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微生物学通报

钩端螺旋体聚Beta羟基丁酸(PHB)生物合成途径分析
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国家自然科学基金项目(No. 81301404, 81071375); 首都医科大学附属北京友谊医院科研启动基金项目(No. 2011_30yykyqd); 首都医科大学基础-临床科研合作基金项目(No. 13JL16); 热带病防治研究北京市重点实验室开放基金项目(No. 100017031017); 北京市自然科学基金项目(No. 7132042)


Pathway analysis of the leptospiral polyhydroxybutyrate (PHB) synthesis
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    摘要:

    【目的】致病型问号钩端螺旋体(问号钩体, Leptospira interrogans)和腐生型双曲钩体(L. biflexa)能够大量合成菌体内贮藏物, 这可能是钩体在营养贫瘠环境中长时间存活的主要原因之一。本研究对钩体聚Beta羟基丁酸(PHB)贮藏物进行定性定量测定, 通过基因组分析补充定义PHB合成主要功能基因, 并采用分子生物学方法初步证明PHB合成途径的完整性, 为进一步研究PHB合成与钩体抗逆能力的关系奠定基础。【方法】采用脂类特异性尼罗红染色法和浓硫酸氧化-紫外分光光度计测定法, 对问号钩体和双曲钩体的PHB贮藏物进行定性定量测定; 采用生物信息学方法(BLAST和InterProscan/InterPro2Go), 通过同源性分析和功能结构域搜索寻找钩体基因组中的PHB合成相关基因; 最后采用克隆测序和定量RT-PCR技术检测相关基因表达情况, 初步验证生物信息学预测结果。【结果】尼罗红染色和氧化后比色定量实验证明钩体合成细菌常见贮藏物PHB, 问号钩体合成量为菌体干重的42%?45%, 双曲钩体合成量为64%?68%。尽管已公布的多个钩体基因组中均没有定义完整的PHB合成途径, 但本研究通过综合生物信息学分析, 在问号钩体和双曲钩体中鉴定了PHB合成途径的主要功能基因(phbC)。克隆测序和定量RT-PCR证实钩体转录表达大部分PHB合成相关基因(phbA/B/C), 说明钩体内该生物途径基本完整, 且部分高水平表达基因可能是钩体主要的PHB合成相关基因。【结论】问号钩体和双曲钩体均可合成PHB贮藏物, 且具有基本完整的PHB合成生物途径。

    Abstract:

    [Objective] Leptospira spp., including the pathogenic Leptospira interrogans and the saprophytic L. biflexa, can synthetise abundant bacterial storage in cell bodies, which may be one of the primary reasons why Leptospira spp. can survive in barren environments for a long time. In this study, the leptospiral polyhydroxybutyrate (PHB) storage was examined qualitatively and quantitatively. The major functional genes for PHB synthesis were complementarily annotated by the genome analysis. The integrity of the PHB synthesis pathway was verified by the molecular biology methods. This study laid foundation for further research on the relationship between the leptospiral storage synthesis and the stress tolerance. [Methods] The leptospiral PHB storage was determined qualitatively and quantitatively by the lipid-specific Nile Red dyeing and the concentrated sulfuric acid oxidation-ultraviolet spectrophotometer method. The storage synthesis genes were identified by the bioinformatics methods (BLAST and InterProscan/InterPro2Go) through the homolog analysis and the functional domain search. The expression of the predicted PHB synthesis genes were verified by gene cloning, sequencing and quantitative RT-PCR. [Results] By the Nile Red dyeing and the oxidation-colorimetric assay method, Leptospira spp. was proved to synthetise PHB, a common bacterial storage. The synthetic amounts of L. interrogans and L. biflexa were 42%?45% and 64%?68% of their dry weights, respectively. Though no integrated PHB synthesis pathways were defined in the leptospiral genomes, the homologs of the major functional genes for PHB synthesis (phbC) were identified in the genomes of L. interrogans and L. biflexa by the comprehensively bioinformatics analysis in this study. By cloning, sequencing and qRT-PCR, most of the PHB synthesis genes (phbA/B/C) were proved to be transcribed in Leptospira spp., which suggested that the leptospiral PHB synthesis pathways were primarily integrated, and some highly-expressed genes may be the main functional genes involved in the leptospiral PHB synthesis. [Conclusion] L. interrogans and L. biflexa all can synthetise PHB storage and have primarily integrated PHB synthesis pathways.

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薛峰,刘媛,洪彩玲,孙岚,辛德莉,温艳,黄敏君,谷俊朝. 钩端螺旋体聚Beta羟基丁酸(PHB)生物合成途径分析[J]. 微生物学通报, 2013, 40(11): 2047-2056

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  • 在线发布日期: 2013-11-04
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