Background/Aims: This work is a study of the ability of three recombinant Zymomonas mobilis strains to release ice nucleators into their growth medium. Methods: The recombinant ice+Z. mobilis cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. Results: Cell-free ice nucleators were obtained from all the recombinant ice+Z. mobilis cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 μm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. Conclusion: These findings indicate that the recombinant ice+Z. mobilis cells could be valuable for future use as a source of active cell-free ice nucleation protein.

1.
Afendra AS, Drainas C: Expression and stability of a recombinant plasmid in Zymomonas mobilis and Escherichia coli. J Gen Microbiol 1987;133:127-134.
[PubMed]
2.
Barlett GR: Phosphorus assay in column chromatography. J Biol Chem 1959;234:466-468.
[PubMed]
3.
Diefenbach RJ, Duggleby RG: Pyruvate decarboxylase from Zymomonas mobilis: structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion. Biochem J 1991;276:439-445.
[PubMed]
4.
Doelle HW, Preusser H-J, Rostek H: Electron microscopic investigations of Zymomonas mobilis cells grown in low and high glucose concentrations. Eur J Appl Microbiol Biotechnol 1982;16:136-141.
5.
Drainas C, Vartholomatos G, Panopoulos NJ: The ice nucleation gene from Pseudomonas syringae as a sensitive gene reporter for promoter analysis in Zymomonas mobilis. Appl Environ Microbiol 1995;61:273-277.
[PubMed]
6.
Govindarajan AG, Lindow SE: Phospholipid requirement for expression of ice nuclei in Pseudomonas syringae and in vitro. J Biol Chem 1988;263:9333-9338.
[PubMed]
7.
Graether SP, Jia Z: Modeling Pseudomonas syringae ice-nucleation protein as a β-helical protein. Biophys J 2001;80:1169-1173.
[PubMed]
8.
Gross DC, Proebsting EL, Maccrindle-Zimmerman H: Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in prunus wood. Plant Physiol 1988;88:915-922.
[PubMed]
9.
Gurian-Sherman D, Lindow SE: Bacterial ice nucleation: significance and molecular basis. FASEB J 1993;7:1338-1343.
[PubMed]
10.
Gurian-Sherman D, Lindow S: Differential effects of growth temperature on ice nuclei active at different temperatures that are produced by cells of Pseudomonas syringae. Cryobiology 1995;32:129-138.
[PubMed]
11.
Kates M: Techniques of lipidology: isolation, analysis and identification of lipids; in Work TS, Work E (eds): Laboratory Techniques in Biochemistry and Molecular Biology. Amsterdam, North-Holland, 1972, pp 347-353.
12.
Kieft TL, Ruscetti T: Characterization of biological ice nuclei from a lichen. J Bacteriol 1990;172:3519-3523.
[PubMed]
13.
Kozloff LM, Turner MA, Arellano F: Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes. J Bacteriol 1991;173:6528-6536.
[PubMed]
14.
Lindow SE, Arny DC, Upper CD: Distribution of ice nucleation-active bacteria on plants in nature. Appl Environ Microbiol 1978;36:831-838.
[PubMed]
15.
Lindow SE, Arny DC, Upper CD: Bacterial ice nucleation: a factor in frost injury to plants. Plant Physiol 1982;70:1084-1089.
[PubMed]
16.
Lindgren PB, Frederick R, Govindarajan AG, Panopoulos NJ, Staskawicz BJ, Lindow SE: An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola. EMBO J 1989;8:1291-1301.
[PubMed]
17.
Lundheim R: Ice nucleation in seaweeds in relation to verticalzonation. J Phycol 1997;33:739-742.
18.
Maki LR, Galyan EL, Chang-Chien MM, Caldwell DR: Ice nucleation induced by Pseudomonas syringae. Appl Microbiol 1974;28:456-459.
[PubMed]
19.
Maki LR, Willoughby KJ: Bacteria as biogenic sources of freezing nuclei. J Appl Meteorol 1978;17:1049-1053.
20.
Margaritis A, Bassi AS: Principles and biotechnological applications of bacterial ice nucleation. Crit Rev Biotechnol 1991;11:277-295.
[PubMed]
21.
Motulsky HJ: Analyzing Data with GraphPad Prism. San Diego, GraphPad Software, 1998. http://www.graphpad.com/faq/file/AnalyzingDataPrism3.pdf.
22.
Motulsky HJ: GraphPad Prism version 4.0 Statistics Guide: Statistical Analyses for Laboratory and Clinical Researchers. San Diego, GraphPad Software, 2003. http://www.graphpad.com/manuals/Prism4/StatisticsGuide.pdf.
23.
Muryoi N, Kawahara H, Obata H: Properties of a novel extracellular cell-free ice nuclei from ice-nucleating Pseudomonas antarctica IN-74. Biosci Biotechnol Biochem 2003;67:1950-1958.
[PubMed]
24.
Phelps P, Giddings TH, Prochoda M, Fall R: Release of cell-free ice nuclei by Erwinia herbicola. J Bacteriol 1986;167:496-502.
[PubMed]
25.
Pouleur S, Richard C, Martin JG, Antoun H: Ice nucleation activity in Fusarium acuminatum and Fusarium avenaceum. Appl Environ Microbiol 1992;58:2960-2964.
[PubMed]
26.
Sahm H, Bringer-Meyer S, Sprenger GA: The Prokaryotes, ed 2. New York, Springer, 2006.
27.
Schnell RC, Vali G: Atmospheric ice nuclei from decomposing vegetation. Nature 1972;236:163-165.
28.
Tegos G, Vargas C, Perysinakis A, Koukkou AI, Christogianni A, Nieto JJ, Ventosa A, Drainas C: Release of cell-free ice nuclei from Halomonas elongata expressing the ice nucleation gene inaZ of Pseudomonas syringae. J Appl Microbiol 2000;89:785-792.
[PubMed]
29.
Turner MA, Arellano F, Kozloff LM: Three separate classes of bacterial ice nucleation structures. J Bacteriol 1990;172:2521-2526.
[PubMed]
30.
Vali G: Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids. J Atmos Sci 1971;28:402-409.
31.
Vali G: Freezing rate due to heterologous nucleation. J Atmos Sci 1994;51:1843-1856.
32.
Varsaki A, Afendra AS, Vartholomatos G, Tegos G, Drainas C: Production of ice nuclei from two recombinant Zymomonas mobilis strains employing the inaZ gene of Pseudomonas syringae. Biotechnol Lett 1998;20:647-651.
33.
Vincent R, Nadeau D: A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay. Anal Biochem 1983;135:355-362.
[PubMed]
34.
Wolanczyk JP, Storey KB, Baust JG: Ice nucleating activity in the blood of the freeze-tolerant frog, Rana sylvatica. Cryobiology 1990;27:328-335.
[PubMed]
35.
Wolber PK, Deininger CA, Southworth MW, Vandekerckhove J, van Montagu M, Warren GJ: Identification and purification of a bacterial ice-nucleation protein. Proc Natl Acad Sci USA 1986;83:7256-7260.
[PubMed]
36.
Wolber P, Warren G: Bacterial ice-nucleation proteins. Trends Biochem Sci 1989;14:179-182.
[PubMed]
37.
Wolber PK: Bacterial ice nucleation. Adv Microb Physiol 1993;34:203-237.
[PubMed]
38.
Zhao J, Orser CS: Conserved repetition in the ice nucleation gene inaX from Xanthomonas campestris pv. translucens. Mol Gen Genet 1990;223:163-166.
[PubMed]
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