Abstract：Listeria monocytogenes (LM) is a human-animal Gram-positive bacterium with a wide range of adaptations to the external environment. In order to understand the regulatory role of sRNA rli82 on the environmental adaptability and biofilm generation of LM. In this study, the rli82 gene deletion mutant strain LM- Δrli82 and the complementation strain LM- Δrli82/rli82 were constructed using homologous recombination technology and the differences in growth and biofilm generation of these strains under different stress environment were determined, respectively. Moreover, bioinformatics was used to predict the potential target genes regulated by rli82, and the transcript levels of genes related to environmental stress and biofilm formation were measured by qPCR. The results showed that rli82 gene owns 70 bp in length, with a secondary structure of 2 neck ring. The potential target gene was opuCC. LM- Δrli82 significantly enhanced environmental adaptation capacity under different osmotic pressure (4% and 8% NaCl) when compared to LM EGD-e and LM- Δrli82/rli82. The environmental adaptation capacity was significantly lower under stress conditions of 3.8% ethanol, pH4, pH9 and 30 ℃ . However, the biofilm formation of the 3 strains was not significantly different. The qPCR showed a significant decrease in the relative expression of genes related to environmental stress but no significant difference in biofilm related genes. The results demonstrate that the sRNA rli82 plays an important role in regulating the adaptive capacity of LM to environmental stress, and set a foundation for studying the molecular mechanism of sRNA rli82 regulating environmental stress in LM.
季春辉, 郭蕴, 王立霞, 宁程程, 李娜, 乔军, 孟庆玲, 才学鹏. sRNA rli82 分子特征及其对单核细胞增生李斯特菌环境应激和生物被膜形成的调控作用[J]. 农业生物技术学报, 2022, 30(5): 990-998.
JI Chun-Hui, GUO Yun, WANG Li-Xia, NING Cheng-Cheng, LI Na, QIAO Jun MENG Qing-Ling, CAI Xue-Peng. Molecular Characterization of sRNA rli82 and Its Regulation Roles in Environmental Stress and Biofilm Formation in Listeria monocytogenes. 农业生物技术学报, 2022, 30(5): 990-998.
[1] 付娇娇 .2016. 环境胁迫应答因子 SigB 影响单增李斯特菌生物被膜形成的初步机制研究[D]. 硕士学位论文, 上海海洋大学, 导师: 赵勇, pp. 1-7. (Fu J J.2016. Preliminary study on mechanism of environmental stress response factor sigb in biofilm formation by Listeria monocytogenes[D]. Thesis for M.S., Shanghai Ocean University, Suppervisor: Zhao Y, pp. 1-7.) [2] 杨丽红, 孟庆玲, 乔军, 等 . 2013. Sigma B 基因缺失对单核细胞增生性李斯特菌毒力的影响[J]. 西北农业学报, 22(08): 92-97. (Yang L H, Meng Q L, Qiao J, et al.2013. Effect of sigmab gene deletion on virulence of Listeria monocytogenes[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 22(08): 92-97.) [3] 于新惠, 张颖, 王文静, 等 . 2017. rmlB 基因在单核细胞增生李斯特菌耐药性、生物被膜形成和毒力方面的作用[J]. 微生物学通报, 44(01): 161-171. (Yu X H, Zhang Y,Wang W J, et al.2017. Contribution of rmlb in envelope-acting antibiotic resistance, biofilm formation and virulence in Listeria monocytogenes[J]. Microbiology, 44(01): 161-171.) [4] Boehm A, Vogel J.2012. The csgD mRNA as a hub for signal integration via multiple small RNAs[J]. Molcular Microbiology, 84(1): 1-5. [5] Gandhi M, Chikindas M L.2007. Listeria: A foodborne pathogen that knows how to survive[J]. International Journal of Food Microbiology, 113(1): 1-15. [6] Jørgensen M G, Pettersen J S, Kallipolitis B H.2020. sRNA-mediated control in bacteria: An increasing diversity of regulatory mechanisms[J]. Gene Regulatory Mechanisms, 1863(5): 194504. [7] Kappes R M, Kempf B, Kneip S, et al.1999. Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant gly‐cine betaine in Bacillus subtilis[J]. Molcular Microbiolo‐gy, 32(1): 203-216. Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods, 25(4): 402-408. [8] Matereke L T, Okoh A I.2020. Listeria monocytogenes viru‐lence, antimicrobial resistance and environmental persistence: A review[J]. Pathogens, 9(7): 528. [9] Matle I, Mbatha K R, Madoroba E.2020. A review of Listeria monocytogenes from meat and meat products: Epidemiology, virulence factors, antimicrobial resistance and diagnosis[J]. Onderstepoort Journal of Veterinary Research, 87(1): e1-e20. [10] Mraheil M A, Billion A, Mohamed W, et al.2011. The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages[J]. Nucleic Acids Research, 39(10): 4235-4248. [11] Nicaogáin K, O'byrne C P.2016. The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain[J]. Frontiers in Microbiology, 7: 1865. [12] Nielsen J S, Larsen M H, Lillebæk E M, et al.2011. A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes[J]. PLOS ONE, 6(4): e19019. [13] Peng Y L, Meng Q L, Qiao J, et al.2016. The regulatory roles of ncRNA Rli60 in adaptability of Listeria monocytogenes to environmental stress and biofilm formation[J].Current Microbiology, 73(1): 77-83. [14] Quereda J J, Cossart P.2017. Regulating bacterial virulence with RNA[J]. Annual Review of Microbiology, 71(1): 263-280. [15] Quereda J J, Ortega A D, Pucciarelli M G, et al.2014. The listeria small RNA Rli27 regulates a cell wall protein in‐side eukaryotic cells by targeting a long 5'-UTR variant[J]. PLOS Genetics, 10(10): e1004765. [16] Schmid B, Klumpp J, Raimann E, et al.2009. Role of cold shock proteins in growth of Listeria monocytogenes un‐der cold and osmotic stress conditions[J]. Applied and Environmental Microbiology, 75(6): 1621-1627. [17] Sleator R D, Wouters J, Gahan C G, et al.2001. Analysis of the role of OpuC, an osmolyte transport system, in salt tolerance and virulence potential of Listeria monocyto‐genes[J]. Applied and Environmental Microbiology, 67(6): 2692-2698.
