广西师范大学学报（自然科学版） ›› 2023, Vol. 41 ›› Issue (2): 161-174.doi: 10.16088/j.issn.1001-6600.2022020901

• 研究论文 • 上一篇    下一篇

铜绿假单胞菌phzR对生物被膜基因转录及细胞运动性能的影响

戴明瑶1, 李亚诗1, 黄新妮1, 肖君1, 黄植清1, 吕春萌1, 陆祖军1,2*   

  1. 1.广西师范大学 生命科学学院,广西 桂林 541006;
    2.珍稀濒危动植物生态与环境保护教育部重点实验室(广西师范大学),广西 桂林 541006
  • 收稿日期:2022-02-09 修回日期:2022-05-30 出版日期:2023-03-25 发布日期:2023-04-25
  • 通讯作者: 陆祖军(1963—),男(壮族),广西百色人,广西师范大学教授,博士。E-mail:1946208364@qq.com
  • 基金资助:
    国家自然科学基金(31860010);广西大学生创新创业训练项目(201910602061, 202010602055, 202010602256)

Effects of phzR Gene of Pseudomonas aeruginosa on Biofilm Gene Expression and Cell Motility

DAI Mingyao1, LI Yashi1, HUANG Xinni1, XIAO Jun1, HUANG Zhiqing1, LÜ Chunmeng1, LU Zujun1,2*   

  1. 1. College of Life Science, Guangxi Normal University, Guilin Guangxi 541006, China;
    2. Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin Guangxi 541006, China
  • Received:2022-02-09 Revised:2022-05-30 Online:2023-03-25 Published:2023-04-25

PDF (PC)

250

摘要： 为预测高产吩嗪类物质铜绿假单胞菌(Pseudomonas aeruginosa, PA)工程菌株PA2019NES在农用开发时的生物安全性,本文采用实时荧光定量PCR技术,研究多拷贝phzR基因的PA2019NES与PA2016NX1在生物被膜状态(被膜态)、浮游状态(浮游态)的生物被膜、群体感应系统、六型分泌系统共9个基因转录量差异,结果显示:在浮游态下,多拷贝phzR基因上调vgrG、algD基因的mRNA转录量(P<0.05),下调pilA基因mRNA合成(P<0.05),但其他6个基因的转录量无显著差异(P>0.05);在被膜态下,仅提高rhlI基因的mRNA表达量(P<0.05),而fimX和wspR基因的mRNA合成受到抑制(P<0.05),但其他6个基因的转录量无显著影响(P>0.05);经24 h培养后,PA2016NX1和PA2019NES的蹭行运动无显著差异(P>0.05),但游泳运动后者强于前者(P<0.05),集群运动后者弱于前者(P<0.05)。此结果证明PA2019NES的条件致病性能可能不低于PA2016NX1,在农用开发该菌株时应杜绝活菌向环境扩散。

关键词: 铜绿假单胞菌, 生物被膜, 群体感应系统, 六型分泌系统, phzR基因

Abstract: In order to prognosis the biosecurity of PA2019NES which is with high yield of phenazines in agricultural exploitation, the real-time fluorescence quantitative PCR was applied to test the difference of transcriptional volume of 9 genes which were associated with biofilm, quorum sensing system and type Ⅵ secretory system, to reveal the transcriptional relationship between phzR and the above mentioned 9 genes, as well as its effect on the cells motility, especially the differeces between PA2016NX1 and PA2019NES, which was with multiple copies of phzR gene, were in biofilm and planktonic state, respectively. The results showed that, in the planktonic state, multiple copies phzR gene up-regulated the mRNA level of vgrG and algD genes (P<0.05), the mRNA level of pilA was down-regulated (P<0.05); however, the mRNA level of the other 6 genes was not significantly affected (P>0.05). In the biofilm state, the mRNA level of rhlI gene was up-regulated (P<0.05), but that of fimX and wspR genes was down-regulated (P<0.05) and that of the other 6 genes was not significantly affected (P>0.05). After 24 h culture, there was no significant difference in twitching ability between PA2016NX1 and PA2019NES (P>0.05), but the swimming, ability of PA2019NES was stronger than that of PA2016NX1 (P<0.05), however, the swarming ability of PA2019NES was weaker than that of PA2016NX1 (P<0.05). These results suggested that the power of conditional pathogenicity of PA2019NES may not be lower than that of PA2016NX1, hence, when PA2019NES was developed for agricultural use, the spread of live bacteria to the environment should be prevented.

Key words: Pseudomonas aeruginosa, bacterial biofilm, quorum sensing system, type Ⅵ secretion system, phzR gene

中图分类号: 

  • Q933
[1] YE R W, THOMAS S M. Microbial nitrogen cycles: physiology, genomics and applications[J]. Current Opinion in Microbiology, 2001, 4(3):307-312. DOI: 10.1016/s1369-5274(00)00208-3.
[2] 张霞. 荧光假单胞菌遗传标记及工程菌生物安全性评价[D]. 保定:河北农业大学,2004. DOI: 10.7666/d.y612503.
[3] 马展,张德纯.假单胞基因工程菌的开发应用现状与展望[J].中国微生态学杂志,2003, 15(3):186-187.DOI: 10.13381/j.cnki.cjm.2003.03.039.
[4] 赵大健,蔡宝立.遗传工程学家A.M.Chakrabarty[J].遗传工程,1982(3):69-71.DOI: 10.13523/j.cb.19820316.
[5] WU C, LI F, YI S W, et al. Genetically engineered microbial remediation of soils co-contaminated by heavy metals and polycyclic aromatic hydrocarbons: Advances and ecological risk assessment[J]. Journal of Environmental Management, 2021, 296:113185. DOI: 10.1016/j.jenvman.2021.113185.
[6] GARCÍA B, OLIVERA E R, MIÑAMBRES B, et al. Novel biodegradable aromatic plastics from a bacterial source: Genetic and biochemical studies on a route of the phenylacetyl-CoA catabolon[J]. Journal of Biological Chemistry, 1999, 274(41): 29228-29241. DOI: 10.1074/jbc.274.41.29228.
[7] LOESCHCKE A, THIES S. Engineering of natural product biosynthesis in Pseudomonas putida[J]. Current Opinion in Biotechnology, 2020, 65:213-224. DOI: 10.1016/j.copbio.2020.03.007.
[8] OLIVERA E R, CARNICERO D, JODRA R, et al. Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications[J]. Environmental Microbiology, 2001, 3(10):612-618. DOI: 10.1046/j.1462-2920.2001.00224.x.
[9] NANDI M, SELIN C, BRAWERMAN G, et al. Hydrogen cyanide, which contributes to Pseudomonas chlororaphis strain PA23 biocontrol, is upregulated in the presence of glycine[J]. Biological Control, 2017, 108:47-54. DOI: 10.1016/j.biocontrol.2017.02.008.
[10] HAAS D, KEEL C. Regulation of antibiotic production in root-colonizing Pseudomonas spp. and relevance for biological control of plant disease[J]. Annual Review of Phytopathology,2003,41:117-153. DOI: 10.1146/annurev.phyto.41.052002.095656.
[11] RAIO A, BRILLI F, BARALDI R, et al. Impact of spontaneous mutations on physiological traits and biocontrol activity of Pseudomonas chlororaphis M71[J]. Microbiological Research,2020,239:126517. DOI: 10.1016/j.micres.2020.126517.
[12] JASIM B, ANISHA C, ROHINI S, et al. Phenazine carboxylic acid production and rhizome protective effect of endophytic Pseudomonas aeruginosa isolated from Zingiber officinale[J]. World Journal of Microbiology and Biotechnology,2014,30(5):1649-1654. DOI: 10.1007/s11274-013-1582-z.
[13] LETOURNEAU M K, MARSHALL M J, CLIFF J B, et al. Phenazine-1-carboxylic acid and soil moisture influence biofilm development and turnover of rhizobacterial biomass on wheat root surfaces[J]. Environmental Microbiology, 2018, 20(6):2178-2194. DOI: 10.1111/1462-2920.14244.
[14] 肖咪云,孙孟龙,阮楚晋,等.生防细菌2016NX1对病原真菌的抑制及发酵条件优化[J].广西师范大学学报(自然科学版),2019,37(2):168-178. DOI: 10.16088/j.issn.1001-6600.2019.02.021.
[15] KHAN S R, MAVRODI D V, JOG G J, et al. Activation of the phz operon of Pseudomonas fluorescens 2-79 requires the luxR homolog phzR,N-(3-OH-Hexanoyl)-L-homoserine lactone produced by the luxI homolog phzI, and a cis-acting phz box[J].Journal of Bacteriology, 2005, 187(18):6517-6527.DOI: 10.1128/jb.187.18.6517-6527.2005.
[16] 刘亭亭. 铜绿假单胞菌抗菌目标活性物质的分离鉴定及其高产工程菌的构建[D]. 桂林:广西师范大学, 2020.
[17] SCHWARZ S, HOOD R D, MOUGOUS J D. What is type Ⅵ secretion doing in all those bugs?[J]. Trends in Microbiology, 2010,18(12): 531-537. DOI: 10.1016/j.tim.2010.09.001.
[18] MANN E E, WOZNIAK D J. Pseudomonas biofilm matrix composition and niche biology[J]. FEMS Microbiology Reviews, 2012, 36 (4): 893-916. DOI: 10.1111/j.1574-6976.2011.00322.x.
[19] WU H J, WANG D, TANG M M, et al. The advance of assembly of exopolysaccharide Psl biosynthesis machinery in Pseudomonas aeruginosa[J]. MicrobiologyOpen, 2019, 8(10):e857. DOI: 10.1002/mbo3.857.
[20] BYRD M S, SADOVSKAYA I, VINOGRADOV E, et al. Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production[J]. Molecular Microbiology, 2009, 73(4): 622-38.DOI: 10.1111/j.1365-2958.2009.06795.x.
[21] ZHANG J C, HE J, ZHAI C H, et al. Effects of pslG on the surface movement of Pseudomonas aeruginosa[J]. Applied and Environmental Microbiology, 2018, 84(13): e00219-18. DOI: 10.1128/AEM.00219-18.
[22] YANG S, CHENG X Y, JIN Z Y, et al. Differential production of Psl in planktonic cells leads to two distinctive attachment phenotypes in Pseudomonas aeruginosa[J]. Applied and Environmental Microbiology, 2018, 84(14):e00700-18. DOI: 10.1128/AEM.00700-18.
[23] VASSEUR P, VALLET-GELY I, SOSCIA C, et al. The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation[J]. Microbiology, 2005, 151(Pt 3):985-997. DOI: 10.1099/mic.0.27410-0.
[24] JENNINGS L K, STOREK K M, LEDVINA H E, et al. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(36):11353-11358. DOI: 10.1073/pnas.1503058112.
[25] WHITFILED G B, MARMONT L S, OSTASZEWSKI A, et al. Pel polysaccharide biosynthesis requires an inner membrane complex comprised of pelD, pelE, pelF, and pelG[J]. Journal of Bacteriology, 2020, 202(8): e00684-19. DOI: 10.1128/JB.00684-19.
[26] FRANKLIN M J, NIVENS D E, WEADGE J T, et al. Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl[J]. Frontiers in Microbiology, 2011, 2: 167. DOI: 10.3389/fmicb.2011.00167.
[27] BAKER P, WHITFIELD G B, HILL P J, et al. Characterization of the Pseudomonas aeruginosa glycoside hydrolase PslG reveals that its levels are critical for Psl polysaccharide biosynthesis and biofilm formation[J]. Journal of Biological Chemistry, 2015, 290(47):28374-28387. DOI: 10.1074/jbc.M115.674929.
[28] O'TOOLE G A, KOLTER R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development[J]. Molecular Microbiology, 1998, 30(2):295-304. DOI: 10.1046/j.1365-2958.1998.01062.x.
[29] 于珊, 马旅雁. 铜绿假单胞菌铁摄取与生物被膜形成研究进展[J]. 生物工程学报,2017,33(9):1489-1512.DOI: 10.13345/j.cjb.170140.
[30] QI L J, CHRISTOPHER G F. Role of flagella, type IV pili, biosurfactants, and extracellular polymeric substance polysaccharides on the formation of pellicles by Pseudomonas aeruginosa[J]. Langmuir, 2019, 35(15):5294-5304. DOI: 10.1021/acs.langmuir.9b00271.
[31] SEMMLER A B, WHITCHUURCH C B, MATTICK J S. A re-examination of twitching motility in Pseudomonas aeruginosa[J]. Microbiology, 1999, 145( Pt 10):2863-2873. DOI: 10.1099/00221287-145-10-2863.
[32] HUESO-GIL A, CALLES B, DE LORENZO V. The Wsp intermembrane complex mediates metabolic control of the swim-attach decision of Pseudomonas putida[J]. Environmental Microbiology,2020,22(8):3535-3547. DOI: 10.1111/1462-2920.15126.
[33] HUANGYUTITHAM V, GÜVENER Z T, HARWOOD C S. Subcellular clustering of the phosphorylated WspR response regulator protein stimulates its diguanylate cyclase activity[J]. mBio, 2013, 4(3): e00242-13. DOI: 10.1128/mBio.00242-13.
[34] 邹雅如,李颖,伍勇,等.铜绿假单胞菌Ⅵ型分泌系统和群体感应系统参与生物被膜形成[J]. 第二军医大学学报, 2019, 40(7):732-736.DOI: 10.16781/j.0258-879x.2019.07.0732.
[35] SANA T G, HACHANI A, BUCIOR I, et al. The second type Ⅵ secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and fur and modulates internalization in epithelial cells[J]. Journal of Biological Chemistry, 2012, 287(32):27095-27105. DOI: 10.1074/jbc.M112.376368.
[36] JANI A J, COTTER P A. Type Ⅵ secretion: not just for pathogenesis anymore[J]. Cell Host & Microbe, 2010, 8(1):2-6. DOI: 10.1016/j.chom.2010.06.012.
[37] 方雪瑶,胡龙华,杭亚平,等. 铜绿假单胞菌VI型分泌系统的研究进展[J]. 中国生物工程杂志, 2018, 38(9):88-93.DOI: 10.13523/j.cb.20180913.
[38] 肖君. 铜绿假单胞菌分泌物对RAW264.7细胞炎症及phzR对生物被膜基因表达的影响[D].桂林:广西师范大学,2021. DOI: 10.27036/d.cnki.ggxsu.2021.000164.
[39] PANG B B, LIU T T, ZHANG W J, et al. Cloning and characterization of phzR gene from Pseudomonas aeruginosa[J]. Current Microbiology, 2021, 78(4): 1482-1487. DOI: 10.1007/s00284-021-02410-2.
[40] 冯春燕. γ-干扰素对铜绿假单胞菌生物膜形成的影响[D]. 青岛:中国海洋大学,2008. DOI: 10.7666/d.y1337222.
[41] SKARIYACHAN S, SRIDHAR V S, PACKIRISAMY S, et al. Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal[J]. Folia Microbiologica, 2018, 63(4): 413-432. DOI: 10.1007/s12223-018-0585-4.
[42] 王珏鑫,余广超. 铜绿假单胞菌群体感应系统研究进展[J]. 中国微生态学杂志,2018,30(7):858-861. DOI: 10.13381/j.cnki.cjm.201807026.
[43] POMPILIO A, PICCOLOMINI R, PICCIANI C, et al. Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: The role of cell surface hydrophobicity and motility[J]. FEMS Microbiology Letters, 2008, 287(1): 41-47. DOI: 10.1111/j.1574-6968.2008.01292.x.
[44] RASHID M H, KORNBERG A. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(9): 4885-4890. DOI: 10.1073/pnas.060030097.
[45] MAVRODI D V, BLANKENFELDT W, THOMASHOW L S. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation[J]. Annual Review of Phytopathology,2006,44:417-445.DOI: 10.1146/annurev.phyto.44.013106.145710.
[46] BRADLEY D E. A function of Pseudomonas aeruginosa PAO polar pili: twitching motility[J]. Canadian Journal of Microbiology, 1980, 26(2): 146-154. DOI: 10.1139/m80-022.
[47] DRISCOLL J A, BRODY S L, KOLLEF M H. The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections[J]. Drugs, 2007, 67(3): 351-368. DOI: 10.2165/00003495-200767030-00003.
[48] STÅHLBERG A, KRZYZANOWSKI P M, EGYUD M, et al. Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing[J]. Nature Protocols,2017,12(4):664-682. DOI: 10.1038/nprot.2017.006.
[49] 王素霞,陈向东,汪辉,等.多尼培南抗铜绿假单胞菌生物被膜的体外活性研究[J].中国抗生素杂志,2016,41(6): 466-472. DOI: 10.3969/j.issn.1001-8689.2016.06.012.
[50] WU Y L, JIANG Y, KAISER D, et al. Social interactions in myxobacterial swarming[J]. PLoS Computational Biology, 2007, 3(12): e253. DOI: 10.1371/journal.pcbi.0030253.
[51] LO Y L, SHEN L D,CHANG C H, et al. Regulation of motility and phenazine pigment production by FliA is cyclic-di-GMP dependent in Pseudomonas aeruginosa PAO1[J]. PLoS One, 2016, 11(5): e0155397. DOI: 10.1371/journal.pone.0155397.
[52] MOROHOSHI T, YAMAGUCHI T, XIE X N, et al. Complete genome sequence of Pseudomonas chlororaphis subsp. aurantiaca reveals a triplicate quorum-sensing mechanism for regulation of phenazine production[J]. Microbes and Environments, 2017, 32(1): 47-53. DOI: 10.1264/jsme2.ME16162.
[53] 杨建社,王帅涛,牛艳婷,等.铜绿假单胞菌T6SS的组装、分泌、功能和调控[J].微生物学报,2021,61(9):2607-2627. DOI: 10.13343/j.cnki.wsxb.20200703.
[54] POMPILIO A,CROCETTA V,DE NICOLA S, et al. Cooperative pathogenicity in cystic fibrosis: Stenotrophomonas maltophilia modulates Pseudomonas aeruginosa virulence in mixed biofilm[J]. Front Microbiol, 2015, 6: 951. DOI: 10.3389/fmicb.2015.00951.
[55] SCHABER J A, HAMMOND A, CARTY N L, et al. Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa[J]. Journal of Medical Microbiology, 2007, 56(Pt 6): 738-748. DOI: 10.1099/jmm.0.47031-0.
[1] 肖咪云, 孙孟龙, 阮楚晋, 陈寿昆, 刘裕华, 陆祖军. 生防细菌2016NX1对病原真菌的抑制及发酵条件优化[J]. 广西师范大学学报（自然科学版）, 2019, 37(2): 168-178.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] 周正春. 互补序列研究进展[J]. 广西师范大学学报（自然科学版）, 2023, 41(1): 1 -16 .
[2] 杨烁祯, 张珑, 王建华, 张恒远. 声音事件检测综述[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 1 -18 .
[3] 杨生龙, 母庆闯, 张志华, 刘葵. 废旧锂离子电池回收利用技术进展[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 19 -26 .
[4] 李康良, 邱彩雄, 何爽, 黄春华, 伍冠一. 白介素-31参与瘙痒的研究进展[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 27 -35 .
[5] 卢许孟, 南新元, 夏斯博. 无模型坐标补偿积分滑模约束的自动驾驶汽车轨迹跟踪控制[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 36 -48 .
[6] 张伟健, 邴其春, 沈富鑫, 胡嫣然, 高鹏. 城市快速路路段行程时间估计方法[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 49 -57 .
[7] 杨秀, 韦笃取. 基于单状态变量的永磁同步电机混沌跟踪控制[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 58 -66 .
[8] 赵媛, 宋树祥, 刘振宇, 岑明灿, 蔡超波, 蒋品群. 一种新型电流镜运算跨导放大器的设计[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 67 -75 .
[9] 王鲁娜, 杜洪波, 朱立军. 基于流形正则的堆叠胶囊自编码器优化算法[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 76 -85 .
[10] 赵明, 罗秋莲, 陈蔚萌, 陈嘉妮. 控制时机和力度对传染病传播的影响[J]. 广西师范大学学报（自然科学版）, 2023, 41(2): 86 -97 .
版权所有 © 广西师范大学学报（自然科学版）编辑部
地址：广西桂林市三里店育才路15号　邮编：541004
电话：0773-5857325　E-mail: gxsdzkb@mailbox.gxnu.edu.cn
本系统由北京玛格泰克科技发展有限公司设计开发