ORIGIN, STRUCTURE, AND REGULATION OF ARGK, ENCODING THE PHASEOLOTOXIN-RESISTANT ORNITHINE CARBAMOYLTRANSFERASE IN PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA, AND FUNCTIONAL EXPRESSION OF ARGK IN TRANSGENIC TOBACCO
ORIGIN, STRUCTURE, AND REGULATION OF ARGK, ENCODING THE PHASEOLOTOXIN-RESISTANT ORNITHINE CARBAMOYLTRANSFERASE IN PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA, AND FUNCTIONAL EXPRESSION OF ARGK IN TRANSGENIC TOBACCO
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DOI：
10.1128/jb.174.18.5895-5909.1992
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发表时间：
1992-09-01
影响因子：
3.2
通讯作者：
PANOPOULOS, NJ
中科院分区：
文献类型：
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作者：
HATZILOUKAS, E;PANOPOULOS, NJ
丁香假单胞菌菜豆毒素（phaseolicola）产生三肽N（δ）（N’-磺基-二氨基膦基）-鸟氨酰-丙氨酰-高精氨酸（菜豆毒素），其通过抑制鸟氨酸氨基甲酰基转移酶（OCT）而在菜豆晕斑病中起着引起绿变性的毒素的作用。该细菌具有重复的OCT基因，其中之一argK编码毒素抗性酶（ROCT）并赋予对菜豆毒素的抗性。我们从菌株NPS 3121中测序了argK基因，确定了其启动子区域，分析了其调控，并表征了其转录本。该基因可能起源于另一种生物体，因为它与编码管家毒素敏感OCT的argF基因关系非常远，并且与细菌基因组整体和来自P. p. lingae pv的其他蛋白质编码基因相比具有低G+C含量。菜豆生。13 OCT序列的优化比对使我们能够定义可能负责毒素抗性的关键残基，并确定ROCT中不同的原核氨基酸特征，这表明argK的原核起源。通过农杆菌介导法将豌豆rbcS基因的argK编码区与叶绿体转运肽片段进行框内融合，转化烟草。通过体外和体内测定证明了转基因植物中ROCT活性的存在。部分植物对毒素具有抗性，表明病原菌对毒素的抗性在病原菌寄主中是可行的。
Pseudomonas syringae pv. phaseolicola produces the tripeptide N(delta)(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl-homoarginine (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.