Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel hup minitransposon

被引:24
作者
Báscones, E
Imperial, J
Ruiz-Argüeso, T
Palacios, JM
机构
[1] Univ Politecn Madrid, Escuela Tecn Super Ingn Agron, Microbiol Lab, E-28040 Madrid, Spain
[2] CSIC, E-28040 Madrid, Spain
关键词
D O I
10.1128/AEM.66.10.4292-4299.2000
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
lHydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the Rhizobium-legume symbiosis, To develop a strategy to generate rhizobial strains with H-2-recycling ability, we have constructed a Tn5 derivative minitransposon (TnHB100) that contains the ca, 18-kb H-2 uptake (hup) gene cluster from Rhizobium leguminosarum by. viciae UPM791, Bacteroids from TnHB100-containing strains of R. leguminosarum by. viciae PRE, Bradyrhizobium japonicum, R. etli, and Mesorhizobium loti expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase In nodules, Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts. In contrast, Sinorhizobium meliloti, M. ciceri, and R, leguminosarum by. viciae UML2 strains showed poor expression of the hup system that resulted in H-2-evolving nodules, For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of hup genes or HupL instability. A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release.
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页码:4292 / 4299
页数:8
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