• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.120-120
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• 1995

The total size of the pF AR1, a genomic clone of Frankia FaCI, was estimated to be about 44Kb by summation of the individual fragment length generated by single or double restriction enzymes. Southern hybridization analyses with Azotobacter vinelandii nif-genes as probes and partial sequencing analyses of the subclones revealed that organization of the nif-gene in the FaCI strain was nifV, H, D, K, E, N, X, W, B. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes but the positioning of the nifV-like gene relative to the nifHDK cluster differs. A consensus nif-promoter-like sequence, found at 5' of nifH, was not detected upstream of the niJV-like gene. nifV-like gene contained a ORF of 1206 NT encoding 401 amino acids. The nucleotide sequence and deduced amino acid sequence of the gene exhibit homology value of 65% and 41% with that from A vinelandii, respectively. The putative Shine-Dargamo sequences were present preceding nitK, nifH, D, K, and nifE, and in nitK gene putative start codon GTG was detected instead of A TG. The nucleotide and amino acid sequence of niIK of FaCI showed 82% and 76% homolgy with those of Frankia HFPCc 13, respectively. Amino acid sequence of niIK showed 69% and 61% homology with those of A vinelandii, Klebsiella pnewnoniae, respectively, while that of nifE 73% and 71%, respecti vely.i vely.

• 한국환경과학회지
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• 제21권9호
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• pp.1139-1147
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• 2012

To find out the growth conditions for the maximum activity of nitrogenase which catalyzes nitrogen fixation in Rhodobacter sphaeroides, the promoter activities of nifA and nifH were analyzed and the results indicated that expression of both nifA and nifH was increased in response to deprivation of both O2 concentration and nitrogen source. The nifA mutant was constructed by deleting the gene to investigate the effect of NifA, the transcriptional regulator, on the nifH and nifA expression in R. sphaeroides. Analysis of expression of nif genes using the nifA::lacZ and nifH::lacZ fusions in the nifA mutant revealed that NifA acts as a positive activator for nifH and an autoregulator in its own expression. The promoter activities of nifA and nifH in the prrA mutant grown under anaerobic and ${NH_4}^+$-free conditions were derepressed, comparing with those of the wild-type grown under the same conditions, indicating that the prrA product acts as a positive regulator in expression of nifA and nifH.

• 생명과학회지
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• 제22권8호
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• pp.1009-1017
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• 2012

Rodobacter sphaeroides에서 orf282 유전자는 cbb3 terminal oxidase를 암호화하는 ccoNOQP 오페론과 혐기적 활성자인 FnrL을 암호화하는 fnrL 유전자 사이에 있으며, 아직은 기능이 잘 알려지지 않았다. orf282 유전자의 기능을 알기 위해 우리는 orf282의 일부를 삭제함으로써 유전자를 붕괴시켜 orf282-minus mutant를 제조하였다. 두개의 FnrL 결합 부위가 orf282의 upstream에 존재한다는 것이 밝혀져 있으며, orf282 유전자가 FnrL에 의해 양성적으로 조절된다는 것이 증명되었다. orf282 유전자는 B875와 B800-850 spectral complexes의 형성과 관련이 없다. orf282 mutant에서의 cbb3 oxidase 활성을 wild type와 비교해보면 orf282 유전자가 ccoNOQP 오페론의 조절과 cbb3 cytochrome c oxidase의 생합성과 무관하다는 것을 알 수 있다. orf282 mutant의 구조 유전자인 nifH와 조절유전자인 nifA의 프로모터 활성이 증가한 것은 orf282 유전자 산물이 nifH와 nifA의 발현에서 음성적 effector로 작용한다는 것을 시사한다.

• 미생물학회지
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• 제26권1호
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• pp.20-26
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• 1988

Three $NIf^{-}$ -mutants were isolated from Enterbacter agglomerans 339 through the transposon umtagenesis using a RP4-mobilising system for its nif-gene characterization. All mutants hadn't acetylene-reduction ability. Then we confirmed that Tn5 was inserted into all conserved nif-plasmids through the Southern Hybridization.

• 미생물학회지
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• 제24권3호
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• pp.211-220
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• 1986

Nitrogen fixation (nif) genes of Rhizobium leguminosarum were transferred to nif Klebsiella pneumoniae and E. coli by conjugation after partial heat induction of $RP_4$ :: Mu cts in Rhizobium $R^+$ transconjugant, and the hybrid plasmids in the transconjugant strains were isolated and characterized. In order to transfer the nif genes from Rhizobium, the hybrid plasmid $RP_4$ :: Mu cts was transferred by conjugation from E. coil to the symbiotic nitrogen fixer, R. leguminosarum. After stabillity test, the $RP_4$ :: Mu cts in Rhixobium $R^+$ transconjugant was subjected to partial heat induction by culturing it statically at $38^{\circ}C$ for 16 hours, and then conjugated with the nif defective mutant strains of K. pneumoniae or nif mutant strains of E. coli having whole nif gene plasmid. Recombinant strains of K. pneumoniae, which could grow in a N-free medium and exhibit the nitrogenase activity were selected. However, in the case of E. coli, they could grow well in a NA medium containing antibiotices, but hardly frow in a N-free medium. The hybrid plasmids in these transconjugal strains were isolated by gel electrophoresis and compared their molecular sizes.

• 미생물학회지
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• 제23권1호
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• pp.20-24
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• 1985

Klebsiella pnellmoniae의 nif-lac융합변이주를 사용하여 질소고정 유전자 발현에 미치는 질소원의 효과를 검토하였다. Klebsiella pnellmoniae UN4482를 heat induction하여 만든 Mudllysate를 K. pnellmoniae U UN2979에 접종시켜 nif-lac융합체 약 80여주를 분리하고 이들을 LX series로 명하였다. 이들의 ${\beta}-galactosidase$ 한성은 $NH_4^+$의 존재하에 크게 억제되었다. Serine, glutamine, asparagine같은 아미노산을 켈소원으로 사용했을때 K. pnellmoniae의 성장은 양호하였으며. NFHM배지에서nif-lac융합주LX-9, LX-22의 ${\beta}-galactosidase$의 환성은 억제 효과도 높았으나, glutamine, histidine, arginine 같은 amino acid는 위와 반대 의 효과플 나타냈다. Casitone, prote-ose peptone같은 유기섣소원(2 mg/ml )의 균성장에 대한효과는 전반적으로양호했으나LX-9, LX-22 의 ${\beta}-galactosidase$활성에 대한 억제효과는 각 칠소원에 따라 다르게 나타났다. 한편, 질소원이 없는 최소배지에서의 LX-9와 L LX-22의 ${\beta}-galactosidase$의 활성은 초기 4 시간내에 급격히 증가하였다.

• 미생물학회지
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• 제22권4호
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• pp.265-269
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• 1984

E. coli $P^{RDI}$에서 DAP-영양요구성 변이주를 분리하였으며 이 변이주가 갖고 있는 plasmid의 안정성과 활성도를 확인하는 살험을 통하여 다음과 같은 결론을 얻었다. 1. Mutagen인 NG를 처리 후 DAP-변이주를 보다 쉽게 분리하기 위하여 항생제처리를 하였다. 이때 사용균주가 갖고 있는 plasmid내의 내성인자들 중에 carbr인자로 인하여 같은 계열에 있는 penicillin유도체들에 대해서는 교차내성을 갖고 있음이 확인되었다. 그러냐 penicillin과 같은 기능을 갖고 있으나 그 구조가 다른 cephalexin, cycloserine에 대해서는 교차 내성을 잘 나타내지 않았으므로 항생제처리로서는 cephalexin을 사용하였다. 2. 세균 접합을 통하여 DAP-균주의 특성을 동정하였다. 즉 nif-gene의 안정성과 활성도는 DAP-균주로 부터 plasmid를 전이반은 전이체에서 6-cyanopurine첨가로 확인하였다.

• 미생물학회지
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• 제15권3호
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• pp.131-138
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• 1977

The effects of various nitrogen soruces on the expression of nif gene were investigated using nif-lac fusants of Klebsiella pneumoniae. K. pneumoniae UK 2979 was infected with Mudl lysate prepared by heat induction of K. pneumoniae UK 4482. About 80 nif-lac fusants were greatly repressed. Amino acids, such as serine, glutamine and asparagine, were found to support the growth of K. pneumoniae M5al quite well, and showed a repressive effect on .betha.-galactosidase activities of nif-lac fusants LX-9 and LX-22 in NFHM. Glutamic acid, histidine and arginine rendered poor growth but high activities of .betha.-galactosidase. Good cell growth and high enzyme activity were observed when complex nitrogen sources, such as casitone, proteose pepone, were employed. .betha.-Galactosidase activities of LX-9 and LX-22 in nitrogen free minimal medium increased sharply within first 4 hours.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1339-1348
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• 2015

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.

• Journal of Microbiology and Biotechnology
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• 제20권5호
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• pp.862-870
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• 2010

Cyperus rotundus L. is a perennial herb that was found to be dominating an area in northeast Brazil previously contaminated with petroleum. In order to increase our knowledge of microorganism-plant interactions in phytoremediation, the bacterial community present in the rhizosphere and roots of C. rotundus was evaluated by culture-dependent and molecular approaches. PCR-DGGE analysis based on the 16S rRNA gene showed that the bacterial community in bulk soil, rhizosphere, and root samples had a high degree of similarity. A complex population of alkane-utilizing bacteria and a variable nitrogen-fixing population were observed via PCR-DGGE analysis of alkB and nifH genes, respectively. In addition, two clone libraries were generated from alkB fragments obtained by PCR of bulk and rhizosphere soil DNA samples. Statistical analyses of these libraries showed that the compositions of their respective populations were different in terms of alkB gene sequences. Using culturedependent techniques, 209 bacterial strains were isolated from the rhizosphere and rhizoplane/roots of C. rotundus. Dot-blot analysis showed that 17 strains contained both alkB and nifH gene sequences. Partial 16S rRNA gene sequencing revealed that these strains are affiliated with the genera Bosea, Cupriavidus, Enterobacter, Gordonia, Mycoplana, Pandoraea, Pseudomonas, Rhizobium, and Rhodococcus. These isolates can be considered to have great potential for the phytoremediation of soil with C. rotundus in this tropical soil area.