Inhibition of bacterial ice nucleation by polyglycerol polymers

The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 microL volume. The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification.     

New Metabolites of Streptomyces alboniger

A mutant unable to grow under acidic conditions was isolated from the acidophilic heterotrophic bacterium Acidiphilium facilis 24R. The growth of the mutant could be fully restored by the addition of spermidine or lysine at the concentration of 100 μm. The HPLC analysis of polyamine composition showed that spermidine and putrescine were major polyamine components in the parental strain. In the mutant strain, putrescine was replaced by cadaverine. It was found that some polyamines in the cells were conjugated with the other cell components. The growth of the bacterium in the medium below pH 4.5 was inhibited in the presence of α-methylornithine or methylglyoxal-bis(guanylhydrazone), which are inhibitors of rate-limiting enzymes involved in the biosynthesis of polyamines. The growth of the bacterium that had been inhibited in the presence of inhibitors could be fully restored by the addition of putrescine or spermidine. On the basis of these results, it was concluded that polyamines have a significant role in the growth of Acidiphilium facilis 24R under acidic conditions.     

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.     

Biochemical modification of plasma ice nucleating activity in a freeze-tolerant frog.

Recently, we reported the presence of ice nucleating activity, apparently proteinaceous, in the plasma of a freeze-tolerant frog, Rana sylvatica, collected in autumn and spring. Although this protein has not been purified, its ice nucleating behavior can act as an internal reference for tests that attempt to modify its ability to nucleate ice formation. If the addition of a chemical reagent alters the temperature of ice crystallization compared with the control, it can be assumed that protein modification may have occurred. The ice nucleating protein in R. sylvatica showed resistance to proteolysis with four different proteases although there was a significant reduction in the temperatures of nucleation with these treatments (ANOVA P less than 0.001). However, ice nucleating activity was lost when plasma was treated with the addition of urea or N-bromosuccinimide. Modification of protein sulphydryl groups with iodoacetamide did not affect the crystallization temperature (Tc) but treatment with iodoacetic acid resulted in a significant increase in Tc of plasma. An abrupt loss of ice nucleating ability was observed in plasma samples after heating above 87 degrees C. Anomalous potentiation of ice nucleating activity occurred when the plasma was heated to and held at temperatures between 67-75 degrees C.     

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion‐weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high‐temperature exotherm (?3°C to ?5°C) corresponded with ice formation and high ice‐nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (?6°C and ?8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low‐temperature exotherm corresponded with SAM injury and the killing temperature in wheat (?21°C) and rye (?27°C). The SAM had lower ice‐nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.     

Components of ice nucleation structures of bacteria

Nonprotein components attached to the known protein product of the inaZ gene of Pseudomonas syringae have been identified and shown to be necessary for the most efficient ice nucleation of supercooled H2O. Previous studies have shown that cultures of Ina+ bacteria have cells with three major classes of ice-nucleating structures with readily differentiated activities. Further, some cells in the culture have nucleating activities intermediate between those of the different classes and presumably have structures that are biosynthetic intermediates between those of the different classes. Since these structures cannot be readily isolated and analyzed, their components have been identified by the use of specific enzymes or chemical probes, by direct incorporation of labeled precursors, and by stimulation of the formation of specific classes of freezing structures by selective additions to the growth medium. From these preliminary studies it appears that the most active ice nucleation structure (class A) contains the ice nucleation protein linked to phosphatidylinositol and mannose, probably as a complex mannan, and possibly glucosamine. These nonprotein components are characteristic of those used to anchor external proteins to cell membranes of eucaryotic cells and suggest that a similar but not identical anchoring mechanism is required for efficient ice nucleation structure. The class B structure has been found to contain protein presumably linked to the mannan and glucosamine moieties but definitely not to the phosphatidylinositol. The class C structure, which has the poorest ice nucleation activity, appears to be the ice nucleation protein linked to a few mannose residues and to be partially imbedded in the outer cell membrane.     

Three separate classes of bacterial ice nucleation structures

Studies of the properties of the ice nucleation structure exposed on the surfaces of various bacteria such as Pseudomonas syringae, Erwinia herbicola, or various strains of Ice+ recombinant Escherichia coli have shown that there are clearly three major related but chemically distinct types of structures on these cells. First, the ability of Ice+ cells to nucleate super-cooled D2O has been examined, and it has been found that this ability (relative to the ability of the same cells to nucleate super-cooled H2O) exhibited three characteristic nucleating patterns. The rarest structure, called class A, is found on only a small fraction of cells in a culture, nucleates H2O at temperatures above -4.4 degrees C, and is an effective nucleator of super-cooled D2O. A second class of structure, called class B, is found on a larger portion of the cells, nucleates H2O between -4.8 and -5.7 degrees C, and is a relatively poor nucleator of super-cooled D2O. The class C structure is found on almost all cells and nucleates at -7.6 degrees C or colder. These three classes of structures were also differentiated by their sensitivities to low concentrations of water-miscible organic solvents such as dioxane or dimethyl sulfoxide. Depending on the specific bacterial strain, the addition of these solvents to bacterial suspensions lowered the nucleation activity of the class A structure by 1,000-fold or more. The nucleation activities of class B structures in the same culture were highly resistant to these compounds and were lowered only by 20 to 40%. The class C structures were more sensitive than Class B structures were, and the nucleation activities decreased 70 to 90%. Finally, the pH sensitivity of these three classes of structures was examined. The class A structure was destroyed in buffers at pH 4.5 lower but was stable in buffers at higher pHs. The class B structure was less sensitive to acidic buffers but was destroyed at pH 5.5 or lower and was stable at higher pHs. However, the class C structure was unaffected by incubation in buffers with pHs of 3.5 to 9.0. Suggestions for the actual nucleation structures of the three classes are proposed.     

Purification and characterization of an insect hemolymph lipoprotein ice nucleator: evidence for the importance of phosphatidylinositol and apolipoprotein in the ice nucleator activity

Summary A lipoprotein with ice nucleator activity was purified from the hemolymph of the freezetolerant larvae of the craneflyTipula trivittata. Characterization of this lipoprotein ice nucleator (LPIN) showed that it differed from other previously described insect hemolymph lipoproteins which lack ice nucleator activity, by the presence of phosphatidylinositol (PI) at 11.0% by weight of the total phospholipid content. The potential roles of PI and other lipoprotein components in the ice nucleating activity were examined using various phospholipases, proteases, LPIN antibodies, borate compounds and various lipid-protein reconstitutions. It was found that phosphatidylinositol specific phospholipase C was the most effective phospholipase in eliminating the activity of the LPIN. Borate compounds effectively depressed activity. Treatment of the LPIN with protease also eliminated ice nucleator activity but the binding of LPIN specific antibody did not. Reconstitutions consisting of the native LPIN lipids, PI specific phospholipase-treated native LPIN lipids, or pure standard phospholipids with the apolipoproteins of the LPIN andManduca sexta larval lipoproteins gave evidence that both the apolipoproteins of the LPIN and PI are necessary for the ice nucleating activity.Abbreviations LPIN polyclonal antibodies to lipoprotein ice nucleator - ANOVA analysis of variance - Apo-I apolipoprotein I - Apo-II apolipoprotein II - LPIN lipoprotein ice nucleator - PAGE polyacrylamide gel electrophoresis - PAS Periodoacetate-Schiff's base - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - SCP supercooling point (ice nucleation temperature) - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography     

Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes

The preliminary finding that nonprotein additions to the protein product of the ice-nucleating gene of Pseudomonas syringae or Erwinia herbicola are essential for ice nucleation at the warmest temperatures has led to experiments aimed at identifying possible linkages between the ice protein and the other components. It appears that the protein is coupled to various sugars through N- and O-glycan linkages. Mannose residues are apparently bound via an N-glycan bond to the amide nitrogen of one or more of the three essential asparagine residues in the unique amino-terminal portion of the protein. In turn, these mannose residues are involved in the subsequent attachment of phosphatidylinositol to the nucleation structure. This phosphatidylinositol-mannose-protein structure is the critical element in the class A nucleating structure. In addition to sugars attached to the asparagine residues, additional sugar residues appear to be attached by O-glycan linkages to serine and threonine residues in the primary repeating octapeptide, which makes up 70% of the total ice protein. These additional sugar residues include galactose and glucosamine and most likely additional mannose residues. These conclusions were based on (i) the changes in ice-nucleating activity due to the action of N- and O-glycanases, alpha- and beta-mannosidoses, and beta-galactosidase; (ii) immunoblot analyses of ice proteins in cell extracts after enzyme treatments; and (iii) the properties of transformed Ice+ Escherichia coli cells containing plasmids with defined amino-terminal and carboxyl-terminal deletions in the ice gene. Finally, evidence is presented that these sugar residues may play a role in aggregating the ice gene lipoglycoprotein compound into larger aggregates, which are the most effective ice nucleation structures.     

Reinitiation of cell division and polyamine and aromatic monoamine levels in alfalfa explants during the induction of somatic embryogenesis

The early cellular events in leaf explants of Medicago sativa L. cultured on somatic embryogenesis- and callogenesis-inducing media (EIM and CIM, respectively) were correlated with the endogenous contents of polyamines (PAs) and aromatic monoamines (AMs). On the second day of culture, replication of DNA occurred in epidermal and subepidermal cells on the edges of explants on EIM and was a prerequisite for proembryonal mass and, later, globular proembryo formation. In explants cultured on CIM, replication occurred at least one day later and in fewer cells, which were randomly spread all over the explant. Transition of leaf explant cells to rapidly dividing meristematic-like cells on EIM or to enlarged, highly vacuolated cells on CIM was observed. The increase in total PA levels in cultured explants was primarily a consequence of increases in putrescine (Put) and spermidine (Spd) contents and was much more pronounced on EIM than on CIM. High Spd levels were characteristic of meristematic cells and might be essential for the development of globular structures. The higher amount of insoluble PA conjugates was determined in explant cells on EIM, as compared with CIM. Proembryogenic cell mass formation was positively correlated with free tyramine and negatively correlated with free phenylethylamine contents.     

Role of Cell Hydrophobicity on Colony Formation in Microcystis (Cyanobacteria)

Colony formation is highly import ant for the competitive advantage of the cyanobacterium Microcystis over other phytoplankton species. The laboratory‐grown colonial Microcystis strains isolated from Lake Taihu (China) maintained colonial forms under the low light condition (10 μE m^–2 s^–1). The cell surface hydrophobicities of the Microcystis colonies were measured by cyanobacterial adherence to xylene in comparison with unicellular Microcystis strains. The cells of the tested colonial strains were all hydrophobic, while the cells of the tested unicellular strains were all hydrophilic. Incubation under the higher light condition (75 μE m^–2 s^–1) leaded to the significant decrease in the cell hydrophobicities of the colonial Microcystis and the transition from colonial forms to unicellular forms. These findings indicated that the cell hydrophobicity of Microcystis may play a role in cell‐cell adherence and colony formation. Phosphate‐limitation, nitrate‐limitation and pH did not affect cell hydrophobicities of colonial Microcystis. Treatment with proteolytic enzymes had no effect on the cell hydrophobicity, indicating that cell surface proteins did not contribute to high cell hydrophobicity. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola.

Chemical and biological properties of the ice nucleating sites of Pseudomonas syringae, strain C-9, and Erwinia herbicola have been characterized. The ice nucleating activity (INA) for both bacteria was unchanged in buffers ranging from pH 5.0 to 9.2, suggesting that there were no essential groups for which a change in charge in this range was critical. The INA of both bacteria was also unaffected by the addition of metal chelating compounds. Borate compounds and certain lectins markedly inhibited the INA of both types of bacterial cells. Butyl borate was not an inhibitor, but borate, phenyl borate, and m-nitrophenyl borate were, in order, increasingly potent inhibitors. These compounds have a similar order of affinity for cis hydroxyls, particularly for those found on sugars. Lentil lectin and fava bean lectin, which have binding sites for mannose or glucose, inhibited the INA of both bacteria. All other lectins examined had no effect. The inhibition of INA by these two types of reagents indicate that sugar-like groups are at or near the ice nucleating site. Sulfhydryl reagents were potent inhibitors of the INA of both bacteria. When treated with N-ethylmaleimide, p-hydroxymercuribenzoate, or iodoacetamide, the INA was irreversibly inhibited by 99%. The kinetics of inactivation with N-ethylmaleimide suggested that E. herbicola cells have at least two separate ice nucleating sites, whereas P. syringae cells have possibly four or more separate sites. The effect of infection with a virulent phage (Erh 1) on the INA of E. herbicola was examined. After multiple infection of a bacterial culture the INA was unchanged until 40 to 45 min, which was midway through the 95-min latent period. At that time, the INA activity began falling and 99% of the INA was lost by 55 min after infection, well before any cells had lysed. This decrease in INA before lysis is attributed to phage-induced changes in the cell wall.     

High-level expression of the ice-nucleating activity of Pseudomonas syringae in relation to its growth characteristics

Optimization of the culture medium is essential for the production of a large biomass of high ice-nucleating-active micro-organisms such as Pseudomonas syringae. Cultures in bioreactors show that optimal substrate concentrations are approximately the same for ice nucleating activity (INA) and total biomass (50–80 g/l of glucose; 28 g/l of peptone) but not for the growth rate. The INA is lowest when the growth rate is highest (50 g/l of glucose, 15 g/l of peptone). We have shown that the maximal biomass production and INA are related to the C/N ratio (optimal ratio: 10) rather than to the substrate concentration. These results also contribute to knowledge on the physiology of these bacteria and support the sequential maturation of the ice nucleating sites.     

Laboratory studies identify a colloidal groundwater tracer: implications for bioremediation

We have identified and tested a new colloidal tracer for use in hazardous waste site characterization and other environmental applications. The tracer is primarily composed of the dead ice nucleating active (INA) bacterium Pseudomonas syringae. Assay conditions are simple and based on the observation of the freezing behavior of 10 μl solution volumes. Under specific assay conditions (−5 to −7°C) these drops freeze only if the tracer is present. The results are available within 3 min of sample collection thus, providing near real-time results. The tracer detection limits are in the range of ng/l, it is stable over a pH range of 2–11, and maintains its high activity in the presence of a variety of contaminant compounds. In a simple column experiment this colloid tracer eluted just prior to NaCl with little or no tailing.     

Purification and composition of an ice nucleating protein from queens of the hornet,Vespula maculata

Summary Hemolymph ice nucleating factors are found in many freeze tolerant insects. These factors function to initiate ice nucleation in the extracellular fluid at fairly high subzero temperatures thereby minimizing the possibility of lethal intracellular ice formation.An ice nucleating protein was purified from the hymolymph of pupal bald faced hornets,Vespula maculata. This is the first ice nucleating protein to be purified. The protein has a molecular weight of 74,000, as determined by SDS-PAGE, and is quite hydrophilic. Glutamate and/or glutamine accounts for 20% of the amino acid residues. It is likely that the hydrophilic nature of the protein is involved in the ability of the protein to function as an ice nucleator.     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Azaphilones from the Fungus Penicillium multicolor LZUC-S2 and Their Antibacterial Activity

Two new azaphilones, penimultiones A and B, together with seven known analogs were isolated from the culture of Penicillium multicolor LZUC-S2. Their structures were elucidated by detailed spectroscopic data analysis and chemical transformation. Penimultiones A and B belong to a rare class of azaphilones possessing a 1,3-dioxolane moiety. In addition, all compounds were evaluated for their antibacterial activity against five clinically bacterial strains in vitro, and three compounds showed potent antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 12.5 to 50.0 μg/mL.     

Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria

An agar plate freezing assay is described based on the incorporation of fluorescein dye in agar medium. Upon addition of fluorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes.     

Polyamine metabolism in an obligately alkalophilic Bacillus alcalophilus that grows at pH 11.0

Bacillus alcalophilus, an obligately alkalophilic bacterium that grows at pH 11.0, has an intracellular pH of 9.5 or less. Unlike all other living organisms, polyamines (putrescine, spermidine and spermine) in B. alcalophilus, if present, will be largely unprotonated. HPLC analysis indicated that spermidine is the major polyamine in B. alcalophilus, accounting for more than 90% of total polyamines, and the level of spermidine varies during growth. Ornithine decarboxylase activity was not detectable in B. alcalophilus under all conditions examined. When [3H]arginine was added to the culture medium, the radioactivity can be recovered from polyamine pool; the distribution is 3% for putrescine, 94% for spermidine, and 3% for spermine, suggesting the presence of arginine pathway for polyamine biosynthesis. The polyamine transport system in B. alcalphilus appears to be Na+-dependent and is highly sensitive to the inhibition of gramicidin S and valinomycin.