Deletion mutations and its phenotypic analysis of two-component genes in Lonsdalea populi
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摘要:
目的 欧美杨细菌性溃疡病是革兰氏阴性细菌Lonsdalea populi引起的杨树枝干病害，其危害严重，已造成欧美杨人工林的重要经济损失。双组分系统是细菌致病过程的关键调控途径之一。目前，欧美杨细菌性溃疡病菌的双组分系统如何调控致病过程仍缺乏系统研究。因此，本研究开展欧美杨细菌性溃疡病菌的双组分编码基因的缺失突变及突变体表型分析，为深入解析其致病机制提供遗传材料。 方法 本研究以欧美杨溃疡病菌菌株N-5-1为研究对象，利用双亲结合方法获得了28个双组分系统基因的缺失突变体，并通过表型测定方法分析了这些基因突变体的致病性、生长、游动性、生物膜形成和抗逆性等表型特征，研究不同双组分系统编码基因对该病菌致病过程的调控。 结果 构建了36个欧美杨溃疡病菌的双组分编码基因的敲除重组载体，获得了28个基因的缺失突变体。致病性测定表明18个双组分基因的敲除降低了病原菌的毒性，其中8个突变体毒性丧失。此外，还获得了调控游动性和生物膜形成能力的突变体以及在逆境胁迫反应（金属离子、盐离子、抗生素等胁迫）有缺陷的突变体。 结论 本研究获得了5个显著影响欧美杨细菌性溃疡病菌毒性及其他生物表型的双组分基因，为后续双组分信号调控致病机制研究提供了遗传材料。 Abstract:Objective The bacterial canker of Populus euramericana is caused by the Gram-negative bacterium Lonsdalea populi. The rapid spread of the disease has seriously threatened the growth and development of P. euramericana and has caused great economic losses to the plantation. Two-component system (TCS) is the important signal transduction mechanism of Lonsdalea populi. Now, how the two-component system of poplar bacterial canker regulates the pathogenic process is still lack of systematic research. Therefore, the large-scale deletion mutation and mutant phenotype analysis of TCS in this study will provide genetic materials for further study on the pathogenic mechanism of poplar bacterial canker. Method In this study, 28 two-component gene deletion mutants of poplar canker pathogen strain L. populi N-5-1 were constructed by parental association, the differences in pathogenicity, growth, motility, biofilm formation and resistance of these mutants were analyzed by phenotypic analysis, and the regulation of two-component system coding genes on the pathogenicity of these mutants was studied. Result In this study, 36 recombinant vectors of two-component coding genes were constructed and 28 deletion mutants were obtained. Phenotypic analysis showed that 18 genes encoding TCS were involved in virulence of L. populi N-5-1. Among them, the pathogenicity of 8 mutants had obviously disappeared. In addition, the deletion mutants regulating motility and biofilm-forming ability and those deficient in stress response (metal ions, salt ions, antibiotic stress, etc.) were also screened. Conclusion In this study, five two-component genes significantly affecting the pathogenicity of L. populi were obtained, providing genetic material for future studies on the pathogenic mechanism of L. populi. -
Key words:
- Lonsdalea populi /
- two-component system /
- pathogenicity /
- adverse stress /
- motility
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图 2 缺失突变体的毒性分析
缺失突变体接种于欧美杨枝条10 d后的病情，NYG为CK对照组。A. 致病力减弱的基因；B. 致病力完全丧失的基因；C. 根据发病程度统计的病情指数。*表示在 P < 0.05 水平上显著性差异。下同。NYG is the control group of CK when the deletion mutants are inoculated on the 10th day. A, blunted genes; B, non-pathogenic genes; C, disease index. * indicates significant difference compared with the wild-type at P < 0.05 level. The same below.
Figure 2. Virulence assay of deletion mutants
图 1 缺失突变体鉴定
M. 250 bp Marker；A. Lqp201突变体，1 ~ 3泳道为Lqp201突变体菌株，4为野生型菌株扩增出Lqp201基因；B. Lqp 200突变体，1为Lqp200突变体菌株，2为野生型菌株扩增出Lqp201基因；C. Lqp1012突变体，1−3为Lqp1012突变体菌株，4为野生型菌株扩增出Lqp1012基因；D. Lqp1011突变体，1−3为Lqp1011突变体菌株基因，4为野生型菌株扩增出Lqp1011基因；E. Lqp2672突变体，1 ~ 3为Lqp2672突变体菌株基因，4为野生型菌株扩增出Lqp2672基因；F. Lqp2671突变体，1、2为Lqp2671突变体菌株基因，3为野生型菌株扩增出Lqp2671基因。M, 250 bp Marker；A, the mutant of Lqp201, lanes 1−3 are Lqp201 mutant strains, 4 is the wild-type strain that amplified the Lqp201; B, the mutant of Lqp200, lane 1 is Lqp200 mutant strains, 2 is the WT strain that amplified the Lqp200; C, the mutant of Lqp1012, lanes 1−3 are Lqp1012 mutant strains, 4 is the wild-type strain that amplified the Lqp1012; D, the mutant of Lqp1011, lanes 1−3 are Lqp1011 mutant strains, 4 is the wild-type strain that amplified the Lqp1011; E, the mutant of Lqp2672, lanes 1−3 are Lqp2672 mutant strains, 4 is the wild-type strain that amplified the Lqp2672; F, the mutant of Lqp2671, lanes 1−3 are Lqp2671 mutant strains, 4 is the wild-type strain that amplified the Lqp2671.
Figure 1. PCR verification of mutants
图 3 缺失突变体对细菌生长的影响
A. 基因组基因缺失突变体的生长速率，每隔6 h记录一次菌株的生长；B 在富营养性培养基NYG上菌株的生长，36 ~ 48 h培养后观察菌落形成状态。A, the growth rate of genomic deletion mutants was recorded every 6 h；B, the growth of NYG strain on the eutrophic medium was observed after 36−48 hours culture.
Figure 3. Effects of genomic gene deletion mutants on bacterial growth
图 4 缺失突变体的游动性分析
A. 野生型菌株和缺失突变体菌株在0.3%NYG琼脂平板上于30 ℃连续培养3 d的游动性分析；B. 接种后3 d，每种缺失突变体菌株在NYG琼脂平板上形成的游动区直径。A, the motility of WT strain and deletion mutant strains cultured at 30 °C for 3 d on 0.3% NYG agar plate; B, three days after inoculation, the motility zone diameter of each mutant strain is formed on NYG agar plate.
Figure 4. Motility of deletion mutants
图 5 缺失突变体的生物膜成膜能力
定量分析野生型菌株和缺失突变体菌株的生物膜形成。在96孔板孔中对每种菌株的48 h菌液进行OD600测量，并用结晶紫染色，使用分光光度计测量OD590的吸光值，计算OD590/OD600。Biofilm formation in WT and deletion mutant strains of quantitative analysis. The OD600 of each strain is measured in 96 holes of plate hole, and stained with crystal violet, the absorption value of OD590 is measured with photometer, and the OD590/OD600 is calculated.
Figure 5. Biofilm formation of deletion mutants
图 6 突变体氧化应激反应及抗性敏感性差异
在含有不同抗性的NYG琼脂平板上，30 ℃，培养36 ~ 48 h后观察菌株的抑菌圈直径。A. 将5 μL菌液添加到含5%H2O2的NYG琼脂平板中央的滤纸片上；B. 3 μL菌液滴加到含有17%氯霉素的NYG琼脂平板上；C.每种菌株接种到含有25%壮观霉素的NYG琼脂平板上。The diameter of bacteriostatic zone is observed after 36−48 h culture at 30 °C on NYG agar plate containing different resistances. A, 5 μL of bacterial solution is added to the filter paper in the middle of NYG agar plate containing 5% H2O2; B, 3 μL of bacterial solution is added to NYG agar plate containing 17% chloramphenicol; C, each strain is inoculated on a NYG agar plate containing 25% spectinomycin.
Figure 6. Differences in oxidative stress response and resistance sensitivity of mutants
图 7 不同金属胁迫对突变体生长的影响
在含有不同金属胁迫的NYG培养基上接种2 d，观察缺失突变体菌落生长情况。A. 0.15 mmol/L CoCl2胁迫对 突变体生长的影响；B. 在含0.2 mmol/L CuSO4胁迫下缺失突变体的生长；C. 在3 mmol/L MnSO4的NYG培养基上生长差异；D. 0.1 mmol/L FeCl3对缺失突变体的影响；E. 0.1 mmol/L FeCl2胁迫下缺失突变体的生长。Inoculate the NYG medium containing different metal stresses for 2 days, and observe the growth of the deletion mutant colony. A, effect of 0.15 mmol/L CoCl2 stress on the growth of mutants; B, growth of deletion mutants under the stress of 0.2 mmol/L CuSO4; C, growth difference on NYG medium containing 3 mmol/L MnSO4; D, the effect of 0.1 mmol/L FeCl3 on deletion mutants; E, growth of deletion mutants under 0.1 mmol/L FeCl2 stress.
Figure 7. Effects of different metal stresses on the growth of mutants
图 8 盐胁迫和渗透胁迫下的变化
A. 4 μL各缺失突变体菌株菌液分别滴加在含有3%NaCl的NYG琼脂平板中，并在30 ℃下培养36 ~ 48 h，以观察细菌菌落的形成；B. 在5%的KCl胁迫下于30 ℃培养36 ~ 48 h，并观察菌落的形成；C. 16% Sorbitol胁迫下培养36 ~ 48 h的菌株的差异。A, 4 μL of the bacterial liquid of each deletion mutant strain is added dropwise to NYG agar plates containing 3% NaCl, and cultured at 30°C for 36−48 h to observe the formation of bacterial colonies; B, cultivate for 36−48 h at 30°C under 5% KCl stress, and observe the formation of colonies; C, difference of strains cultured for 36−48 h under 16% Sorbitol stress.
Figure 8. Changes in environment with salt stress and osmotic stress
表 1 本研究所用引物序列
Table 1. Primer sequences used in this study
用途 Function 引物 Primer name 序列(5′—3′) Sequence (5′−3′) 载体构建 Constructing vector 0779-F CGGGATCCCCATTCCAGCCGTTGAAGAC 0779-R CCGCTCGAGGACCGCCAGCACATAGTGC 0778-F CGGGATCCTCCTCGCCCTGGAAATG 0778-R CCGCTCGAGCTTTCTGAGCCGGTGCTAC 1304-F CGGGATCCTCTGGATGAGCGTCTAACCC 1304-R CCGCTCGAGAATGGCATCACGCAGACG 1305-F CGGGATCCAGGCGTGCCAGGAGAACAG 1305-R CCGCTCGAGGCTCCGCTGGCTTTAACTAC 0290-F CGGGATCCTGATGCTGGATGCTCTGC 0290-R CCGCTCGAGCCGAATCTGGACGGGTAC 0289-F AAGGAAAAAAGCGGCCGCAAATGTTCCGCGACGATGAG 0289-R TCCCCCGGGGTGGACCCGTCGATTCTGG 201-F CGGGATCCGTCTCAGCCTGGACCCTTCG 201-R CCGCTCGAGTCGAAGTGCCACCAAGACC 200-F CGGGATCCAGAAACGCCTGATGTTCTGC 200-R CCGCTCGAGGCAAATGTTGAGCCAGACG 2576-F CGGGATCCGGAACTGGTAGCGCGTATTC 2576-R CCGCTCGAGGTAAGTCTGCACACCCGTTC 2575-F CGGGATCCCTGTTGGCGTTGCTGCTC 2575-R CCGCTCGAGGGTTGATGAAGTCGGACTGC 2989-F CGGGATCCTGGAAATTGGTGCAGACGAC 2989-R TCCCCCGGGCTGGCTCAACGCTTTATCG 2988-F CGGGATCCTCGGTTCTCATACGTCTCGG 2988-R CCGCTCGAGCGGGAGACAGCCAAGTTTTC 1012-F CGGGATCCGTCGTAACAGCGGATTGG 1012-R CCGCTCGAGCTCATGCCAATGATCTACCG 1011-F CGGGATCCACCCCTGCCAACTATATCGG 1011-R CCGCTCGAGCAGTTTTCCGCCATGATCGT 2312-F CGGGATCCTGGCGGTGATTATGCTGTC 2312-R CCGCTCGAGGGAGCAGCCGTAAGTGATG 2424-F CGGGATCCGTCTACCATCGCATCTCCG 2424-R CCGCTCGAGGCCGCTGATCTTTGTCC 1702-F CGGGATCCTGGATGGACAAGCAGCAAC 1702-R CCGCTCGAGCGCACGACCTGTCTAACG 1217-F AAGGAAAAAAGCGGCCGCGGTGGTGCTCAGTATTCAGG 1217-R TCCCCCGGGGTTTACGAATGCCGATGC 0545-F CGGGATCCGCTGGCTTCAATGGTTC 0545-R CCGCTCGAGCCGGAAGTTTGGAAAGC 1393-F CGGGATCCCGCCCTGAGGCACAACT 1393-R CCGCTCGAGCGAGGTTGGTAACGCTTG 2653-F CGGGATCCGGCGTTGGGAGAAGAAGG 2653-R CCGCTCGAGGTGAACCCACGGTGATGG 1080-F CGGGATCCGCTTTCGGCTTGACGACC 1080-R CCGCTCGAGCCGGATGAACTGTCGATGC 1704-F CGGGATCCTTGATGGCACGGTAGATG 1704-R CCGCTCGAGCCGGTATGTCTTCGCTCT 2671-F CGGGATCCCTTTCGCCACAGTCGTTACG 2671-R CCGCTCGAGGGTGAAGCCATCGTGTCG 2672-F CGGGATCCTCGGGTTCAGGGATTACAGG 2672-R CCGCTCGAGCGGCGGTTTACTTCTCCC 500-F CGGGATCCCCCGATTGATTTACGACG 500-R CCGCTCGAGGCGGTAAAGCCGTATTGG 0938-F AAGGAAAAAAGCGGCCGCCGAACGACTTCACTTGGGAG 0938-R TCCCCCGGGGCCTGCCGCAATACATTC 2313-F CGGGATCCCCTGTGCCTGTCCAATCTACAC 2313-R CCGCTCGAGCAAAGGCCACTGCCACTCC 3150-F CGGGATCCCTGGCTGGCGAAATCAGT 3150-R CCGCTCGAGAACGGGCGGTATCAAAGG 3347-F CGGGATCCCGAACGACTTCACTTGGGAG 3347-R CCGCTCGAGGCCGTTTATTCAGAGGGGTG 0029-F CGGGATCCGAACGCTACCGCAAACCC 0029-R CCGCTCGAGCCAGAATATCGAGCTGACGC 0030-F CGGGATCCGCAGCGGTATTTATGGGGTG 0030-R CCGCTCGAGGAACGCGATTTCCTGCTC 0541-F CGGGATCCCAAGCAGACGCAGGAAAG 0541-R CCGCTCGAGCGCGGTCAAAGACAGCAC 0546-F CGGGATCCCGTCCTTCCGTTGATGT 0546-R CCGCTCGAGGCGTCGTCAGGGATTTAG 1862-F CGGGATCCGGTCACCTACGGCGAAC 1862-R CCGCTCGAGCCGTCATCATGCCTTCG 1863-F CGGGATCCGGACTTTATCGCCCAATG 1863-R CCGCTCGAGCCATCGCAGGCTTATCAC CXF ATGTGCTGCAAGGCGATTAAGTTG CXR TTTATGCTTCCGGCTCGTATGTTG 突变体验证 Mutant verification SacB 1 GGCTTGTATGGGCCAGTTAAAG SacB 2 GTCTTTGCATTAGCCGGAGATC MCS1 GGCTCGTATGTTGTGTGGAATTG MCS2 TGTGCTGCAAGGCGATTAAGTTG 注：引物中的下划线表示酶切位点；引物代号即为缺失突变体基因代号。Notes: underline in the primers indicates digestion site. The primer code is the gene code of deletion mutant. 
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