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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 ...

Enterotoxigenic Escherichia coli strains express fimbriae which mediate binding to intestinal mucosal cells. The F17 fimbriae mediate binding to N-acetylglucosamine-containing receptors present on calf intestinal mucosal cells. These fimbriae consist of F17-A subunit peptides. Analysis of the F17 gene cluster indicated that at least the F17-A, F17-C, F17-D, and F17-G genes are indispensable to obtain adhesive F17 fimbriae (unpublished data). Genetic evidence is presented that the F17-G protein, a minor fimbrial component, is required for the binding of the F17 fimbriae to the intestinal villi. The F17-G gene was cloned and sequenced. An open reading frame of 1,032 bp encoding a polypeptide of 344 amino acids, starting with a signal sequence of 22 residues, was localized. The F17-G mutant strain produced F17 fimbriae which were morphologically identical to the fimbriae purified from strains which contained the intact F17 gene cluster. However, this F17-G mutant could no longer adhere to calf villi. The F17-G locus was shown to act in trans: transformation of the F17-G mutant strain, still expressing the genes F17-A, F17-C, and F17-D, with a vector expressing the F17-G gene restored the binding activity of this mutant strain.

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