The structure of the biocrystalline nucleoid and its role in the regulation of dissociative phenotypic heterogeneity of microbial populations

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Abstract

The survival of the microbial population in constantly changing environmental conditions, including those unfavorable for growth, is ensured by: (1) the formation of a subpopulation of persister cells (P), maturing into ametabolic quiescent forms (RF); (2) protection of chromosomal DNA of stationary cells using the physicochemical mechanism of its co-crystallization with the nucleoid-associated protein Dps and the formation of a biocrystalline nucleoid (BN); (3) the ability of RF to germinate in a fresh environment with a mixed population of phenotypically different dissociators, one of which will be the most adaptive to it. This study addressed two questions: (1) how BN is structurally organized in prokaryotic RFs, and (2) how nucleoid biocrystallization is related to the phenotypic heterogeneity of populations growing from RFs. The work proposes a new model of BN decrystallization/recrystallization during heating/cooling of RF at sublethal temperatures in a non-growth environment, which reproduces the dynamics of BN formation in the model of nucleoid organization as a folded globule. Electron microscopic analysis of structural changes in BN in heated/cooled RFs, together with the determination of the dissociative spectra of the populations growing from them, allowed us to obtain the following new information. Biocrystallization of the nucleoid occurs in the following sequence: (1) the beginning co-crystallization of DNA-Dps is accompanied by the division of the nucleoid volume with the formation of a compacted nucleoid from superfolded DNA in the central region of the cell and loops of superfolded linear DNA extending from it; (2) co-crystallization of looped DNA-Dps with its different geometric arrangement – toroidal, lamellar, etc.; (3) crystallization of Dps-Dps, repeating the template folding of looped DNA-Dps and the formation of a multilayer structure of the Dps-Dps crystalline array. It was found that the actual heating of the PF (45‒700C, 15 min), leading to decrystallization of looped DNA-Dps while maintaining the structure of the compacted nucleoid, does not affect the dissociative (colonial-morphological) spectrum of the population growing from the PF. The change in its dissociative spectrum is influenced by the process of DNA-Dps recrystallization, during which, apparently, Dps binds not only to the former, but also to other DNA sites, also affinity for Dps and, possibly, partially occupied by other nucleoid-associated proteins, which influences changes in DNA topology and its transcription.

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About the authors

G. I. El’-Registan

FRC Fundamental of Biotechnology RAS

Email: elenademkina@mail.ru

Winogradsky Institute of Microbiology

Russian Federation, Moscow, 119071

N. E. Suzina

Pushchino Scientific Center for Biological Research RASciences

Email: elenademkina@mail.ru

Scryabin Institute of Biochemistry and Physiology of Microorganisms

Russian Federation, Pushchino, 142290

Е. V. Demkina

FRC Fundamental of Biotechnology RAS

Author for correspondence.
Email: elenademkina@mail.ru

Winogradsky Institute of Microbiology

Russian Federation, Moscow, 119071

Yu. A. Nikolaev

FRC Fundamental of Biotechnology RAS

Email: elenademkina@mail.ru

Winogradsky Institute of Microbiology

Russian Federation, Moscow, 119071

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2. Fig. 1. Thin sections of intact PF E. coli (3 months): a – thin-walled type I PF; b – multilayered type II PF. Visible: compacted nucleoid region (a, b, e); multilayered membrane of type II PF (b); lamellar DNA-Dps and/or Dps-Dps packing (d); toroidal DNA-Dps and/or Dps-Dps packing (c). Scale bar – 0.2 µm.

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3. Fig. 2. Thin sections of intact PF of P. extremaustralis (3 months): a – PF type I; b – PF type II. Visible: lamellar packing of DNA-Dps and/or Dps-Dps (c, d); toroidal packing of DNA-Dps (d). Scale bar – 0.2 µm.

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4. Fig. 3. Thin sections of intact PF of R. erythropolis (3 months): a, b – PF type I; c – PF type II. Visible: compacted nucleoid (a, b); liquid crystalline structure of DNA of compacted nucleoid of isotropic type (b); membrane-like linear structures (b); multilayer packing and loop of linear DNA-Dps (c). Scale bar – 0.2 µm.

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5. Fig. 4. Thin sections of E. coli PF heated to 50°C, instant fixation. Visible: melt of biocrystalline nucleoid (BN); liquid crystal structure of compacted isotropic nucleoid. Scale bar – 0.2 µm.

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6. Fig. 5. Thin sections of E. coli PF heated to 55°C, instant fixation: melting of the toroidal structure in type I PF. Visible: toroids that retained the biocrystalline structure and DNA-Dps strands. Magnification – DNA-Dps strand extending from the toroid (b). Scale mark – 0.2 µm.

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7. Fig. 6. Thin sections of E. coli PF heated to 65°C, instant fixation: cell with melted BN. Individual islands of dense BN packing are visible (a, b, c); DNA-Dps strands (b); fragments of melted BN Dps-Dps (b, c, d). Scale mark – 0.2 µm.

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8. Fig. 7. Thin sections of E. coli PF heated to 55°C, rapid cooling. Visible: expanded periplasmic space in heated PF (a); linear DNA-Dps strand (b); liquid crystal structure of DNA of cholesteric-type biocrystalline nucleoid. Scale bar – 0.2 µm.

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9. Fig. 8. Thin sections of E. coli PF heated to 65°C, rapid cooling: a-e – protoplast plasmolysis and BN melting. Visible: enlargement of the periplasmic space (a, b); fragments of the Dps-Dps packing in the periplasmic space (b); remnants of dense packing of the biocrystal (a). Scale mark – 0.2 μm.

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10. Fig. 9. Heating of E. coli PF heated to 55°C, slow cooling: a, b – plasmolysis, beginning of BN recrystallization. In cooled PF the following are visible: compacted BN areas (a); formation of toroids (a, b). Scale mark – 0.2 µm.

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11. Fig. 10. Thin sections of R. erythropolis PF heated to 60°C, rapid cooling: a–g – compacted nucleoid. Visible: areas of retained structure of liquid crystalline DNA of cholesteric (c, d) and isotropic type of compacted nucleoid (b); packing of DNA-Dps strands (a, b), including toroidal type (b). Scale bar – 0.2 µm.

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12. Fig. 11. Thin sections of R. erythropolis PF heated to 50°C, rapid cooling: a – cell with fairly well-preserved BN. Visible: liquid crystalline DNA of the compacted nucleoid that has retained its structure. Scale bar – 0.2 µm.

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13. Fig. 12. Thin sections of R. erythropolis PF heated to 60°C, slow cooling. Visible: a, b – compacted nucleoid, which has practically restored its BN structure (a, b); toroidal structures of DNA-Dps (a); loop of the linear DNA-Dps strand (b). Scale mark – 0.2 µm.

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14. Fig. 13. Thin sections of R. erythropolis PF heated to 70°C, rapid cooling: a, b – restoration of BN. Visible: formation and packing of linear DNA-Dps strands (a); preserved packing of supercoiled DNA strands of compacted cholesteric nucleoid (b). Scale bar – 0.2 µm.

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15. Fig. 14. Thin sections of P. extremaustralis PF heated to 60°C, slow cooling (a–d). Visible: intact PF type II (a) and type I (b) with inclusions of poly-β-hydroxybutyric acid (POMC) (b); DNA strands of the compact nucleoid that has lost its structure (c); self-assembly of DNA-Dps strands into “toroidal” structures (d, d). Scale bar – 0.2 μm.

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