Mutsuyasu Nakajima

Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metallothionein gene (CUP1)

Cauliflower (Brassica oleracea var. botrytis) tolerates treatment with 25 µM CdCl2 for eight days, but is killed by that with a 50 µM concentration. However, even 15 µM CdCl2 is toxic in the presence of 1 mM L-buthionine sulfoximine (BSO), suggesting the presence of a Cd-inducible phytochelatin and its involvement in Cd-tolerance in cauliflower. To develop heavy metal-tolerant transgenic plants, we ligated the structural gene of yeast metallothionein gene (CUP1) downstream of CaMV35S promoter and introduced the fused gene into cauliflower. A Cd-tolerant transgenic cauliflower was selected, which grew well in the presence of 400 µM or less Cd, whereas the non-transformed cauliflower tolerated only up to 25 µM Cd. The transgenic cauliflower accumulated more Cd, especially in the upper leaves, than the non-transformed plant.In conclusion, by transfer of the yeast metallothionein gene into cauliflower increased Cd-tolerance and Cd-accumulating ability can be conferred to the plant.

Extracellular Polysaccharides of Rhodococcus rhodochrous S-2 Stimulate the Degradation of Aromatic Components in Crude Oil by Indigenous Marine Bacteria

ABSTRACT Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing d-glucose, d-galactose, d-mannose, d-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.

Mucilaginibacter kameinonensis sp., nov., isolated from garden soil.

An extracellular polysaccharide-producing bacterium, strain SCK(T), was isolated from a soil sample taken from Kameino, Fujisawa, Japan. The isolate was Gram-negative and cells were non-motile, irregular-shaped rods that grew optimally at 25 degrees C and grew between pH 5 and 8. Strain SCK(T) contained MK-7 as the major isoprenoid quinone, iso-C(15:0) and C(16:1)omega7c and/or iso-C(15:0) 2-OH as the major fatty acids and sphingolipids, with d-17:0 as the main dihydrosphingosine. Flexirubin-type pigments were also present. The DNA G+C content was 43.7 mol%. On the basis of 16S rRNA gene sequence analysis, strain SCK(T) was shown to belong to the genus Mucilaginibacter. The 16S rRNA gene sequence similarity between strain SCK(T) and the two type strains of Mucilaginibacter was 93%. The results of physiological and biochemical tests allowed phenotypic differentiation of the strain from published Mucilaginibacter species. Therefore, strain SCK(T) represents a novel species, for which the name Mucilaginibacter kameinonensis sp. nov. is proposed. The type strain is SCK(T) (=NBRC 102645(T) =KCTC 22227(T)).

Rapid method utilizing the polymerase chain reaction for detection of canine parvovirus in feces of diarrheic dogs

By using primers based on the sequence of the VP2 gene of canine parovirus (CPV), we established a rapid and specific assay for identification of the virus from fecal specimens based on the polymerase chain reaction (PCR). By use of a pair of primers, a specific 226-bp sequence was amplified by the PCR. All strains of CPV tested gave a specific amplification product by the PCR, while neither porcine parovirus nor host cell did so. The PCR assay can detect fewer particles of CPV than the conventional methods, being able to detect CPV from fecal specimens in a rapid manner, provided that gel filtration of the samples through a spun column was done to remove inhibitory substances from the fecal specimens. These results suggest that the PCR assay can detect the presence of CPV in dogs early enough to prevent secondary infection by CPV in veterinary hospitals.

Relationships between Colony Morphotypes and Oil Tolerance in Rhodococcus rhodochrous

ABSTRACT A mucoidal strain of Rhodococcus rhodochrous was resistant to 10% (vol/vol) n-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance ton-hexadecane. Thus, EPS confer tolerance ton-hexadecane in members of the genusRhodococcus.

Cell-surface hydrophobicity and scum formation of Rhodococcus rhodochrous strains with different colonial morphologies.

Rhodococcus rhodochrous has been reported to be one of the micro‐organisms responsible for the formation of scum which is thick and viscous biological foam in activated sludge plants. The hydrophobicity of mycolic acids present on the cell surface and the long‐branched shape of the hyphae have been thought to contribute to the scum formation. Cell surface hydrophobicity and scum formation of four R. rhodochrous strains with different colony morphologies were determined, and the results showed that the two rough strains had strong cell surface hydrophobicity and produced scum, whereas the weakly hydrophobic smooth strain and the hydrophilic mucoidal strain did not. All four strains displayed long, branched hyphae, and their electrophoretic mobilities were similar, between pH 4 and 9. These data suggest that changes in the cell surface hydrophobicity of the R. rhodochrous result in changes in the culture characteristics and the formation of scum.

Rapid and efficient method to eliminate substances inhibitory to the polymerase chain reaction from animal fecal samples

To detect pathogenic viruses in animal fecal specimens by polymerase chain reaction (PCR) assays, it is important to remove or inactivate PCR-inhibitory substances. Recently, it was reported that such inhibitory substances in human feces could be efficiently eliminated by a cationic surfactant, Catrimox-14 (Iowa Biotechnology, Iowa) during extraction of viral RNA. In the present report, Catrimox-14 was successfully applied to detect pathogenic viruses in fecal specimens from a variety of animals. By extraction of viral DNA in the presence of this cationic surfactant, the PCR assay could detect canine parvovirus (CPV) in all fecal specimens prepared from 13 kinds of animals, i.e., cat, chicken, cow, dog, gerbil, goat, golden hamster, horse, mouse, pig, rat, rabbit, or sheep. Pretreatment by gel-filtration or boiling failed to remove or inactivate the PCR-inhibitory substances in fecal specimens from mouse, goat, rat, and sheep.

Relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus

To elucidate the function of extracellular polysaccharide (EPS) produced by bacteria with hydrophobic surfaces we investigated relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus. Rough strains adhered well to various materials and formed cell aggregates, while their mucoidal derivatives did so poorly. Cell surfaces of the rough strains were more hydrophobic than those of the corresponding mucoidal strains. Production of the EPS by the rough strains was far less than that by the corresponding mucoidal strains. Adhesion of the rough strains to quartz, glass or hydrocarbon was inhibited by the addition of EPS. Thus, rhodococcal EPS plays an important role in determination of the cell-surface hydrophobicity by acting as a hydrophilin, and its addition to the rough strains resulted in inhibition of their adhesion to the substrata.

Inhibitory effects of odor substances, geosmin and 2-methylisoborneol, on early development of sea urchins

Geosmin (1α,10β-dimethyl-9α-decanol) and 2-methylisoborneol ((1-R-exo)-1,2,7,7-tetramethyl-bicyclo-(2,2,1)-heptan-2-ol) (MIB) are volatile terpene derivatives, and have received a great deal of attention because they can cause musty/muddy off-flavor in water and food resources. By the Ames test, these metabolites showed no mutagenicity but antimicrobial activity toward tester strains. While these compounds are produced by various organisms living in aquatic environments, there are few reports of their effects on aquatic organisms. The effects of geosmin and MIB on sea urchin development were examined. The estimated IC50 (50% inhibitory concentration) values for the formation of the fertilization membrane were 16.67 mg geosmin l−1 and 68.77 mg MIB l−1; those for the cell cleavage were 16.58 mg geosmin l−1 and 66.86 mg MIB l−1, suggesting that the toxicity of geosmin and MIB toward sea urchins are comparable to their toxicity toward Salmonella tester strains in the Ames test. These values are far greater than concentrations of these substances observed in aquatic environments with severe muddy off-flavor problems.

Burkholderia acidipaludis sp. nov., aluminium- tolerant bacteria isolated from Chinese water chestnut (Eleocharis dulcis) growing in highly acidic swamps in South-East Asia

Two strains of aluminium-tolerant bacteria, SA33(T) and 7A078, were isolated from Chinese water chestnut (Eleocharis dulcis) growing in highly acidic swamps (pH 2-4) in actual acid sulfate soil areas of Vietnam (SA33(T)) and Thailand (7A078). The strains were Gram-negative, aerobic, non-spore-forming rods, 0.6-0.7 mum wide and 1.3-1.7 mum long. These strains showed good growth at pH 3.0-8.0 and 17-37 degrees C. The organisms contained ubiquinone Q-8 as the predominant isoprenoid quinone and C(16 : 0), C(18 : 1) ω 7c and C(17 : 0) cyclo as the major fatty acids. Their fatty acid profiles were similar to those reported for other Burkholderia species. The DNA G+C content of these strains was 64 mol%. On the basis of 16S rRNA gene sequence similarity, the strains were shown to belong to the genus Burkholderia. Although the 16S rRNA gene sequence similarity values calculated for strain SA33(T) to 7A078 and the type strains of Burkholderia kururiensis, B. sacchari and B. tuberum were 100, 97.3, 97.1 and 97.0 %, respectively, strains SA33(T) and 7A078 formed a group that was distinct in the phylogenetic trees; the DNA-DNA relatedness of strain SA33(T) to 7A078 and these three type strains were respectively 90, 47, 46 and 45 %. The results of physiological and biochemical tests, including whole-cell protein pattern analysis, allowed phenotypic differentiation of these strains from described Burkholderia species. Therefore, strains SA33(T) and 7A078 represent a novel species, for which the name Burkholderia acidipaludis sp. nov. is proposed. The type strain is SA33(T) (=NBRC 101816(T) =VTCC-D6-6(T)). Strain 7A078 (=NBRC 103872 =BCC 36999) is a reference strain.