转座子随机插入鉴定肠炎沙门氏菌生物膜形成相关基因
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高等学校博士学科点专项基金(20061117005)


Identification of genes for biofilm formation in a Salmonella enteritidis strain by transposon mutagenesis
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Supported by the Research Fund for the Doctoral Program of Higher Education(20061117005)

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    摘要:

    【目的】生物膜在沙门氏菌的致病性和引起沙门氏菌食物中毒等方面起着重要作用,本研究为了鉴定影响沙门氏菌生物膜形成的基因。【方法】利用结晶紫染色定量法对74株鸡源的肠炎、鸡白痢和鸡伤寒沙门氏菌进行生物膜测定,选择生物膜生长较好的肠炎沙门氏菌C050041,采用转座子随机插入法构建突变株库。【结果】84%的鸡源沙门氏菌菌株可在塑料表面形成生物膜;通过转座子插入获得1924个突变株,筛选的生物膜降低突变株经生长曲线测定、测序和序列比对及Southern blot分析鉴定出15个插入基因,它们分别为metE、ompR、rpoS、rfaG、rfaJ、rfaK、rfaP、rfbH、rhlE、spiA、steB、tpx、ybdN和2个未知功能的基因。【结论】我们鉴定出了多个影响生物膜形成的新基因,这些基因的发现为进一步研究沙门氏菌生物膜形成的调控机制,研制减毒沙门氏菌疫苗奠定了基础。

    Abstract:

    [Objective] Biofilm plays an important role in pathogenicity of Salmonella and food poisoning caused by Salmonella. Our aim was to identify genes associated with Salmonella biofilm formation. [Methods] Seventy-four strains of Salmonella enterica serovar enteritidis (S. enteritidis), S. pullorum and S, gallinarum isolated from chickens were determined for biofilm using crystal violet staining, and the strain C050041 with well biofilm formation was chosen to construct a mutant library using transposon mutagenesis. [Results] Eighty-four percent of these Salmonella strains produced biofilm on the plastic surface. We screened 1924 mutants with transposon insertion, and 15 inserted genes were identified by growth curve determination of mutants, sequence analysis of the chromosomal DNA, and further confirmed by southern blot. These genes included metE, ompR, rpoS, rfaG, rfaJ, rfaK, rfaP, rfbH, rhlE, spiA, steB, tpx, ybdN and other two genes with unknown function. [Conclusion] We identified some new genes associated with Salmonella biofilm formation, these findings may help understand the regulation mechanism of biofilm formation and develop an attenuated Salmonella vaccine.

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董洪燕,张小荣,潘志明,彭大新,刘秀梵. 转座子随机插入鉴定肠炎沙门氏菌生物膜形成相关基因[J]. 微生物学报, 2008, 48(7): 869-873

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