• Microbiology and Biotechnology Letters
• /
• v.19 no.3
• /
• pp.222-227
• /
• 1991

Rhizobium fredii USDA193 is one of the causal organism for root nodule formation in soybean (peking). Previously we cloned the pectate lyase gene (SY1) of R. fredii USDA193. The $pel^-$ mutants (SY1$\Omega$ and SY1$\Omega$1) of SY1 were obtained using the in vitro insertional omega mutagenesis of RpelB (of Rhizobium pel) and fill-in reaction of RpelE (of Rhizobium pel) gene respectively, and we constructed two mutants (R, fredii USDA193$\Omega$ and R. fredii USDA193$\Omega$1) in pectate lyase function by marker-exchange with pe1B::$\Omega$ and R. fredii USDA193 strain (rif). The pectate lyase activity of two pel- mutant of R. fredii USDA193 was determined by spectrophotometric method. However, all pectate lyase activity of these mutants was not lost upon the mutagenesis by marker-exchange. This suggests that other pectate lyase genes may be present on the plasmid or the chromosome of R. fredii. As yet we do not have evidence linking RpelB and RpelE genes of R. fredii directly to the early nodulation process.

• Applied Biological Chemistry
• /
• v.30 no.2
• /
• pp.163-168
• /
• 1987

A method, based upon the separation of cellular proteins by one-and two-dimensional electrophoresis was used for distinguishing butween Bradyrhizobium japonicum strains and Rhizobium fredii strains. Significant differences in protein pattern of one-dimensional SDS-PAGE vs-ere observed between Rhizobium fredii strains and Bradyrhizobium japonicum strains. The differences in six distinct main lands were observed among total 52 kinds of protein bands. Furthermore, the distribution of proteins in two groups by two-dimensional polyacrylamide gel electrophoresis was very different. The majority of visible proteins of Rhizobium fredii were acidic, whereas those of Bradyrhizobium japonicum were basic. In addition, amino acid composition was analyzed to detect the differences between two groups. No significant differences in amino acid composition were observed between Bradyrhizobium japonicum strains and Rhizobium fredii strains. The results indicate that one-and two-dimensional polyacrylamide gel electrophoresis were useful for identifying rhizobia isolates. One-dimensional SDS-PAGE of rhizobia proteins provided a rapid method for screening a large number of isolates, whereas two-dimensional electrophoresis was more of resolution and easiness for analyzing protein spots.

• Korean Journal Plant Pathology
• /
• v.12 no.3
• /
• pp.351-357
• /
• 1996

콩으로부터 분리한 140균주의 근류균은 25균주(17.9%)가 Rhizobium fredii로 115 균주(82.1%)가 Brady-rhizobium japonicum으로 동정되었다. R. fredii에 속하는 분리 균주의 생존 pH 범위는 4..5∼9.0이었고 B. japonicum의 생육 pH 범위는 5.5∼8.0로 비교적 좁게 나타났다. B. japonicum에 속하는 98균주 중에서는 53균주(54%)가 IAA를 생산하지 않는 GT I group으로, 45균주(46%)는 IAA를 생산하는 GT II group으로 명확하게 구분되었으며, 항생물질에 대한 내성 유무에 의해 10개의 group으로 구분되었다.

• Korean Journal of Microbiology
• /
• v.31 no.1
• /
• pp.27-36
• /
• 1993

Rhizobium fredii USDA191 은 대기 중의 질소를 환원하여 식물체의 생육에 필요한 질소원을 공급해주는 세균으로 다량의 체외 다당류를 합성한다. 전위요소 Tn5의 삽입에 의한 돌연변이 유도로 다당류결핍 변이주 R. fredii YKL293 가 분리되었으며 이 변이주로부터 Tn5 에 인접한 DNA 단편이 pUC19 에 클로닝되었고(plyk5293),이 DNA 단편을 탐침으로 하여 .lambda.NM1149 에 구성되 USDA191 genomic library 로부터 야생형체외다당류 합성관련 유전자(exo) 를 함유한 클론 .lambda. NM1149 22E 를 plaque 혼성화에 의하여 분리하였다. 클론 NM1149.22E 에 들어있는 exo 유전자를 pBR322 에 옮겨서 pJW33을 만들고, 재조합체 pJW33 을 Escherichia coli POII734 에 도입시켜 lacZ 구조유전자를 함유한 MudI 1734 가 exo 유전자의 프러모토와 융합되어 lacZ 구조유전자의 전사가 이루어지도록 하였다. 위와 같이 만들어진 재조합체 플라스미드 pUM21을 함유한 E. coli JM83 은 .betha.-galactosidase 를 합성하였으며, 야생형 tacZ 유전자를 갖고 있는 E. coli LE392 에 비해서 14-25배 정도 낮은 역가를 보였다.

• Microbiology and Biotechnology Letters
• /
• v.23 no.3
• /
• pp.275-281
• /
• 1995

From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and pYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37$\circ$C. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase- negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS- PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.

• Applied Biological Chemistry
• /
• v.30 no.2
• /
• pp.153-162
• /
• 1987

Soybean rhizobia were isolated from 101 soybean (Glycine max.) cultivar which had been grown for the breeding experiment in Korea. Seven strains of the fast-growing soybean rhizobia and nine strains of the slow-growing soybean rhizobia were selected on the basis of their growth rate in AMA medium and their high ability of nodulation. The slow-growing soybean rhizobia were identified as Bradyrhizobium japonicum in the acetylene-reducing activity, microbial characteristics, and biochemical characteristics whereas the fast-growing soybean rhizobia were very similar to Rhizobium fredii.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.41 no.6
• /
• pp.718-724
• /
• 1996

Two soybean cultivars, ‘Lee’ and ‘Peking’, were used to evaluate the competition and interaction of rhizobium strains PRC205 (R. fredii, fast-grower) and USDA110 (B. japonicum, slow-grower). Strains were inoculated separately on the root parts of a split-root growth system. Both root sides were inoculated simultaneously with four combinations of strain treatment to evaluate the competition of strains. And to evaluate the interaction of strains one side of split-root system was inoculated a week prior to the other side. Nodule mass and dry weight of the plants were measured 3 weeks after treatments. PRC205 showed no effective nodulation and no competing ability with USDA110 on Lee cultivar, however, contrary results on Peking cultivar. Top dry weight of Lee inoculated with PRC205 was much lower than that of any other inoculation treatments, however, in Peking that with PRC205 was higher than that with USDA110. There were no differences in root dry weight among the inoculation treatments. USDA110 used as primary inoculant suppressed nodule mass of opposite side, secondary inoculant, severely in both cultivars. PRC205 showed same tendency as USDA110 in Peking, but revealed little suppression effects on USDA110 used as secondary inoculant in Lee. USDA110 used as primary inoculant in Lee and PRC205 in Peking showed much more dry weight of soybean plants than that of other treatments.

• Korean Journal of Soil Science and Fertilizer
• /
• v.20 no.3
• /
• pp.275-283
• /
• 1987

In order to find out the effectiveness of nitrogen fixation in rhizobia-legume symbiotic relationship, ecological and physiological characteristics of indigenous rhizobia distributed in Korean soils, that is, dissimilatory nitrate reduction patterns of indigenous soybean rhizobia isolated from four different soils, and differences in one-and two-dimensional polyacrylamide gel electrophoretic pattern of proteins among the each subgroups of Bradyrhizobium japonicum and Rhizobium fredii, were investigated. The results were summarized as follows: 1. The indigenous soybean rhizobia isolated from four different soils could be classified into 4 groups depending on growth rate and dissimilatory nitrate reduction pattern, that is, $S_1$ (slow-grower; Bradyrhizobium japonicum and nitrate denitrifier), $S_2$ (slow-grower; Bradyrhizobium japonicum and nitrate respirer), $F_1$ (fast-grower; Rhizobium fredii and denitrifier), and $F_2$ (fast-grower; Rhizobium fredii and nitrate respirer). 2. The population ratio of fast- to slow-growing R. japonicum was 39% to 61%, and the ratio of denitrifier to nitrate respirer was 31% to 69% and 89% to 11% in fast and slow-grower, respectively. Some differences were observed between fast- and slow-growing R. japonicum but no significant difference was observed between denitrifier and nitrate respirer within same growth type by one and two dimensional SDS-polyacrylamide gel electrophoretic patterns.

• Applied Biological Chemistry
• /
• v.29 no.2
• /
• pp.175-181
• /
• 1986

Eightyseven strains of indigenous Rhizobia were isolated from the nodule of soybean cultivar, Danyup, cultivated in four different soils sampled from continuously soybean cultivated and newly reclaimed fields. The strains were grouped into Bradyrhizobium japanicum (slow-grower:55 strains) and Rhizobium fredii (fast-grower: 32 strains). The both groups could be divided into two sub-groups according to the denitrification characteristics, that is, denitrifying fast-grower (F-I), nitrate respiring fast-grower (F-II), denitrifying slow-grower (S-I). and nitrate respiring slow-grower (S-II). Among the 87 isolates, F-I, F-II, S-I and S-II sub-groups were 10, 22, 48 and 7 strains, respectively. The one-and two-dimensional polyacrylamide gel electrophoretic pattern of the four sub-groups were compared and discernible difference was observed between fast and slow-grower, but the difference was not discernible between subgroups within the same growth rate group.