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Volume 164, Issue 9

Editor's Choice Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation Open Access

Abstract

Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): c.f.u. , colony formation , Escherichia coli , fabB , fatty acid and MPN
© 2018 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2018-07-20
2024-04-16
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References

  1. Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 1985; 39:321–346 [View Article][PubMed]
     [Google Scholar]
  2. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 1995; 59:143–169[PubMed]
     [Google Scholar]
  3. Ohta H, Hattori T. Bacteria sensitive to nutrient broth medium in terrestrial environments. Soil Sci Plant Nutr 1980; 26:99–107
     [Google Scholar]
  4. Bussmann I, Philipp B, Schink B. Factors influencing the cultivability of lake water bacteria. J Microbiol Methods 2001; 47:41–50 [View Article][PubMed]
     [Google Scholar]
  5. Button DK, Schut F, Quang P, Martin R, Robertson BR. Viability and isolation of marine bacteria by dilution culture: theory, procedures, and initial results. Appl Environ Microbiol 1993; 59:881–891[PubMed]
     [Google Scholar]
  6. Schut F, de Vries EJ, Gottschal JC, Robertson BR, Harder W et al. Isolation of typical marine bacteria by dilution culture: growth, maintenance, and characteristics of isolates under laboratory conditions. Appl Environ Microbiol 1993; 59:2150–2160[PubMed]
     [Google Scholar]
  7. Connon SA, Giovannoni SJ. High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microbiol 2002; 68:3878–3885[PubMed]
     [Google Scholar]
  8. Mizunoe Y, Wai SN, Takade A, Yoshida S. Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds. Arch Microbiol 1999; 172:63–67 [View Article][PubMed]
     [Google Scholar]
  9. Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean's surface. PLoS One 2011; 6:e16805 [View Article][PubMed]
     [Google Scholar]
  10. Tanaka T, Kawasaki K, Daimon S, Kitagawa W, Yamamoto K et al. A hidden pitfall in the preparation of agar media undermines microorganism cultivability. Appl Environ Microbiol 2014; 80:7659–7666 [View Article][PubMed]
     [Google Scholar]
  11. Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and VerrucomicrobiaImproved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ Microbiol 2002; 68:2391–2396
     [Google Scholar]
  12. Tamaki H, Hanada S, Sekiguchi Y, Tanaka Y, Kamagata Y. Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment. Environ Microbiol 2009; 11:1827–1834 [View Article][PubMed]
     [Google Scholar]
  13. Tamaki H, Sekiguchi Y, Hanada S, Nakamura K, Nomura N et al. Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake by molecular and improved cultivation-based techniques. Appl Environ Microbiol 2005; 71:2162–2169 [View Article][PubMed]
     [Google Scholar]
  14. Suzuki S, Horinouchi S, Beppu T. Growth of a Tryptophanase-producing Thermophile, Symbiobacterium thermophilum gen. nov., sp. nov., Is Dependent on Co-culture with a Bacillus sp. J Gen Microbiol 1988; 134:2353–2362
     [Google Scholar]
  15. Tanaka Y, Hanada S, Manome A, Tsuchida T, Kurane R et al. Catellibacterium nectariphilum gen. nov., sp. nov., which requires a diffusible compound from a strain related to the genus Sphingomonas for vigorous growth. Int J Syst Evol Microbiol 2004; 54:955–959 [View Article][PubMed]
     [Google Scholar]
  16. Morris JJ, Kirkegaard R, Szul MJ, Johnson ZI, Zinser ER. Facilitation of robust growth of Prochlorococcus colonies and dilute liquid cultures by "helper" heterotrophic bacteria. Appl Environ Microbiol 2008; 74:4530–4534 [View Article][PubMed]
     [Google Scholar]
  17. D'Onofrio A, Crawford JM, Stewart EJ, Witt K, Gavrish E et al. Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chem Biol 2010; 17:254–264 [View Article][PubMed]
     [Google Scholar]
  18. Bruns A, Cypionka H, Overmann J. Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea. Appl Environ Microbiol 2002; 68:3978–3987[PubMed]
     [Google Scholar]
  19. Bruns A, Nübel U, Cypionka H, Overmann J. Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton. Appl Environ Microbiol 2003; 69:1980–1989[PubMed]
     [Google Scholar]
  20. Zengler K, Toledo G, Rappe M, Elkins J, Mathur EJ et al. Cultivating the uncultured. Proc Natl Acad Sci USA 2002; 99:15681–15686 [View Article][PubMed]
     [Google Scholar]
  21. Ingham CJ, Sprenkels A, Bomer J, Molenaar D, van den Berg A et al. The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms. Proc Natl Acad Sci USA 2007; 104:18217–18222 [View Article][PubMed]
     [Google Scholar]
  22. Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A et al. Use of ichip for high-throughput in situ cultivation of "uncultivable" microbial species. Appl Environ Microbiol 2010; 76:2445–2450 [View Article][PubMed]
     [Google Scholar]
  23. Eun YJ, Utada AS, Copeland MF, Takeuchi S, Weibel DB. Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation. ACS Chem Biol 2011; 6:260–266 [View Article][PubMed]
     [Google Scholar]
  24. Ma L, Kim J, Hatzenpichler R, Karymov MA, Hubert N et al. Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa. Proc Natl Acad Sci USA 2014; 111:9768–9773 [View Article][PubMed]
     [Google Scholar]
  25. Torsvik V, Goksøyr J, Daae FL. High diversity in DNA of soil bacteria. Appl Environ Microbiol 1990; 56:782–787[PubMed]
     [Google Scholar]
  26. Gans J, Wolinsky M, Dunbar J. Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 2005; 309:1387–1390 [View Article][PubMed]
     [Google Scholar]
  27. Rappé MS, Giovannoni SJ. The uncultured microbial majority. Annu Rev Microbiol 2003; 57:369–394 [View Article][PubMed]
     [Google Scholar]
  28. Achtman M, Wagner M. Microbial diversity and the genetic nature of microbial species. Nat Rev Microbiol 2008; 6:431–440 [View Article][PubMed]
     [Google Scholar]
  29. Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 2013; 499:431–437 [View Article][PubMed]
     [Google Scholar]
  30. Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ et al. A new view of the tree of life. Nat Microbiol 2016; 1:16048 [View Article][PubMed]
     [Google Scholar]
  31. Kogure K, Simidu U, Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 1979; 25:415–420[PubMed]
     [Google Scholar]
  32. Shigematsu T, Ueno S, Tsuchida Y, Hayashi M, Okonogi H et al. Comparative analyses of viable bacterial counts in foods and seawater under microplate based liquid- and conventional agar plate cultivation: increased culturability of marine bacteria under liquid cultivation. Biosci Biotechnol Biochem 2007; 71:3093–3097 [View Article][PubMed]
     [Google Scholar]
  33. Shigematsu T, Hayashi M, Kikuchi I, Ueno S, Masaki H et al. A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment. FEMS Microbiol Lett 2009; 293:240–247 [View Article][PubMed]
     [Google Scholar]
  34. Rappé MS, Connon SA, Vergin KL, Giovannoni SJ. Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 2002; 418:630–633 [View Article][PubMed]
     [Google Scholar]
  35. Isono K, Krauss J, Hirota Y. Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins. Mol Gen Genet 1976; 149:297–302[PubMed]
     [Google Scholar]
  36. Saka K, Tadenuma M, Nakade S, Tanaka N, Sugawara H et al. A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies. DNA Res 2005; 12:63–68[PubMed]
     [Google Scholar]
  37. Cronan JE, Birge CH, Vagelos PR. Evidence for two genes specifically involved in unsaturated fatty acid biosynthesis in Escherichia coli. J Bacteriol 1969; 100:601–604[PubMed]
     [Google Scholar]
  38. Garwin JL, Klages AL, Cronan JE. β-ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. J Biol Chem 1980; 255:3263–3265[PubMed]
     [Google Scholar]
  39. Jones KL, Keasling JD. Construction and characterization of F plasmid-based expression vectors. Biotechnol Bioeng 1998; 59:659–665[PubMed]
     [Google Scholar]
  40. Halvorson HO, Ziegler NR. Application of statistics to problems in bacteriology: I. A means of determining bacterial population by the dilution method. J Bacteriol 1933; 25:101–121[PubMed]
     [Google Scholar]
  41. Garwin JL, Klages AL, Cronan JE. Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli. J Biol Chem 1980; 255:11949–11956[PubMed]
     [Google Scholar]
  42. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006; 2:2006.0008 [View Article][PubMed]
     [Google Scholar]
  43. Cronan JE, Gelmann EP. An estimate of the minimum amount of unsaturated fatty acid required for growth of Escherichia coli. J Biol Chem 1973; 248:1188–1195[PubMed]
     [Google Scholar]
  44. Broekman JH, Steenbakkers JF. Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J Bacteriol 1974; 117:971–977[PubMed]
     [Google Scholar]
  45. D'Agnolo G, Rosenfeld IS, Awaya J, Omura S, Vagelos PR. Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of beta-ketoacyl-acyl carrier protein synthetase. Biochim Biophys Acta 1973; 326:155–166[PubMed]
     [Google Scholar]
  46. Price AC, Choi K-H, Heath RJ, Li Z, White SW et al. Inhibition of β-ketoacyl-acyl carrier protein synthases by thiolactomycin and cerulenin. J Biol Chem 2001; 276:6551–6559
     [Google Scholar]
  47. Omura S. The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis. Bacteriol Rev 1976; 40:681–697[PubMed]
     [Google Scholar]
  48. Schujman GE, Choi KH, Altabe S, Rock CO, de Mendoza D. Response of Bacillus subtilis to cerulenin and acquisition of resistance. J Bacteriol 2001; 183:3032–3040 [View Article][PubMed]
     [Google Scholar]
  49. Trajtenberg F, Altabe S, Larrieux N, Ficarra F, de Mendoza D et al. Structural insights into bacterial resistance to cerulenin. FEBS J 2014; 281:2324–2338 [View Article][PubMed]
     [Google Scholar]
  50. Heath RJ, Yu YT, Shapiro MA, Olson E, Rock CO. Broad spectrum antimicrobial biocides target the FabI component of fatty acid synthesis. J Biol Chem 1998; 273:30316–30320 [View Article][PubMed]
     [Google Scholar]
  51. Xu HS, Roberts N, Singleton FL, Attwell RW, Grimes DJ et al. Survival and viability of non culturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb Ecol 1982; 8:313–323 [View Article][PubMed]
     [Google Scholar]
  52. Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA et al. Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Bio/Technology 1985; 3:817–820
     [Google Scholar]
  53. Li L, Mendis N, Trigui H, Oliver JD, Faucher SP. The importance of the viable but non-culturable state in human bacterial pathogens. Frontiers Microbiol 2014; 5:258
     [Google Scholar]
  54. Silbert DF, Vagelos PR. Fatty acid mutant of E. coli lacking a beta-hydroxydecanoyl thioester dehydrase. Proc Natl Acad Sci USA 1967; 58:1579–1586[PubMed]
     [Google Scholar]
  55. Heath RJ, Rock CO. Roles of the FabA and FabZ β-Hydroxyacyl-Acyl carrier protein dehydratases in Escherichia coli fatty acid biosynthesis. J Biol Chem 1996; 271:27795–27801[PubMed]
     [Google Scholar]
  56. Nosho K, Fukushima H, Asai T, Nishio M, Takamaru R et al. cAMP-CRP acts as a key regulator for the viable but non-culturable state in Escherichia coli. Microbiology 2018; 164:410–419 [View Article][PubMed]
     [Google Scholar]
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Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation
Microbiology 164, 1122 (2018); https://doi.org/10.1099/mic.0.000673
/content/journal/micro/10.1099/mic.0.000673
/content/journal/micro/10.1099/mic.0.000673
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