Sequencing of whole genome of Bacillus velezensis LG37 and screening of inorganic nitrogen metabolism candidate genes
-
摘要: 前期研究发现贝莱斯芽孢杆菌 (Bacillus velezensis) LG37可高效同化无机氮，但其机理尚不清楚。为解读其高效同化无机氮的机理，结合三代PacBio RS II 和二代Illumina HiSeq 2000 测序技术对贝莱斯芽孢杆菌LG37进行全基因组测序，在此基础上利用NR、KEGG、eggNOG、GO和CARD数据库进行序列注释、分析，并通过本地Blast+对无机氮代谢相关基因进行挖掘。测序结果表明：1) 贝莱斯芽孢杆菌LG37的基因组为3 929 697 bp的环状染色体，GC含量为46.5%，包含3 854个蛋白质编码基因、86个tRNA 基因和27个rRNA基因。2) 共筛选出无机氮代谢相关候选基因94个，主要涉及编码感应蛋白、转录调控因子、转运蛋白、氧化还原酶和同化酶等，并对这些基因的GO功能进行了注释分析。综上，LG37 全基因组测序及无机氮代谢相关基因的分析为芽孢杆菌降低养殖水体中无机氮的研究提供了基因水平数据，为芽孢杆菌微生态制剂降低水体中无机氮的应用研究提供了理论依据。Abstract: It has been found that Bacillus velezensis can assimilate inorganic nitrogen efficiently. However, the underlying mechanism of inorganic nitrogen assimilation remains enigmatic. In order to elucidate the mechanism, we sequenced the complete genome of LG37 by PacBio RS II and Illumina HiSeq 2000, and then annotated and analyzed the sequence by the database of NR, KEGG, eggNOG, GO and CARD. Finally, we screened the genes related to inorganic nitrogen metabolism by local Blast+. The results show that: 1) The genome contained one circular chromosomal with a size of 3 929 697 bp and a GC-content of 46.5%. Gene prediction and annotation was performed to acquire a total of 3 854 protein-coding genes, 86 tRNA genes and 27 rRNA genes. 2) A total of 94 inorganic nitrogen metabolism candidate genes were screened by local Blast+. These genes were involved into coding sensing protein, transcriptional regulator, transporter, oxidoreductase and assimilator, etc.. In conclusion, the whole genome sequencing and data analysis of LG37 provide data at gene level and theoretical basis for functional study and application of Bacillus in reducing inorganic nitrogen in aquaculture water.
-
Key words:
- Bacillus velezensis /
- Whole genome sequence /
- Inorganic nitrogen /
- Metabolic pathway
-
图 2 贝莱斯芽孢杆菌基于16S rDNA 序列构建的系统进化树
Figure 2. Neighbor-joining tree of B. velezensis LG37 based on 16S rDNA sequences
表 1 贝莱斯芽孢杆菌LG37 基因组特性
Table 1. Genome features of B. velezensis LG37 strain
特性
Feature数值
Value基因组大小 Genome size/bp 3 929 697 GC-含量 GC-content 46.5% 质粒数量 Plasmid number 0 总基因 Total genes 3 967 蛋白编码基因 Protein-coding genes 3 854 转运 RNA tRNA 86 核糖体 RNA rRNA 27 编码区域大小 Coding region size/bp 3 495 864 编码区域GC-含量 GC-content of coding region 47.3% 编码区域/全基因组 Coding region/Genome length 89.0% 间隔区域大小 Intergenic region size/bp 433 833 间隔区域占比 Ratio of intergenic region 11.0% 
下载: 导出CSV
表 2 LG37 基因组氮代谢通路及其相关基因
Table 2. Related genes of nitrogen metabolism pathways of LG37
通路
Pathway IDKEGG 描述
KEGG description基因
Geneko00910 氮代谢 Nitrogen metabolism orf00490, orf00539, orf00544, orf00545, orf00546, orf00547, orf00817, orf01213, orf01254, orf01968, orf02226, orf02227, orf02368, orf03807, orf03808, orf03809, orf03810, orf03811 M00531 同化硝酸盐还原 Assimilatory nitrate reduction orf00539, orf03807, orf03809 M00530 异化硝酸盐还原 Dissimilatory nitrate reduction orf00544, orf00545, orf00547, orf03808, orf03809, orf03810, orf03811 M00529 反硝化 Denitrification orf00544, orf00545, orf00547, orf03809 M00804 完全硝化 Complete nitrification orf03809
下载: 导出CSV
表 3 无机氮代谢候选基因
Table 3. Candidate genes of inorganic nitrogen metabolism
基因
Gene大小
Size/bp蛋白
ProteinGO-分子功能
GO-Molecular functionorf00084 741 Type III pantothenate kinase YacB 泛酸激酶活性 orf00106 246 Putative septation protein SpoVG 分子功能的负调控 orf00148 885 Pyridoxal 5'-phosphate synthase subunit PdxS 谷氨酰胺水解活性 orf00173 2 004 Nitrate reductase YyaE 硝酸还原酶活性 orf00246 1 416 Arginine utilization regulatory protein RocR 转录因子结合 orf00250 1 206 Ornithine aminotransferase RocD 鸟氨酸氧酸转氨酶活性 orf00316 963 Iron(3+)-hydroxamate-binding protein YxeB 无机离子转运与代谢 orf00379 1 017 Respiratory nitrate reductase NarI 硝酸还原酶活性 orf00490 1 287 Glutamate dehydrogenase RocG 谷氨酸脱氢酶 (NAD+) 活性 orf00535 1 377 Cytochrome cd1-nitrite reductase-like YwhL 亚硝酸盐还原酶活性 orf00539 1 131 Nitrate transporter NarT 跨膜转运 orf00544 3 687 Nitrate reductase alpha chain NarG 硝酸还原酶活性 orf00545 1 464 Nitrate reductase beta chain NarH 硝酸还原酶活性 orf00546 558 Nitrate reductase NarJ 未折叠蛋白结合 orf00547 672 Nitrate reductase gamma chain NarI 硝酸还原酶活性 orf00608 318 Urease subunit gamma 氨基酸转运与代谢 orf00609 375 Urease subunit beta 氨基酸转运与代谢 orf00610 1 710 Urease subunit alpha 氨基酸转运与代谢 orf00623 351 Nitrogen regulatory protein P-II GlnB 酶调节活性 orf00624 1 212 Ammonium transporter NrgA 铵跨膜转运蛋白活性 orf00630 282 Stage III sporulation protein D SpoIIID DNA结合转录因子活性 orf00783 726 Glucosamine-6-phosphate deaminase NagB 葡萄糖胺-6-磷酸脱氨酶活性 orf00817 582 YvdA 无机离子转运与代谢 orf00959 2 112 YvgW 阳离子转运ATP酶活性 orf00966 1 716 Sulfite reductase [NADPH] CysI 亚硫酸盐还原酶 (NADPH) 活性 orf00998 1 047 ABC transporter permease protein YvrB 转运体活性 orf01042 357 Uncharacterized protein YusI 氧化还原酶活性 orf01058 1 398 ABC transporter ATP-binding protein ATP结合 orf01105 237 Nitrogen-fixing NifU domain-containing protein 铁硫簇结 orf01175 2 403 Cation:proton antiporter 单价无机阳离子跨膜转运蛋白活性 orf01213 1 032 Nitronate monooxygenase Ncd2 硝酸单加氧酶活性 orf01254 549 YtiB 无机离子转运与代谢 orf01262 813 Nitrate transport system permease protein YtlD 跨膜转运蛋白活性 orf01263 783 Nitrate ABC transporter permease YtlC 跨膜转运 orf01264 1 005 Nitrate ABC transporter periplasmic protein YtlA ATP酶活性 orf01290 753 Quaternary-amine-transporting ATPase 传输ATP酶活性的季铵盐化合物 orf01357 1 191 Nitric oxide dioxygenase 一氧化氮双加氧酶活性 orf01435 1 962 Threonine--tRNA ligase 1 ThrS ATP结合 orf01491 225 Spore germination protein GerE DNA结合 orf01591 657 GlnP 氨基酸转运与代谢 orf01592 651 GlnM 氨基酸转运与代谢 orf01593 828 GlnH 氨基酸转运与代谢 orf01617 801 Formate/nitrite transporter 跨膜转运蛋白活性 orf01659 726 RNA polymerase sigma factor DNA结合转录因子活性 orf01864 855 Nitrogen assimilation regulatory protein nac DNA结合转录因子活性 orf01911 450 Ferric uptake regulation protein DNA结合转录因子活性 orf01968 1 275 Glutamate dehydrogenase RocG 谷氨酸脱氢酶硝酸还原酶活性 orf02215 1 983 Nitrate reductase 硝酸还原酶活性 orf02225 903 HTH-type transcriptional regulator GltC DNA结合转录因子活性 orf02226 903 Glutamate synthase (NADPH/NADH) GltB 谷氨酸合酶 (NADPH) 活性 orf02227 4 560 Glutamate synthase (NADPH/ NADH) GltD 谷氨酸合酶 (NADPH) 活性 orf02368 1 335 Glutamine synthetase GlnA 谷氨酸氨连接酶活性 orf02369 405 HTH-type transcriptional regulator GlnR DNA结合 orf02384 315 Ammonium compound efflux SMR transporter 膜的组成部分 orf02385 354 Ammonium compound efflux SMR transporter 膜的组成部分 orf02417 261 Stage V sporulation protein S 核酸结合 orf02418 795 2',3'-cyclic-nucleotide 2'-phosphodiesterase YmdB 2',3'-环核苷酸2'-磷酸二酯酶活性 orf02465 723 Uridylate kinase PyrH ATP结合 orf02483 363 Chemotaxis protein CheY 磷脂酶信号转导系统 orf02588 783 RNA polymerase sigma factor DNA结合转录因子活性 orf02638 930 Glutaminase 氨基酸转运与代谢 orf02680 666 Potassium uptake protein KtrA 阳离子跨膜转运蛋白活性 orf02747 1 914 YkvW 阳离子转运ATP酶活性 orf02756 342 Putative transcriptional regulator 转录调控，DNA模板 orf02787 1 353 YkrM 阳离子跨膜转运蛋白活性 orf02794 783 Uncharacterized membrane protein YkoY 膜的组成部分 orf02804 742 HTH-type transcriptional regulator TnrA 核心启动子结合 orf02828 315 Ammonium compound efflux SMR transporter 膜的组成部分 orf02830 339 Ammonium compound efflux SMR transporter 膜的组成部分 orf02854 999 Anion permease 无机磷酸盐跨膜转运蛋白活性 orf03008 396 ArsC family transcriptional regulator SpxA 电子转移活性 orf03161 1 215 Cation/H (+) antiporter YhaU 溶质：质子逆向转运活性 orf03186 396 Putative fluoride ion transporter CrcB 无机阴离子跨膜转运蛋白活性 orf03187 360 Putative fluoride ion transporter CrcB 无机阴离子跨膜转运蛋白活性 orf03209 435 HTH-type transcriptional regulator NsrR DNA结合 orf03262 834 ABC-type nitrate transport system 离子跨膜转运 orf03263 990 Putative binding protein SsuA ATP酶活性 orf03264 768 Aliphatic sulfonates import protein SsuB 阴离子跨膜转运蛋白活性 orf03448 720 Probable transcriptional regulatory protein 转录调控，DNA模板 orf03546 330 Ammonium compound efflux SMR transporter 膜的组成部分 orf03547 315 Ammonium compound efflux SMR transporter 膜的组成部分 orf03597 963 Arsenic resistance protein 无机阴离子跨膜转运蛋白活性 orf03677 822 Probable manganese catalase YdbD 无机离子转运与代谢 orf03682 366 Ammonium compound efflux SMR transporter 膜的组成部分 orf03807 1 206 Assimilatory nitrate reductase NasA 硝酸铁氧还蛋白还原酶活性 orf03808 2 328 Nitrite reductase large subunit NasB 亚硝酸盐还原酶 [NAD(P)H] 活性 orf03809 2 133 Assimilatory nitrate reductase NasC 硝酸还原酶活性 orf03810 2 418 Nitrite reductase [NAD(P)H] NasD 亚硝酸盐还原酶 [NAD(P)H] 活性 orf03811 321 Assimilatory nitrite reductase [NAD(P)H] NasE 亚硝酸盐还原酶 [NAD(P)H] 活性 orf03812 1 440 NasF 辅酶代谢 orf03840 1 257 Transport system atp-binding protein opuaa 传输ATP酶活性的季铵盐化合物 orf03889 984 Glutaminase 1 GlsA1 谷氨酰胺酶活性 orf03890 1 437 GlnT 假定的钠/谷氨酰胺转运体 orf03963 1 434 Sodium-independent anion transporter 次级活性硫酸盐跨膜转运蛋白活性
下载: 导出CSV
-
[1] 胡晓娟, 文国樑, 田雅洁, 等. 不同培养条件下菌株NB5 对氨氮的去除效果研究[J]. 南方水产科学, 2020, 16(6): 89-96. [2] 方金龙, 王元, 房文红, 等. 氨氮胁迫下白斑综合征病毒对凡纳滨对虾的致病性[J]. 南方水产科学, 2017, 13(4): 52-58. doi: 10.3969/j.issn.2095-0780.2017.04.007 [3] 肖炜, 李大宇, 徐杨, 等. 慢性氨氮胁迫对吉富罗非鱼幼鱼生长、免疫及代谢的影响[J]. 南方水产科学, 2015, 11(4): 81-87. doi: 10.3969/j.issn.2095-0780.2015.04.012 [4] 郭国强, 孙红飞, 张永耀. 分子氨对草鱼鱼种红细胞渗透脆性的影响[J]. 水产科学, 2010, 29(8): 489-491. doi: 10.3969/j.issn.1003-1111.2010.08.011 [5] 孙侦龙, 朱永祥, 刘大勇, 等. 非离子氨氮和亚硝酸盐氮对暗纹东方鲀稚鱼的急性毒性试验[J]. 水产科学, 2015, 34(3): 135-139. [6] KUEBUTORNYE F K A, ABARIKE E D, LU Y. A review on the application of Bacillus as probiotics in aquaculture[J]. Fish Shellfish Immunol, 2019, 87: 820-828. doi: 10.1016/j.fsi.2019.02.010 [7] LIU G X, VIJAYARAMAN S B, DONG Y J, et al. Bacillus velezensis LG37: transcriptome profiling and functional verification of GlnK and MnrA in ammonia assimilation[J]. BMC Genom, 2020, 21(1): 215. doi: 10.1186/s12864-020-6621-1 [8] GOODWIN S, MCPHERSON J D, MCCOMBIE W R. Coming of age: ten years of next-generation sequencing technologies[J]. Nat Rev Genet, 2016, 17(6): 333-51. [9] GALPERIN M Y, KRISTENSEN D M, MAKAROVA K S, et al. Microbial genome analysis: the COG approach[J]. Brief Bioinform, 2019, 20(4): 1063-1070. doi: 10.1093/bib/bbx117 [10] CASTAÑEDA C D, GAMBLE J N, WAMSLEY K G S, et al. In ovo administration of Bacillus subtilis serotypes effect hatchability, 21-day performance, and intestinal microflora[J]. Poult Sci, 2021, 100(6): 101125. doi: 10.1016/j.psj.2021.101125 [11] TAKAMI H, TOYODA A, UCHIYAMA I, et al. Complete genome sequence and expression profile of the commercial lytic enzyme producer Lysobacter enzymogenes M497-1[J]. DNA Res, 2017, 24(2): 169-177. [12] MOLTON J S, LEE I R, BERTRAND D, et al. Stool metagenome analysis of patients with Klebsiella pneumoniae liver abscess and their domestic partners[J]. Int J Infect Dis, 2021, 107: 1-4. doi: 10.1016/j.ijid.2021.04.012 [13] 朱玉玲, 彭晶, 唐诗哲, 等. 宏基因组技术在极端环境酯酶挖掘中的应用[J]. 生命科学研究, 2021, 25(2): 169-175. [14] 魏大鹏, 单洪伟, 马甡, 等. 混料设计优化复合菌剂比例的研究[J]. 南方水产科学, 2014, 10(1): 86-91. doi: 10.3969/j.issn.2095-0780.2014.01.013 [15] TAMURA K, STECHER G, PETERSON D, et al. MEGA6: molecular evolutionary genetics analysis version 6.0[J]. Mol Biol Evol, 2013, 30(12): 2725-2729. doi: 10.1093/molbev/mst197 [16] NAKANO K, SHIROMA A, SHIMOJI M, et al. Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area[J]. Hum Cell, 2017, 30(3): 149-161. doi: 10.1007/s13577-017-0168-8 [17] GAN Y Q, ZHANG T, GAN Y Q, et al. Complete genome sequences of two Enterococcus faecium strains and comparative genomic analysis[J]. Exp Ther Med, 2020, 19(3): 2019-2028. [18] BENNETT J P Jr, KEENEY P M, BROHAWN D G. RNA sequencing reveals small and variable contributions of infectious agents to transcriptomes of postmortem nervous tissues from amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease subjects, and increased expression of genes from disease-activated microglia[J]. Front Neurosci, 2019, 13: 235. doi: 10.3389/fnins.2019.00235 [19] EMSLEY C, KING S, NYULASI I, et al. A GLIMmer of insight into lung transplant nutrition: enhanced detection of malnutrition in lung transplant patients using the GLIM criteria[J]. Clin Nutr, 2021, 40(5): 2521-2526. doi: 10.1016/j.clnu.2021.02.047 [20] CHAN P P, LOWE T M. tRNAscan-SE: searching for tRNA genes in genomic sequences[J]. Methods Mol Biol, 2019, 1962: 1-14. [21] LAGESEN K, HALLIN P, RØDLAND E A, et al. RNAmmer: consistent and rapid annotation of ribosomal RNA genes[J]. Nucleic Acids Res, 2007, 35(9): 3100-8. doi: 10.1093/nar/gkm160 [22] CAMACHO C, COULOURIS G, AVAGYAN V, et al. BLAST+: architecture and applications[J]. BMC Bioinformatics, 2009, 10: 421. doi: 10.1186/1471-2105-10-421 [23] NAYAK V S. Nonelectrolytic production of caustic soda and hydrochloric acid from sodium chloride[J]. Ind Eng Chem Res, 1996, 35(10): 3808-3811. doi: 10.1021/ie960043h [24] 张静, 高婷婷, 李勇, 等. 蛋白营养对工业养殖大菱鲆 (Scophthatmus maximus L.)幼鱼生长、氨氮排泄及肌肉氨基酸的效应[J]. 渔业科学进展, 2016, 37(6): 34-41. doi: 10.11758/yykxjz.20151124001 [25] EBELING J M, TIMMONS M B, BISOGNI J J. Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia-nitrogen in aquaculture systems[J]. Aquaculture, 2006, 257(1/2/3/4): 346-358. [26] 雷阳, 张倩, 陈钰, 等. 对虾养殖高效降解氨氮微生态菌的筛选与鉴定[J]. 福建农业科技, 2019(10): 16-20. [27] ZHU X H, ZHANG S M, ZHOU L Y, et al. Probiotic potential of Bacillus velezensis: antimicrobial activity against non-O1 Vibrio cholerae and immune enhancement effects on Macrobrachium nipponense[J]. Aquaculture, 2021, 541: 736817. doi: 10.1016/j.aquaculture.2021.736817 [28] 张德锋, 高艳侠, 可小丽, 等. 贝莱斯芽孢杆菌LF01基因组序列分析及其代谢产物的生防作用[J]. 水产学报, 2022, 46(2): 196-206. [29] FAN B, BLOM J, KLENK H P, et al. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an "operational group B. amyloliquefaciens" within the B. subtilis species complex[J]. Front Microbiol, 2017, 8: 22. [30] MURUKE M S H, CAMP H J O D, SEMESI A K, et al. The level of enzymes involved in the allantoin metabolism of Bacillus fastidiosus grown under different conditions[J]. Curr Microbiol, 1995, 30(1): 45-7. doi: 10.1007/BF00294523 [31] NIU T, LV X, LIU Z, et al. Synergetic engineering of central carbon and nitrogen metabolism for the production of N-acetylglucosamine in Bacillus subtilis[J]. Biotechnol Appl Biochem, 2020, 67(1): 123-132. doi: 10.1002/bab.1845 [32] WANG B, ZHANG D, CHU S, et al. Genomic analysis of Bacillus megaterium NCT-2 reveals its genetic basis for the bioremediation of secondary salinization soil[J]. Int J Genomics, 2020, 2020: 4109186. [33] MASSAD R S, LOUBET B, TUZET A, et al. Relationship between ammonia stomatal compensation point and nitrogen metabolism in arable crops: current status of knowledge and potential modelling approaches[J]. Environ Pollut, 2008, 154(3): 390-403. [34] TANG Y, YE Z, WEI Y, et al. Vertebrate paralogous CRMPs in nervous system: evolutionary, structural, and functional interplay[J]. J Mol Neurosci, 2015, 55(2): 324-34. doi: 10.1007/s12031-014-0327-2 [35] 顾志良, 耿拓宇. 鸡重要性状主效基因和QTL 的研究进展[J]. 中国家禽, 2003(S1): 130-134. [36] AWADA H, THAPA B, VISCONTE V. The genomics of myelodysplastic syndromes: origins of disease evolution, biological pathways, and prognostic implications[J]. Cells, 2020, 9(11): 2512. doi: 10.3390/cells9112512 [37] 丁忠涛, 张锐, 郭三堆. 棉花抗逆相关基因GhDr1的克隆及生物信息学分析[J]. 生物技术通报, 2011(1): 99-106. -
点击查看大图
图(5) / 表(3)
计量
- 文章访问数: 566
- HTML全文浏览量: 135
- PDF下载量: 35
- 被引次数: 0
粤公网安备 44010502001741号
