Identification and purification of a bacterial ice-nucleation protein.

Abstract

The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl 1-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process.Some bacteria of the genera Pseudomonas, Erwinia, and Xanthomonas can nucleate the crystallization of ice from supercooled water (1-3). Genes encoding the ice-nucleation active (Ina') phenotype have been cloned from Erwinia herbicola, Pseudomonas syringae, and Pseudomonas fluorescens (4-7). Theoretical considerations (8), as well as the actual sequence of the inaZ gene from P. syringae (6), suggest that the bacteria synthesize a template for ice-crystal formation, rather than an enzyme. The translation product predicted from the inaZ sequence is a protein with repetitive primary structure; its tertiary structure, which might also be repetitive, could provide considerable insight into the mechanism of ice nucleation. The ice nuclei of P. syringae are associated with its outer membrane (9) and are believed to contain both protein (9-11) and lipid (11) components. One report has suggested that the inaZ product is a phosphatidylinositol synthase, and that the lipid phosphatidylinositol is a key component of the water-binding template (12).Here, we argue that the active component of bacterial ice nuclei is a protein, the product of inaZ. We have reached this conclusion after purifying the InaZ protein. To Ice-Nucleation Spectra. Ice-nucleation frequencies were measured by a drop-freezing method with an instrument constructed as described by Vali (16). For analysis of column fractions, 20 drops of 10 ,u1 per dilution were examined at a dilution interval of 10-2; in all other cases, 40 drops of 10 p.1 per dilution were tested, with a dilution interval of 10-1. All spectra of subcellular fractions were normalized to the frequency per cell, by dividing the frequency per ml value for a given sample by the ratio (volume of sample)/(number of cells used to prepare sample).Construction of pMWS10. The region of DNA encoding the P. syringae S203 inaZ gene (6) was digested with restriction enzymes Aha III (cutting at nucleotide 775) and EcoRI (cutting at nucleotide 4453), resulting in a fragment beginning 23 base pairs 5' of the initiator codon. The EcoRI end was converted to a HindIII end by addition of a linker, and the fragment was inserted into pKK223.3 (17), so that inaZ was placed downstream of the tac promoter. The construct retained the original ribosome binding site of inaZ. The plasmid pMWS10 ...

Immersion freezing of ice nucleation active protein complexes

“…For particles in the size range where intact bacteria are to be expected (> 600 nm), we found that less than 50 % of the particles were ice nucleation active despite the low temperatures (down to −38 • C), indicating that maybe only half of the bacteria were able to nucleate ice, i.e., were carrying an INA protein complex. This finding supports data available in the literature suggesting that the majority of P. syringae cells have none, one or only little more than one ice forming site on their surface (e.g., Orser et al, 1985;Wolber et al, 1986;Lindow et al, 1989;Attard et al, 2012).…”

“…Unfortunately in many studies (Orser et al, 1985;Wolber et al, 1986;Lindow et al, 1989;Attard et al, 2012), the concentrations of bacterial cells in the examined droplets are not reported, and therefore the cumulative ice nuclei concentrations given in these studies cannot be converted to ice fractions (f ice ), and hence they cannot be included in our comparison.…”

“…The ability of INA bacteria to nucleate ice is caused by a special protein anchored in the outer membrane of the bacterial cell wall (Wolber et al, 1986;Warren and Wolber, 1987). This INA protein is encoded by a single gene (Orser et al, 1985;Green and Warren, 1985), which is highly conserved among the INA bacterial species (Warren and Wolber, 1991).…”

Expression of a Bacterial Ice Nucleation Gene in Plants

“…To facilitate the constructions described below, a 3.7-kb EcoRI fragment carrying the inaZ gene from Pseudomonas syringae without its promoter from plasmid pMWS10 (33) was first cloned in the vector pUC1 19 (22) digested with EcoRI. A derivative designated pUZl 19 was selected that had the inaZ fragment in the appropriate orientation to be reexcised by digestion with BamHI.…”

“…These bacterial species were shown to be the predominant icenucleating agent on the surface of plants and the cause of frost injury in many plant species (18). The molecular basis for ice nucleation activity of P. syringae has been extensively investigated (5,20,33). Genes (ice, ina) encoding ice nucleation proteins in different strains of P. syringae have been cloned and structurally characterized (8,25).…”