天山根瘤菌群体感应调控蛋白MrtR与百脉根根瘤菌MrlI1/MrlR1群体感应调控体系的功能研究
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摘要
群体感应体系是细菌调控基因表达的重要调控方式,在根瘤菌与植物的相互作用过程中,群体感应体系发挥着重要作用。在革兰氏阴性细菌中,群体感应体系由自体诱导物(AI)分子,自体诱导物合成酶以及自体诱导物受体蛋白组成。本研究通过对天山根瘤菌MrtR蛋白结构与功能的关系以及百脉根根瘤菌MrlI1/MrlRl群体感应体系的研究,探讨群体感应体系在根瘤菌与植物共生过程中的作用。
天山根瘤菌HMZ0菌株的培养上清液浓缩物进行电喷雾电离质谱(ESI-MS/MS)分析可知,天山根瘤菌主要合成2种AHL分子,进行公式计算并同时结合根癌农杆菌R10(pCF218)菌株产生的AHLs作为对照,推测这2种AHLs分子分别是3-oxodocecanoyl-HSL(3-O-C12-HSL MW 300)和3-oxotetradecenoyl-HSL(3-O-C14-HSLMW 326)。而当MrtR蛋白与3-O-C12-HSL结合后,既可在体内诱导mrtI基因的表达,也可以在体外与mrtl基因的启动子区域结合。
为研究MrtR蛋白的性质,把mrtR基因克隆到表达载体pET28,得到受T7启动子调控的His-MrtR融合蛋白表达质粒pMH61。把表达质粒转入大肠杆菌BL21λDE3,表达蛋白经过Ni+柱纯化后,对His-MrtR蛋白性质进行研究发现,His-MrtR蛋白在不与天山根瘤菌自体诱导物(MAI)结合的条件下,仍然可以以可溶性蛋白存在,这与其它一些典型的LuxR类蛋白有着明显的区别。对MrtR蛋白与mrtl基因的调控方式研究发现,只有当MrtR-MAI与mrt box结合后，mrtl基因才可以被转录和表达,这说明MrtR与MAI的作用,及其与mrt box的结合是mrtl基因表达所必需的。
对MrtR蛋白进遗传学分析其结构与功能的关系发现,当MrtR蛋白氮端前50个氨基酸缺失后,对mrtl基因的诱导调控功能没有改变,而且当缺失40或50个氨基酸后,MrtR (A2-50)蛋白具有不依赖于MAI的调控作用,能在不含有MAI的条件下诱导mrtl的表达。而当加入MAI分子后,其诱导能力相当于野生型的2倍多。这可能是因为在40-50区域内的某些氨基酸缺失后,改变了MrtR蛋白的结构,使其表现出不依赖于MAI的蛋白活性。
当MrtR缺失氮端80,120和140个氨基酸时,MrtR对mrtI的诱导调控功能完全丧失,推测这可能是由于当这些氨基酸缺失后,MrtR的蛋白结构被严重的改变所致。MrtR(Δ2-160)突变子尽管在加入MAI的条件下,对mrtl的诱导能力只是野生型的20%,但这与其在没有MAI的条件下的诱导能力相当,也就是MrtR (Δ2-160)突变子也表现出不依赖于MAI的诱导能力,推测这与LuxR-type蛋白的结构特点有关,即其氮端结构域与碳端结构域的结构与功能相对独立。。而当MrtR蛋白羧基末端40个氨基酸被缺失后,蛋白功能几乎完全丧失,这也跟多数LuxR类蛋白结构类似,说明MrtR蛋白具有典型的LuxR类蛋白结构特征。
为研究MrtR蛋白关键氨基酸的功能,利用大肠杆菌XL1Red菌株,构建得到mrtR基因突变子文库,以LacZ作为报告基因筛选得到11株具有不同的突变位点的突变株,在自体诱导物不存在的条件下,都可以诱导mrtI基因的表达,即其MrtR蛋白活性均表现出的不依赖于与天山根瘤菌自体诱导物分子(MAI)的作用。与LuxR和TraR蛋白的同源比较发现,发生在氮端结构域的突变位点(V105A,G114S,M135I和H142R)位于蛋白的AI结合位点结构域附近,而S6L和R182C这两个位点则位于与蛋白形成二聚体相关的氨基酸序列附近,这些位点的突变有可能改变了蛋白与MAI分子的相互作用,从而使得MrtR蛋白在MAI分子不存在的条件下,仍然具有诱导mrtI基因表达的功能。在碳端结构域的突变位点(N204,P208,H219,T221和Y227)推测都是位于负责与DNA结合的HTH结构域周围,这些位点的突变可能影响蛋白与DNA的结合效率,从而提高蛋白的活性。对其中一个突变子MrtR G114S进行凝胶阻滞分析发现,在没有自体诱导物分子存在的条件下,该突变子仍然能与mrtI基因的启动子区域在体外结合。这说明这些突变株的表型特征是受其基因型的改变而产生的。
本论文的第二部分是对百脉根根瘤菌NZP2213菌株的群体感应系统的研究,利用电喷雾电离质谱(ESI-MS/MS)的方法对百脉根根瘤菌自体诱导物分子进行分析得知,百脉根根瘤菌能合成4种自体诱导物分子,进行公式计算后,推测出这4种AHL分子分别是3-Oxohexanoyl-homoserine lactone (3-O-C6-HSL, MW 214), Octanoyl-HSL (C8-HSL, MW 228), decanoyl-HSL(C10-HSL, MW 256)和docecanoyl-HSL (C12-HSLMW 282)。通过研究百脉根根瘤菌MrlI1/MrlR1群体感应体系发现,MrlI1负责合成一种碳链长度较长的AHL分子,结合ESI-MS/MS方法,推测该种AHL分子为C12-HSL.
百脉根根瘤菌MAFF30399菌株的基因组全序列已经公布,参考mlr5638和mlr5637基因的序列,设计引物,利用PCR的方法扩增得到百脉根根瘤菌NZP2213的mrlll和mrlRl基因。通过基因序列分析发现,mrlll和mrlR1基因与mlr5638和mlr5637基因的氨基酸序列同源性分别为95%和92%。在大肠杆菌中异源表达mrlll发现,mrlll在大肠杆菌中也可以合成自体诱导物分子,而且通过薄层层析(TLC)分析表明mrlll在大肠杆菌中合成的自体诱导物分子与其在根瘤菌中合成的分子相似。
对mrlll的表达调控方式分析发现,mrlll的表达依赖于其自身合成的AHL分子和MrlR1的共同作用,并表现出典型的群体感应调控方式,即其表达与细菌的浓度相关,当细菌生长达到一定阈值时,mrtI的表达表现出明显的提高。而且,当MrlI1/MrlR1群体感应体系突变后,突变株在与植物结瘤的前期结瘤数量表现出明显的减少,推测MrlI1/MrlR1群体感应体系影响到百脉根根瘤菌与植物之间的结瘤效率。
As one of the most important signal transduction pathways in bacteria, quorum sensing system is involved in many regulatory circuits in rhizobia, especially the communication between rhizobia and their plant hosts. The typical QS system in Gram-negative bacteria is the marine symbiotic bacteria Vibrio fisheri LuxI/R-type regulatory system. The key regulatory components of these signaling systems are LuxI-type AHL synthases, and LuxR-type AHL receptors and AHL-dependent transcription factors. Here, both of the regulatory mechanism of quorum sensing regulator MrtR of Mesorhizobium tianshanense CCBAU 3306 and Mrlll/MrlRl quorum sensing system of Mesorhizbium loti NZP 2213 were studied.
Mesorhizobium tianshanense, a nitrogen-fixing symbiont for at least eight different plant species, including Glycyrrhiza (licorice), produces most diversity of AHL signaling molecules. Interestingly, the MrtI/R quorum sensing system in M. tianshanense seems to be responsible for the synthesis of all of the AHL molecules and controls its symbiotic nodulation. To further study the role of MrtI/MrtR QS system during the symbiosis process of M. tianshanense and Glycyrrhiza, the structure and function of MrtR was analyzed in detailed. Detected by the electrospray ionization mass spectrometry (ESI MS/MS), the acyl homoserine lactone (AHL) signal molecules produced by M. tianshanense (MAI) were suspected to be 3-oxodocecanoyl-HSL (3-O-C12-HSL, MW300) and 3-oxotetradecenoyl-HSL (3-O-C14-HSL,MW 326). And MrtR can specifically bind with the mrt box inside the promoter region of mrtI in vitro and activate the expression of mrtI in vivo by binding with the autoinducer,3-O-C12-HSL (MAI).
The way of mrtI expression regulated by MrtR shown that mrtI could only be expressed when the predicted mrt box, the invert repeated sequence located in the-69 to-52 region of mrtI promoter, was bound by MrtR-MAI complex. That is MrtR, MAI and mrt box were indispensable for expression of mrtI, which is also the typical characteristics of regulatory mechanism of LuxR-type protein.
To determine the regions of MrtR necessary for transcriptional activation of mrtI, a serial of deletion mutations of MrtR were generated. Eight MrtR mutant encoding truncated MrtR proteins with deletions of residues of 2-30,2-40,2-50,2-80,2-120,2-140,2-160 and 230-272 were constructed by PCR amplying and cloning into pBBR-MCS5 vector under the control of Plac promoter. All of these constructs were transformed into a M. tianshanense MrtR/MrtI quorum sensing system deleted mutant MHT9 strain, with PmrtI-lacZ (pMH1) report gene. Based on analysis of the activity of LacZ, the result showed that similar to C-terminal truncations of LuxR, RhlR, and TraR, MrtRΔ233-271 which lacks the conserved HTH DNA binding domain, had no activity. And MrtRΔ2-30 was similar to that of full-length MrtR. However, deletion of up to 50 N-terminal amino acids enhanced MrtR activity even in the absence of 3OC12-HSL. MrtRΔ2-80,Δ2-120, andΔ2-140 showed no activity; however, MrtRΔ2-160 displayed a low level of autoinducer-independent activity. Previous studies of LuxR and TraR suggest that in activators of the LuxR-family, the N-terminal domain masks the C-terminal DNA binding domain in the absence of autoinducer, thus interfering with DNA binding, and the C-terminal domain alone was shown to function in AHL-independent activation.
To further characterize the structure and function of MrtR, we screened for MrtR constitutive mutants. We used E. coli mutator strain XL-1 Red to introduce mutations to the Plac-mrtR plasmid. The mutated plasmids were then transformed into M. tianshanense MHT9 strain containing a PmrtI-lacZ plasmid, and the transformants were spread on X-gal plates without any autoinducer. Approximately 25,000 colonies were screened, of which exhibited a LacZ+ phenotype in the absence of autoinducer and had point mutations in the mrtR gene. Compared to wild-type MrtR, all eleven MrtR mutants had enhanced ability to activate the expression of mrtI in the absence of 3OC12-HSL. However, a cluster of mutations located in the C-terminal DNA binding domain (N204D, P208S, H219R, T221M, and Y227C) were hypersensitive mutants as they still responded to the 3OC12-HSL stimulation. The constitutive MrtR mutants that activated mrtI independent of and with no further stimulation by 3OC12-HSL were clustered in the N-terminal autoinducer binding domain (S6L, V105A, G114S, M135I, H142R, and R182C). We further characterized the mutant, MrtRG114S, with the highest activity in the absence of 3OC12-HSL by purifying it as His6-tagged recombinant protein. The MrtRG114S protein could efficiently retard mrtI promoter DNA in the absence of 3OC12-HSL.
The second part of this desertation is about the study of MrlRl/Mrlll quorum sensing system of Mesorhizobium loti NZP2213. Detected by ultrasensitive bioassay strain JZA1, AHLs could be found in the supernatant of M. loti NZP2213 culture in TY medium (not, in MM medium) and displayed a typical cell density-dependent pattern, which indicated that there was quorum sensing system in M. loti NZP2213. The supernatant of M.loti culture was extracted with ethyl acetate and subjected to TLC and electrospray ionization mass spectrometry (ESI MS/MS) analysis, and the results showed that M. loti could produce at least four distinct AHL-like molecules, which were suspected to be3-Oxohexanoyl-homoserine lactone (3-O-C6-HSL, MW 214), Octanoyl-HSL (C8-HSL, MW 228), decanoyl-HSL(C10-HSL, MW 256) and docecanoyl-HSL (C12-HSL MW 282)。
Based on the sequence of mlr5638 and mlr5637 of M. loti MAFF303099 strain, autoinducer synthase gene mrlI1 and relevant transcriptional regulator mrlR1 from the M. loti NZP2213 were successfully amplified by PCR. mrlI1 and mrlR1 were 95% and 92% homologus to mlr5638 and mlr5637 at amino acid level respectively. By hetro-expressing mrlI1 in E. coli and constructing a mrlll deletion mutant in M. loti genetic background, we found that MrlI1 was responsible for synthesizing AHL molecules with long carbon train (C12-HSL) in M. loti and the expression of mrlI1 was shown to be growth phase-dependent, regulated by MrlR1 and the autoinducer produced by the protein itself, which is of typical autoinducing and quorum sensing charateristics. To examine whether the MrlR1/MrlI1 quorum sensing system would affect nodulation, nodulation assays of AHL-deficient mutants on their native plant host Lotus japonicus was performed and it was found that the efficiency of nodulation was reduced in mutation of mrlI1, suggesting that quorum sensing system in M.loti may play an important role in successful establishment of a rhizobium-legume symbiosis.
天山根瘤菌HMZ0菌株的培养上清液浓缩物进行电喷雾电离质谱(ESI-MS/MS)分析可知,天山根瘤菌主要合成2种AHL分子,进行公式计算并同时结合根癌农杆菌R10(pCF218)菌株产生的AHLs作为对照,推测这2种AHLs分子分别是3-oxodocecanoyl-HSL(3-O-C12-HSL MW 300)和3-oxotetradecenoyl-HSL(3-O-C14-HSLMW 326)。而当MrtR蛋白与3-O-C12-HSL结合后,既可在体内诱导mrtI基因的表达,也可以在体外与mrtl基因的启动子区域结合。
为研究MrtR蛋白的性质,把mrtR基因克隆到表达载体pET28,得到受T7启动子调控的His-MrtR融合蛋白表达质粒pMH61。把表达质粒转入大肠杆菌BL21λDE3,表达蛋白经过Ni+柱纯化后,对His-MrtR蛋白性质进行研究发现,His-MrtR蛋白在不与天山根瘤菌自体诱导物(MAI)结合的条件下,仍然可以以可溶性蛋白存在,这与其它一些典型的LuxR类蛋白有着明显的区别。对MrtR蛋白与mrtl基因的调控方式研究发现,只有当MrtR-MAI与mrt box结合后，mrtl基因才可以被转录和表达,这说明MrtR与MAI的作用,及其与mrt box的结合是mrtl基因表达所必需的。
对MrtR蛋白进遗传学分析其结构与功能的关系发现,当MrtR蛋白氮端前50个氨基酸缺失后,对mrtl基因的诱导调控功能没有改变,而且当缺失40或50个氨基酸后,MrtR (A2-50)蛋白具有不依赖于MAI的调控作用,能在不含有MAI的条件下诱导mrtl的表达。而当加入MAI分子后,其诱导能力相当于野生型的2倍多。这可能是因为在40-50区域内的某些氨基酸缺失后,改变了MrtR蛋白的结构,使其表现出不依赖于MAI的蛋白活性。
当MrtR缺失氮端80,120和140个氨基酸时,MrtR对mrtI的诱导调控功能完全丧失,推测这可能是由于当这些氨基酸缺失后,MrtR的蛋白结构被严重的改变所致。MrtR(Δ2-160)突变子尽管在加入MAI的条件下,对mrtl的诱导能力只是野生型的20%,但这与其在没有MAI的条件下的诱导能力相当,也就是MrtR (Δ2-160)突变子也表现出不依赖于MAI的诱导能力,推测这与LuxR-type蛋白的结构特点有关,即其氮端结构域与碳端结构域的结构与功能相对独立。。而当MrtR蛋白羧基末端40个氨基酸被缺失后,蛋白功能几乎完全丧失,这也跟多数LuxR类蛋白结构类似,说明MrtR蛋白具有典型的LuxR类蛋白结构特征。
为研究MrtR蛋白关键氨基酸的功能,利用大肠杆菌XL1Red菌株,构建得到mrtR基因突变子文库,以LacZ作为报告基因筛选得到11株具有不同的突变位点的突变株,在自体诱导物不存在的条件下,都可以诱导mrtI基因的表达,即其MrtR蛋白活性均表现出的不依赖于与天山根瘤菌自体诱导物分子(MAI)的作用。与LuxR和TraR蛋白的同源比较发现,发生在氮端结构域的突变位点(V105A,G114S,M135I和H142R)位于蛋白的AI结合位点结构域附近,而S6L和R182C这两个位点则位于与蛋白形成二聚体相关的氨基酸序列附近,这些位点的突变有可能改变了蛋白与MAI分子的相互作用,从而使得MrtR蛋白在MAI分子不存在的条件下,仍然具有诱导mrtI基因表达的功能。在碳端结构域的突变位点(N204,P208,H219,T221和Y227)推测都是位于负责与DNA结合的HTH结构域周围,这些位点的突变可能影响蛋白与DNA的结合效率,从而提高蛋白的活性。对其中一个突变子MrtR G114S进行凝胶阻滞分析发现,在没有自体诱导物分子存在的条件下,该突变子仍然能与mrtI基因的启动子区域在体外结合。这说明这些突变株的表型特征是受其基因型的改变而产生的。
本论文的第二部分是对百脉根根瘤菌NZP2213菌株的群体感应系统的研究,利用电喷雾电离质谱(ESI-MS/MS)的方法对百脉根根瘤菌自体诱导物分子进行分析得知,百脉根根瘤菌能合成4种自体诱导物分子,进行公式计算后,推测出这4种AHL分子分别是3-Oxohexanoyl-homoserine lactone (3-O-C6-HSL, MW 214), Octanoyl-HSL (C8-HSL, MW 228), decanoyl-HSL(C10-HSL, MW 256)和docecanoyl-HSL (C12-HSLMW 282)。通过研究百脉根根瘤菌MrlI1/MrlR1群体感应体系发现,MrlI1负责合成一种碳链长度较长的AHL分子,结合ESI-MS/MS方法,推测该种AHL分子为C12-HSL.
百脉根根瘤菌MAFF30399菌株的基因组全序列已经公布,参考mlr5638和mlr5637基因的序列,设计引物,利用PCR的方法扩增得到百脉根根瘤菌NZP2213的mrlll和mrlRl基因。通过基因序列分析发现,mrlll和mrlR1基因与mlr5638和mlr5637基因的氨基酸序列同源性分别为95%和92%。在大肠杆菌中异源表达mrlll发现,mrlll在大肠杆菌中也可以合成自体诱导物分子,而且通过薄层层析(TLC)分析表明mrlll在大肠杆菌中合成的自体诱导物分子与其在根瘤菌中合成的分子相似。
对mrlll的表达调控方式分析发现,mrlll的表达依赖于其自身合成的AHL分子和MrlR1的共同作用,并表现出典型的群体感应调控方式,即其表达与细菌的浓度相关,当细菌生长达到一定阈值时,mrtI的表达表现出明显的提高。而且,当MrlI1/MrlR1群体感应体系突变后,突变株在与植物结瘤的前期结瘤数量表现出明显的减少,推测MrlI1/MrlR1群体感应体系影响到百脉根根瘤菌与植物之间的结瘤效率。
As one of the most important signal transduction pathways in bacteria, quorum sensing system is involved in many regulatory circuits in rhizobia, especially the communication between rhizobia and their plant hosts. The typical QS system in Gram-negative bacteria is the marine symbiotic bacteria Vibrio fisheri LuxI/R-type regulatory system. The key regulatory components of these signaling systems are LuxI-type AHL synthases, and LuxR-type AHL receptors and AHL-dependent transcription factors. Here, both of the regulatory mechanism of quorum sensing regulator MrtR of Mesorhizobium tianshanense CCBAU 3306 and Mrlll/MrlRl quorum sensing system of Mesorhizbium loti NZP 2213 were studied.
Mesorhizobium tianshanense, a nitrogen-fixing symbiont for at least eight different plant species, including Glycyrrhiza (licorice), produces most diversity of AHL signaling molecules. Interestingly, the MrtI/R quorum sensing system in M. tianshanense seems to be responsible for the synthesis of all of the AHL molecules and controls its symbiotic nodulation. To further study the role of MrtI/MrtR QS system during the symbiosis process of M. tianshanense and Glycyrrhiza, the structure and function of MrtR was analyzed in detailed. Detected by the electrospray ionization mass spectrometry (ESI MS/MS), the acyl homoserine lactone (AHL) signal molecules produced by M. tianshanense (MAI) were suspected to be 3-oxodocecanoyl-HSL (3-O-C12-HSL, MW300) and 3-oxotetradecenoyl-HSL (3-O-C14-HSL,MW 326). And MrtR can specifically bind with the mrt box inside the promoter region of mrtI in vitro and activate the expression of mrtI in vivo by binding with the autoinducer,3-O-C12-HSL (MAI).
The way of mrtI expression regulated by MrtR shown that mrtI could only be expressed when the predicted mrt box, the invert repeated sequence located in the-69 to-52 region of mrtI promoter, was bound by MrtR-MAI complex. That is MrtR, MAI and mrt box were indispensable for expression of mrtI, which is also the typical characteristics of regulatory mechanism of LuxR-type protein.
To determine the regions of MrtR necessary for transcriptional activation of mrtI, a serial of deletion mutations of MrtR were generated. Eight MrtR mutant encoding truncated MrtR proteins with deletions of residues of 2-30,2-40,2-50,2-80,2-120,2-140,2-160 and 230-272 were constructed by PCR amplying and cloning into pBBR-MCS5 vector under the control of Plac promoter. All of these constructs were transformed into a M. tianshanense MrtR/MrtI quorum sensing system deleted mutant MHT9 strain, with PmrtI-lacZ (pMH1) report gene. Based on analysis of the activity of LacZ, the result showed that similar to C-terminal truncations of LuxR, RhlR, and TraR, MrtRΔ233-271 which lacks the conserved HTH DNA binding domain, had no activity. And MrtRΔ2-30 was similar to that of full-length MrtR. However, deletion of up to 50 N-terminal amino acids enhanced MrtR activity even in the absence of 3OC12-HSL. MrtRΔ2-80,Δ2-120, andΔ2-140 showed no activity; however, MrtRΔ2-160 displayed a low level of autoinducer-independent activity. Previous studies of LuxR and TraR suggest that in activators of the LuxR-family, the N-terminal domain masks the C-terminal DNA binding domain in the absence of autoinducer, thus interfering with DNA binding, and the C-terminal domain alone was shown to function in AHL-independent activation.
To further characterize the structure and function of MrtR, we screened for MrtR constitutive mutants. We used E. coli mutator strain XL-1 Red to introduce mutations to the Plac-mrtR plasmid. The mutated plasmids were then transformed into M. tianshanense MHT9 strain containing a PmrtI-lacZ plasmid, and the transformants were spread on X-gal plates without any autoinducer. Approximately 25,000 colonies were screened, of which exhibited a LacZ+ phenotype in the absence of autoinducer and had point mutations in the mrtR gene. Compared to wild-type MrtR, all eleven MrtR mutants had enhanced ability to activate the expression of mrtI in the absence of 3OC12-HSL. However, a cluster of mutations located in the C-terminal DNA binding domain (N204D, P208S, H219R, T221M, and Y227C) were hypersensitive mutants as they still responded to the 3OC12-HSL stimulation. The constitutive MrtR mutants that activated mrtI independent of and with no further stimulation by 3OC12-HSL were clustered in the N-terminal autoinducer binding domain (S6L, V105A, G114S, M135I, H142R, and R182C). We further characterized the mutant, MrtRG114S, with the highest activity in the absence of 3OC12-HSL by purifying it as His6-tagged recombinant protein. The MrtRG114S protein could efficiently retard mrtI promoter DNA in the absence of 3OC12-HSL.
The second part of this desertation is about the study of MrlRl/Mrlll quorum sensing system of Mesorhizobium loti NZP2213. Detected by ultrasensitive bioassay strain JZA1, AHLs could be found in the supernatant of M. loti NZP2213 culture in TY medium (not, in MM medium) and displayed a typical cell density-dependent pattern, which indicated that there was quorum sensing system in M. loti NZP2213. The supernatant of M.loti culture was extracted with ethyl acetate and subjected to TLC and electrospray ionization mass spectrometry (ESI MS/MS) analysis, and the results showed that M. loti could produce at least four distinct AHL-like molecules, which were suspected to be3-Oxohexanoyl-homoserine lactone (3-O-C6-HSL, MW 214), Octanoyl-HSL (C8-HSL, MW 228), decanoyl-HSL(C10-HSL, MW 256) and docecanoyl-HSL (C12-HSL MW 282)。
Based on the sequence of mlr5638 and mlr5637 of M. loti MAFF303099 strain, autoinducer synthase gene mrlI1 and relevant transcriptional regulator mrlR1 from the M. loti NZP2213 were successfully amplified by PCR. mrlI1 and mrlR1 were 95% and 92% homologus to mlr5638 and mlr5637 at amino acid level respectively. By hetro-expressing mrlI1 in E. coli and constructing a mrlll deletion mutant in M. loti genetic background, we found that MrlI1 was responsible for synthesizing AHL molecules with long carbon train (C12-HSL) in M. loti and the expression of mrlI1 was shown to be growth phase-dependent, regulated by MrlR1 and the autoinducer produced by the protein itself, which is of typical autoinducing and quorum sensing charateristics. To examine whether the MrlR1/MrlI1 quorum sensing system would affect nodulation, nodulation assays of AHL-deficient mutants on their native plant host Lotus japonicus was performed and it was found that the efficiency of nodulation was reduced in mutation of mrlI1, suggesting that quorum sensing system in M.loti may play an important role in successful establishment of a rhizobium-legume symbiosis.
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