CHARACTERIZATION OF A GENOMIC REGION REQUIRED FOR PRODUCTION OF THE ANTIBIOTIC PYOLUTEORIN BY THE BIOLOGICAL-CONTROL AGENT PSEUDOMONAS-FLUORESCENS PF-5
CHARACTERIZATION OF A GENOMIC REGION REQUIRED FOR PRODUCTION OF THE ANTIBIOTIC PYOLUTEORIN BY THE BIOLOGICAL-CONTROL AGENT PSEUDOMONAS-FLUORESCENS PF-5
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DOI：
10.1128/aem.61.3.849-854.1995
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发表时间：
1995-03-01
影响因子：
4.4
通讯作者：
LOPER, JE
中科院分区：
文献类型：
--
作者：
KRAUS, J;LOPER, JE
鉴定并克隆了一个21-kb的区域，该区域是由生防菌荧光假单胞菌Pf-5生物合成聚酮抗生素绿脓菌素所必需的。从三个Plt（-）突变体的基因组DNA中克隆了Tn 5插入片段的侧翼序列，并将其用作探针从Pf-5基因组文库中鉴定pit基因座的野生型等位基因。含有基因组DNA的重叠区域的五个互补序列与一个或多个探针杂交。一种粘粒pJEL 1938含有完整的21-kb区域，当引入Plt（-）突变体中时，在生产中部分恢复了溶血。为了研究细菌生物合成中所需基因的表达，将含有无启动子的冰核基因（inaZ）和I型新霉素磷酸转移酶基因的转座子Tn 3-nice导入Pf-5的基因组凹坑区域。影响Pf-5产生细菌的碳源对含有基因组plt：：Tn 3-nice融合体的Pf-5的冰核活性具有平行的影响，表明inaZ是由pit区的启动子转录的。含有基因组plt：：Tn 3-nice融合体的Pf-5的细胞在田间土壤中种植的棉花和黄瓜种子上表达冰核活性。Pf-5在黄瓜精原细胞中的表达比在棉花精原细胞中的表达要慢。本研究表明，生防菌Pf-5在黄瓜精原细胞中原位表达了黄瓜生产中所需的基因。
A 21-kb region required for the biosynthesis of the polyketide antibiotic pyoluteorin by the biological control agent Pseudomonas fluorescens Pf-5 was identified and cloned. Seven previously isolated mutants deficient in pyoluteorin production (Plt(-)) had Tn5 insertions spanning the 21-kb region, Sequences flanking Tn5 inserts were cloned from genomic DNA of three Plt(-) mutants and used as probes to identify wild-type alleles of the pit loci from a genomic library of Pf-5. Five cosmids containing overlapping regions of genomic DNA hybridized to one or more of the probes. One cosmid, pJEL1938, contained the entire 21-kb region and, when introduced into a Plt(-) mutant, partially restored pyoluteorin production. To study the expression of the genes required for pyoluteorin biosynthesis, the transposon Tn3-nice, which contains a promoterless ice nucleation gene (inaZ) and a type I neomycin phosphotransferase gene, was introduced into the genomic pit region of Pf-5. Carbon sources that influenced pyoluteorin production by Pf-5 had parallel effects on ice nucleation activity of Pf-5 containing a genomic plt::Tn3-nice fusion, indicating that inaZ was transcribed from a promoter of the pit region, Cells of Pf-5 containing a genomic plt::Tn3-nice fusion expressed ice nucleation activity on cotton and cucumber seeds planted in field soil. The expression of plt genes by Pf-5 in the cucumber spermosphere was delayed in comparison with expression in the cotton spermosphere, This study demonstrates that genes required for pyoluteorin production were expressed in situ by the biological control bacterium.