Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Mar;85(5):1334–1338. doi: 10.1073/pnas.85.5.1334

Size of bacterial ice-nucleation sites measured in situ by radiation inactivation analysis

Arepura G Govindarajan 1, Steven E Lindow 1
PMCID: PMC279765  PMID: 16593912

Abstract

Four bacterial species are known to catalyze ice formation at temperatures just below 0°C. To better understand the relationship between the molecular structure of bacterial ice-nucleation site(s) and the quantitative and qualitative features of the ice-nucleation-active phenotype, we determined by γ-radiation analysis the in situ size of ice-nucleation sites in strains of Pseudomonas syringae and Erwinia herbicola and in Escherichia coli HB101 carrying the plasmid pICE1.1 (containing a 4-kilobase DNA insert from P. syringae that confers ice-nucleation activity). Lyophilized cells of each bacterial strain were irradiated with a flux of γ radiation from 0 to 10.2 Mrad (1 Mrad = 106 J/kg). Differential concentrations of active ice nuclei decreased as a first-order function of radiation dose in all strains as temperature was decreased from -2°C to -14°C in 1°C intervals. Sizes of ice nuclei were calculated from the γ-radiation flux at which 37% of initial ice nuclei active within each 1°C temperature interval remained. The minimum mass of a functional ice nucleus, active only between -12°C and -13°C, was about 150 kDa for all strains. The size of ice nuclei increased logarithmically with increasing temperature from -12°C to -2°C, where the estimated nucleant mass was 19,000 kDa. The ice nucleant in these three bacterial species may represent an oligomeric structure, composed at least in part of an ice gene product that can self-associate to assume many possible sizes.

Keywords: Pseudomonas syringae, Erwinia herbicola, ice gene, γ radiation

Full text

PDF
1334

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Angelides K. J., Nutter T. J., Elmer L. W., Kempner E. S. Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel. J Biol Chem. 1985 Mar 25;260(6):3431–3439. [PubMed] [Google Scholar]
  2. Goldkorn T., Rimon G., Kempner E. S., Kaback H. R. Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1021–1025. doi: 10.1073/pnas.81.4.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. HUTCHINSON F., PRESTON A., VOGEL B. Radiation sensitivity of enzymes in wet and in dry yeast cells. Radiat Res. 1957 Nov;7(5):465–472. [PubMed] [Google Scholar]
  4. JAGGER J., WILSON D. Deuteron cross section of beta-amylase in vitro and in vivo. Radiat Res. 1955 Oct;3(2):127–134. [PubMed] [Google Scholar]
  5. Kempner E. S., Haigler H. T. The influence of low temperature on the radiation sensitivity of enzymes. J Biol Chem. 1982 Nov 25;257(22):13297–13299. [PubMed] [Google Scholar]
  6. Kempner E. S., Miller J. H. Radiation inactivation of glutamate dehydrogenase hexamer: lack of energy transfer between subunits. Science. 1983 Nov 11;222(4624):586–589. doi: 10.1126/science.6635656. [DOI] [PubMed] [Google Scholar]
  7. Kempner E. S., Miller J. H., Schlegel W., Hearon J. Z. The functional unit of polyenzymes. Determination by radiation inactivation. J Biol Chem. 1980 Jul 25;255(14):6826–6831. [PubMed] [Google Scholar]
  8. Kempner E. S., Schlegel W. Size determination of enzymes by radiation inactivation. Anal Biochem. 1979 Jan 1;92(1):2–10. doi: 10.1016/0003-2697(79)90617-1. [DOI] [PubMed] [Google Scholar]
  9. Kepner G. R., Macey R. I. Membrane enzyme systems. Molecular size determinations by radiation inactivation. Biochim Biophys Acta. 1968 Sep 17;163(2):188–203. doi: 10.1016/0005-2736(68)90097-7. [DOI] [PubMed] [Google Scholar]
  10. Kozloff L. M., Lute M., Westaway D. Phosphatidylinositol as a Component of the Ice Nucleating Site of Pseudomonas syringae and Erwinia herbiola. Science. 1984 Nov 16;226(4676):845–846. doi: 10.1126/science.226.4676.845. [DOI] [PubMed] [Google Scholar]
  11. Kozloff L. M., Schofield M. A., Lute M. Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola. J Bacteriol. 1983 Jan;153(1):222–231. doi: 10.1128/jb.153.1.222-231.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lad P. M., Preston M. S., Welton A. F., Nielsen T. B., Rodbell M. Effects of phospholipase A2 and filipin on the activation of adenylate cyclase. Biochim Biophys Acta. 1979 Mar 8;551(2):368–381. doi: 10.1016/0005-2736(89)90013-8. [DOI] [PubMed] [Google Scholar]
  13. Lindow S. E., Arny D. C., Upper C. D. Distribution of ice nucleation-active bacteria on plants in nature. Appl Environ Microbiol. 1978 Dec;36(6):831–838. doi: 10.1128/aem.36.6.831-838.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lindow S. E., Hirano S. S., Barchet W. R., Arny D. C., Upper C. D. Relationship between Ice Nucleation Frequency of Bacteria and Frost Injury. Plant Physiol. 1982 Oct;70(4):1090–1093. doi: 10.1104/pp.70.4.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maki L. R., Galyan E. L., Chang-Chien M. M., Caldwell D. R. Ice nucleation induced by pseudomonas syringae. Appl Microbiol. 1974 Sep;28(3):456–459. doi: 10.1128/am.28.3.456-459.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nielsen T. B., Lad P. M., Preston M. S., Kempner E., Schlegel W., Rodbell M. Structure of the turkey erythrocyte adenylate cyclase system. Proc Natl Acad Sci U S A. 1981 Feb;78(2):722–726. doi: 10.1073/pnas.78.2.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Noël H., Beauregard G., Potier M., Bleau G., Chapdelaine A., Roberts K. D. The target sizes of the in situ and solubilized forms of human placental steroid sulfatase as measured by radiation inactivation. Biochim Biophys Acta. 1983 Jul 5;758(1):88–90. doi: 10.1016/0304-4165(83)90013-2. [DOI] [PubMed] [Google Scholar]
  18. Orser C., Staskawicz B. J., Panopoulos N. J., Dahlbeck D., Lindow S. E. Cloning and expression of bacterial ice nucleation genes in Escherichia coli. J Bacteriol. 1985 Oct;164(1):359–366. doi: 10.1128/jb.164.1.359-366.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. POWELL W. F., POLLARD E. Radiation inactivation of enzymes inside and outside intact cells. Radiat Res. 1955 Apr;2(2):109–118. [PubMed] [Google Scholar]
  20. Saccomani G., Sachs G., Cuppoletti J., Jung C. Y. Target molecular weight of the gastric (H+ + K+)-ATPase functional and structural molecular size. J Biol Chem. 1981 Aug 10;256(15):7727–7729. [PubMed] [Google Scholar]
  21. Schlegel W., Kempner E. S., Rodbell M. Activation of adenylate cyclase in hepatic membranes involves interactions of the catalytic unit with multimeric complexes of regulatory proteins. J Biol Chem. 1979 Jun 25;254(12):5168–5176. [PubMed] [Google Scholar]
  22. Schwartz A. L., Steer C. J., Kempner E. S. Functional size of the human asialoglycoprotein receptor as determined by radiation inactivation. J Biol Chem. 1984 Oct 10;259(19):12025–12029. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES