Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases.

Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti-viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild-type and drug-resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs-displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug-resistant influenza virus.

Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry.

Astaxanthin possesses higher antioxidant activity than other carotenoids and, for this and other reasons, has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. The basidiomycetous yeast Xanthophyllomyces dendrorhous is one of the best natural producers of astaxanthin, but wild-type cells accumulate only a small amount of astaxanthin. In this study, we developed an efficient flow cytometry method to screen for astaxanthin-overproducing mutants of X. dendrorhous. We first examined the relationship between cellular astaxanthin content and the intensity of fluorescence emitted from the cell. Although the fluorescence emission maximum of astaxanthin dissolved in acetone occurred at 570 nm, intracellular astaxanthin content correlated better with emission at around 675 nm in different X. dendrorhous strains. Using this emission wavelength, we screened cells mutagenized with ethyl methanesulfonate and successfully isolated mutants that produced 1.5-3.8-fold more astaxanthin than parent cells. This method enabled us to obtain overproducers five times more efficient than conventional screening from plate culture.

Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase.

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.

Screening of carbon dioxide-requiring extreme oligotrophs from soil.

We screened soil samples for CO(2)-requiring extreme oligotrophs similar to Rhodococcus erythropolis N9T-4, which can grow on a basal salt agar medium without an organic carbon source. From 387 soil samples, three isolates were obtained and identified as Streptomyces spp. by 16S rDNA analysis. The isolates required gaseous CO(2) for growth and grew on a basal salt medium solidified by silica gel. These results suggest that such CO(2)-requiring oligotrophs occur widely in nature.

An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil.

Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO(2)-limiting conditions but could grow on a medium containing NaHCO(3) under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO(2). Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO(2) fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4.

Bacterial communities in petroleum oil in stockpiles.

Bacterial communities in crude-oil samples from Japanese oil stockpiles were investigated by 16S rRNA gene cloning, followed by denaturing gradient gel electrophoresis (DGGE) analysis. 16S rRNA genes were successfully amplified by PCR after isooctane treatment from three kinds of crude-oil sample collected at four oil stockpiles in Japan. DGGE profiles showed that bacteria related to Ochrobactrum anthropi, Burkholderia cepacia, Stenotrophomonas maltophilia, Propionibacterium acnes, and Brevundimonas diminuta were frequently detected in most crude-oil samples. The bacterial communities differed in the sampling time and layer. Among the predominant bacteria detected in the crude oil, only three species were found for bacteria isolated on agar plates and were related to Burkholderia, Stenotrophomonas, and Propionibacterium, while Ochrobactrum sp. could not be isolated although this species seemed to be the most abundant bacterium in crude oil from the DGGE profiles. Using an archaea-specific primer set, methanogens were found in crude-oil sludge but not in crude-oil samples, indicating that methanogens might be involved in sludge formation in oil stockpiles.

Fructosyl-amino acid oxidases of Aspergillus oryzae are induced by the reaction product, glucosone.

Aspergillus oryzae has two fructosyl-amino acid oxidase (FAOD) isozymes (AoFao1 and AoFao2), which are different in the substrate specificities. Northern blot analysis showed both FAO genes were induced by autoclave-browned medium containing l-lysine or l-valine. Studies with a mutant, that had a disrupted AoFAO2 gene, revealed that the expression of AoFAO1 by fructosyl l-valine depended on the expression of AoFAO2. Both genes were also induced by one of the FAOD-reaction products, glucosone. In contrast, other alpha-dicarbonyl compounds, which display a similar structure to that of glucosone were not able to induce the genes expression. These results imply that glucosone may contribute to the expression of FAO genes.

Immobilization of individual cells by local photo-polymerization on a chip.

A novel separation method for random screening of target cells from a large heterogeneous population by using a local photo-polymerization is developed. A photo-crosslinkable resin solution is mixed with the sample liquid and we controlled the state from sol to gel by irradiating the near ultraviolet (UV) light with the mercury lamp and He-Cd laser near the target cell. We applied three types of immobilization methods such as direct immobilization method, caging method, and direct immobilization with position control method. The selected cell is immobilized in the cured resin directly or inside the cage of the cured resin. In the position control method, laser tweezers are employed to manipulate the target cell indirectly by using the droplet of the resin as a microtool. The cell is positioned properly by the laser manipulation system and is immobilized in the polymerized resin. After the selected cells are immobilized we can easily remove the other objects by the cleaning flow in the microchannel since the polymerized resin strongly binds with the cover glass and resists more than 466 mm s(-1) flow speed in the microchannel (microchannel size: width is 500 micron and depth is 100 micron). We tested the mercury lamp as well as the He-Cd laser for UV-light irradiation at the local area and confirmed improvement of resolution of the cured area by using the He-Cd laser (from 7 micron to 5 micron). Based on this method, we succeeded in single cell immobilization and basic experiments such as culture and fluorescent dyeing of immobilized yeast cells.

Occurrence of fructosyl-amino acid oxidase-reactive compounds in fungal cells.

Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.

Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae.

Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N(epsilon)-fructosyl N(alpha)-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain Delta faoAo2 did not grow. Addition of glucose or (NH(4))(2)SO(4) to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO(2) as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.

Characterization of two fructosyl-amino acid oxidase homologs of Schizosaccharomyces pombe.

Two putative fructosyl-amino acid oxidase genes, FAP1 and FAP2, found in the Schizosaccharomyces pombe genome were cloned and expressed. Both of the gene products (Fap1 and Fap2) were flavoproteins and have no activity for fructosyl-amino acids. It was suggested that Fap1 and Fap2 are an L-pipecolic acid oxidase and L-saccharopine oxidase, respectively.

Isolation and extraction of target microbes using thermal sol-gel transformation.

We developed a novel separation method for random screening of target microorganisms from a large heterogeneous population by using a local viscosity control. A thermal sol-gel transformation material is mixed with the sample liquid and we controlled the state from sol to gel and gel to sol reversibly based on the temperature change controlled by heating of the microelectrode with the electric current and focused laser irradiation near the target. The selected microorganisms are fixed on the bottom plate by gel, since the viscosity around the target is temporally increased by the local heating by the focused laser. The other objects are easily washed away by the cleaning flow in the microchamber. Process of fixation, cleaning, isolation and extraction of the target microbe was possible in very short time. Based on this method, two separation systems are developed and basic experimental results of fixation and isolation of targets are shown.

Investigation of 1-deoxy-D-xylulose 5-phosphate synthase and transketolase of Bacillus subtilis in relation to vitamin B6 biosynthesis.

In Escherichia coli, 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose 5-phosphate are believed to be direct precursors of vitamin B6 (B6), and 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) and transketolase could catalyze the formation of each precursor. In this report, the possible involvement Dxs and transketolase (Tkt) in B6 biosynthesis in Bacillus subtilis was investigated. The gene disruptant of tkt and conditional mutants of dxs were constructed, and their ability of B6 biosynthesis was examined. It was found that the tkt disruptants retain the ability to synthesize B6. The conditional mutant of dxs synthesized the same amount of B6 per dry cell weight as the wild-type strain. Therefore, it is very likely that neither Dxs nor transketolase is involved in B6 biosynthesis in B. subtilis.

A novel NAD-dependent dehydrogenase, highly specific for 1,5-anhydro-D-glucitol, from Trichoderma longibrachiatum strain 11-3.

A novel NAD-dependent dehydrogenase highly specific for 1,5-anhydro-D-glucitol (1,5-AG) was found in the cell extract of an imperfect fungus, Trichoderma longibrachiatum strain 11-3. This fungus used 1,5-AG as a sole carbon source for growth and transformed 1,5-AG into glucose. 1,5-AG dehydrogenase (AGH) was purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 36 and 141 kDa by SDS-PAGE and by gel filtration, respectively, suggesting that the enzyme was homotetrameric. The enzyme was highly specific for 1,5-AG and did not exhibit activity with any sugar or sugar alcohol tested in this study other than 1,5-AG. A linear relationship between the initial rate of the enzyme reaction and the concentration of 1,5-AG at the physiological level was observed. The presence of glucose in abundance did not interfere with the relationship. The optimum temperature for the enzyme reaction was 50 degrees C, and the enzyme was stable at temperatures up to 70 degrees C. These results suggested that AGH is a novel enzyme and is useful for specifically diagnosing diabetes mellitus.

Mechanism for degradation of poly(sodium acrylate) by bacterial consortium no. L7-98.

Poly(sodium acrylate) (PSA) can be degraded by consortia of several bacterial species. We investigated the degradation mechanism for PSA (average molecular weight, 2100) by consortium no. L7-98. PSA was used as the sole carbon source in a mineral salt medium. After cultivation, the PSA had a range of molecular weights, including low-molecular-weight compounds, which were purified by gel-permeation and reversed-phase column chromatography. One purified compound, B1, with the molecular weight of 200, had a carbonyl group next to the terminus, according to 1H and 13C nuclear magnetic resonance spectrometry and X-ray analysis of the crystal structure. Two categories of metabolites of PSA were detected in the culture by electrospray ionization mass spectrometry. Results of high-resolution mass spectrometry (HR-MS) suggested that one kind of compounds had a carbonyl group and that the other kind of compounds had an aldehyde group and a double bond. Compounds having the molecular weights of 200 and 272 were rapidly produced from an acrylic acid oligomer with the molecular weight of 258 by resting cells of the consortium. HR-MS showed that a methylene group at the terminal unit was oxidized to a carbonyl group and that the compound with the molecular weight of 200 was compound B1. From these results, we propose that the degradation pathway of PSA involves (i) oxidation of a methylene group to a carbonyl group next to the terminus, (ii) decarboxylation to form an aldehyde group and dehydrogenation to form a double bond between the terminal unit and the next unit, and (iii) oxidation of the aldehyde group to a carboxyl group followed by elimination of an acetic acid.

yaaD and yaaE are involved in vitamin B6 biosynthesis in Bacillus subtilis.

We show that yaaD and yaaE are involved in vitamin B6 (B6) biosynthesis in Bacillus subtilis. This is the first report which identifies genes involved in B(6) biosynthesis in B. subtilis. Based on homology, yaaD and yaaE belong to the highly conserved SNZ and SNO families, respectively. Disruptants of yaaD and yaaE required pyridoxal (PL) or pyridoxine (PN), and grew in the same way as the wild type in a minimal medium supplemented with 0.05 mM PL. The SNZ family is considered to be involved in singlet-oxygen resistance. Singlet-oxygen quenchers, L(+)-ascorbic acid and reduced glutathione, did not support the growth of these disruptants. Both yaaD and yaaE were transcribed at the highest level during the middle- to late-exponential phase and at a much lower level during the stationary phase. Neither PL nor PN affected the transcriptional rates of yaaD and yaaE. It is concluded that yaaD and yaaE are involved in B6 biosynthesis in B. subtilis, and are transcribed at the highest level during the middle- to late-exponential phase.

serC is involved in vitamin B6 biosynthesis in Escherichia coli but not in Bacillus subtilis.

The possible involvement of serC in vitamin B6 (B6) biosynthesis in Bacillus subtilis was investigated and compared with that in Escherichia coli. The genes of E. coli and B. subtilis were disrupted with pBEN66 and pMutin1-derived integration vectors, respectively. Nutrient requirement analyses showed that the serC-disrupted E. coli mutant required pyridoxine (PN) and L-serine, and lacked the ability to synthesize B6. Glycolaldehyde (GA), a confirmed precursor of B6, could replace PN and support the growth of the disruptant. However, the serC-disrupted E. coli mutant grown in a minimal medium supplemented with L-serine and GA synthesized B6 at a level less than 20% of that synthesized by the wild type. In contrast to E. coli, the serC-disrupted B. subtilis mutant required L-serine or glycine for growth, but did not require PN. The serC disruptant retained its ability for B6 biosynthesis and produced almost the same amount of PN as the wild type. GA had no effect on the growth and level of B6 biosynthesis of both the wild type and the serC disruptant. These results lead to the conclusion that serC is directly involved in B6 biosynthesis in E. coli, but not in B. subtilis.