Construction and biological characterization of a cps2D gene knockout strain of Streptococcus suis serotype 2
LIU Xu-miao1,2, WANG Qiao-qiao1,2, LIN Miao1,2, NI Hua3, ZHENG Feng2, WANG Yi-wen2, WANG Chun-hui2, PAN Xiu-zhen1,2, CAO Xiang-rong1
1. College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; 2. Hua Dong Research Institute for Medicine and Biotechnics, Nanjing 210002, China; 3. Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygui Autonomous Kegion, College of Life and Geographic Sciences, Kashi University, Kashi 844000, China
Abstract：A cps2D gene knockout strain was constructed to study the effects of this gene on basic biological characteristics. The gene is located in the capsular polysaccharide locus of Streptococcus suis serotype 2. The cps2D upstream and downstream homology arms were amplified and named the L and R fragment respectively. The SpcR resistance gene was amplified and named the S fragment. The three fragments were then ligated into the pUC19 linear vector to construct the knockout plasmid pUC19::cps2D, which was then electrotransformed into 05ZYH33 competent cells. We screened the knockout strains with Spc resistance plates and performed further verification with combined PCR and RT-PCR. We preliminarily assessed the differences in basic biological characteristics between 05ZYH33 and Δcps2D, including growth curve determination, hemolytic activity observation, Gram staining, bacterial chain length statistics and transmission electron microscopy observation. L(1 030 bp), R(1 030 bp) and S(1 130 bp) gene fragments were amplified by the L1/L2, R1/R2, and Spc1/Spc2 sets of primers respectively. The knockout plasmid pUC19::cps2D was successfully constructed through In-fusion cloning. After electroporation into competent cells, 69 clones were obtained, and 26 suspected knockout strains were screened. After combined PCR and RT-PCR screening, a Δcps2D gene knockout strain was obtained. Compared with 05ZYH33, Δcps2D grew slowly in the logarithmic phase, and its cell numbers per chain were significantly smaller. The capsule structure of Δcps2D was also looser and thinner than that of 05ZYH33. In conclusion, we successfully constructed a cps2D gene knockout strain, thus laying a foundation for further research on its mechanism of regulating capsule synthesis.
刘旭苗, 王悄悄, 林苗, 倪华, 郑峰, 王怡雯, 汪春晖, 潘秀珍, 曹祥荣. 2型猪链球菌cps2D基因敲除株的构建及其基本生物学特性的研究[J]. 中国人兽共患病学报, 2022, 38(4): 285-290.
LIU Xu-miao, WANG Qiao-qiao, LIN Miao, NI Hua, ZHENG Feng, WANG Yi-wen, WANG Chun-hui, PAN Xiu-zhen, CAO Xiang-rong. Construction and biological characterization of a cps2D gene knockout strain of Streptococcus suis serotype 2. Chinese Journal of Zoonoses, 2022, 38(4): 285-290.
[1] Goyette-Desjardins G, Auger JP, Xu J, et al.Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing[J]. Emer Microbes Infect, 2014, 3(6):e45. DOI:10.1038/emi.2014.45 [2] Feng Y, Zhang H, Wu Z, et al.Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases[J]. Virulence, 2014, 5(4):477-497. DOI:10.4161/viru.28595 [3] Segura M, Fittipaldi N, Calzas C, et al.Critical Streptococcus suis virulence factors: are they all really critical[J]. Trends Microbiol, 2017, 25(7): 585-599. DOI:10.1016/j.tim.2017.02.005 [4] Huong VT, Ha N, Huy NT, et al.Epidemiology, clinical manifestations, and outcomes of Streptococcus suis infection in humans[J]. Emerg Infect Dis, 2014, 20(7):1105-1114. DOI:10.3201/eid2007.131594 [5] Tang J, Wang C, Feng Y, et al.Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2[J]. PLoS Med, 2006, 3(5):e151. DOI:10.1371/journal.pmed.0030151 [6] Yu H, Jing H, Chen Z, et al.Human Streptococcus suis outbreak, Sichuan, China[J]. Emerg Infect Dis, 2006, 12(6):914-920. DOI:10.3201/eid1206.051194 [7] Gottschalk M, Xu J, Calzas C, et al.Streptococcus suis: a new emerging or an old neglected zoonotic pathogen[J]. Future Microbiol, 2010, 5(3):371-391. DOI:10.2217/fmb.10.2 [8] Feng Y, Zhang H, Wu Z, et al.Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases[J]. Virulence, 2014, 5(4):477-497. DOI:10.4161/viru.28595 [9] Jacek D, Violetta Z, Jacek S, et al.Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II-Pathogenesis[J]. Ann Agric Environ Med, 2018, 25:186-203. DOI:10.26444/aaem/85651 [10] Calsteren MV, Gagnon F, Calzas C, et al.Structure determination of Streptococcus suis serotype 2 capsular polysaccharide[J]. Med Biochem Cell, 2013, 91(2):49-58.DOI:10.1139/bcb-2012-0036 [11] Getz LJ, Runte CS, Rainey JK, et al.Tyrosine phosphorylation as a widespread regulatory mechanism in prokaryotes[J]. J Bacteriol, 2019;201(19):e00205-19. DOI:10.1128/JB.00205-19 [12] Chen C, Tang J, Wei D, et al.A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates[J]. PLoS One, 2007, 2(3): e315. DOI:10.1371/journal.pone.0000315 [13] 胡丹. 猪链球菌2型荚膜缺失株的构建及其致病性研究[D]. 重庆:第三军医大学, 2009. [14] 吴倩倩. 猪链球菌酪氨酸磷酸化系统相关蛋白的制备及功能研究[D]. 温州:温州医科大学, 2018. [15] 王悄悄. 2型猪链球菌酪氨酸磷激酶基因cps2C对荚膜合成及细菌毒力的调控研究[D]. 南京:南京师范大学, 2021. [16] Takamatsu D, Osaki M, Sekizaki T.Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors[J]. Plasmid, 2001, 45(2): 101-113. DOI:10.1006/plas.2000.1510 [17] Smith HE, Wisselink HJ, Vecht U, et al.High-efficiency transformation and gene inactivation in Streptococcus suis type 2[J]. Microbiology, 1995, 141(1):181-188. DOI:10.1099/00221287-141-1-181 [18] Julien N, Morten K, M Chryslène, et al. Autophosphorylation of the bacterial tyrosine-kinase CpsD connects capsule synthesis with the cell cycle in Streptococcus pneumoniae[J]. PLoS Genetics, 2015, 11(9):e1005518. DOI:10.1371/journal.pgen.1005518 [19] Toniolo C, Balducci E, Romano MR, et al.Streptococcus agalactiae capsule polymer length and attachment is determined by the proteins CpsABCD[J]. J Biol Chem, 2015, 290(15):9521-9532. DOI:10.1074/jbc.M114.631499 [20] Evans K, Stone V, Chen L, et al.Systematic study of genes influencing cellular chain length in Streptococcus sanguinis[J]. Microbiology (Reading, England), 2014, 160(2): 307-315. DOI:10.1099/mic.0.071688-0
2022, Vol. 38 
