Activity Determination of 4 Glycosyltransferases and Protein Interaction Analysis of Erwinia beijingensis

doi:10.13304/j.nykjdb.2022.0736

Abstract

Abstract:

Erwinia beijingensis can cause the bacterial soft rot disease of Pleurotus eryngii. In order to clarify the function of glycosyltransferase in E. beijingensis， a recombinant expression vector was constructed to express and purify the glycosyltransferase in this pathogen. The protein activity was measured， and the interactions between proteins were analyzed. The results showed that the prokaryotic expression vector was successfully constructed， 4 soluble proteins were obtained， and MshA， WbnH2， EpsH and TuaG proteins were purified by affinity chromatography column. The activity assay showed that four proteins preferentially used UDP galactose. GST pull down results confirmed that WbnH2 and TuaG proteins interacted with MshA and EpsH proteins in vitro， respectively. Above results laid a foundation for the subsequent study on the function of the glycosyltransferase gene in E. beijingensis.

Key words: Erwinia beijingensis, soft rot, glycosyltransferase, prokaryotic expression

摘要：

北京欧文氏菌（Erwinia beijingensis）可引起刺芹侧耳细菌性软腐病，为明确该病原菌中糖基转移酶的功能，以糖基转移酶基因为研究对象，构建重组表达载体进行表达纯化；并测定蛋白活性，分析蛋白间相互作用。结果表明，成功构建了原核表达载体，获得4个可溶性蛋白。经亲和层析柱纯化获得MshA、WbnH2、EpsH及TuaG蛋白，活性测定显示4个蛋白均可以利用UDP-糖，并优先利用UDP-半乳糖。GST pull down证实WbnH2及TuaG蛋白分别与MshA及EpsH蛋白在体外具有相互作用。以上研究结果为进一步研究北京欧文氏菌糖基转移酶基因功能奠定了基础。

关键词: 北京欧文氏菌, 软腐病, 糖基转移酶, 原核表达

CLC Number:

Q933

Xiaoning CHANG, Jinying GUO, Chengbo RONG, Tongtong GU, Yu LIU. Activity Determination of 4 Glycosyltransferases and Protein Interaction Analysis of Erwinia beijingensis[J]. Journal of Agricultural Science and Technology, 2024, 26(1): 125-132.

常晓宁, 郭金英, 荣成博, 谷彤彤, 刘宇. 北京欧文氏菌4个糖基转移酶活性测定及蛋白相互作用分析[J]. 中国农业科技导报, 2024, 26(1): 125-132.

Figures/Tables 6

Table 1 Primers in this experiment

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp
mshA-F	GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCGAATAGTAACTTTGTTGTATGTTTGC	1 058
mshA-R	TCTCAGTGGTGGTGGTGGTGGTGCTCGAGGATGTTACCGCCCTCTAAAAGATACGATCTAAAATATTCTT
wbnH1 F	GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGTAATGCCGGAAGGAAAATAATTTATAT	1 044
wbnH1 R	CAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTAATGGAATCAACTCAAGATCTTTTTGGGCTTC
wbnH2 F	CCCCGGGAATTTCCGGTGGTGGTGGTGGAATTCACTTGATCGTGATCCATAATGGTGTTCCTCCTCATGC	642
wbnH2 R	CGTCAGTCAGTCACGATGAATTAAGCTTGAGCTCGAGCTACCGAAGAAATATTTCCTGATACAATTTATTC
EpsH F	AAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAATGAGAAGATCGCATCCCCAAAACTCTCT	936
EpsH R	CAGTGGTGGTGGTGGTGGTGCTCGAGTTTTATAGTTAGCACTCTAGTTTTACGCAACAAAAAACGTAAAA
tuaG F	CTGGAAGTTCTGTTCCAGGGGCCCCTGGGATCCATGAAAATAACAATAGTTACTGCTACATATAATTCTG	685
tuaG R	AGATCGTCAGTCAGTCACGATGCGGCCGCTCGAGTCATCCCCTTAGAATAGCTCTTAGAGCATAATTCAT
His1422 F	AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGACGCCTGTTTATAATAGAGCAGACTTAC	732
His1422 R	AGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCATCTTTCTAATTCCAGAACCGACTCTAATTATG
GST1422 F	GGAAGTTCTGTTCCAGGGGCCCCTGGGATCCATGACGCCTGTTTATAATAGAGCAGACTTACTTAAAAAT	714
GST1422 R	TCGTCAGTCAGTCACGATGCGGCCGCTCGAGTTACATCTTTCTAATTCCAGAACCGACTCTAATTATGTT

Fig. 1 Screening results of soluble protein induced by a small amount of induced expression.A： E. coli BL21/ pET-30a-mshA； B： E. coli BL21/pET-30a-EpsH；C： E. coli BL21/pGEX-6p-tuaG；D： E. coli BL21/pGEX-wbnH2. 1~4 are whole bacteria before induction， whole bacteria after induction， broken supernatant after induction and broken sediment after induction with 0.1 mmol·L-1 IPTG induced， respectively； M is marker； 5~8 are the whole bacteria before induction， whole bacteria after induction， broken supernatant after induction and broken sediment after induction induced with 1.0 mmol·L-1 IPTG， respectively

Fig. 2 Protein purification resultsA： MshA protein purification， in which M is marker， 1 is running through liquid， 2 is buffer A elution， 3~9 are the eluents of 20， 40， 60， 80， 100， 150， 200 mmol L-1 imidazole concentration， respectively； B： EpsH protein purification， in which 1 is the flow through solution， 2~6 are 60， 80， 100， 150， 200， 300 mmol L-1 imidazole concentration eluates， respectively， M is marker； C： WbnH2 protein purification， in which M is marker， 1 is running through liquid， 2~6 are 20 mmol L-1 GSH elution； D： TuaG protein purification， in which M is marker， 1 is running through liquid， 2~4 is 10 mmol L-1 GSH elution， 5-6 is 10 mmol·L-1 GSH+5 mmol L-1 DTT elution

Fig. 3 Hydrolysis ability of four glycosyltransferases to different UDP sugars

Fig. 4 GST pull down resultA： Binding between WbnH2 and MshA proteins， M—Marker， 1—GST binds to MshA protein， 2—MshA protein， 3—WbnH2 binds to MshA protein； B： Binding between TuaG and MshA proteins， M—Marker， 1—GST binds to MshA proteins， 2—MshA protein， 3—TuaG binds to MshA protein； C： Binding between WbnH2 and TuaG proteins to EpsH protein， respectively， M—Marker， 1—WbnH2 binds to EpsH protein， 2—GST binds to EpsH protein， 3—EpsH protein， 4—GST binds to EpsH protein， 5—TuaG binds to EpsH protein

Fig. 5 Western blot test resultA： Binding between WbnH2 and MshA proteins. 1—GST binds to MshA protein； 2—MshA protein； 3—WbnH2 binds to MshA protein； B： Binding between TuaG and MshA proteins. 1—GST binds to MshA protein； 2—MshA protein； 3—TuaG binds to MshA protein； C： Binding between WbnH2 and EpsH proteins， 1—GST binds to EpsH protein； 2—EpsH protein； 3—WbnH2 binds to EpsH protQein； D： Binding between TuaG and EpsH proteins， 1—GST binds to EpsH protein， 2—EpsH protein； 3—TuaG binds to MshA protein

References 27

1	WANG Q, ZHANG C, WU X, et al.. Chitosan augments tetramycin against soft rot in kiwifruit and enhances its improvement for kiwifruit growth, quality and aroma [J]. Biomolecules, 2021, 11(9):1257-1270.
2	LIU M, WU F, WANG S, et al.. Comparative transcriptome analysis reveals defense responses against soft rot in Chinese cabbage [J]. Hortic. Res., 2019, 6:68-86.
3	SUN M, LIU H, HUANG J, et al.. A loop-mediated isothermal amplification assay for rapid detection of Pectobacterium aroidearum that causes soft rot in konjac [J]. Int. J. Mol. Sci., 2019, 20(8):1937-1950.
4	YAKOVLIEVA L, WALVOORT M. Processivity in bacterial glycosyltransferases [J]. ACS Chem. Biol., 2019, 15(1):3-16.
5	KIM M K, LEE S M, SEUK S W, et al.. Characterization of the rcsA gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii [J]. Plant Pathol. J., 2017, 33(3):276-287.
6	XU F, YAN H, LIU Y, et al.. A re-evaluation of the taxonomy and classification of the type Ⅲ secretion system in a pathogenic bacterium causingsoft rot disease of Pleurotus eryngii [J]. Curr. Microbiol., 2021, 78(1):179-189.
7	赖亮民,胡晶晶,王艳,等.刺芹侧耳软腐病病原菌鉴定及其致病机制[J].食用菌学报,2021,28(2):89-99.
	LAI L M, HU J J, WANG Y, et al.. Identification and pathogenic mechanisms of pathogens causing soft rot disease in Pleurotus eryngii [J]. Acta Edulis Fungi, 2021, 28(2):89-99.
8	张瑞颖,胡丹丹,顾金刚,等.刺芹侧耳细菌性软腐病病原菌分离鉴定[J].食用菌学报,2013,20(3):43-49.
	ZHANG R Y, HU D D, GU J G, et al.. Identification and characterization of an Erwinia sp. causing bacterial soft-rot disease on pleurotuseryngii cultivated in China [J]. Acta Edulis Fungi, 2013, 20(3):43-49.
9	马元伟,马康,王守现,等.一株刺芹侧耳软腐病致病菌新种的分离与鉴定[C]//第十届全国食用菌学术研讨会论文汇编,北京,2014:382-389.
	MA Y M, MA K, WANG S X, et al.. Isolation and identification of a novel pathogenic bacteria,causing soft-rot diease of Pleurotus eryngii [C]// Proceedings of the 10th National Symposium on edible fungi, Beijing, 2014:382-389.
10	LERMINIAUX N A, MACKENZIE K D, CAMERON A. Salmonella pathogenicity island 1 (SPI-1): the evolution and stabilization of a core genomic type three secretion system [J]. Microorganisms, 2020, 8(4):2-22.
11	KLEE S M, SINN J P, CHRISTIAN E, et al.. Virulence genetics of an Erwinia amylovora putative polysaccharide transporter family member [J]. J. Bacteriol., 2020, 202(22):1-19.
12	ROBERTS, IAN S. The biochemistry and genetics of capsular polysaccharide production in bacteria [J]. Annu. Rev. Microbiol., 1996, 50(1):285-315.
13	ASKARIAN F, UCHIYAMA S, MASSON H, et al.. The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection [J]. Nat. Comm., 2021, 12(1):1-19.
14	BAE N, PARK H J, PARK H, et al.. Deciphering the functions of the outer membrane porin OprBXo involved in virulence, motility, exopolysaccharide production, biofilm formation and stress tolerance in Xanthomonas oryzae pv. Oryzae [J]. Mol. Plant Pathol., 2018, 19(12):2527-2542.
15	JEON J G, ROSALEN P L, FALSETTA M L, et al.. Natural products in caries research: current (limited) knowledge, challenges and future perspective [J]. Caries Res., 2011, 45(3):243-263.
16	BISWAS A, THATTAI M. Promiscuity and specificity of eukaryotic glycosyltransferases [J]. Portland Press Open Access., 2020, 48(3):891-900.
17	LU Q, LI S, SHAO F. Sweet talk: protein glycosylation in bacterial interaction with the host [J]. Trends Microbiol., 2015, 23(10):630-641.
18	MIDDLETON D R, ACEIL J, MUSTAFA S, et al.. Glycosyltransferases within the psrP locus facilitate pneumococcal virulence [J]. J. Bacteriol., 2021, 203(7):389-400.
19	JIANG Y L, JIN H, YANG H B, et al.. Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP [J]. J. Biol. Chem., 2017, 292(15):6213-6224..
20	DENG Q, WU H, GU Q, et al.. Glycosyltransferase FvCpsA regulates fumonisin biosynthesis and virulence in Fusarium verticillioides [J]. Toxins, 2021, 13(10):718-730.
21	ECHEVERZ M, GARCÍA B, SABALZA A, et al.. Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host [J]. PLoS Genetics, 2017, 13(5):816-843.
22	EMI L, TYKESSO N, YAN G, et al.. Deciphering the mode of Action of the processive polysaccharide modifying enzyme dermatan sulfate epimerase 1 by hydrogen-deuterium exchange mass spectrometry [J]. Chem. Sci., 2016, 7(2):1447-1456.
23	DENG S, SUN W, DONG L, et al.. MoGT2 is essential for morphogenesis and pathogenicity of Magnaporthe oryzae [J]. Msphere, 2019, 4(5):309-319.
24	PÉREZ-PASCUAL D, GÓMEZ E, GUIJARRO J A. Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence [J]. Veterinary Res., 2015, 46(1):1-9.
25	DABRAL N, JAIN-GUPTA N, SELEEM M N, et al.. Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide [J]. Front. Cellular Infect. Microbiol., 2015, 5(4):54-68.
26	SUSAN G, KATHERINE, et al.. Structural determinants of the interaction between the TpsA and TpsB proteins in the Haemophilus influenzae HMW1 two-partner secretion system [J]. J. Bacteriol., 2015, 197(10):1769-1781.
27	ECHLIN H, ZHU F, LI Y, et al.. Gap2 promotes the formation of a stable protein complex required for mature fap1 biogenesis [J]. J. Bacteriol., 2013, 195(10):2166-2176.

[1]	SUI Fu1,2, LIU Xiaolin1,2, XIE Zhihong1,3*. Sensing Mechanism of Receptor TlpA1 to Succinic Acid in Azorhizobium caulinodans ORS571 [J]. Journal of Agricultural Science and Technology, 2020, 22(10): 77-84.
[2]	YU Xiao-qing1, XIE Hua1, WANG Xiu-ling2, MA Rong-cai1. Research Progress on Molecular Mechanisms of Plant Defense to Soft Rot Disease [J]. , 2012, 14(6): 49-53.
[3]	CUI Yan-fang, SUN Pei-long, LIU Xin-qi. Effect Comparison of EIAV gp45 and HIV gp120 Genes Using Different Expressional Systems [J]. , 2011, 13(4): 72-78.
[4]	WANG Jian-she1,2, BAI Ying-guo2, YIN Jun1, YAO Bin2. Cloning, Expression and Characterization Studies on Xylanase Gene, xynA15, from Alicyclobacillus sp.A15 [J]. , 2010, 12(6): 114-119.
[5]	LIU Yu1,2, YAN Cai-xia1, ZHANG Ting-ting1, LI Chun-juan1, . Cloning and Prokaryotic Expression of Peanut NBS-LRR Resistant Gene [J]. , 2010, 12(3): 73-78.
[6]	LI Rui-qin1, FENG Shu-dan1, YU Ying2, LV Zhao-zhi1, LI Li1, XU Ming-hua1, YIN Yu. Cloning, Bioinformatics Analysis and Prokaryotic Expression of Chitinase ClassⅡ Gene in Leymus chinensis [J]. , 2010, 12(2): 103-110.
[7]	YAO Guo-yu, LI Ning, SHI Peng-jun, CHEN Qiang, YAO Bin. Cloning, Expression and Characterization of a Xylanase Gene, xynBS9, from Streptomyces sp. S9 [J]. , 2009, 11(4): 64-70.
[8]	LIU Quan, LI Guang-yue, ZENG Hong-mei, YANG Xiu-fen, QIU De-wen. Acquisition of Microbial Protein Elicitor PeaT1 and Preliminary Research on Inducing Drought Resistance of Rice [J]. , 2009, 11(3): 51-55.
[9]	DUAN Wen-juan, YANG Jiao-yan, LI Zhuo-fu, ZHANG Wei\| ZHANG Zhi-fang . Expression of β-Galactosidase from Pyrococcus furiosus \|in E. coli and Analysis of Lactase Properties [J]. , 2008, 10(2): 76-81.