Paul K. Wolber

Bacterial Ice Nucleation

Publisher Summary This chapter investigates the ability of certain biological systems to initiate physical processes in metastable systems. The best-characterized biological initiators are bacterial ice nuclei that trigger crystallization of ice from supercooled water. The ice-nucleating bacteria are members of plant epiphytic communities. Three mechanisms of ice nucleation are (1) homogeneous ice nucleation, (2) heterogeneous ice nucleation, and (3) secondary ice nucleation. Nucleation activity is calculated from the frequency of freezing observed in multiple, small volumes (often droplets) of a sample diluted in water or a weak buffer so that, at the measurement temperature, some but not all of the replicate volumes freeze. In every microorganism, the Ina + phenotype is the result of expression of a single ice-nucleation ( ina ) gene, to yield a single ice-nucleation (Ina) protein. The genetic basis of the Ina + phenotype, sizes, and locations of the nucleation sites, and the source of the heterogeneity of nucleation-threshold temperatures exhibited by clonal populations of bacteria are discussed. Ice-nucleating bacteria are the chief initiators of frost damage to many economically important crop plants. This non-intuitive effect is the result of the ability of many air-exposed plant parts to supercool to temperatures of -6°C or lower before nucleation sites intrinsic to the plant material become active. The chapter concludes with the applications of bacterial ice nucleation.

Detection of bacteria by transduction of ice nucleation genes

A recent addition to the array of test strategies available for bacterial pathogen surveillance is the Bacterial Ice Nucleation Diagnostic (BIND TM ) assay. This system combines the recognition specificity of a bacteriophage with the sensitivity of a unique reporter gene: the gene encoding a bacterial ice nucleation protein

Clustering of ice nucleation protein correlates with ice nucleation activity

Antibodies raised against a synthetic peptide specifically detect ice nucleation proteins from Pseudomonas species in Western blots. In immunofluorescent staining of whole bacteria, the antibodies reveal the protein in clusters, as indicated by patches of intense fluorescence in Escherichia coli cells heterologously expressing Pseudomonas ice nucleation genes. The abundance, size, and brightness of the clusters vary considerably from cell to cell. Their varying sizes may explain the variability in activity of bacterial ice nuclei. Growth at lower temperatures produces more ice nuclei, and gives brighter and more frequent patches, than growth at 37 degrees C. The observed clustering may thus reflect formation of functional ice nucleation sites in vivo. The presence of ice nucleation protein in clusters is also correlated with alterations in cell morphology.

New rapid method for the detection of Salmonella in foods

Abstract The ideal to which quality-control bacteriologists aspire is to detect very small numbers of target bacteria, in the presence of much larger numbers of non-target bacteria, after they have been injured by disinfectants or manufacturing processes and rapidly enough to prevent manufacture or sale of a contaminated product. In addition, the ideal test should never report false negatives, never report false positives, not use hazardous chemicals and not produce hazardous waste. All real assays represent some compromise between such conflicting goals. A recent addition to the array of test strategies available for bacterial pathogen surveillance is the bacterial ice nucleation diagnostic (‘BIND’) assay. This measurement system combines the recognition specificity of a bacteriophage with the extreme sensitivity of a unique reporter gene: the gene encoding a bacterial ice nucleation protein.

Further characterization of the lipoprotein ice nucleator from freeze tolerant larvae of the cranefly Tipula trivittata

Abstract 1. 1. Using Western blots, immunological similarities were found between the hemolymph lipoprotein ice nucleator (LPIN) from freeze tolerant larvae of the cranefly Tipula trivittata and the ice nucleator protein from the bacteria Pseudomonas fluorescens , indicating the presence of some amount of the well known repeat structure of the bacterial protein in the LPIN. 2. 2. Delipidated LPIN apoproteins are inactive, but activity can be regained by reconstitution of the two apoprotein components of the LPIN with phosphatidylinositol (PI) in proteoliposomes. Substitution of PI-4,5-diphosphate or PI-4-monophosphate for PI in reconstitutions resulted in greatly reduced activity. 3. 3. Treatment of the LPIN with periodate eliminated activity. 4. 4. Thus, the presence of both apoproteins plus the integrity of the hydroxyls of the inositol head group of PI seem to be essential for ice nucleator activity of the LPIN.