Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy

Autoři: Nandhini Ashok aff001;  Carl E. Bauer aff002
Působiště autorů: Department of Biology, Indiana University, Bloomington, Indiana, United States of America aff001;  Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America aff002
Vyšlo v časopise: Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy. PLoS Genet 16(3): e1008660. doi:10.1371/journal.pgen.1008660
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008660


Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.

Klíčová slova:

Bacterial spores – Cell walls – DNA replication – Flagella – Gene expression – Gene regulation – Gram negative bacteria – Protein synthesis


1. Berleman JE, Bauer CE. Characterization of cyst cell formation in the purple photosynthetic bacterium Rhodospirillum centenum. Microbiology, 2004; 150: 383–390. doi: 10.1099/mic.0.26846-0 14766916

2. Socolofsky MD, Wyss O. Resistance of the Azotobacter cyst. J Bacteriol, 1962; 84: 119–124. 13914732

3. Higgins D, Dworkin J. Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev, 2012; 36: 131–148. doi: 10.1111/j.1574-6976.2011.00310.x 22091839

4. Narula J, Fujita M, Igoshin OA. Functional requirements of cellular differentiation: lessons from Bacillus subtilis. Curr Opin Microbiol, 2016. 34: 38–46. doi: 10.1016/j.mib.2016.07.011 27501460

5. Marden JN, et al., Cyclic GMP controls Rhodospirillum centenum cyst development. Mol Microbiol, 2011; 79: 600–615. doi: 10.1111/j.1365-2958.2010.07513.x 21214648

6. Roychowdhury S, Dong Q, Bauer CE. DNA binding properties of a cGMP binding CRP homolog that controls development of metabolically dormant cysts of Rhodospirillum centenum. Microbiology, 2015; 161: 2256–2264. doi: 10.1099/mic.0.000172 26362215

7. Berleman JE, Hasselbring BM, Bauer CE. Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation. J Bacteriol, 2004; 186: 5834–5841. doi: 10.1128/JB.186.17.5834-5841.2004 15317789

8. Berleman JE, Bauer CE. Involvement of a Che-like signal transduction cascade in regulating cyst cell development in Rhodospirillum centenum. Mol Microbiol, 2005; 56:1457–1466. doi: 10.1111/j.1365-2958.2005.04646.x 15916598

9. Dong Q, Fang M, Roychowdhury S, Bauer CE. Mapping the CgrA regulon of Rhodospirillum centenum reveals a hierarchal network controlling Gram-negative cyst development. BMC Genomics, 2015; 16:1066 doi: 10.1186/s12864-015-2248-z 26673205

10. He K, Marden JN, Quardokus EM, Bauer CE. Phosphate flow between hybrid histidine kinases CheA3 and CheS3 controls Rhodospirillum centenum cyst formation. PLOS Genetics, 2013; 9: e1004002. doi: 10.1371/journal.pgen.1004002 24367276

11. Din N, Shoemaker CJ, Akin KL, Frederick C, Bird TH. Two putative histidine kinases are required for cyst formation in Rhodospirillum centenum. Arch Microbiol, 2011; 193: 209–222. doi: 10.1007/s00203-010-0664-7 21184217

12. Setlow P. Germination of Spores of Bacillus Species: What We Know and Do Not Know. J Bacteriol, 2014; 196: 1297–1305. doi: 10.1128/JB.01455-13 24488313

13. Setlow P, Wang S, Li YQ. Germination of Spores of the Orders Bacillales and Clostridiales. Annu Rev Microbiol, 2017; 71: 459–477. doi: 10.1146/annurev-micro-090816-093558 28697670

14. Driks A, Eichenberger P.,The Bacterial Spore: From Molecules to Systems. 2016; Washington, DC: ASM Press.

15. Bhattacharjee D, McAllister KN, Sorg JA. Germinants and their receptors in Clostridia. J Bacteriol, 2016; 198: 2767–2775. doi: 10.1128/JB.00405-16 27432831

16. Lin LP, Pankratz S, Sadoff HL. Ultrastructural and physiological changes occurring upon germination and outgrowth of Azotobacter vinelandii cysts. J bacteriol 1978; 135: 641–646. 681284

17. Wyss O, Neumnn MG, Socolofsky MD. Development and germination of the Azotobacter cyst. J biophys biochem cytology, 1961; 10: 555–565.

18. Loperfido B, Sadoff HL. Germination of Azotobacter vinelandii Cysts: Sequence of Macromolecular Synthesis and Nitrogen Fixation. J Bacteriol, 1973; 113: 841–846. 4690966

19. Stevenson LH, Socolofsky MD. 1966. Cyst formation and poly-b-hydroxybutyric acid accumulation in Azotobacter. J Bact., 1966; 91: 304–310. 5903098

20. Hitchins AD, Gould GE, Hurst A. The swelling of bacterial spores during germination and outgrowth. J Gen Microbiol, 1963; 30: 445–453. doi: 10.1099/00221287-30-3-445 13954805

21. Dong Q, Bauer CE. Transcriptome analysis of cyst formation in Rhodospirillum centenum reveals large global changes in expression during cyst development. BMC Genomics, 2015. 16: 68. doi: 10.1186/s12864-015-1250-9 25758168

22. Sinai L, Rosenberg A, Smith Y, Segev E, Ben-Yehuda. The molecular timeline of a reviving bacterial spore. Molecular Cell, 2015; 57: 695–707. doi: 10.1016/j.molcel.2014.12.019 25661487

23. Koch HG, Moser M, Muller M. Signal recognition particle-dependent protein targeting, universal to all kingdoms of life. Rev Physiol Biochem Pharmacol, 2003; 146: 55–94. doi: 10.1007/s10254-002-0002-9 12605305

24. Wild K, Rosendal KR, Sinning I. A structural step into the SRP cycle. Mol Microbiol, 2004. 53: 357–363. doi: 10.1111/j.1365-2958.2004.04139.x 15228518

25. Keijser BJ, et al., Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth. J Bacteriol, 2007; 189: 3624–3634. doi: 10.1128/JB.01736-06 17322312

26. Reusch RN, Sadoff HL. Lipid metabolism during encystment of Azotobacter vinelandii. J Bacteriol, 1981; 145: 889–895. 7462162

27. Reusch RN, Sadoff HL. Novel lipid components of the Azotobacter vinelandii cyst membrane. Nature, 1983; 302: 268–270. doi: 10.1038/302268a0 6835364

28. O'Leary WM. The fatty acids of bacteria. Bacteriological Reviews, 1962; 26: 421–447. 16350179

29. Touz ET, Mengin-Lecreulx D. Undecaprenyl phosphate synthesis. EcoSal Plus, 2008; 3.

30. Park JT, Uehara T. How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol Mol Biol Rev, 2008; 72: 211–227. doi: 10.1128/MMBR.00027-07 18535144

31. Bignell C, Thomas CM. The bacterial ParA-ParB partitioning proteins.J Biotechnol, 2001; 91: 1–34. doi: 10.1016/s0168-1656(01)00293-0 11522360

32. Cooper S, Helmstetter CE. Chromosome replication and the division cycle of Escherichia coli B/r. J Mol Biol, 1968; 31: 519–540. doi: 10.1016/0022-2836(68)90425-7 4866337

33. Fossum S, Crooke E, Skarstad K. Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli. EMBO J, 2007; 26: 4514–4522. doi: 10.1038/sj.emboj.7601871 17914458

34. Setlow P. Deoxyribonucleic acid synthesis and deoxynucleotide metabolism during bacterial spore germination. J Bacteriol, 1973; 114: 1099–1107. 4197265

35. Chakrabarty AM, Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis. Mol Microbiol, 1998; 28: 875–882. doi: 10.1046/j.1365-2958.1998.00846.x 9663675

36. Setlow P. Percent charging of transfer ribonucleic acid and levels of ppGpp and pppGpp in dormant and germinated spores of Bacillus megaterium. J Bacteriol, 1974; 118: 1067–1074. 4208410

37. Chen J, et al., Pyrophosphatase is essential for growth of Escherichia coli. Journal of bacteriology, 1990; 172: 5686–5689. doi: 10.1128/jb.172.10.5686-5689.1990 2170325

38. Heinonen JK. Biological role of inorganic pyrophosphate. 2001; Boston: Kluwer Academic Publishers. viii, 250 p.

39. Poon WW, et al., Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis. J Bacteriol, 2000; 182: 5139–5146. doi: 10.1128/jb.182.18.5139-5146.2000 10960098

40. Setlow P, Primus G. Protein metabolism during germination of Bacillus megaterium spores. I. Protein synthesis and amino acid metabolism. J Biol Chem, 1975; 250: 623–630. 803494

41. Kiyasu T, et al., Contribution of cysteine desulfurase (NifS protein) to the biotin synthase reaction of Escherichia coli. J Bacteriol, 2000; 182: 2879–2885. doi: 10.1128/jb.182.10.2879-2885.2000 10781558

42. Zheng L, White RH, Cash VL, Jack RF, Dean DR. Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis. Proc Natl Acad Sci, 1993; 90: 2754–2758. doi: 10.1073/pnas.90.7.2754 8464885

43. Nickens D, Fry CJ, Ragatz L, Bauer CE, Gest H. Biotype of the purple nonsulfur photosynthetic bacterium, Rhodospirillum centenum. Arch Microbiol, 1996; 165: 91–96.

44. Berleman JE, Bauer CE A che-like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum. Mol Microbiol, 2005; 55:1457–1466.

45. Lu YK, et al., Metabolic flexibility revealed in the genome of the cyst-forming alpha-1 proteobacterium Rhodospirillum centenum. BMC Genomics, 2010; 11: 325. doi: 10.1186/1471-2164-11-325 20500872

46. McClain J, Rollo DR, Rushing BG, Bauer CE. Rhodospirillum centenum utilizes separate motor and switch components to control lateral and polar flagellum rotation. J Bacteriol, 2002; 184; 2429–2438. doi: 10.1128/JB.184.9.2429-2438.2002 11948156

47. Ragatz L, Jiang Z-Y, Bauer C, Gest H. Macroscopic phototactic behavior of the purple photosynthetic bacterium Rhodospirillum centenum. Arch Microbiol, 1995. 163; 1–6. doi: 10.1007/bf00262196 7710317

48. Jiang Z-Y, Bauer CE. Analysis of a chemotaxis operon from Rhodospirillum centenum. J Bacteriol, 1997; 179: 5712–5719. doi: 10.1128/jb.179.18.5712-5719.1997 9294426

49. Jiang Z-Y, Gest H, Bauer CE. Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components. J Bacteriol, 1997; 179: 5720–5727. doi: 10.1128/jb.179.18.5720-5727.1997 9294427

50. He K, Dragnea V, Bauer CE. Adenylate charge regulates sensor kinase CheS3 to control cyst formation in Rhodospirillum centenum. MBio, 2015; 6: e00546–15. doi: 10.1128/mBio.00546-15 25944862

51. Pond FR., et al., R-body-producing bacteria. Microbiological Reviews, 1989; 53: 25–67. 2651865

52. Setlow B, Wahome PG, and Setlow P. Release of small molecules during germination of spores of Bacillus Species. Journal of bacteriology, 2008; 190: 4759–4763. doi: 10.1128/JB.00399-08 18469112

53. Hamamoto T, Hashimoto M, Hino M, Kitada M, Seto Y, Kudo T, Horikoshi K. Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C-125. Molecular Microbiology, 1994; 14: 939–946. doi: 10.1111/j.1365-2958.1994.tb01329.x 7715455

54. Hiramatsu T, Kodama K, Kuroda T, Mizushima T, Tsuchiya T. A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus. J Bacteriol, 1998; 180: 6642–6648. 9852009

55. Baykov AA, et al., Pyrophosphate-fueled Na+ and H+ transport in prokaryotes. Microbiol Mol Biol Rev, 2013; 77: 267–276. doi: 10.1128/MMBR.00003-13 23699258

56. Wu J, Dragnea V, Bauer CE. Redox responding sensor kinases, in Two-component systems in bacteria, Gross R. and Beier D., Editors. 2012, Horizon Scientific Press. p. 41–56.

57. Hobby G.L., Meyer K., and Chaffee E. Observations on the mechanism of action of penicillin. Exp. Biol. Med. 1942; 50, 281–285.

58. Joers Jõers A., Kaldalu N., and Tenson T. The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy. J. Bacteriol. 2010; 192, 3379–3384. doi: 10.1128/JB.00056-10 20435730

59. Van den Bergh B., Fauvart M., and Michiels J. Formation, physiology, ecology, evolution and clinical importance of bacterial persisters. FEMS Microbiol. Rev. 2017; 41, 219–251. doi: 10.1093/femsre/fux001 28333307

60. Roberts M. E. and Stewart P. S., Modelling protection from antimicrobial agents in biofilms through the formation of persister cells. Microbiology 2005. 151: p. 75–80 doi: 10.1099/mic.0.27385-0 15632427

61. Wood T.K., Song S., and Yamasaki R., Ribosome dependence of persister cell formation and resuscitation. J Microbiol, 2019. 57(3): p. 213–219. doi: 10.1007/s12275-019-8629-2 30806978

62. Song S. and Wood T.K., Persister cells resuscitate via ribosome modification by 23S rRNA pseudouridine synthase RluD. Environ Microbiol, 2019. https://doi.org/10.1111/1462-2920.14828

63. Fischer AH, et al., Mounting live cells attached to coverslips for microscopy. Cold Spring Harbor Protocols, 2008; 2008: pdb.prot4927. doi: 10.1101/pdb.prot4927 21356764

64. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014. 30: 2114–2120. doi: 10.1093/bioinformatics/btu170 24695404

65. Langmead B, et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol, 2009; 10: p. R25. doi: 10.1186/gb-2009-10-3-r25 19261174

66. Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics (Oxford, England), 2015; 31: 166–169.

67. Edgar R, Domrachev M, Lash AE. Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res, 2002; 30: 207–210. doi: 10.1093/nar/30.1.207 11752295

68. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 2014; 15: 550. doi: 10.1186/s13059-014-0550-8 25516281

69. Huerta-Cepas J, et al., eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Research, 2015; 44: D286–D293. doi: 10.1093/nar/gkv1248 26582926

70. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res, 2000; 28: 27–30. doi: 10.1093/nar/28.1.27 10592173

71. Kanehisa M, et al., KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res, 2017; 45: D353–d361. doi: 10.1093/nar/gkw1092 27899662

72. Kanehisa M, et al., New approach for understanding genome variations in KEGG. Nucleic Acids Res, 2019; 47: D590–d595. doi: 10.1093/nar/gky962 30321428

73. Kumka JE, Bauer CE. Analysis of the FnrL regulon in Rhodobacter capsulatus reveals limited regulon overlap with orthologues from Rhodobacter sphaeroides and Escherichia coli. BMC Genomics, 2015; 16: 895. doi: 10.1186/s12864-015-2162-4 26537891

74. Roychowdhury S., Dong Q., and Bauer C.E., DNA-binding properties of a cGMP-binding CRP homologue that controls development of metabolically dormant cysts of Rhodospirillum centenum. Microbiology, 2015. 161(11): p. 2256–2264. doi: 10.1099/mic.0.000172 26362215

75. Pfaffl M.W., et al., Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett, 2004. 26(6): p. 509–15. doi: 10.1023/b:bile.0000019559.84305.47 15127793

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