College of Pharmacy, Duksung Women's Univerisity, Seoul, 132-714, Republic of Korea.
J Microbiol. 2012 Feb;50(1):79-90. doi: 10.1007/s12275-012-1353-9. Epub 2012 Feb 27.
An ATP-binding cassette (ABC) transporter, called the PseEF efflux system, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseEF efflux system was located within a 3.3-kb operon that encodes a periplasmic membrane fusion protein (PseE), and an ABC-type cytoplasmic membrane protein (PseF). The PseEF efflux system exhibited amino acid homology to a putative ABC efflux system (MacAB) of E. coli W3104 with identities of 47.2% (i.e., PseE to MacA) and 57.6% (i.e., PseF to MacB). A nonpolar mutation within the pseF gene was generated by nptII insertional mutagenesis. The resultant mutant strain showed significant reduction in secretion of syringomycin (74%) and syringopeptin (71%), as compared to parental strain B301D. Quantitative real-time RT-PCR was used to determine transcript levels of the syringomycin (syrB1) and syringopeptin (sypA) synthetase genes in strain B301D-HK7 (a pseF mutant). Expression of the sypA gene by mutant strain B301D-HK7 was approximately 6.9% as compared to that of parental strain B301D, while the syrB1 gene expression by mutant strain B301D-HK7 was nearly 14.6%. In addition, mutant strain B301D-HK7 was less virulent by approximately 67% than parental strain B301D in immature cherry fruits. Mutant strain B301D-HK7 was not reduced in resistance to any antibiotics used in this study as compared to parental strain B301D. Expression (transcript levels) of the pseF gene was induced approximately six times by strain B301D grown on syringomycin minimum medium (SRM) supplemented with the plant signal molecules arbutin and D-fructose (SRMAF), as compared to that of strain B301D grown on SRM (in the absence of plant signal molecules). In addition, during infection of bean plants by P. syringae pv. syringae strain B728a, expression of the pseF gene increased at 3 days after inoculation (dai). More than 180-fold induction was observed in transcript levels of the pseF gene by parental strain B728a as compared to strain B728a-SL7 (a salA mutant). Thus, the PseEF efflux system, an ABC-type efflux system, has an important role in secretion of syringomycin and syringopeptin, and is required for full virulence in P. syringae pv. syringae.
一个 ATP 结合盒(ABC)转运蛋白,称为 PseEF 外排系统,在丁香假单胞菌 pv. 丁香假单胞菌菌株 B301D 的 syr-syp 基因组岛的左侧边界被鉴定出来。PseEF 外排系统位于一个编码周质膜融合蛋白(PseE)和 ABC 型细胞质膜蛋白(PseF)的 3.3kb 操纵子内。PseEF 外排系统与大肠杆菌 W3104 的一个假定的 ABC 外排系统(MacAB)具有 47.2%(即 PseE 与 MacA)和 57.6%(即 PseF 与 MacB)的氨基酸同源性。通过 nptII 插入诱变在 pseF 基因内产生了一个非极性突变。与亲本菌株 B301D 相比,所得突变株的丁香菌霉素(74%)和丁香肽(71%)分泌显著减少。使用定量实时 RT-PCR 确定了菌株 B301D-HK7(pseF 突变体)中丁香菌霉素(syrB1)和丁香肽(sypA)合成酶基因的转录水平。与亲本菌株 B301D 相比,突变株 B301D-HK7 的 sypA 基因表达约为 6.9%,而突变株 B301D-HK7 的 syrB1 基因表达几乎为 14.6%。此外,与亲本菌株 B301D 相比,突变株 B301D-HK7 在不成熟樱桃果实中的毒力降低了约 67%。与亲本菌株 B301D 相比,突变株 B301D-HK7 对本研究中使用的任何抗生素的耐药性均未降低。与在没有植物信号分子的 SRM 上生长的 B301D 相比,在添加植物信号分子熊果苷和 D-果糖的 SRM (SRMAF)上生长的 B301D 的 pseF 基因表达增加了约六倍。此外,在丁香假单胞菌 pv. 丁香假单胞菌菌株 B728a 感染菜豆植物时,pseF 基因的表达在接种后 3 天(dai)增加。与亲本菌株 B728a 相比,B728a-SL7(salA 突变体)中 pseF 基因的转录水平增加了 180 多倍。因此,ABC 型外排系统 PseEF 外排系统在丁香菌霉素和丁香肽的分泌中具有重要作用,并且是丁香假单胞菌 pv. 丁香假单胞菌完全毒力所必需的。