一株抗革兰阳性菌的戈登氏菌WA4-43全基因组测序与分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0612

摘要/Abstract

摘要：

蜚蠊肠道来源WA4-43为首次发现具有抗革兰阳性菌的戈登氏菌，对其进行全基因组测序和比较基因组分析，获得菌株与其他戈登氏菌的共性和差异性，为后续研究提供基础。菌株WA4-43仅对革兰阳性菌具有抑制活性；通过形态特征和分子生物学鉴定，初步鉴定菌株WA4-43为Gordonia terrae；全基因组生物信息学分析表明该菌株基因组大小为5.44 Mb，GC含量为67.76%，含有4 963个蛋白编码基因，其中GO、COG、KEGG数据库中分别注释3 563、4 178、1 870个基因；antiSMASH预测获得8个类型共13个次级代谢产物基因簇，同源性平均值为（22.67±24.72）%，并运用PRISM 4预测了基因簇合成化合物；比较基因组分析发现G. terrae基因簇较为保守，但同源性均显著低于已知基因簇，具有合成新颖化合物的潜力。

关键词: 戈登氏菌, 全基因组测序, 次级代谢产物, 抗革兰阳性菌, 比较基因组

Abstract:

WA4-43 from the intestinal tract of cockroaches was the first Gordonia that was resistant to gram-positive bacteria. Whole-genome sequencing and comparative genome analysis of WA4-43 revealed the commonness and difference between WA4-43 and other Gordonia, providing a basis for subsequent research. Strain WA4-43 had inhibitory activity only against gram-positive bacteria. The strain WA4-43 was identified as G. terrae by morphological characteristics and molecular biological identification. Genome-wide bioinformatics analysis showed that the strain had a genome size of 5.44 Mb, GC content of 67.76%, and 4 963 protein coding genes, including 3 563, 4 178 and 1 870 genes annotated in GO, COG and KEGG databases, respectively. AntiSMASH predicted 13 secondary metabolite gene clusters in 8 types with an average homology of（22.67±24.72）%. PRISM 4 was used to predict compounds in gene clusters. Comparative genome analysis showed that the G. terrae gene cluster was conserved, but its homology was significantly lower than that of the known gene cluster, and it had the potential to synthesize new compounds.

Key words: Gordonia, whole genome sequencing, secondary metabolites, resistant to gram-positive bacteria, comparative genome

和梦颖, 刘文彬, 林震鸣, 黎尔彤, 汪洁, 金小宝. 一株抗革兰阳性菌的戈登氏菌WA4-43全基因组测序与分析[J]. 生物技术通报, 2023, 39(2): 232-242.

HE Meng-ying, LIU Wen-bin, LIN Zhen-ming, LI Er-tong, WANG Jie, JIN Xiao-bao. Whole Genome Sequencing and Analysis of an Anti Gram-positive Bacterium Gordonia WA4-43[J]. Biotechnology Bulletin, 2023, 39(2): 232-242.

图/表 14

图1 菌株WA4-43菌株特征 A：菌株高氏一号平板生长状态；B：菌株革兰染色光学显微镜（1 000×）；C-D分别为：菌株扫描电子显微镜10 000×、20 000×观察结果

Fig. 1 Characteristics of strain WA4-43 A: Growth state of strain Gao’s No.1 medium. B: Strain gram staining light microscope（1 000×）. C to D: Observed results of 10 000× and 20 000× by scanning electron microscope

图2 菌株WA4-43系统发育树

Fig. 2 Phylogenetic tree of strain WA4-43

表1 菌株WA4-43生理生化特征

Table 1 Physiological and biochemical characteristics of strain WA4-43

检测项目 Test item	结果 Result	检测项目 Test item	结果 Result
明胶水解	-	氧化酶	-
鼠李糖	+	过氧化氢酶	+
葡萄糖	+	尿素酶	+
木糖	+	生长耐受NaCl范围	1%-5%
甘露醇	+	H₂S产生	-

图3 管碟法测定菌株WA4-43抗菌活性 A：耐甲氧西林金黄色葡萄球菌；B：金黄色葡萄球菌；C：枯草芽孢杆菌；D：表皮葡萄球菌。n=3

Fig. 3 Determination of antibacterial activity of strain WA4-43 by tube and plate method A: Methicillin-resistant Staphylococcus aureus. B: Staphylococcus aureus. C: Bacillus subtilis. D: Staphylococcu epidermidis. n=3

表2 菌株WA4-43功能注释的基因数量和大小

Table 2 Number and size of functionally annotated genes in strain WA4-43

数据库 Database	数量 Amount	100≤长度<300 100≤Length <300	长度≥300 Length≥300
eggnog Annotation	4 178	1 847	2 181
GO Annotation	3 563	1 539	1 892
Kegg Annotation	1 870	686	1 117
Nr Annotation	4 835	2 230	2 311
Pfam Annotation	3 239	1 398	1 735
Swissprot Annotation	2 591	954	1 570
TrEMBL Annotation	2 591	954	1 570
All Annotated	4 837	2 230	2 311

图4 菌株WA4-43基因组圈图外圈：基因组大小; 第2圈：基因组所有正链基因大小; 第3圈：基因组所有负链基因大小; 第4圈：基因组序列重复序列; 第5圈：蓝色TRNA49个、紫色RRNA; 第6圈：GC的含量; 最内圈：GC-SKEw

Fig. 4 Genome circle map of strain WA4-43 Outer circle: Genome size. The second circle: Size of all plus-strand genes in the genome.The third circle: The size of all negative-strand genes in the genome.The fourth circle: Genome sequence repeats.The fifth circle: 49 blue TrNAand purple RRNA.The sixth circle: GC content. Innermost circle: GC-SKEw

图5 菌株WA4-43基因组GO功能注释

Fig. 5 GO functional annotation on the genome of strain WA4-43

图6 菌株WA4-43基因组KEGG的功能注释

Fig. 6 KEGG functional annotation of strain WA4-43 genome

图7 菌株WA4-43基因组COG功能注释

Fig. 7 Functional annotation of COG in the genome of strain WA4-43

表3 菌株WA4-43基因组中次级代谢基因簇的预测

Table 3 Prediction of secondary metabolic gene clusters in the genome of strain WA4-43

基因簇 Region	类型 Type	预测产生的化合物 Predicited production	同源性 Similarity/%
BGC1	Ectoine	Ectoine	75
BGC2	Terpene	SF2575	6
BGC3	Arylpolyene
BGC4	NRPS, betalactone
BGC5	NRPS	Ishigamide	11
BGC6	redox-cofactor
BGC7	NRPS
BGC8	NAPAA
BGC9	Terpene	Oxalomycin B	6
BGC10	RiPP-like	Kanglemycin A/Kanglemycin V1/Kanglemycin V2	5
BGC11	T1PKS, NRPS-like
BGC12	NRPS, siderophore	Desferrioxamine	33
BGC13	NRPS

图8 菌株WA4-43生物合成基因簇化合物预测 A-C：预测基因簇4化合物；D-E：预测基因簇5化合物；F：预测基因簇6化合物；G：预测基因簇7化合物；H-J：预测基因簇11化合物；K预测基因簇12化合物

Fig. 8 Prediction of biosynthetic gene cluster compounds of strain WA4-43 A to C: Predict gene cluster 4 compound. D to E: Predict gene cluster 5 compound. F: Predict gene cluster 6 compound. G: Predict gene cluster 7 compound. H-J: Predict gene cluster 11 compound. K: Predict gene cluster 12 compound

表4 BGC10基因预测及数据库分析

Table 4 BGC10 gene prediction and database analysis

基因编号 Gene ID	基因功能 Gene function	长度 Length/bp	Nr数据库 Nr database	GO数据库 GO database	TrEMBL数据库 TrEMBL database
GE002623	Additional biosynthetic genes	1 479	Gordonia sp. IITR100	Transferase activity;carbon-nitrogen ligase activity, with glutamine as amido-N-donor	Amidase
GE002624	Other genes	1 674	Gordonia sp. UCD-TK1	oxidoreductase activity, acting on the CH-CH group of donors; oxidation-reduction process	FAD-binding dehydrogenase
GE002625	Other genes	1 164	Gordonia sp. v-85	peroxidase activity; heme binding; oxidation-reduction process; cellular oxidant detoxification	Putative Iron-dependent peroxidase
GE002626	Core biosynthetic genes	810	Nocardia farcinica	proteolysis; peptidase activity; defense response to bacterium	Bacteriocin
GE002627	Transport-related genes	1 734	Nocardia farcinica	ATP binding; integral component of membrane; ATPase activity, coupled to transmembrane movement of substances; transmembrane transport	Multidrug ABC transporter ATP-binding protein
GE002628	Transport-related genes	1 920	Gordonia sp. IITR100	ATP binding; integral component of membrane; ATPase activity, coupled to transmembrane movement of substances;transmembrane transport	ATP-binding cassette, subfamily B
GE002629	Other genes	573	Gordonia sp. UCD-TK1		Uncharacterized protein

表5 10种Gordonia terrae基因组特征比对

Table 5 Comparison of genomic characteristics of 10 Gordonia terrae species

菌株 Strain	序号 No.	基因组大小 Size/Mb	GC含量 GC/%	染色体 Contigs	rRNA数量 rRNA genes	tRNA数量 tRNA genes	编码蛋白区数量 Number of coding sequence
WA4-43	GCA 020520305.1	5.44	67.76	1	9	49	4 963
NCTC10669	GCA 901542405.1	5.71	67.8	2	12	46	4 974
NRRL B-16283	GCA 003183825.1	5.71	67.8	1	9	46	4 979
3612	GCA 001698225.1	5.70	67.8	1	9	46	4 977
RL-JC02	GCA 011290605.1	5.31	67.87	2	9	47	4 627
NRRL B-16283	GCA 000716975.1	5.67	67.8	108	7	46	4 938
UMB0777	GCA 002847865.1	5.70	67.7	180	3	46	5 007
K	GCA 005502725.1	5.19	68	155	9	45	4 480
C-6	GCA 000390025.1	5.17	67.9	130	5	45	4 572
NBRC 100016	GCA 000248035.2	5.67	67.8	130	6	46	4 956

图9 AntiSMASH预测Gordonia生物合成基因簇

Fig. 9 Predicting the Gordonia biosynthetic gene cluster by AntiSMASH

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