根瘤菌Bd1的全基因组分析及TetR3对细胞生长和结瘤的负调控功能

doi:10.13560/j.cnki.biotech.bull.1985.2025-0215

摘要/Abstract

摘要：

目的从基因组水平了解大豆根瘤菌Bd1的遗传背景，探索促进Bd1生长和结瘤的有效途径。方法利用Illumina+PacBio三代测序平台对Bd1进行全基因组测序，采用生物信息学方法进行物种鉴定、功能基因注释、固氮相关基因和TetR家族转录因子分析，并利用同源重组双交换法构建TetR3基因敲除突变株，研究TetR3在Bd1生长和结瘤过程中的功能。结果 Bd1与Bradyrhizobiumdiazoefficiens USDA110的基因组平均核苷酸一致性（ANI）和数字DNA-DNA杂交值分别为99.98%和98.7%。Bd1基因组大小为9 009 469 bp，平均GC含量为64.1%，共8 403个编码基因，基因平均长度为927 bp，含有51个tRNA编码基因和3套核糖体RNA基因；含共生结瘤固氮相关nod、nif和fix基因数量分别为9、12和10个；含结构多样的TetR家族转录因子编码基因58个。TetR3基因失活突变株增强了Bd1的生长和结瘤能力，且谷胱甘肽S-转移酶活性提高了41.0%。结论 Bd1为B. diazoefficiens的1株新菌，具备与大豆共生结瘤固氮的能力，TetR3基因通过平衡细胞氧化还原能力负调控Bd1的生长和结瘤性能。

关键词: 慢生根瘤菌, 全基因组测序, TetR, 大豆, 谷胱甘肽S-转移酶

Abstract:

Objective This research is aimed to understand the genetic background of Bradyrhizobium sp. Bd1 at the genome level, and explore effective ways to promote the growth and nodules of Bd1. Method Illumina + PacBio third-generation sequencing platform was used to sequence the whole genome of Bd1. Bioinformatics method was employed to analyze the species identification, functional gene annotation, nitrogen fixation-related genes and TetR family transcription factors. TetR3 gene knockout mutant was constructed by homologous recombination double-exchange method to investigate the function of TetR3 in the growth and nodulation of Bd1. Result The genomic average nucleotide consistency (ANI) and digital DNA-DNA hybridization values between Bd1 and Bradyrhizobium diazoefficiens USDA110 were 99.98% and 98.7%, respectively. Bd1 genome size was 9 009 469 bp with 64.1% guanine-cytosine content, 8 403 coding sequences, within them 51 tRNA coding genes and 3 sets of ribosomal RNA genes. The number of nod, nif and fix genes were 9, 12 and 10, respectively. There were 58 genes encoding TetR family transcription factors with diverse structure. Compared with the wild Bd1, the growth and nodulation capacity were significantly enhanced, and glutathione S-transferase activity increased by 41.0% in TetR3-inactive mutant (Bd1ΔTetR3). Conclusion Bd1 is a novel strain belonging to B. diazoefficiens, which has the ability of nitrogen fixation in symbiotic nodulation with soybean. TetR3 negatively regulates the growth and nodulation in Bd1 by balancing cell REDOX capacity.

Key words: Bradyrhizobium, whole genome sequencing, TetR, soybeans, glutathione S-transferase

李亚涛, 张志鹏, 赵梦瑶, 吕镇, 甘恬, 魏浩, 吴书凤, 马玉超. 根瘤菌Bd1的全基因组分析及TetR3对细胞生长和结瘤的负调控功能[J]. 生物技术通报, 2025, 41(9): 289-301.

LI Ya-tao, ZHANG Zhi-peng, ZHAO Meng-yao, LYU Zhen, GAN Tian, WEI Hao, WU Shu-feng, MA Yu-chao. Whole Genome Analysis of Bradyrhizobium sp. Bd1 and the Negative Regulating Function of TetR3 during Cell Growth and Nodulation[J]. Biotechnology Bulletin, 2025, 41(9): 289-301.

图/表 8

参考文献 32

[1]	向星宇, 吴雪许, 许建双, 等. 中国大豆进口贸易现状及对策研究 [J]. 黑龙江粮食, 2024, (11): 26-28.
	Xiang XY, Wu XX, Xu JS, et al. China's soybean import trade: current status and policy recommendations [J]. Heilongjiang Grain, 2024, (11): 26-28.
[2]	王善明. 华癸中慢生根瘤菌7653R全基因组测序及比较基因组学研究 [D]. 武汉: 华中农业大学, 2014.
	Wang SM. Whole-genome sequencing and comparative genomic analysis of Mesorhizobium huakuii strain7653R [D]. Wuhan: Huazhong Agricultural University, 2014.
[3]	Margaret I, Becker A, Blom J, et al. Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean [J]. J Biotechnol, 2011, 155(1): 11-19.
[4]	Yang SH, Chen WH, Wang ET, et al. Rhizobial biogeography and inoculation application to soybean in four regions across China [J]. J Appl Microbiol, 2018, 125(3): 853-866.
[5]	Tian CF, Zhou YJ, Zhang YM, et al. Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations [J]. Proc Natl Acad Sci USA, 2012, 109(22): 8629-8634.
[6]	Balhana RJC, Singla A, Sikder MH, et al. Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions [J]. BMC Genomics, 2015, 16(1): 479.
[7]	Ramos JL, Martínez-Bueno M, Molina-Henares AJ, et al. The TetR family of transcriptional repressors [J]. Microbiol Mol Biol Rev, 2005, 69(2): 326-356.
[8]	吴攀攀, 李博文, 陈克涛, 等. TetR家族转录调控因子配体的研究进展 [J]. 生物工程学报, 2021, 37(7): 2379-2392.
	Wu PP, Li BW, Chen KT, et al. Ligands of TetR family transcriptional regulators: a review [J]. Chin J Biotechnol, 2021, 37(7): 2379-2392.
[9]	Patil RS, Sharma S, Bhaskarwar AV, et al. TetR and OmpR family regulators in natural product biosynthesis and resistance [J]. Proteins, 2025, 93(1): 38-71.
[10]	Ohkama-Ohtsu N, Honma H, Nakagome M, et al. Growth Rate of and Gene Expression in Bradyrhizobium diazoefficiens USDA110 due to a Mutation in blr7984, a TetR Family Transcriptional Regulator Gene [J]. Microbes Environ, 2016, 31(3): 249-259.
[11]	Han F, He XQ, Chen WW, et al. Involvement of a novel TetR-like regulator (BdtR) of Bradyrhizobium diazoefficiens in the efflux of isoflavonoid genistein [J]. Mol Plant Microbe Interact, 2020, 33(12): 1411-1423.
[12]	Luo RB, Liu BH, Xie YL, et al. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler [J]. Gigascience, 2012, 1(1): 18.
[13]	Krzywinski M, Schein J, Birol I, et al. Circos: an information aesthetic for comparative genomics [J]. Genome Res, 2009, 19(9): 1639-1645.
[14]	Lombard V, Golaconda Ramulu H, Drula E, et al. The carbohydrate-active enzymes database (CAZy) in 2013 [J]. Nucleic Acids Res, 2014, 42(Database issue): D490-D495.
[15]	Zhang XX, Guo HJ, Jiao J, et al. Pyrosequencing of rpoB uncovers a significant biogeographical pattern of rhizobial species in soybean rhizosphere [J]. J Biogeogr, 2017, 44(7): 1491-1499.
[16]	张星星. 大豆慢生根瘤菌分子进化学分析及土著大豆根瘤菌生物地理分布的高通量研究 [D]. 北京: 中国农业大学, 2014.
	Zhang XX. Molecular evolution of soybean nodulating Bradyrhizobium and biogeography of indigenous soybean rhizobia revealed by amplicon pyrosequencing [D]. Beijing: China Agricultural University, 2014.
[17]	Wang CH, Li M, Zhao Y, et al. SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean [J]. Proc Natl Acad Sci USA, 2022, 119(3): e2108641119.
[18]	Bender FR, Nagamatsu ST, Delamuta JRM, et al. Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation [J]. Microb Genom, 2022, 8(4): 000795.
[19]	Colclough AL, Scadden J, Blair JA. TetR-family transcription factors in Gram-negative bacteria: conservation, variation and implications for efflux-mediated antimicrobial resistance [J]. BMC Genomics, 2019, 20(1): 731.
[20]	Singh P, Jain A, Chhabra R, et al. TetR family transcriptional regulators: Lipid metabolism and drug resistance in mycobacteria [J]. Gene Rep, 2024, 36: 101938.
[21]	Henríquez T, Stein NV, Jung H. Resistance to bipyridyls mediated by the TtgABC efflux system in Pseudomonas putida KT2440 [J]. Front Microbiol, 2020, 11: 1974.
[22]	Watanabe S, Shimada N, Tajima K, et al. Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism. Novel evolutionary insight into sugar metabolism [J]. J Biol Chem, 2006, 281(44): 33521-33536.
[23]	Nguyen Le Minh P, de Cima S, Bervoets I, et al. Ligand binding specificity of RutR, a member of the TetR family of transcription regulators in Escherichia coli [J]. FEBS Open Bio, 2015, 5: 76-84.
[24]	Kendall SL, Burgess P, Balhana R, et al. Cholesterol utilization in mycobacteria is controlled by two TetR-type transcriptional regulators: kstR and kstR2 [J]. Microbiology, 2010, 156(Pt 5): 1362-1371.
[25]	Erni B, Siebold C, Christen S, et al. Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases [J]. Cell Mol Life Sci, 2006, 63(7/8): 890-900.
[26]	Li YJ, Wang CH, Zheng L, et al. Natural variation of GmRj2/Rfg1 determines symbiont differentiation in soybean [J]. Curr Biol, 2023, 33(12): 2478-2490.e5.
[27]	Wani SR, Dubey AA, Jain V. Ms6244 is a novel Mycobacterium smegmatis TetR family transcriptional repressor that regulates cell growth and morphophysiology [J]. FEBS Lett, 2023, 597(10): 1428-1440.
[28]	Lei YK, Asamizu S, Ishizuka T, et al. Regulation of multidrug efflux pumps by TetR family transcriptional repressor negatively affects secondary metabolism in Streptomyces coelicolor A3(2) [J]. Appl Environ Microbiol, 2023, 89(3): e0182222.
[29]	Schmacht M, Lorenz E, Senz M. Microbial production of glutathione [J]. World J Microbiol Biotechnol, 2017, 33(6): 106.
[30]	Luo S, Yin J, Peng Y, et al. Glutathione is involved in detoxification of peroxide and root nodule symbiosis of Mesorhizobium huakuii [J]. Curr Microbiol, 2020, 77(1): 1-10.
[31]	Van Laar TA, Esani S, Birges TJ, et al. Pseudomonas aeruginosa gshA mutant is defective in biofilm formation, swarming, and pyocyanin production [J]. mSphere, 2018, 3(2): e00155-18.
[32]	Ku JWK, Gan YH. New roles for glutathione: Modulators of bacterial virulence and pathogenesis [J]. Redox Biol, 2021, 44: 102012.

Family	Gene ID	Name	KEGG gene ID	KO description	Location
fix	gene5832	fixK	bja：bll3466	CRP/FNR family transcriptional regulator	6070103-6070786
	gene6542	fixK	bja：bll2757	CRP/FNR family transcriptional regulator	6868843-6869541
	gene6539	fixL	bja：bll2760	LuxR family， sensor kinase FixL	6866141-6867658
	gene6540	fixJ	bja：bll2759	LuxR family， response regulator FixJ	6867662-6868279
	gene7359	fixA	bja：blr2038	Flavoprotein beta subunit	7803669-7804535
	gene7895	fixA	bja：blr1377	Flavoprotein beta subunit	8425108-8425857^C
	gene7566	fixX	bja：bsr1775	Ferredoxin like protein	8067228-8067524^C
	gene7567	fixC	bja：blr1774	Flavoprotein-quinone oxidoreductase	8067563-8068870^C
	gene7568	fixB	bja：blr1773	Flavoprotein alpha subunit	8068882-8069991^C
	gene7894	fixB	bja：blr1378	Flavoprotein alpha subunit	8424164-8425108^C
nod	gene5597	nodT	bsym：CIT39_22430	Multidrug efflux system	5805752-5807308
	gene7363	nodU	bja：blr2034	Carbamoyltransferase	7809897-7811504^C
	gene7367	nodU	bjp：RN69_37980	Carbamoyltransferase	7813893-7815602^C
	gene7365	nodJ	bja：blr2031	Lipooligosaccharide transport system permease	7812179-7812967^C
	gene7366	nodI	bja：blr2030	Lipooligosaccharide transport system ATP-binding protein	7812971-7813891^C
	gene7368	nodC	bja：blr2027	N-acetylglucosaminyltransferase	7816172-7817629^C
	gene7369	nodB	bja：blr2026	Chitooligosaccharide deacetylase	7817644-7818303^C
	gene7370	nodA	bja：blr2025	Nodulation protein A	7818300-7818932^C
	gene7372	nodD	bja：bll2023	LysR family transcriptional regulator	7819726-7820670
nif	gene7360	nifA	bja：blr2037	Nif-specific regulatory protein	7805053-7806801^C
	gene7569	nifW	bja：blr1771	Nitrogenase-stabilizing/protective protein	8070942-8071283^C
	gene7571	nifQ	bju：BJ6T_80490	Nitrogen fixation protein NifQ	8071938-8072714^C
	gene7572	nifH	bja：blr1769	Nitrogenase iron protein NifH	8072861-8073745
	gene7580	nifZ	bjp：RN69_39140	Nitrogen fixation protein NifZ	8078154-8078468^C
	gene7582	nifB	bju：BJ6T_80610	Nitrogen fixation protein NifB	8078954-8080537^C
	gene7583	nifT	bja：bsr1757	Nitrogen fixation protein NifT	8081201-8081425^C
	gene7594	nifX	bja：blr1747	Nitrogen fixation protein NifX	8090471-8090866^C
	gene7595	nifN	bja：blr1746	Nitrogenase molybdenum-iron protein NifN	8090863-8092272^C
	gene7596	nifE	bja：blr1745	Nitrogenase molybdenum-cofactor synthesis protein NifE	8092282-8093925^C
	gene7597	nifK	bja：blr1744	Molybdenum-iron protein beta chain	8094018-8095574^C
	gene7598	nifD	bja：blr1743	Molybdenum-iron protein alpha chain	8095640-8097142^C

Family	Gene ID	Name	KEGG gene ID	KO description	Location
fix	gene5832	fixK	bja：bll3466	CRP/FNR family transcriptional regulator	6070103-6070786
	gene6542	fixK	bja：bll2757	CRP/FNR family transcriptional regulator	6868843-6869541
	gene6539	fixL	bja：bll2760	LuxR family， sensor kinase FixL	6866141-6867658
	gene6540	fixJ	bja：bll2759	LuxR family， response regulator FixJ	6867662-6868279
	gene7359	fixA	bja：blr2038	Flavoprotein beta subunit	7803669-7804535
	gene7895	fixA	bja：blr1377	Flavoprotein beta subunit	8425108-8425857^C
	gene7566	fixX	bja：bsr1775	Ferredoxin like protein	8067228-8067524^C
	gene7567	fixC	bja：blr1774	Flavoprotein-quinone oxidoreductase	8067563-8068870^C
	gene7568	fixB	bja：blr1773	Flavoprotein alpha subunit	8068882-8069991^C
	gene7894	fixB	bja：blr1378	Flavoprotein alpha subunit	8424164-8425108^C
nod	gene5597	nodT	bsym：CIT39_22430	Multidrug efflux system	5805752-5807308
	gene7363	nodU	bja：blr2034	Carbamoyltransferase	7809897-7811504^C
	gene7367	nodU	bjp：RN69_37980	Carbamoyltransferase	7813893-7815602^C
	gene7365	nodJ	bja：blr2031	Lipooligosaccharide transport system permease	7812179-7812967^C
	gene7366	nodI	bja：blr2030	Lipooligosaccharide transport system ATP-binding protein	7812971-7813891^C
	gene7368	nodC	bja：blr2027	N-acetylglucosaminyltransferase	7816172-7817629^C
	gene7369	nodB	bja：blr2026	Chitooligosaccharide deacetylase	7817644-7818303^C
	gene7370	nodA	bja：blr2025	Nodulation protein A	7818300-7818932^C
	gene7372	nodD	bja：bll2023	LysR family transcriptional regulator	7819726-7820670
nif	gene7360	nifA	bja：blr2037	Nif-specific regulatory protein	7805053-7806801^C
	gene7569	nifW	bja：blr1771	Nitrogenase-stabilizing/protective protein	8070942-8071283^C
	gene7571	nifQ	bju：BJ6T_80490	Nitrogen fixation protein NifQ	8071938-8072714^C
	gene7572	nifH	bja：blr1769	Nitrogenase iron protein NifH	8072861-8073745
	gene7580	nifZ	bjp：RN69_39140	Nitrogen fixation protein NifZ	8078154-8078468^C
	gene7582	nifB	bju：BJ6T_80610	Nitrogen fixation protein NifB	8078954-8080537^C
	gene7583	nifT	bja：bsr1757	Nitrogen fixation protein NifT	8081201-8081425^C
	gene7594	nifX	bja：blr1747	Nitrogen fixation protein NifX	8090471-8090866^C
	gene7595	nifN	bja：blr1746	Nitrogenase molybdenum-iron protein NifN	8090863-8092272^C
	gene7596	nifE	bja：blr1745	Nitrogenase molybdenum-cofactor synthesis protein NifE	8092282-8093925^C
	gene7597	nifK	bja：blr1744	Molybdenum-iron protein beta chain	8094018-8095574^C
	gene7598	nifD	bja：blr1743	Molybdenum-iron protein alpha chain	8095640-8097142^C