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Volume 147, Issue 10

Relationship between nucleic acid ratios and growth in Free

Abstract

is a pathogen whose distribution in a range of foodstuffs requires the development of methods for sensitive and rapid detection. Molecular biological methods usually rely on specific detection of rDNA directly amplified by the application of PCR to DNA extracts. Information on the metabolic status of populations would be valuable and can, in theory, be provided by quantitative detection of rRNA itself. Both fluorometry and oligonucleotide probe assays were applied to cultures to quantify RNA and DNA and produced more meaningful data than previous estimates for bacteria based on eukaryotic nucleic acid standards. In batch culture, the RNA–DNA ratio was found to be greatest at the end of exponential growth, after which RNA became degraded in accordance with the rapid decrease in viability. When the pH of the medium was controlled at neutrality, culture viability was dramatically extended and although RNA was degraded, intact DNA was maintained for the duration of the experiment. Ribosome numbers per cell were estimated to decrease from about 25000 observed during mid-exponential growth to about 600 during stationary phase, under pH-controlled conditions. Like , therefore, loses viability and rRNA rapidly once exponential growth has ceased in batch culture. However, much improved survival of a culturable population when pH is controlled has clear implications for the persistence of this species in buffered environments such as dairy products.

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Keyword(s): fluorometry , RNA–DNA ratio and rRNA
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2001-10-01
2024-04-20
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References

  1. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. 1990a; Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analysing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
     [Google Scholar]
  2. Amann R. I., Krumholz L., Stahl D. A. 1990b; Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J Bacteriol 172:762–770
     [Google Scholar]
  3. Bessesen M. T., Luo Q., Rotbart H. A., Blaser M., Ellison R. T. 1990; Detection of Listeria monocytogenes by using the polymerase chain reaction. Appl Environ Microbiol 56:2930–2932
     [Google Scholar]
  4. Blais B. W., Turner G., Sooknanan R., Malek L. T. 1997; A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences. Appl Environ Microbiol 63:310–313
     [Google Scholar]
  5. Blumberg D. B. 1987; Creating a ribonuclease free environment . . Methods Enzymol 15. 20–24
     [Google Scholar]
  6. Bremer H., Dennis P. P. 1987; Modulation of chemical composition and other parameters of the cell by growth rate. . In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology pp 1527–1542 Edited by Neidhardt F. C., Ingraham J. L., Low K. B., Magasanik B., Shaechter M., Umbarger H. E. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  7. Cangelosi G. A., Brabant W. H. 1997; Depletion of pre-16S rRNA in starved Escherichia coli cells. J Bacteriol 179:4457–4463
     [Google Scholar]
  8. Cano R. J., Norton D. M., Inzunza A. E., Sanchez J. G., Oste C. 1995; Polymerase chain reaction assay coupled with fluorescence detection on microwell plates for Listeria monocytogenes in foods. J Food Prot 58:614–620
     [Google Scholar]
  9. Collins M. D., Wallbanks S., Lane D. J., Shah J., Nietupski R., Smida J., Dorsch M., Stackebrandt E. 1991; Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S ribosomal-RNA. Int J Syst Bacteriol 41:240–246 [CrossRef]
     [Google Scholar]
  10. Cooray K. J., Nishibori T., Xiong H., Matsuyama T., Fujita M., Mitsuyama M. 1994; Detection of multiple virulence-associated genes of Listeria monocytogenes by PCR in artificially contaminated milk samples. Appl Environ Microbiol 60:3023–3026
     [Google Scholar]
  11. Davis B. D., Luger S. M., Tai P. C. 1986; Role of ribosome degradation in the death of starved Escherichia coli cells. J Bacteriol 166:439–445
     [Google Scholar]
  12. Dell’Anno A., Fabiano M., Duineveld G. C. A., Kok A., Danovaro R. 1998; Nucleic acid (DNA, RNA) quantification and RNA/DNA ratio determination in marine sediments: comparison of spectrophotometric, fluorometric, and high performance liquid chromatography methods and estimation of detrital DNA. Appl Environ Microbiol 64:3238–3245
     [Google Scholar]
  13. Edwards U., Rogall T., Blocker H., Emde M., Bottger E. C. 1989; Isolation and direct complete nucleotide sequence of entire genes. Characterisation of a gene coding for 16S ribosomal RNA . Nucleic Acids Res 17:7843–7853 [CrossRef]
     [Google Scholar]
  14. Farber J. M., Peterkin P. I. 1991; Listeria monocytogenes , a food-borne pathogen. Microbiol Rev 55:476–511
     [Google Scholar]
  15. Ferron P., Michard J. 1993; Distribution of Listeria spp. in confectioners’ pastries from western France: comparison of enrichment methods. Int J Food Microbiol 18:289–303 [CrossRef]
     [Google Scholar]
  16. Flardh K., Cohen P. S., Kjelleberg S. 1992; Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956. J Bacteriol 174:6780–6788
     [Google Scholar]
  17. Gausing K. 1977; Regulation of ribosome production in Escherichia coli : synthesis and stability of ribosomal RNA at different growth rates. J Mol Biol 115:335–354 [CrossRef]
     [Google Scholar]
  18. George S. M., Lund B. M. 1992; The effect of culture medium and aeration on growth of Listeria monocytogenes at pH 4·5. Lett Appl Microbiol 15:49–52 [CrossRef]
     [Google Scholar]
  19. Gilbert R. J., Miller K. L., Roberts D. 1989; Listeria monocytogenes and chilled foods. Lancet i:383–384
     [Google Scholar]
  20. Hiorns W. D., Hastings R. C., Head I. M., McCarthy A. J., Saunders J. R., Pickup R. W., Hall G. H. 1995; Amplification of 16S ribosomal RNA genes of autotrophic ammonia oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment. Microbiology 141:2793–2800 [CrossRef]
     [Google Scholar]
  21. Hof H., Rocourt J. 1992; Is any strain of Listeria monocytogenes detected in food a health risk?. Int J Food Microbiol 16:173–181 [CrossRef]
     [Google Scholar]
  22. Kaplan R., Apirion D. 1975a; The fate of ribosomes in Escherichia coli cells starved of a carbon source. J Biol Chem 250:1854–1863
     [Google Scholar]
  23. Kaplan R., Apirion D. 1975b; Decay of ribosomal ribonucleic acid in Escherichia coli cells starved for various nutrients. J Biol Chem 250:3174–3178
     [Google Scholar]
  24. Kerkhof L., Kemp P. 1999; Small ribosomal RNA content in marine Proteobacteria during non-steady-state growth. FEMS Microbiol Ecol 30:253–260 [CrossRef]
     [Google Scholar]
  25. Kerkhof L., Ward B. B. 1993; Comparison of nucleic acid hybridization and fluorometry for measurement of the relationship between RNA/DNA ratio and growth rate in a marine bacterium. Appl Environ Microbiol 59:1303–1309
     [Google Scholar]
  26. Kjellgaard N. O., Kurland C. G. 1963; The distribution of soluble and ribosomal RNA as a function of growth rate. J Mol Biol 6:341–348 [CrossRef]
     [Google Scholar]
  27. Klein P. G., Juneja V. K. 1997; Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Appl Environ Microbiol 53:4441–4448
     [Google Scholar]
  28. Kramer J. G., Singleton F. L. 1992; Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery. Appl Environ Microbiol 58:201–207
     [Google Scholar]
  29. LePecq J., Paoletti C. 1966; A new fluorometric method for RNA and DNA determination. Anal Biochem 17:100–107 [CrossRef]
     [Google Scholar]
  30. McLauchlin J., Pini P. N. 1989; The rapid demonstration and presumptive identification of Listeria monocytogenes in food using monoclonal antibodies in a direct immunofluoresence test (DIFT. Lett Appl Microbiol 8:25–27 [CrossRef]
     [Google Scholar]
  31. Makino S.-I., Okada Y., Maruyama T. 1995; A new method for direct detection of Listeria monocytogenes from foods by PCR. Appl Environ Microbiol 61:3745–3747
     [Google Scholar]
  32. Michel C. A., Cossart P. 1992; Physical map of the Listeria monocytogenes chromosome. J Bacteriol 174:7098–7103
     [Google Scholar]
  33. Muttray A. F., Mohn W. W. 1998; RNA/DNA ratio as an indicator of metabolic activity in resin acid-degrading bacteria. Water Sci Technol 37:89–93
     [Google Scholar]
  34. Neidhardt F. C., Ingraham J. L., Schaechter M. 1990 Physiology of the Bacterial Cell: a Molecular Approach Sunderland, MA: Sinauer Associates;
     [Google Scholar]
  35. Nørrung B., Skovgaard N. 1993; Application of multilocus enzyme electrophoresis in studies of the epidemiology of Listeria monocytogenes in Denmark. Appl Environ Microbiol 59:2817–2822
     [Google Scholar]
  36. Pine L., Malcolm G. B., Brooks J. B., Danshevar M. I. 1989; Physiological studies on the growth and utilization of sugars by Listeria species. Can J Microbiol 35:245–254 [CrossRef]
     [Google Scholar]
  37. Powell H. A., Gooding C. M., Garett S. D., Lund B. M., McKee R. A. 1994; Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction. Lett Appl Microbiol 18:59–61 [CrossRef]
     [Google Scholar]
  38. Rosset R., Julien J., Monier R. 1966; Ribonucleic acid composition of bacteria as a function of growth rate. J Mol Biol 18:308–320 [CrossRef]
     [Google Scholar]
  39. Scheu P., Gasch A., Berghof K. 1999; Rapid detection of Listeria monocytogenes by PCR-ELISA. Lett Appl Microbiol 29:416–420 [CrossRef]
     [Google Scholar]
  40. Smith J. L., Buchanan R. L. 1990; Identification of supplements that enhance the recovery of Listeria monocytogenes on modified Vogel–Johnson agar. J Food Saf 10:155–163
     [Google Scholar]
  41. Sokal R. R., Rohlf F. J. 1995 Biometry: The Principles and Practice of Statistics in Biological Research , 3rd edn. New York: Freeman;
     [Google Scholar]
  42. ter-Steeg P. F., Pieterman F. H. 1991; Effects of aeration conditions, temperature and pH on growth rate and maintenance-energy demands of Listeria innocua (the physiological substitute of L. monocytogenes ) in a (semi-) chemically defined medium. Confidential Laboratory Progress Report Vlaardingen, The Netherlands: Unilever Research;
     [Google Scholar]
  43. Verheul A., Hagting A., Amezaga M.-R., Booth I. R., Rombouts F. M., Abee T. 1995; A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth. Appl Environ Microbiol 61:225–233
     [Google Scholar]
  44. Wada A., Yamazaki Y., Fujita N., Ishihama A. 1990; Structure and probable genetic location of a ribosome modulation factor associated with 100S ribosome ribosomes in stationary phase Escherichia coli cells. Proc Natl Acad Sci USA 87:2657–2661 [CrossRef]
     [Google Scholar]
  45. Wang R. F., Cao W.-W., Johnson M. G. 1992; 16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods. Appl Environ Microbiol 58:2827–2831
     [Google Scholar]
  46. Young K. M., Fogeding P. M. 1993; Acetic, lactic and citric acids and pH inhibition of Listeria monocytogenes Scott A and the effect on intracellular pH. J Appl Bacteriol 74:515–520
     [Google Scholar]
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Relationship between nucleic acid ratios and growth in Listeria monocytogenes
Microbiology 147, 2689 (2001); https://doi.org/10.1099/00221287-147-10-2689
/content/journal/micro/10.1099/00221287-147-10-2689
/content/journal/micro/10.1099/00221287-147-10-2689
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