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荧光假单胞菌WCS365的NADH脱氢酶的特性及其在根部定殖竞争中的作用

Characterization of NADH dehydrogenases of Pseudomonas fluorescens WCS365 and their role in competitive root colonization.

作者信息

Camacho Carvajal Margarita M, Wijfjes André H M, Mulders Ine H M, Lugtenberg Ben J J, Bloemberg Guido V

机构信息

Leiden University, Institute of Molecular Plant Sciences, The Netherlands.

出版信息

Mol Plant Microbe Interact. 2002 Jul;15(7):662-71. doi: 10.1094/MPMI.2002.15.7.662.

Abstract

The excellent-root-colonizing Pseudomonas fluorescens WCS365 was selected previously as the parental strain for the isolation of mutants impaired in root colonization. Transposon mutagenesis of WCS365 and testing for root colonization resulted in the isolation of mutant strain PCL1201, which is approximately 100-fold impaired in competitive tomato root colonization. In this manuscript, we provide evidence that shows that the lack of NADH dehydrogenase I, an enzyme of the aerobic respiratory chain encoded by the nuo operon, is responsible for the impaired root-colonization ability of PCL1201. The complete sequence of the nuo operon (ranging from nuoA to nuoN) of P. fluorescens WCS365 was identified, including the promoter region and a transcriptional terminator consensus sequence downstream of nuoN. It was shown biochemically that PCL1201 is lacking NADH dehydrogenase I activity. In addition, the presence and activity of a second NADH dehydrogenase, encoded by the ndh gene, was identified to our knowledge for the first time in the genus Pseudomonas. Since it was assumed that low-oxygen conditions were present in the rhizosphere, we analyzed the activity of the nuo and the ndh promoters at different oxygen tensions. The results showed that both promoters are up-regulated by low concentrations of oxygen and that their levels of expression vary during growth. By using lacZ as a marker, it was shown that both the nuo operon and the ndh gene are expressed in the tomato rhizosphere. In contrast to the nuo mutant PCL1201, an ndh mutant of WCS365 appeared not to be impaired in competitive root tip colonization.

摘要

优良的根定殖荧光假单胞菌WCS365先前被选作亲本菌株,用于分离根定殖受损的突变体。对WCS365进行转座子诱变并测试根定殖能力,得到了突变菌株PCL1201,其在番茄根定殖竞争中受损约100倍。在本论文中,我们提供的证据表明,由nuo操纵子编码的有氧呼吸链酶NADH脱氢酶I的缺失,是PCL1201根定殖能力受损的原因。确定了荧光假单胞菌WCS365的nuo操纵子(从nuoA到nuoN)的完整序列,包括启动子区域和nuoN下游的转录终止子共有序列。生化分析表明PCL1201缺乏NADH脱氢酶I活性。此外,据我们所知,首次在假单胞菌属中鉴定出由ndh基因编码的第二种NADH脱氢酶的存在和活性。由于假定根际存在低氧条件,我们分析了在不同氧张力下nuo和ndh启动子的活性。结果表明,两个启动子均在低浓度氧气下上调,且其表达水平在生长过程中有所变化。通过使用lacZ作为标记,表明nuo操纵子和ndh基因均在番茄根际表达。与nuo突变体PCL1201不同,WCS365的ndh突变体在根尖定殖竞争中似乎未受损。

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