尿嘧啶从头合成酶ODCase在产酶溶杆菌中的独特功能

doi:10.1016/j.jia.2023.11.047

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (9): 3066-3077.DOI: 10.1016/j.jia.2023.11.047

尿嘧啶从头合成酶ODCase在产酶溶杆菌中的独特功能

收稿日期:2023-07-16 接受日期:2023-10-17 出版日期:2024-09-20 发布日期:2024-08-21

A unique role of the pyrimidine de novo synthesis enzyme ODCase in Lysobacter enzymogenes

Mingming Yang^{1, 2}, Yunxiao Tan², Jiabing Ma², Yingjia Zhao³, Xia Yan⁴, Nana Wang³, Pingping Wang⁵, Jiaqi Tan⁵, Suilong Ai⁵, Xiaofei Liang², Bangshuai Chang³, Obadah E. A. Yousif³, Chao Zhao², Bo Wang⁶, Guoliang Qian⁷, Lili Huang^{1, 2#}

¹ State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Northwest A&F University, Yangling 712100, China
² College of Plant Protection, Northwest A&F University, Yangling 712100, China
³College of Life Sciences, Northwest A&F University, Yangling 712100, China
⁴College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China
⁵ Shaanxi Provincial Tobacco Corporation of China National Tobacco Corporation, Xi’an 710061, China
⁶Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
⁷College of Plant Protection, Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing 210095, China

Received:2023-07-16 Accepted:2023-10-17 Online:2024-09-20 Published:2024-08-21
About author:Mingming Yang, E-mail: yangming862@126.com; #Correspondence Lili Huang, Tel: +86-29-87091312, E-mail: huanglili@nwsuaf.edu.cn
Supported by:
This study was supported by the National Natural Science Foundation of China (32102283 to Mingming Yang), the Science and Technology Major Project of China National Tobacco Corporation (110202101056(LS-16)), the Science and Technology Project of Shaanxi Branch of China National Tobacco Corporation (KJ-2021-02 and KJ-2022-04).

摘要/Abstract

摘要：

产酶溶杆菌（Lysobacter enzymogenes）OH11分泌的热稳定性抗真菌因子HSAF具有广谱高效的抑菌活性。目前无法利用化学合成和异源表达等方法生产HSAF。OH11菌株中HSAF原始产量低，是规模化生产的主要瓶颈。从分子层面阐明HSAF生物合成的调控网络可为提升活性代谢物产量，研发绿色高效的新型生防产品奠定理论基础。本研究发现一个编码乳清酸核苷-5'-单磷酸脱羧酶（ODCase）的基因Le0752，该基因在尿嘧啶从头合成途径中催化乳清酸核苷酸（OMP）生成尿嘧啶核苷酸（UMP）。Le0752缺失突变株的HSAF产量及其发酵粗提液对辣椒疫霉（Phytophthora capsici）的皿内抑菌活性均显著降低，而用Le0752缺失突变体发酵液处理过的辣椒疫霉对本氏烟（Nicotiana benthamiana）的毒力较野生型显著提高。Le0752突变体在1/10 TSA平板上生长减缓，但蹭行运动（twitching motility）能力不受影响。qRT-PCR结果表明，Le0752缺失突变后HSAF合成关键基因lafB及其关键调控因子clp的基因表达水平显著降低，遗传学试验证明Le0752通过转录因子Clp调控lafB的转录进而影响HSAF的生物合成。生物信息学分析显示，溶杆菌属中的ODCases归于Group III类群，而与溶杆菌属亲缘关系较近的黄单胞菌属和寡养单胞菌属的ODCases归属Group I类群。基因染色体同源替换实验中，稻黄单胞菌水稻致病变种（Xanthomonas oryzae pv. oryzae）中的ODCase（pyrF）能够完全恢复Le0752缺失突变表型。本研究阐明尿嘧啶从头合成酶ODCase对HSAF合成的关键调控作用，将为改造HSAF高产菌株提供理论支撑，并拓展对细菌ODCase功能与进化的认识。

关键词: ODCase , Pyrimidine de novo synthesis , Secondary metabolite HSAF , Lysobacter enzymogenes

Abstract:

Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential. Heat-stable antifungal factor (HSAF) secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient antifungal activity. Studying the biosynthetic regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production. In this work, we demonstrate that Le0752, an orotidine-5´-phosphate decarboxylase enzyme (ODCase) catalyzing a pivotal step of the UMP de novo biosynthesis pathway, is vital for HSAF-mediated antimicrobial activities and growth of L. enzymogenes OH11, but not for twitching motility. This gene regulates the production of HSAF by affecting the expression of lafB, a key gene in the HSAF biosynthesis operon, through the transcription factor Clp. Interestingly, bioinformatics analysis revealed that Le0752 belongs to the Group III ODCases, whereas its homologs in the closely related genera Xanthomonas and Stenotrophomonas belong to Group I, which contains most ODCases from Gram-positive bacteria, Gram-negative bacteria and cyanobacteria. Moreover, the Group I ODCase PXO_3614 from the Xanthomonas oryzae pv. oryzae PXO99^Astrain complemented the Le0752 mutant in regulating HSAF-mediated antagonistic activity. Together, these results highlight the important requirement of de novo pyrimidine biosynthetic enzymes for antibiotic HSAF production in L. enzymogenes, which lays an important foundation for improving HSAF production via metabolic flow design and for dissecting the regulatory functions of bacterial ODCases.

Key words: ODCase , Pyrimidine de novo synthesis , Secondary metabolite HSAF , Lysobacter enzymogenes

Mingming Yang, Yunxiao Tan, Jiabing Ma, Yingjia Zhao, Xia Yan, Nana Wang, Pingping Wang, Jiaqi Tan, Suilong Ai, Xiaofei Liang, Bangshuai Chang, Obadah E. A. Yousif, Chao Zhao, Bo Wang, Guoliang Qian, Lili Huang. 尿嘧啶从头合成酶ODCase在产酶溶杆菌中的独特功能[J]. Journal of Integrative Agriculture, 2024, 23(9): 3066-3077.

Mingming Yang, Yunxiao Tan, Jiabing Ma, Yingjia Zhao, Xia Yan, Nana Wang, Pingping Wang, Jiaqi Tan, Suilong Ai, Xiaofei Liang, Bangshuai Chang, Obadah E. A. Yousif, Chao Zhao, Bo Wang, Guoliang Qian, Lili Huang. A unique role of the pyrimidine de novo synthesis enzyme ODCase in Lysobacter enzymogenes[J]. Journal of Integrative Agriculture, 2024, 23(9): 3066-3077.

参考文献

Al Ahmar R, Kirby D B, Yu D H. 2019. Pyrimidine biosynthesis regulates the small-colony variant and mucoidy in Pseudomonas aeruginosa through sigma factor competition. Journal of Bacteriology, 201, e00575-18.

Banerjee S, van der Heijden M G A. 2023. Soil microbiomes and one health. Nature Reviews Microbiology, 21, 6–20.

Becker P, Dohmann A, Wohlbrand L, Thies D, Hinrichs C, Buschen R, Wunsch D, Neumann-Schaal M, Schomburg D, Winklhofer M, Reinhardt R, Rabus R. 2022. Complex and flexible catabolism in Aromatoleum aromaticum pCyN1. Environmental Microbiology, 24, 3195–3211.

Boeke J D, LaCroute F, Fink G R. 1984. A positive selection for mutants lacking orotidine-5´-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Molecular Genetics and Genomics, 197, 345–346.

de Bruijn I, Cheng X, de Jager V, Exposito R G, Watrous J, Patel N, Postma J, Dorrestein P C, Kobayashi D, Raaijmakers J M. 2015. Comparative genomics and metabolic profiling of the genus Lysobacter. BMC Genomics, 16, 991.

Capone R F, Ning Y, Pakulis N, Alhazzazi T, Fenno J C. 2007. Characterization of Treponema denticola pyrF encoding orotidine-5´-monophosphate decarboxylase. FEMS Microbiology Letters, 268, 261–267.

Chen J, Shen D, Odhiambo B O, Xu D, Han S, Chou S H, Qian G. 2018. Two direct gene targets contribute to Clp-dependent regulation of type IV pilus-mediated twitching motility in Lysobacter enzymogenes OH11. Applied Microbiology Biotechnology, 102, 7509–7519.

Dunne C, Moënne-Loccoz Y, de Bruijn J, O’Gara F. 2000. Overproduction of an inducible extracellular serine protease improves biological control of Pythium ultimum by Stenotrophomonas maltophilia strain W81. Microbiology, 146, 2069–2078.

Edgar R C. 2004. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797.

Fujihashi M, Mnpotra J S, Mishra R K, Pai E F, Kotra L P. 2015. Orotidine monophosphate decarboxylase - A fascinating workhorse enzyme with therapeutic potential. Journal of Genetics and Genomics, 42, 221–234.

Galvao T C, de Lorenzo V. 2005. Adaptation of the yeast URA3 selection system to gram-negative bacteria and generation of a {delta}betCDE Pseudomonas putida strain. Applied and Environmental Microbiology, 71, 883–892.

Garavaglia M, Rossi E, Landini P. 2012. The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli. PLoS ONE, 7, e31252.

Han S, Shen D, Wang Y C, Chou S H, Gomelsky M, Gao Y G, Qian G. 2020. A YajQ-LysR-like, cyclic di-GMP-dependent system regulating biosynthesis of an antifungal antibiotic in a crop-protecting bacterium, Lysobacter enzymogenes. Molecular Plant Pathology, 21, 218–229.

Horzempa J, O’Dee D M, Shanks R M, Nau G J. 2010. Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo. Infection Immunity, 78, 2607–2619.

Hurek T, Reinhold-Hurek B. 2003. Azoarcus sp. strain BH72 as a model for nitrogen-fixing grass endophytes. Journal of Biotechnology, 106, 169–178.

Kilstrup M, Hammer K, Ruhdal Jensen P, Martinussen J. 2005. Nucleotide metabolism and its control in lactic acid bacteria. FEMS Microbiology Reviews, 29, 555–590.

Kobayashi D Y, Reedy R M, Bick J, Oudemans P V. 2002. Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control. Applied and Environmental Microbiology, 68, 1047–1054.

Liautard J P, Jubier-Maurin V, Boigegrain R A, Kohler S. 2006. Antimicrobials: Targeting virulence genes necessary for intracellular multiplication. Trends in Microbiology, 14, 109–113.

Lin L, Yang Z, Tao M, Shen D, Cui C, Wang P, Wang L, Jing M, Qian G, Shao X. 2023. Lysobacter enzymogenes prevents Phytophthora infection by inhibiting pathogen growth and eliciting plant immune responses. Frontiers in Plant Science, 14, 1116147.

Lou L, Qian G, Xie Y, Hang J, Chen H, Zaleta-Rivera K, Li Y, Shen Y, Dussault P H, Liu F, Du L. 2011. Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. Journal of the American Chemical Society, 133, 643–645.

Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer S V, Machado M A, Toth I, Salmond G, Foster G D. 2012. Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology, 13, 614–629.

Mattick J S. 2002. Type IV pili and twitching motility. Annual Review Microbiology, 56, 289–314.

Meza-Avina M E, Wei L, Buhendwa M G, Poduch E, Bello A M, Pai E F, Kotra L P. 2008. Inhibition of orotidine 5´-monophosphate decarboxylase and its therapeutic potential. Mini-Reviews in Medicinal Chemistry, 8, 239–247.

Naushad S, Adeolu M, Wong S, Sohail M, Schellhorn H E, Gupta R S. 2015. A phylogenomic and molecular marker based taxonomic framework for the order Xanthomonadales: Proposal to transfer the families Algiphilaceae and Solimonadaceae to the order Nevskiales ord. nov. and to create a new family within the order Xanthomonadales, the family Rhodanobacteraceae fam. nov., containing the genus Rhodanobacter and its closest relatives. Antonie van Leeuwenhoek, 107, 467–485.

Qian G, Wang Y, Liu Y, Xu F, He Y W, Du L, Venturi V, Fan J, Hu B, Liu F. 2013. Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology. Applied and Environmental Microbiology, 79, 6604–6616.

Qian G, Wang Y, Qian D, Fan J, Hu B, Liu F. 2012. Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11. World Journal of Microbiology and Biotechnology, 28, 549–557.

Qian G, Xu F, Venturi V, Du L, Liu F. 2014. Roles of a solo LuxR in the biological control agent Lysobacter enzymogenes strain OH11. Phytopathology, 104, 224–231.

Ralli P, Srivastava A C, O’Donovan G. 2007. Regulation of the pyrimidine biosynthetic pathway in a pyrD knockout mutant of Pseudomonas aeruginosa. Journal of Basic Microbiology, 47, 165–173.

Rossi E, Motta S, Aliverti A, Cossu F, Gourlay L, Mauri P, Landini P. 2017. Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli. Environmental Microbiology, 19, 4551–4563.

von Rozycki T, Nies D H. 2009. Cupriavidus metallidurans: Evolution of a metal-resistant bacterium. Antonie van Leeuwenhoek, 96, 115–139.

Shen F, Yin W, Song S, Zhang Z, Ye P, Zhang Y, Zhou J, He F, Li P, Deng Y. 2020. Ralstonia solanacearum promotes pathogenicity by utilizing L-glutamic acid from host plants. Molecular Plant Pathology, 21, 1099–1110.

Su Z, Chen H, Wang P, Tombosa S, Du L, Han Y, Shen Y, Qian G, Liu F. 2017a. 4-Hydroxybenzoic acid is a diffusible factor that connects metabolic shikimate pathway to the biosynthesis of a unique antifungal metabolite in Lysobacter enzymogenes. Microbial Biotechnology, 104, 163–178.

Su Z, Han S, Fu Z Q, Qian G, Liu F. 2017b. Heat-Stable Antifungal Factor (HSAF) biosynthesis in Lysobacter enzymogenes is controlled by the interplay of two transcription factors and a diffusible molecule. Applied and Environmental Microbiology, 84, e01754-17.

Ueda A, Attila C, Whiteley M, Wood T K. 2009. Uracil influences quorum sensing and biofilm formation in Pseudomonas aeruginosa and fluorouracil is an antagonist. Microbial Biotechnology, 2, 62–74.

Wang P, Chen H, Qian G, Liu F. 2017. LetR is a TetR family transcription factor from Lysobacter controlling antifungal antibiotic biosynthesis. Applied Microbiology and Biotechnology, 101, 3273–3282.

Wang Y, Zhao Y, Zhang J, Zhao Y, Shen Y, Su Z, Xu G, Du L, Huffman J M, Venturi V, Qian G, Liu F. 2014. Transcriptomic analysis reveals new regulatory roles of Clp signaling in secondary metabolite biosynthesis and surface motility in Lysobacter enzymogenes OH11. Applied Microbiology and Biotechnology, 98, 9009–9020.

Xu G, Han S, Huo C, Chin K H, Chou S H, Gomelsky M, Qian G, Liu F. 2018. Signaling specificity in the c-di-GMP-dependent network regulating antibiotic synthesis in Lysobacter. Nucleic Acids Research, 46, 9276–9288.

Xu K, Shen D, Yang N, Chou S H, Gomelsky M, Qian G. 2021. Coordinated control of the type IV pili and c-di-GMP-dependent antifungal antibiotic production in Lysobacter by the response regulator PilR. Molecular Plant Pathology, 22, 602–617.

Yang M, Ren S, Shen D, Chou S H, Qian G. 2020a. ClpP mediates antagonistic interaction of Lysobacter enzymogenes with a crop fungal pathogen. Biological Control, 140, 104125.

Yang M, Ren S, Shen D, Yang N, Wang B, Han S, Shen X, Chou S H, Qian G. 2020b. An intrinsic mechanism for coordinated production of the contact-dependent and contact-independent weapon systems in a soil bacterium. PLoS Pathogens, 16, e1008967.

Yu F, Zaleta-Rivera K, Zhu X, Huffman J, Millet J C, Harris S D, Yuen G, Li X C, Du L. 2007. Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action. Antimicrobial Agents and Chemotherapy, 51, 64–72.

Zhang X, de Maat V, Guzman Prieto A M, Prajsnar T K, Bayjanov J R, de Been M, Rogers M R C, Bonten M J M, Mesnage S, Willems R J L, van Schaik W. 2017. RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum. BMC Genomics, 18, 893.

Zhao Y, Cheng C, Jiang T, Xu H, Chen Y, Ma Z, Qian G, Liu F. 2019. Control of wheat fusarium head blight by heat-stable antifungal factor (HSAF) from Lysobacter enzymogenes. Plant Disease, 103, 1286–1292.

尿嘧啶从头合成酶ODCase在产酶溶杆菌中的独特功能

A unique role of the pyrimidine de novo synthesis enzyme ODCase in Lysobacter enzymogenes

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics