Stabilization of astaxanthin by a novel biosurfactant produced by Rhodotorula mucilaginosa KUGPP-1.

We found that a novel biosurfactant from the cultured broth of red yeast, Rhodotorula mucilaginosa KUGPP-1, originating in the Antarctic, has dispersive power against astaxanthin. The novel biosurfactant was purified from extracts to the ultrafiltration state by acetone precipitation and chromatography on a DEAE-Toyopearl 650 M, and gel filtration on a Sephacryl S-400 HR. The molecular mass of the novel biosurfactant was estimated to be about 730,000 by gel filtration chromatography. The novel biosurfactant was comprised of sugar and protein in an approximate molar ratio of 9 : 1. The sugars were comprised of mannose, galactose and glucose. The particle size of the astaxanthin (0.13 µ g/ml) micelle was about 410 nm. Astaxanthin was stable to oxidation in the novel biosurfactant micelles. To our knowledge, this is the first report on a glycoprotein type of biosurfactant with astaxanthin-stabilizing ability.

Identification of the N-terminal region of TjZNT2, a Zrt/Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity.

The Zrt/Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn(2+) and Mn(2+) transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn(2+) uptake of yeast cells expressing them. As a result, the extracellular N-terminal ends were identified as regions involved in Zn(2+) selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn(2+) uptake activity. This suggests that the extended N-terminal region inhibits Zn(2+) transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn(2+) transport. Furthermore, the N-terminal truncated TjZNT2 lacked Mn(2+) uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters.

Cryoprotection and cryosterilization effects of type I antifreeze protein on E. coli cells.

To establish the effects of type I antifreeze protein (AFP) on E. coli cells, we have focused on the survival rate of the E. coli cells using type I AFP at various concentrations under rapid cooling conditions using liquid N2 at atmospheric or low pressure. The survival rate of E. coli was enhanced by the addition of type I AFP at a concentration of 10 microg/ml, and its value shifted from 0.73% to 2.96%. When the concentration of type I AFP was 100 microg/ml, the cell survival rate markedly decreased to 0.090%. This low survival rate was further decreased (0.022%) by the application of the same freeze-thaw treatment for four times. Also, the effect of type I AFP as a bactericidal agent did not vary according to the varying initial cell densities from 10(4) to 10(8) cells / ml. Furthermore, the effects of using type I AFP at 1.0 MPa with N2 gas under conditions of low pressure and low oxygen tension using a simple device were examined. When the actions of type I AFP as a cryoprotectant were stimulated, the survival rate of the E. coil cells increased to 57.8%. In addition, the bactericidal effect of type I AFP at 100 micro g/ml of protein concentration could also be enhanced. The survival rate using 100 g/ml of type I AFP under low pressure was 0.35% of that using 10 microg/ml under the same conditions. This is the first report on the cryoprotectant and cryosterilization effects of type I AFP of E. coli cells under various conditions.

Antifreeze activity of cold acclimated Japanese radish and purification of antifreeze peptide.

Japanese radish tuber and leaf produced antifreeze proteins (AFPs) having thermal hysteresis activity (TH) and ice recrystallization inhibiting activity (RI). Upon cold acclimation, the apoplastic fluid of the Japanese radish exhibited hexagonal crystal growth, indicating the presence of an antifreeze protein. The induction patterns of protein and the TH activity of apoplastic fraction from both samples were different. The TH activities of apoplastic fraction from tuber and leaves were 0.20 +/- 0.03 and 0.18 +/- 0.02 degree C, respectively. Also, the TH and RI activities of apoplastic fraction of leaves were activated by autoclave treatment at pH 10.0. An antifreeze peptide (molecular weight 1320), was purified using chromatography. Furthermore, the chitinase and beta-1, 3-glucanase activities in the apoplastic fraction of its tuber were induced by the cold acclimation. Some proteins in this apoplastic fraction were reacted with the anti-glucanase-like protein (GLP) antiserum and anti-chitinase-like protein (CLP) antiserum produced against isolated winter rye AFPs. This is the first report on the presence and characterization of AFPs from Japanese radish tuber.

Cryoprotective activity of mannoprotein from the cell membrane of Pichia anomala.

Some organisms like bacteria and plants have a cryoprotective protein to cryopreserve the freeze-labile enzyme under stable conditions having high activity. By screening of the cryoprotective activities of various food-industrial yeasts, we found that the cell membrane component that was a glucanase extractable component in Pichia anomala NBRC 141 had a high level of cryoprotective activity against freeze-labile lactate dehydrogenase (LDH). The absorption of the active compound in the crude extract by ConA-Sepharose chromatography suggested that this active compound might be a glycoprotein (COGP). Strain NBRC had the COGP constantly without the treatment of cold acclimation. The active compound, that is, a COGP, could be homogeneously purified using DEAE-TOYOPEARL and Sephacryl S-400 chromatography. The purified COGP had a cryoprotective activity of 50.9% at a sugar concentration of 17.9 microg/ml. The molecular weight of purified COGP was 83,000, which was composed of one protein with a molecular weight of 22,000 and polysaccharide. Furthermore, the constituent sugar of COGP was only D-mannose based on HPLC analysis of its acid hydrolysate. Also, we confirmed that the cryoprotective activity of COGP was higher than those of the commercial cell membrane components. The CP50 of COGP was 0.28 microM, which was half to the CP50 of BSA. This is the first report, to our knowledge, that the cell membrane component of Pichia anomala had a high level of cryoprotective activity against a freeze-labile enzyme.

Involvement of histidine-rich domain of ZIP family transporter TjZNT1 in metal ion specificity.

The Zrt/Irt-like protein (ZIP) family generally contributes to metal homeostasis by regulating cation transport into the cytoplasm. Most ZIP members have a long variable loop between transmembrane domains III and IV, and these loops are predicted to be located in the cytoplasm. The loops contain a histidine-rich domain (HRD) postulated to serve as a metal ion binding site; however, its role has not yet been determined. We previously determined that deletion of the HRD did not affect the Ni tolerance ability of TjZNT1-a ZIP transporter that confers high Ni tolerance to yeast. In this study, we investigated the effect of HRD deletion on the ion transport ability of TjZNT1. The deletion of HRD increased the specificity for Zn2+, but not for Cd2+. In addition, we confirmed subcellular localizations of TjZNT1 and HRD-deleted mutants by green fluorescence protein (GFP)-fused proteins, indicating that the deletion of HRD did not affect the localization of TjZNT1. From these results, we propose that the HRD could be involved in the ion specificity of TjZNT1.

Isolation and characterization of arbuscules from roots of an increased-arbuscule-forming mutant of Lotus japonicus.

BACKGROUND AND AIMS: Previous methods for isolation of arbuscules from mycorrhizal roots are time-consuming, complex and expensive. Therefore, a simple, rapid and inexpensive method for the isolation of metabolically active arbuscules from plant root of an increased-arbuscule-forming mutant of Lotus japonicus (Ljsym78-2) is described. METHODS: Roots of the L. japonicus mutant plants Ljsym78-2 colonized by Glomus sp. were separated from soil, washed with water, immersed in CaSO(4) before being cut into 5-mm pieces and homogenized with a Waring blender at 6000 rpm for 30 s. The arbuscules were purified by separation from plant tissues with a 50-mum nylon mesh, finally collecting on a 30-mum nylon mesh. Enzyme histochemical staining showed that the collected arbuscules had succinate dehydrogenase, alkaline phosphatase and acid phosphatase activities. KEY RESULTS AND CONCLUSIONS: The enzymic activity of the arbuscules was not affected after the isolation process. The establishment of this simple, rapid and inexpensive method for the isolation of metabolically active arbuscules will be useful to clarify the biochemical processes occurring in nutrient exchange at the arbuscular interface.

Investigation of the basis for Ni tolerance conferred by the expression of TjZnt1 and TjZnt2 in yeast strains.

Introduction of ZIP family transporter gene homologues TjZnt1 and TjZnt2 (metal ion transporters) into yeast strains conferred increased Ni(II) tolerance in that species. The action of ZIP family transporter homologues, however, could not explain the Ni resistance of yeast strains transformed with TjZnt1 and TjZnt2. To elucidate the mechanism of Ni tolerance conferred by TjZnt1 and TjZnt2 in yeast strains, we made a series of investigations based upon three hypotheses, including (1) cellular Ni efflux, (2) exclusion of Ni due to competitive uptake of other metals, and (3) Ni binding to histidine-rich domains (chelation). The critical Ni tolerance level of TjZnt2 expressing yeast strains was 1.4mM, whereas, the TjZnt1 expressing yeast strains were tolerant of Ni concentrations as high as 2.0mM. The TjZnt1 expressing yeast strain had significantly lower Ni content and significantly higher Zn content than the control and TjZnt2 expressing yeast strain. Effects of the deletion of histidine-rich domain HRD1 or HRD2, or deletion of the region from HRD1 to HRD2, resulted in the same or slightly less Ni(II) tolerance in the TjZnt1 expressing yeast strain. These data indicate that Ni tolerance of the TjZnt2 expressing yeast strain is not correlated with binding to HRDs (Hypothesis 3). Ni tolerance of TjZnt1 expressing yeast strain was, however, partially correlated with Zn influx, which suppressed Ni influx, therefore Ni influx (Hypothesis 1) and competitive inhibition of Ni influx by other metals (Hypothesis 2), remain viable hypotheses which will be subject to further testing.

A novel, intracellular antifreeze protein in an antarctic bacterium, Flavobacterium xanthum.

One strain of Antarctic bacteria, Flavobacterium xanthum IAM12026, has a highly active antifreeze protein (AFP) in the intracellular space. The cell-free extract from strain IAM12026 after culturing at 4 degree C for 7 days in TSB medium, had activity of 0.04 degree C at a concentration of 0.7 mg/ml. The ice crystals formed do not have distinct facets without typically rounded shape and the changes of their morphology during the course of the thermal hysteresis (TH) measurement. The ice crystal 'burst' occurring at the end-point of the TH is dendritic with hexagonal symmetry. Also, this activity was not affected by the treatment of dialysis and the addition of EDTA. Furthermore, this cell-free extract had high levels of ice recrystallization-inhibiting (RI) activity like those of Fish AFPs. The AFP (FlAFP) was homogeneity purified using chromatography. A relative molecular mass of approximately 59,000 was calculated from gel filtration and SDS-PAGE data. The thermal stability of FlAFP was below 50 degree C, and TH value was absent above 60 degree C. The TH value of FlAFP was activated at 5.2 degree C by the addition of 0.5 M malate. This activation was decreased with increasing protein concentration. To our knowledge this is the first report on the high level of TH and RI activities of bacterial intracellular AFP.

Biochemical and genetic analysis of the gamma-resorcylate (2,6-dihydroxybenzoate) catabolic pathway in Rhizobium sp. strain MTP-10005: identification and functional analysis of its gene cluster.

We identified a gene cluster that is involved in the gamma-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium Rhizobium sp. strain MTP-10005. The cluster consists of the graRDAFCBEK genes, and graA, graB, graC, and graD were heterologously expressed in Escherichia coli. Enzymological studies showed that graD, graA, graC, and graB encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC 1.3.1.32), and a hydroxyquinol 1,2-dioxygenase (GraB) (EC 1.13.11.37). Bioinformatic analyses suggested that graE, graR, and graK encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of graF, which encodes gamma-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 +/- 0.82] x 10(-4)) of graF was detected in the total RNA of the cells after one hour of cultivation when gamma-resorcylate was used as the sole carbon source. Reverse transcription-PCR of graDAFCBE showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by gamma-resorcylate. These results suggested that the graDAFCBE genes are responsible as an operon for the growth of Rhizobium sp. strain MTP-10005 on gamma-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the gamma-resorcylate catabolic pathway in an aerobic bacterium.

Cloning of three ZIP/Nramp transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+-transport abilities.

Ni homeostasis is essential for plant cell activity, but the mechanisms of Ni-transport and delivery are unknown. To elucidate the role of ZIP and NRAMP metal-transporters for Ni2+-transport and homeostasis, we cloned their homologous genes from the Ni hyperaccumulator Thlaspi japonicum, and investigated their Ni-transporting abilities by expression in yeast. The deduced amino acid sequences of the two Zip transporter genes (TjZnt1, TjZnt2) and one Nramp transporter gene cloned had high homologies with TcZNT1 and TcZNT2 of Thlaspi caerulescens and AtNRAMP4 of Arabidopsis thaliana, respectively, and were predicted as integral membrane proteins with 6 or 12 transmembrane domains. TjZNT1 and TjZNT2 had two long histidine-rich domains in the putative cytoplasmic domain between transmembrane domains III and IV. TjNRAMP4 conserved a consensus transporter motif between transmembrane domains VIII and IX. The yeast transformed with TjZNT1 or TjZNT2 showed a marked increase in Ni2+ tolerance with the gene expression. In contrast, the expression of TjNramp4 caused elevation of Ni2+ sensitivity and Ni2+ concentration. These data suggest that ZIP/NRAMP transporters participate in Ni2+ homeostasis of Ni hyperaccumulator plants. TjZNT1 had Zn2+-, Cd2+- and Mn2+-transporting abilities and TjZNT2 also had Zn2+- and Mn2+-transporting abilities, but TjNRAMP4 could transport Ni2+ but not Zn2+, Cd2+ or Mn2+.

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2.

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.

Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686.

The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.

Production of two types of ice crystal-controlling proteins in Antarctic bacterium.

Pseudomonas fluorescens KUAF-68, which was isolated from Antarctica, had both ice-nucleating protein and antifreeze protein activities in the culture broth. We found that both proteins were separately produced based on the results of column chromatography, SDS-PAGE analysis and Southern hybridization. The activity of the ice-nucleating protein was stimulated by the addition of glycine (0.020 N%), whereas the activity of the antifreeze protein was stimulated by the addition of L-asparagine (0.025 N%). This is the first report on the production of two types of ice crystal-controlling proteins in one bacterial strain.

Properties of a novel extracellular cell-free ice nuclei from ice-nucleating Pseudomonas antarctica IN-74.

Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium. A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica. Cell-free ice nuclei from P. antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization. Cell-free ice nuclei were purified by centrifugation, filtration (0.45 microm), ultrafiltration, and gel filtration. In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei. Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH. It was demonstrated that the components of cell-free ice nuclei were protein (33%), saccharide (12%), and lipid (55%), indicating that cell-free ice nuclei were lipoglycoproteins. Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.

Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis).

A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.

Differential scanning calorimetry of the effects of Ca2+ on the thermal unfolding of Pseudomonas cepacia lipase.

Thermal unfolding of P. cepacia lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme. The temperature of unfolding was higher at higher concentrations of Ca2+. From the Ca2+ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one. These results confirmed the validity of the unfolding mechanism proposed previously: NCa2+ < = => D + Ca2+, where N and D represent the native and denatured states, respectively, of the enzyme.

Purification and properties of an ice-nucleating protein from an ice-nucleating bacterium, Pantoea ananatis KUIN-3.

An ice-nucleating protein (INP) from the extracellular ice-nucleating matter (EIM) of Pantoea ananatis (Erwinia uredovora) KUIN-3 was purified and characterized. The EIM produced by the strain KUIN-3 was purified by ultrafiltration, sucrose density-gradient ultracentrifugation and gel filtration. The INP was purified using of column chromatography on hydroxyapatite and Superdex 200 in the nondenaturing detergent of 0.1% (w/v) Triton X-100. The purified INP was composed of one subunit of 117 kDa according to SDS-PAGE. It has become apparent that the INP was the ice-nucleating lipoglycoprotein based on the reaction of carbohydrate stain and lipid stain with the INP. It was inhibited by p-mercuribenzoate and N-bromosuccinimide. The activity of the INP gradually decreased from 65 degrees C. The pH stability was held between pH 7.0 and pH 11.0. The INP had a lower ice-nucleating temperature below pH 6.0. It has become apparent that the INP consisted of the class C structure in the EIM based on its freezing difference spectrum in D2O versus H2O.

Identification of a novel anti-ice-nucleating polysaccharide from Bacillus thuringiensis YY529.

Strain YY529, capable of producing some anti-ice-nucleating materials (ANM), was isolated from the surface of a camphor leaf. Strain YY529 was identified as Bacillus thuringiensis from its characteristics and taxonomy; the optimum temperature and pH for producing these ANMs were 30 degrees C and 7.0, respectively. One of the ANM with the highest activities among them was purified from the culture. The molecular weight of the ANM was approximately 130 kDa based on a gel filtration analysis. We confirmed that this ANM was a polysaccharide based on the results of the treatment with a mannosidase and the molish reaction. In addition, the LCMS analysis showed that this anti-ice-nucleating polysaccharide (ANPS) had the polyacetyl-D-glucosamine moiety in its structure. Furthermore, this ANPS showed its ability as a non-freeze agent in a preservative solution for the cryopreservation of cock liver. This is the first report of ANPS as a novel ANM from Bacillus thuringiensis YY529.

Response of the ice-nucleating bacterium Pantoea ananas KUIN-3 during cold acclimation.

The ice-nucleating bacterium Pantoea ananas KUIN-3 accumulated glucose in cells following a shift in temperature (10 degrees C) from the optimum growth temperature (30 degrees C). This accumulation might be caused by the activation of glucose-6-phosphatase. Although this strain after culturing at 30 degrees C was harmed by freezing, the cryotolerance of this strain was reached about 80% after cold acclimation at 10 degrees C.