation of intrinsically disordered proteins. Methods Enzymol. 611, 1-30 (2018).
F. Erdel, K. Rippe, Formation of chromatin subcompartments by phase separation. Biophys. J. 114, 2262-2270 (2018).
T. Ali Azam, A. Iwata, A. Nishimura, S. Ueda, A. Ishihama, Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid. J. Bacteriol. 181, 6361-6370 (1999).
T. B. Updegrove, J. J. Correia, R. Galletto, W. Bujalowski, R. M. Wartell, E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain. Biochim. Biophys. Acta 1799, 588-596 (2010).
T. B. Updegrove, A. Zhang, G. Storz, Hfq: The flexible RNA matchmaker. Curr. Opin. Microbiol. 30, 133-138 (2016).
S. Panja, S. A. Woodson, Fluorescence reporters for Hfq oligomerization and RNA annealing. Methods Mol. Biol. 1259, 369-383 (2015).
H. H. Tuson, J. S. Biteen, Unveiling the inner workings of live bacteria using superresolution microscopy. Anal. Chem. 87, 42-63 (2015).
A. S. Hansen, M. Woringer, J. B. Grimm, L. D. Lavis, R. Tjian, X. Darzacq, Robust model-based analysis of single-particle tracking experiments with Spot-On. eLife 7, e33125 (2018).
M. Yousuf, I. Iuliani, R. T. Veetil, A. S. N. Seshasayee, B. Sclavi, M. C. Lagomarsino, Early fate of exogenous promoters in E. coli. Nucleic Acids Res. 48, 2348-2356 (2020).
W. W. Navarre, S. Porwollik, Y. Wang, M. McClelland, H. Rosen, S. J. Libby, F. C. Fang, Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella. Science 313, 236-238 (2006).
B. A. Shen, R. Landick, Transcription of bacterial chromatin. J. Mol. Biol. 431, 4040-4066 (2019).
K. Singh, J. N. Milstein, W. W. Navarre, Xenogeneic silencing and its impact on bacterial genomes. Annu. Rev. Microbiol. 70, 199-213 (2016).
T. A. Azam, A. Ishihama, Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J. Biol. Chem. 274, 33105-33113 (1999).
A. Malabirade, K. Jiang, K. Kubiak, A. Diaz-Mendoza, F. Liu, J. A. van Kan, J. F. Berret, V. Arluison, J. R. C. van der Maarel, Compaction and condensation of DNA mediated by the C-terminal domain of Hfq. Nucleic Acids Res. 45, 7299-7308 (2017).
E. Fortas, F. Piccirilli, A. Malabirade, V. Militello, S. Trepout, S. Marco, A. Taghbalout, V. Arluison, New insight into the structure and function of Hfq C-terminus. Biosci. Rep. 35, e00190 (2015).
A. G. Larson, D. Elnatan, M. M. Keenen, M. J. Trnka, J. B. Johnston, A. L. Burlingame, D. A. Agard, S. Redding, G. J. Narlikar, Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236-240 (2017).
A. R. Strom, A. V. Emelyanov, M. Mir, D. V. Fyodorov, X. Darzacq, G. H. Karpen, Phase separation drives heterochromatin domain formation. Nature 547, 241-245 (2017).
F. R. Blattner, G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, Y. Shao, The complete genome sequence of Escherichia coli K-12. Science 277, 1453-1462 (1997).
K. A. Datsenko, B. L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645 (2000).
J. Salvatier, T. V. Wiecki, C. Fonnesbeck, Probabilistic programming in Python using PyMC3. Peer J. Comput. Sci. 2, e55 (2016).
A. Ducret, E. M. Quardokus, Y. V. Brun, MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol. 1, 16077 (2016).
E. Laskowska, J. Bohdanowicz, D. Kuczynska-Wisnik, E. Matuszewska, S. Kȩdzierska, A. Taylor, Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells. Microbiology 150, 247-259 (2004).
J. Miller, A Short Course in Bacterial Genetics-A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992).
N. L. Bray, H. Pimentel, P. Melsted, L. Pachter, Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525-527 (2016).
H. Pimentel, N. L. Bray, S. Puente, P. Melsted, L. Pachter, Differential analysis of RNA-seq incorporating quantification uncertainty. Nat. Methods 14, 687-690 (2017).
C. R. Harris, K. J. Millman, S. J. van der Walt, R. Gommers, P. Virtanen, D. Cournapeau, E. Wieser, J. Taylor, S. Berg, N. J. Smith, R. Kern, M. Picus, S. Hoyer, M. H. van Kerkwijk, M. Brett, A. Haldane, J. F. del Rio, M. Wiebe, P. Peterson, P. Gerard-Marchant, K. Sheppard, T. Reddy, W. Weckesser, H. Abbasi, C. Gohlke, T. E. Oliphant, Array programming with NumPy. Nature 585, 357-362 (2020).
H. Wickham, ggplot2 - Elegant Graphics for Data Analysis (Springer, 2009).
B. Demeler, G. E. Gorbet, Analytical ultracentrifugation data analysis with UltraScan-III, in Analytical Ultracentrifugation, S. Uchiyama, F. Arisaka, W. F. Stafford, T. Laue, Eds. (Springer, 2016), pp. 119-143.
B. Demeler, E. Brookes, L. Nagel-Steger, Analysis of heterogeneity in molecular weight and shape by analytical ultracentrifugation using parallel distributed computing. Methods Enzymol. 454, 87-113 (2009).
E. Brookes, W. Cao, B. Demeler, A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. 39, 405-414 (2010).
J. Lempart, E. Tse, J. A. Lauer, M. I. Ivanova, A. Sutter, N. Yoo, P. Huettemann, D. Southworth, U. Jakob, Mechanistic insights into the protective roles of polyphosphate against amyloid cytotoxicity. Life Sci. Alliance 2, e201900486 (2019).
S. A. Smith, J. H. Morrissey, Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4', 6-diamidino-2-phenylindol. Electrophoresis 28, 3461-3465 (2007).
C. Wong, S. Sridhara, J. C. Bardwell, U. Jakob, Heating greatly speeds Coomassie blue staining and destaining. Biotechniques 28, 426-428, 430, 432 (2000).
B. P. Isaacoff, Y. Li, S. A. Lee, J. S. Biteen, SMALL-LABS: Measuring single-molecule intensity and position in obscuring backgrounds. Biophys. J. 116, 975-982 (2019).
L. M. Guzman, D. Belin, M. J. Carson, J. Beckwith, Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121-4130 (1995).
