Construction and Biofilm Formation of yibT-and-csgD-deleted Salmonella typhimurium
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摘要:
目的 通过构建鼠伤寒沙门菌（Salmonella typhimurium）yibT、csgD双基因缺失株及缺失株的回补株，探究其对鼠伤寒沙门菌生物膜形成的影响，以期为沙门菌的有效防控提供新策略。 方法 以鼠伤寒沙门菌野生株CVCC541（wild-type, WT）为研究对象，利用λ-red同源重组技术构建yibT和csgD基因缺失株；利用重组载体技术构建其基因回补株；利用结晶紫染色法比较鼠伤寒沙门菌突变株生物膜形成能力的差异；通过苯酚-硫酸法测定胞外多糖含量、半固体平板测定运动能力的变化；比较不同突变菌株自聚集能力的变化；通过扫描电镜观察生物膜结构；最后利用荧光定量PCR技术鉴定生物膜形成过程中关键基因的mRNA表达水平。 结果 成功构建了鼠伤寒沙门菌yibT和csgD的双基因缺失株WTΔyibTΔcsgD和csgD基因单缺失株WTΔcsgD及基因缺失株的回补株WTΔcsgDΔyibT/pcsgD和WTΔcsgD/pcsgD。 yibT和csgD基因的缺失降低了生物膜的形成能力、胞外多糖含量和自聚集能力，增强了运动能力；invF和sdiA基因的mRNA表达水平下降。 结论 yibT和csgD基因缺失会降低鼠伤寒沙门菌的生物膜形成能力。 Abstract:Objective Strains of Salmonella typhimurium with yibT and/or csgD deleted were created to observe the effect on its biofilm formation for effective prevention and control of the foodborne pathogen. Method The λ-red homologous recombination technique was applied to create the gene-deleted mutants, WTΔyibTΔcsgD and WTΔcsgD, of WT S. typhimurium (CVCC541). Subsequently, the gene-complemented strains were also obtained using recombinant vector technology. Differences of the mutants in biofilm formation were determined by crystal violet staining, content of exopolysaccharide measured by phenol-sulfuric acid, mobility tested on semi-solid agar plates, and self-aggregation monitored. Under a scanning electron microscope, biofilm structure was observed. Finally, mRNA expressions of the target genes in the biofilm were identified by fluorescence quantitative PCR. Result A double-gene deleted WTΔyibTΔcsgD, a WTΔcsgD without csgD, and their respective complemented strains, WTΔcsgDΔyibT/pcsgD and WTΔcsgD/pcsgD, were successfully obtained. The double-gene deletion significantly reduced the ability in forming biofilm, the content of exopolysaccharide, and the aggregation of the genes to targets, and the mRNA expressions of invF and sdiA in WT S. typhimurium. Conclusion Deletion of yibT and csgD significantly impacted some critical biofunctions of S. typhimurium that could become a new approach for control of the foodborne disease. -
Key words:
- Salmonella typhimurium /
- yibT /
- csgD /
- biofilm
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图 1 菌株WTΔyibTΔcsgD:Kan和WTΔcsgD:Kan的PCR鉴定
M：D2000 Marker；1：WTΔyibTΔcsgD:Kan PCR产物；2： WTΔcsgD:Kan PCR产物；3：WT PCR产物。
Figure 1. PCR identification for WTΔyibTΔcsgD:Kan and WTΔcsgD:Kan
M: D2000 marker; 1: PCR product of WTΔyibTΔcsgD:Kan; 2: PCR product of WTΔcsgD:Kan; 3: PCR product of WT.
图 2 菌株WTΔyibTΔcsgD和WTΔcsgD的PCR鉴定
M：D2000 Marker；1：WT 的 PCR 产物；2：WTΔyibTΔcsgD 的 PCR 产物；3：WTΔcsgD 的 PCR 产物。
Figure 2. PCR identification for WTΔyibTΔcsgD and WTΔcsgD
M: D2000 marker; 1: PCR product of WT; 2: PCR product of WTΔyibTΔcsgD; 3: PCR product of WTΔcsgD.
图 3 WTΔyibTΔcsgD/pcsgD和WTΔcsgD/pcsgD的PCR鉴定
M：D2000 Marker；1：WTΔyibTΔcsgD/pcsgD的PCR产物；2：WTΔcsgD/pcsgD的PCR产物；3：转入空质粒pET28a的菌 PCR产物。
Figure 3. PCR identification for WTΔyibTΔcsgD/pcsgD and WTΔcsgD/pcsgD
M: D2000 marker; 1: PCR product of WTΔyibTΔcsgD/pcsgD; 2: PCR product of WTΔcsgD/pcsgD; 3: PCR product of bacteria transferred to empty plasmid pET28a.
图 4 鼠伤寒沙门菌突变株的生物膜形成
A：试管法测定生物膜能力；B：二十四孔板测定生物膜能力；C：生物膜的结晶紫量化。*表示与WT差异显著（P＜0.05），**表示与WT差异极显著（P＜0.01）。下同。
Figure 4. Biofilm formation of S. typhimurium mutants
A: test tube method for determining biofilm formation; B: 24-well plates for determining biofilm formation; C: crystal violet quantization of biofilm. Results are comparisons with WT; *: significant difference at P＜0.05; **: extremely significant difference at P＜0.01. Same for below.
图 6 鼠伤寒沙门菌突变株胞外多糖含量
A：葡萄糖标准曲线；B：胞外多糖含量。
Figure 6. Exopolysaccharide contents in S. typhimurium mutants
A: glucose standard curve; B: exopolysaccharide content.
图 7 鼠伤寒沙门菌突变株的运动能力
A：鼠伤寒沙门菌突变株在半固体平板的运动性；B：鼠伤寒沙门菌突变株的运动直径。
Figure 7. Motility of S. typhimurium mutants
A: motility of S. typhimurium mutants on semi-solid plates; B: diameters of moving area of S. typhimurium mutants.
表 1 PCR扩增引物
Table 1. Primers applied for PCR
引物名称
Primer names引物序列5′-3′
Primer sequences引物长度
Primer length/bp反应长度
Reaction length/bpP1 TTTCATCATGTTTAATGAAGTCCATAGTAGTCATGGTCAGTGTAGGCTGGAGCTGCTTC 59 1600 P2 ATCTTTTTGAAAAGATTATAAAGATGTGTCTTAACCGTACATATGAATATCCTCCTTAG 59 P3 GCTGTCAGATGTGCGATT 18 723 P4 TGCTACAATCCAGGTCAGA 19 P5 CCGCTCGAGCCGCCTGAGATTATCGTTTG 29 649 P6 CGCGGATCCATGTTTAATGAAGTCCATAG 29 P7 CAGCCAGGCGTTCCGTGAAT 20 469 P8 AGCCGCCGGTAATATTCCAGAC 22 
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表 2 荧光定量PCR引物序列
Table 2. Primers applied for qRT-PCR
引物名称
Primer names引物序列5′-3′
Primer sequences引物长度
Primer length/bpluxS-F ACTGATGGGCTGCCTGTATC 20 luxS-R
sdiA-F
sdiA-R
invF-F
invF-R
16S-F
16S-RGCCTCTTCGCTATTACGCCA
ATGAAGCGAAGGCGATGT
CGAGGAGCAGCGTAAACT
ACGATGAGAATGCTGGGAGA
TATGTGAAGGCGATGAGTAAC
TTACCCGCAGAAGAAGCACC
CTCAAGGGCACAACCTCCAA20
18
18
20
20
20
20
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