天知 誠吾

Alphaproteobacterium strain Q-1 is able to oxidize iodide (I-) to molecular iodine (I-2) by an oxidase-like enzyme. One of the two isoforms of the iodide-oxidizing enzyme (IOE-II) produced by this strain was excised from a native polyacrylamide gel, eluted, and purified. TOE-It appeared as a single band (51 kDa) and showed significant in-gel iodide-oxidizing activity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heat treatment. However, at least two bands with much higher molecular masses (150 and 230 kDa) were observed with heat treatment (95 degrees C, 3 min). IOE-II was inhibited by NaN3, KCN, EDTA, and a copper chelator, o-phenanthroline. In addition to iodide, IOE-II showed significant activities toward phenolic compounds such as syringaldazine, 2,6-dimethoxy phenol, and p-phenylenediamine. IOE-II contained copper atoms as prosthetic groups and had UV/VIS absorption peaks at 320 and 590 nm. Comparison of several internal amino acid sequences obtained from trypsin-digested IOE-II with a draft genome sequence of strain Q-1 revealed that the products of two open reading frames (loth and IoxC), with predicted molecular masses of 62 and 71 kDa, are involved in iodide oxidation. Furthermore, subsequent tandem mass spectrometric analysis repeatedly detected peptides from IoxA and IoxC with high sequence coverage (32 to 40%). IoxA showed homology with the family of multicopper oxidases and included four copper-binding regions that are highly conserved among various multicopper oxidases. These results suggest that IOE-II is a multicopper oxidase and that it may occur as a multimeric complex in which at least two proteins (IoxA and IoxC) are associated.

Iodine-129 (I-129) is a fission product with a half-life of 15.7 million years. Because of its long half-life, high mobility, and high affinity to the human body, I-129 is considered as one of the most problematic radionuclides in nuclear waste disposals in deep geological formation. In this study, 16 strains of anaerobic microorganisms, including nitrate-reducing bacteria, iron-reducing bacteria, sulfate-reducing bacteria, and methanogens, were cultured anaerobically with a radioiodine tracer (I-125) to determine whether they possess the ability to associate with iodine. We evaluated it by association efficiency, that was determined by measuring the decrease in I-125 activity in the culture supernatant. It was found that the efficiency of about half of the strains was below detection limit (1.0%), and that of the remaining strains varied from 1.0% to 6.3%, although it was not statistically significant. Similar experiments were conducted by using anaerobic microbial communities inhabiting the iodine-rich subsurface formation water collected from the Minami-kanto gas field in Japan. The specific uptake of iodine by the microbial communities was estimated to be 0.71-2.0 mu g g(-1) dry weight of biomass, indicating that the association ability was, if present, very limited. These results suggest that anaerobic microorganisms, in contrast with aerobic microorganisms, neither enhance nor repress the mobility of I-129, in the case of discharge of this radionuclide from disposal facilities into the surrounding environment. (C) 2010 Elsevier Ltd. All rights reserved.

Malic enzyme (ME) was purified as an electrophoretically homogenous protein from Rhodopseudomonas palustris No. 7. The molecular weight of ME was estimated to be 650 kDa and that of its subunit, 86 kDa. ME activity was remarkably enhanced by di- and mono-valent cations, and the K-a values for Mg2+ and NH4+ were 0.26 and 0.56 mM respectively. Purified ME used both NAD(+) and NADP(+) as electron acceptors, with K-m values of 0.11 and 1.8 mM. The K-m value for L-malate was 1.7 mM using NAD(+) as electron acceptor. Gene cloning of the ME indicated that the ME from R. palustris strain No. 7 was composed of 774 amino acids encompassing the ME and phosphotransacetylase domains, although purified ME displayed no phosphotransacetylase activity. ME activity was inhibited by acetyl-CoA, oxaloacetate, and fructose-6-phosphate. These results suggest that ME plays an important role in the metabolic regulation of R. palustris No. 7 under photoheterotrophic conditions.

A culture filtrate of Bacillus sp. KT12 was used to prepare polyphenyl beta-oligoxylosides from xylan and polyphenols in a one-step reaction. One oligoxyloside transfer enzyme was purified from multiple xylanolytic enzymes in the culture filtrate. N-terminal amino acid sequence determination classified the enzyme as a glycosyl hydrolase family 11 (endo-xylanase). The xylanolytic enzyme activities could be markedly altered; its hydrolytic activity was almost entirely inhibited at acidic pH, whereas near constant transxylosylation activity was observed at pH 4-11. Further, metal ions activated transxylosylation and almost completely inhibited hydrolysis. The enzyme specifically induced a beta-xylosyl transfer reaction to acceptor molecules, such as divalent and trivalent phenolic hydroxyl groups, and displayed no activity toward alcoholic compounds. The Bacillus sp. KT12 xylanolytic enzyme was a suitable enzyme for the synthesis of polyphenyl beta-oligoxylosides.

Fructooligosaccharides (FOS) were produced from sucrose by using crude enzyme preparations of beta-fructofuranosidases (FFases) obtained from sucrose-cultured cells of Aureobasidium pullulans DSM 2404. When the preparation mainly consisted of FFase I, that has high transfructosylating activity, the FOS yield was 62%. When the reaction was carried out with additional commercial glucose isomerase (GI) at an activity ratio of FFase and GI of 1:2, the maximum FOS yield reached 69%. This value was higher than those obtained previously using other Aureobasidium spp. (53-59%).

We developed a method for direct DNA isolation from phyllosphere microbial communities, designated Direct-DIP, This method comprises DNA extraction from non-shredded leaves with benzyl chloride, and DNA purification by gel filtration. Scanning electron microscopy showed that epiphytic microorganisms were completely removed from the leaf surface after benzyl chloride treatment, while microstructures of the leaf were not damaged. Clear DGGE profiles were obtained regardless of the plant species. Shannon diversity indices of DGGE profiles by Direct-DIP were higher than those by a conventional method. Our findings suggest that Direct-DIP is a rapid, simple, and cost-effective method of extracting DNA from phyllosphere microbial communities.

Iodine is an essential trace element for humans and animals because of its important role as a constituent of thyroid hormones. If the anthropogenic iodine-129 ((129)I, half-life: 1.6x 10(7) years), which is released from nuclear facilities into the environment and has a long half-life, participates in the biogeochemical cycling of iodine, it potentially accumulates in the human thyroid gland and might cause thyroid cancer. Therefore, it is necessary to obtain better information on the behavior of iodine in the environment for accurate safety assessments of (129)I. Major pathways of iodine cycling are the volatilization of organic iodine compounds into the atmosphere, accumulation of iodine in living organisms, oxidation and reduction of inorganic iodine species, and sorption of iodine by soils and sediments. Considerable geochemical evidence has indicated that these processes are influenced or controlled by microbial activities, although the precise mechanisms involved are still unclear. This review summarizes current knowledge on interactions between microorganisms and iodine, with special emphasis on newly isolated bacteria possibly contributing to the cycling of iodine on a global scale.

The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I-). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, I-125(-). In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.

Bacterial iodate (IO3-) reduction is poorly understood largely due to the limited number of available isolates as well as the paucity of information about key enzymes involved in the reaction. In this study, an iodatereducing bacterium, designated strain SCT, was newly isolated from marine sediment slurry. SCT is phylogenetically closely related to the denitrifying bacterium Pseudomonas stutzeri and reduced 200 lLM iodate to iodide (I-) within 12 h in an anaerobic culture containing 10 mM nitrate. The strain did not reduce iodate under the aerobic conditions. An anaerobic washed cell suspension of SCT reduced iodate when the cells were pregrown anaerobically with 10 mM nitrate and 200 mu M iodate. However, cells pregrown without iodate did not reduce it. The cells in the former category showed methyl viologen-dependent iodate reductase activity (0.31 U mg(-1)), which was located predominantly in the periplasmic space. Furthermore, SCT was capable of anaerobic growth with 3 mM iodate as the sole electron acceptor, and the cells showed enhanced activity with respect to iodate reductase (2.46 U mg(-1)). These results suggest that SCT is a dissimilatory iodate-reducing bacterium and that its iodate reductase is induced by iodate under anaerobic growth conditions.

beta-Fructofuranosidase I (FFase I) formed by Aureobasidium pullulans DSM 2404 was purified. The enzyme had a molecular weight of about 430 kDa, was not affected by various metal ions and showed high transfructosylating activity. The yield of fructooligosaccharides production using purified FFase I was 62%.

Strobilurus ohshimae is an edible mushroom, and it specifically forms its fruiting bodies on buried sugi (Cryptomeria japonica) twigs. In this research, we studied lignindegrading activity of S. ohshimae. We isolated 18 strains of S. ohshimae from various regions of Japan, and determined their lignin degradation rates on sugi wood meal medium. All the strains of S. ohshimae degraded approximately 6%-12% of sugi lignin in 30 days, and these lignin degradation rates were 1.5-3 times higher than those of Trametes versicolor, which is a typical lignin-degrading fungus. Among the three main lignin-degrading enzymes, activity of lignin peroxidase and manganese peroxidase was not observed, while 4340 U/g of laccase was produced in 30 days. To investigate the effect of wood species on lignin degradation by S. ohshimae, the lignin degradation rate and laccase productivity on sugi wood meal medium were compared with those on beech (Fagus crenata). In T. versicolor, both lignin degradation rate and laccase productivity were higher on beech than on sugi. Conversely, in S. ohshimae, lignin degradation rate and laccase productivity were higher on sugi than on beech. Therefore, it was suggested that coniferous lignin is not always difficult to degrade for the fungi that inhabit softwood.

Five strains of basidiomycetes (Lentinula edodes, Coprinus phlyctidosporus, Hebelorna vinosophyllum, Pleurotus ostreatus and Agaricus bisporus), one strain of ascomycete (Hormoconis resinae) and six strains of imperfect fungi (Penicillium chrysogenum, Penicillium roque-fortii, Cladosporium cladosporioides, Alternaria alternata, Aspergillus niger and Aspergillus oryzae) were cultured in a liquid medium containing a radioactive iodine tracer (I-125), and were tested for their abilities to volatilize or accumulate iodine. Of the fungal strains tested, 11 strains volatilized a considerable amount of iodine, with L. edodes showing the highest volatilization rate of 3.4%. The volatile organic iodine species emitted from imperfect fungi cultures was identified as methyl iodide (CH3I)- In contrast, six fungal strains in 12 strains accumulated a considerable amount of iodine from the medium with concentration factors of more than 1.0. Among these, Alt. alternata and Cl. cladosporioides accumulated more than 40% of the iodine in their hyphae, and showed high concentration factors of 22 and 18, respectively. These results suggest that filamentous fungi have a potential to influence the mobility and speciation of iodine by volatilization and accumulation. Considering their great biomass in soils, filamentous fungi may contribute to the global circulation of stable iodine and also the long-lived radioiodine, I-129 (half-life: 1.6 x 10(7) years), released from nuclear facilities into the environment. (c) 2006 Elsevier Ltd. All rights reserved.

At least five types of beta-fructofuranosidases (FFases I, II, III, IV and V) were found in the cell wall of Aureobasidium pullulans DSM2404 grown in a sucrose medium. The fungus first catalyzed the transfructosylation of sucrose, and produced fructooligosaccharide (FOS) and glucose in the culture. FOS was then consumed together with glucose, and finally fructose was produced. In the FOS-producing period, the fungus expressed FFase I as a dominant FFase. However, in the FOS-degrading period, the levels of FFases II, III, IV and V increased. The ratios of transfructosylating activity to hydrolyzing activity by FFases I-V were 14.3, 12.1, 11.7, 1.28 and 8.11, respectively. When glucose was used as a carbon source, only FFase I showed significant activity. On the other hand, the activities of all five FFases were detected when FOS or fructose was used as a carbon source. These results suggested that the expression of FFase I was not repressed by glucose, but those of FFases II-V were strongly inhibited in the presence of glucose. It is considered that FFase I plays a key role in FOS production by this fungus, whereas FFase IV may function as a FOS-degrading enzyme with its strong hydrolyzing activity.

We report the fruiting-body formation and cultivation properties of Asterophora lycoperdoides, a fungicolous fungus. Asterophora lycoperdoides formed fruiting bodies on potato dextrose agar medium in approximately 1 week, although this fungus shows high host specificity to Russula nigricans in nature. Optimal temperature of mycelial growth and fruiting-body formation was 25°C. Mannitol or soluble starch was preferably used as a carbon source, and amino nitrogen was preferably used as the nitrogen source. For a better understanding of the relationship between A. lycoperdoides and R. nigricans, we cultivated A. lycoperdoides on media supplemented with freeze-dried fruiting bodies of various fungi. The germination rate was approximately 2.5 times higher on the medium containing freeze-dried R. nigricans than that on the PDA medium. The mycelia extended most rapidly in the presence of R. nigricans. Furthermore, the stipe length of its fruiting body was the longest on the medium containing R. nigricans. These results indicated that A. lycoperdoides can grow faster by utilizing certain substances that are abundantly contained in R. nigricans, such as mannitol, or by utilizing R. nigricans itself. It is considered that the constituents of R. nigricans might contribute to the host specificity of A. lycoperdoides. © 2006 The Mycological Society of Japan and Springer-Verlag.

An efficient method for the isolation of iodine-accumulating bacteria was established. A total number of 138 bacterial strains were gown on agar media containing I-125, and the strains having strong radioactivity were chosen by autoradiography. They were then grown in liquid media containing I-125, and the radioactivity of the cells and the supernatants was determined after centrifugation through silicone oil. Eight strains were found to remove 78 to 90% of iodine from the media within 24 hours, and corresponding amount of iodine was detected in their cells. The advantage of this method is in its simplicity and the low detection limit.

Long-lived radioactive iodine (¹²⁹I, half-life: 1.57×10⁷ y) has been released into the environment from nuclear fuel reprocessing plants. ¹²⁹I may also be released from ground storage of nuclear waste. Given its long half-life, a better understanding of the behavior of iodine in the environment is necessary to ensure the safety of humans and the health of the environment. In this report, we summarize our recent results and new experimental data about microbial influences on the mobility and transformation of iodine. Microbial volatilization of organic iodine was observed in soil slurries and seawater samples, and various species of aerobic bacteria were considered to play a significant role through methylation of iodide (I^-) to form methyl iodide (CH₃I). The volatilization of iodine was also found in iodide-rich natural gas brine water, where iodide concentration is approximately 2, 000 times higher than that in seawater. In this case, however, a significant amount of molecular iodine (I₂) was produced together with organic iodine compounds. Iodide-oxidizing bacteria, which oxidize iodide to I₂, were isolated from seawater and natural gas brine water. Phylogenetically, they were divided into two groups within the α-subclass of the Proteobacteria (Roseovarius sp. and unidentified bacteria), and they produced not only I₂ but also diiodomethane (CH₂I₂) and chloroiodomethane (CH₂C1I). Iodide-accumulating bacteria, which accumulate iodide to concentrations 5, 500-fold over that of the medium, were also isolated from marine sediment. They were closely related to Arenibacter troitsensis, and iodide uptake was mediated by an active transport system. Our results suggest that the fate of iodine can be affected by microorganisms, particularly by bacteria, through processes such as volatilization, oxidation, and accumulation.

Iodide-oxidizing bacteria (IOB), which oxidize iodide (Gamma) to molecular iodine (I-2), were isolated from iodide-rich (63 mu M to 1.2 mM) natural gas brine waters collected from several locations. Agar media containing iodide and starch were prepared, and brine waters were spread directly on the media. The IOB, which appeared as purple colonies, were obtained from 28 of the 44 brine waters. The population sizes of IOB in the brines were 10(2) to 10(5) colony-forming units (CFU) mL(-1). However, IOB were not detected in natural seawaters and terrestrial soils (fewer than 10 CFU mL(-1) and 10(2) CFU g wet weight of soils) 1, respectively). Interestingly, after the enrichment with 1 mM iodide, IOB were found in 6 of the 8 seawaters with population sizes of 10(3) to 10(5) CFU mL(-1). 16S rDNA sequencing and phylogenetic analyses showed that the IOB strains are divided into two groups within the a-subclass of the Proteobacteria. One of the groups was phylogenetically most closely related to Roseovarius tolerans with sequence similarities between 94% and 98%. The other group was most closely related to Rhodothalassium salexigens, although the sequence similarities were relatively low (89% to 91%). The iodide-oxidizing reaction by IOB was mediated by an extracellular enzyme protein that requires oxygen. Radiotracer experiments showed that IOB produce not only I-2 but also volatile organic iodine, which were identified as diiodomethane (CH2I2) and chloroiodomethane (CH2ClI). These results indicate that at least two types of IOB are distributed in the environment, and that they are preferentially isolated in environments in which iodide levels are very high. It is possible that IOB oxidize iodide in the natural environment, and they could significantly contribute to the biogeochemical cycling of iodine.

Intracellular and extracellular alcohol oxidases (AOint and AOext) were purified from the liquid and solid cultures of a thermophilic fungus, Thermoascus aurantiacus NBRC 31693, as electrophoretically and isoelectrophoretically homogeneous proteins, respectively. Both enzymes contained a flavin adenine dinucleotide (FAD) cofactor and were stained with Schiff's reagent. The molecular weight of AOint was estimated to be about 320 kDa and its subunit was 75 kDa. The molecular weight of AOext was about 560 kDa, and it was composed of two types of subunits (75 kDa and 59 kDa). The pIs of AOint and AOext were 5.88 and 6.08, respectively. AOint and AOext were stable up to 60 degrees C and 55 degrees C, respectively. The enzymes were stable over a wide range of pH from 6 to 11. AOint oxidized short straight-chain alcohols (K-m for methanol, 13.5 mM and K-m for ethanol, 15.8 mM). On the other hand, AOext could oxidize secondary alcohols and aromatic alcohols (veratryl alcohol and benzyl alcohol) in addition to straight-chain alcohols (K. for methanol, 0.5 mM and K-m for ethanol, 10.2 mM).

Thermoascus aurantiacus showed the best growth on medium containing pectin as a carbon source. The enzyme involved in the production of catalase in the fungus was alcohol oxidase. Formaldehyde dehydrogenase and formate dehydrogenase, in addition to alcohol oxidase and catalase, were detected in the cells grown on pectin. Alcohol oxidase was alkali resistant (pH 7 to 11), and was comparatively heat stable (55° C).

Iodide (I-)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive (125)1-. When they were grown in a liquid medium containing 0.1 muM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 muM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 +/-3.6 (mean +/- standard deviation) pmol of iodide per mg of dry cells and 5.5 x 10(3), respectively. In the presence of much higher concentrations of iodide (1 muM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 p,M and 0.55 ptnol min(-1) mg of dry cells(-1), respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (10(3)(-)), was not transported.

Biological volatilization of iodine from seawaters was studied using a radiotracer technique. Seawater samples were incubated aerobically in serum bottles with radioactive iodide tracer (I-125), and volatile organic and inorganic iodine were collected with activated charcoal and silver wool trap, respectively. Iodine was volatilized mainly as organic iodine, and inorganic iodine volatilization was not observed. Influence of light intensity on the volatilization was determined, but no significant differences were observed under light (70,000 lux) and dark conditions. The effect of the chemical form of iodine on the volatilization was determined, and the results suggested that volatilization preferentially occurs from iodide (I-) but not from iodate (IO3-). Volatilization did not occur when the samples were autoclaved or filtered through a 0.22-mum pore size membrane filter. Incubation of the samples with antibiotics caused decreased volatilization. Conversely, enhanced volatilization was observed when the samples were incubated with yeast extract. Fifty-nine marine bacterial strains were then randomly isolated from marine environments, and their iodine-volatilizing capacities were determined. Among these, 19 strains exhibited significant capacities for volatilizing iodine. 16S ribosomal RNA gene comparisons indicated that these bacteria are members of Proteobacteria (alpha and gamma subdivisions) and Cytophaga-Flexibacter-Bacteroides group. One of the strains, strain C-19, volatilized 1 to 2% of total iodine during cultivation, and the gaseous organic iodine was identified as methyl iodide (CH3I). These results suggest that organic iodine volatilization from seawaters occurs biologically, and that marine bacteria participate in the process. Considering that volatile organic iodine emitted from the oceans causes atmospheric ozone destruction, biological iodine volatilization from seawater is of great importance. Our results also contribute to prediction of movement and diffusion of long-lived radioactive iodine (I-129) in the environment.

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M-r, about 235,000; subunit M-r about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50degreesC, respectively. The Km of the enzyme for D-lactate was 0.8 mm. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxy-butyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mm). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.

Iodine is an important trace element in geological and biological processes. We summarize here recent results and new data of experiments and observations carried out to improve the understanding of concentration levels and behavior of natural and anthropogenic, iodine nuclides in the global environment. The distribution of stable iodine in the Earth's crust was estimated using concentration data in a suite of representative samples and the influence of subduction on the marine iodine cycle was investigated using I-129 systematics on iodine-rich brines from Japan. The importance of microorganisms for the natural iodine cycle is shown in recent studies of iodine sorption on soil and of iodine volatilization from terrestrial and marine environments. Levels of anthropogenic I-129 were measured in samples collected around a spent fuel reprocessing plant in Japan. (C) 2004 Elsevier Ltd. All rights reserved.

The roles of microorganisms in iodine volatilization from soils were studied. Soils were incubated with iodide ion (I-), and volatile organic iodine species were determined with a gas chromatograph. Iodine was emitted mainly as methyl iodide (CH3I), and CH3I emission was sometimes enhanced by the addition of glucose. Soils were then incubated with a radioactive iodinetracer (I-125), and radioiodine emitted from soils was determined. The emission of iodine was enhanced in the presence of yeast extract but was inhibited by autoclaving of soils. The addition of streptomycin and tetracycline, antibiotics that inhibit bacterial growth, strongly inhibited iodine emission, while a fungal inhibitor cycloheximide caused little effect. Forty bacterial strains were randomly isolated from soils, and their capacities for volatilizing iodine were determined. Among these, 14 strains volatilized significant amounts of iodine when they were cultivated with iodide ion. Phylogenetic analysis based on 16S ribosomal DNA sequences showed that these bacteria are widely distributed through the bacterial domain. Our results suggest that iodine in soils is methylated and volatilized as CH3I by the action of soil bacteria and that iodine-volatilizing bacteria are ubiquitous in soil environments. The pathway of iodine volatilization by soil bacteria should be important for understanding the biogeochemical cycling of iodine as well as for the assessment of long-lived radioactive iodine (I-129) in the environment.

Glycine aminotransferase (EC 2.6.1.4; GlyAT) was presumed to be an enzyme concerning the supply of glycine for the extracellular porphyrin production by Rhodopseudomonas palustris No. 7. GlyAT was purified from strain No. 7 as an electrophoretically homogeneous protein. The enzyme was a monomer protein with the molecular weight of about 42,000. From the absorption spectrum of the enzyme (350 run, 410 nm), it was indicated that the enzyme had pyridoxal phosphate as a prosthetic group. The enzyme showed high substrate specificity for glutamate as an amino group donor. Apparent K(m)s for glutamate and glyoxylate were 6.20 mm and 3.75 mm, respectively. The V-max and K-cat for glutamate were 66.8 mumol/min/mg protein and 46.8 s(-1), respectively. The V-max and K-cat for glyoxylate were 68.8 mumol/min/mg protein and 48.2 s(-1). The optimum temperature and pH were 40-45degreesC and 7.0similar to7.5, respectively. The enzyme activity lowered to about 50% in the presence of 15 mm ammonium chloride.

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activivities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.

Lactic acid bacteria (LAB) are among the integral microflora of ragi tape, a dry starter of Balinese rice wine, brem. The species diversity and population level of LAB present in different types of ragi tape were studied by colony hybridization using 16S and 23S rDNA targeted oligonucleotide probes. These probes were DB6, Lbc, Wgp, and Rpt, which were specific for Enterococcus faecium, Lactobacillus curvatus, Weissella spp., and Pediococcus pentosaceus, respectively. Results revealed that P. pentosaceus and Weissella spp. were the predominant LAB in ragi tape, whereas L. curvatus and E. faecium were associated specific to types of ragi tape. A 21-mer species-specific oligonucleotide probe, Rpt, that targets the 16S rDNA of P. pentosaceus was developed in this study and found to be highly specific to be used as an effective tool to enumerate population of this species in ragi tape and its population changes during rice wine production. It was detected that LAB showed active growth during the early stage of brem fermentation. A succession of growth of LAB population during the fermentation was observed in which the heterofermentative LAB, Weissella spp., grew first, followed by the proliferation of P. pentosaceus.

Methyl iodide (CH,I) plays an important role in the natural iodine cycle and participates in atmospheric ozone destruction. However, the main source of this compound in nature is still unclear. Here we report that a wide variety of bacteria including terrestrial and marine bacteria are capable of methylating the environmental level of iodide (0.1 muM). Of the strains tested, Rhizobium sp, strain MRCD 19 was chosen for further analysis, and it was found that the cell extract catalyzed the methylation of iodide with S-adenosyl-L-methionine as the methyl donor. These results strongly indicate that bacteria contribute to iodine transfer from the terrestrial and marine ecosystems into the atmosphere.

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.

A reliable method using I-125 tracer for direct determination of volatile iodine formed in aqueous environmental samples was established Soil solution, seawater and bacterial cell suspension were selected as model samples, and incubated with I-125(-). Volatile inorganic and organic iodine species produced during incubation were collected in silver wool and activated charcoal traps, separately the efficiency of the traps, the storage conditions of I-125(-) stock solution and the procedures to expel the dissolved volatile iodine from the sample solutions were examined. Formation of biological volatile iodine was observed in all samples, and the dominant iodine species was found to be organic iodine. The advantages of this method are its simplicity, low cost and low detection limit.

A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic on near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermenters were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0, while the level of increase in the mutant was only 1.6. Northern and western blot analyses found that at pH 7.0 the transcriptional level and the amount of F(1)beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F(1)beta subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.

Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp, lactis C2 as spontaneous neomycin-resistant mutants, Characteristics of the representative mutant, No, 1016-51, were compared with the parental strain in cultures using a jar fermenter with the pH controlled at various values, At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical, At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24h decreased to 60, 36, and 60% of the parental strain, respectively, During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80h, On the other hand, at pH 4.0, the viable count of the strain No. 1016-51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability, Therefore, it was concluded that the membrane-bound ATPase is essential for this organism to survive at low pH, probably through its function of proton pumping for maintaining cytoplasmic pH levels.