无色杆菌77的基因组构成及其趋化和耐药特性

doi:10.13560/j.cnki.biotech.bull.1985.2022-0112

摘要/Abstract

摘要：

无色杆菌77（Achromobacter sp. 77）是黄瓜枯萎病菌（Fusarium oxysporum f. sp. cucumerinum,Foc）菌丝际优势细菌,可随Foc菌丝迁移且状态活跃。为明确菌株77在Foc菌丝际的定殖机理,本研究在分析菌株77全基因组构成基础上,研究了其趋化特性和耐药性。Illumina Hiseq+PacBio测序分析表明,菌株77基因组全长5 868 070 bp,仅含1条染色体,GC含量为65.89%。在KEGG、COG和GO数据库分别注释到2 696、4 862和4 212个基因。菌株77基因组中含mcp、che、mot等重要趋化基因;通过CARD数据库注释,发现含有drr、emr等抗生素耐药外排泵基因、抗生素耐药基因簇以及多种抗生素的抗性基因。抗生素敏感性实验表明,菌株77对浓度低于300 µg/mL的氯霉素、卡那霉素、氨苄青霉素以及浓度低于50 µg/mL的四环素均有抗性,是一株多重耐药菌株。采用改良的游动平板法测定菌株77的趋化特性,结果表明,菌株77对1 mmol/L对羟基苯乙酸、水杨酸、α-酮戊二酸、延胡索酸、琥珀酸、苹果酸等多种根分泌物和真菌分泌物组分具有明显的趋化响应;0.5 mmol/L的上述化合物和0.5-1 mmol/L的镰刀菌酸可促进菌株77的生长。上述研究结果说明菌株77对根分泌物或真菌分泌物具有趋化和利用能力,是连作条件下无色杆菌属细菌在枯萎病菌菌丝际丰度升高的重要原因。

关键词: 无色杆菌, 黄瓜枯萎病菌, 全基因组分析, 趋化, 耐药

Abstract:

Achromobacter sp. 77,the dominant hyphosphere bacterium of Fusarium oxysporum f. sp. cucumerinum（Foc）,actively migrates along with Foc hyphae growth. To clarify the colonization mechanism of strain 77 in Foc hyphosphere,the whole genome of strain 77 was sequenced by using Illumina Hiseq+PacBio sequencing method,and its characteristics on chemotaxis and antibiotic resistance were examined. The genome of strain 77 was 5 868 070 bp with one chromosome and GC content of 65.89%. Total of 2 696,4 862 and 4 212 genes were annotated against KEGG,COG and GO database,respectively. Gene annotation demonstrated that strain 77 contained multiple important chemotactic genes such as mcp,che and mot in its genome. Annotation of its genome against CARD database showed that it harbored antibiotic resistance efflux pump genes such as drr,emr and gene clusters resistant to multiple antibiotics. Antibiotic sensitivity tests showed that strain 77 was resistant to chloramphenicol,kanamycin and ampicillin at the concentration < 300 µg/mL,and resistant to tetracycline at the concentration < 50 µg/mL,indicating that strain 77 was a multi-drug resistant strain. The chemotactic test indicated that strain 77 showed obviously chemotactic response to 1 mmol/L p-hydroxyphenylacetic acid,salicylic acid,α-ketoglutaric acid,fumaric acid,succinic acid and malic acid. Moreover,the growth of strain 77 was promoted by the above compounds at the concentration of 0.5 mmol/L and by 0.5-1.0 mmol/L of fusaric acid. These results indicated that strain 77 showed chemotaxis toward root or fungal exudates and utilization them,which might be the reason that the abundance of Achromobacter increased in Foc hyphosphere after continuous cultivation of cucumber in Foc infested soil.

Key words: Achromobacter, Fusarium oxysporum f. sp. cucumerinum, whole genome sequencing, chemotaxis, antibiotic resistance

李霁虹, 荆玉玲, 马桂珍, 郭荣君, 李世东. 无色杆菌77的基因组构成及其趋化和耐药特性[J]. 生物技术通报, 2022, 38(9): 136-146.

LI Ji-hong, JING Yu-ling, MA Gui-zhen, GUO Rong-jun, LI Shi-dong. Genome Construction of Achromobacter 77 and Its Characteristics on Chemotaxis and Antibiotic Resistance[J]. Biotechnology Bulletin, 2022, 38(9): 136-146.

图/表 14

图1 细菌77对真菌分泌物及根分泌物的趋化响应示意图上下两部分为两个平行实验。A和B分别为待测趋化化合物和无菌水的接种位置,C为细菌77菌液的接种位置

Fig. 1 Demonstration of chemotactic response of strain 77 to fungal and root exudates The plate was divided into two parts for two parallel tests of the same treatment. A and B shows the inoculation site of the candidate chemotactic compounds and sterilized water,respectively,and C shows the inoculation site of the bacterial suspension of strain 77

表1 菌株77基因组组装

Table 1 Genome assembly of strain 77

名称Name	数据Data
基因组大小Genome size	5 868 070 bp
Scaffold条数Number of Scaffolds	1
染色体数Number of chromosomes	1
质粒数Number of plasmids	0
GC含量GC content	65.89%
CRISPR-Cas系统数量Number of CRISPR-Cas system	7
tRNA数目Number of tRNA	55
rRNA数量Number of rRNA	13
基因组岛数量Number of genome island	12
基因数目Number of CDS	5 475

图2 菌株77的基因组圈图最外面一圈为基因组大小的标识;第二圈和第三圈为正链、负链上的CDS,不同的颜色表示CDS不同的COG的功能分类;第四圈为rRNA和tRNA;第五圈为GC含量,向外的红色部分表示该区域GC含量高于全基因组平均GC含量,峰值越高表示与平均GC含量差值越大,向内的蓝色部分表示该区域GC含量低于全基因组平均GC含量,峰值越高表示与平均GC含量差值越大;最内一圈为GC-Skew值

Fig. 2 Circle map of the genome of strain 77 The outside circle of the map shows the genome size. The second and third circles are CDS on the positive and negative chains. Different colors indicate the functional classification of different COG of CDS. The fourth circle is rRNA and tRNA. The fifth circle is GC content. The outward red part indicates that GC content in this region is higher than the average GC content in the whole genome. The higher the peak value,the greater the difference between GC content in this region and the average GC content. The interior blue part indicates that GC content in this region is lower than the average GC content in the whole genome. The higher the peak value,the greater the difference between GC content in this region and the average GC content. The interior circle is GC-SKEW

图3 菌株77基因组的KEGG注释途径纵坐标表示KEGG pathway的level2层级分类,横坐标表示在注释该分类下基因的数目。不同柱子颜色代表KEGG pathway的level1层级分类。最右侧柱子表示不同level1分类下的基因数目。由于同一个基因可能会被注释到多个level2分类中,因此计算level1分类的基因数目时会进行去冗余处理

Fig. 3 KEGG annotated pathways of strain 77's genome The vertical coordinate represent the level2 classification of KEGG pathway,and the horizontal coordinate represents the number of genes under this classification. Different colors represent the level1 classification of KEGG pathway. The right column represents the number of genes under different level1 classifications. Since the same gene may be annotated into more than one level2 categories,de-redundancy is performed when counting the number of genes in level1 categories

图4 菌株77基因组的COG功能注释分类

Fig. 4 Annotated classification of COG functions of strain 77's genome

图5 菌株77基因组的GO功能注释分类 BP、CC、MF分别为GO功能中的生物过程（biological process）、细胞组分（cellular component）、分子功能（molecular function）,每个饼图展示TOP 10的GO功能

Fig. 5 Classification of GO functions annotation of strain 77's genome Each category shows TOP 10 GO annotations

表2 菌株77基因组的GO注释中分解代谢与趋化相关基因统计

Table 2 Statistics of catabolism and chemotaxis related genes in GO annotation of strain 77's genome

GO注释GO annotation	基因数Number of genes
代谢过程Metabolic process	158
三羧酸循环Tricarboxylic acid cycle	18
苹果酸代谢Malate metabolic process	4
细胞氨基酸分解代谢 Cellular amino acid catabolic process	5
胁迫应答Stress response	22
抗生素分解Antibiotic decomposition	2
药物跨膜运输Transmembrane transport of drugs	2
细胞氧化解毒Cellular oxidant detoxification	33
趋化性Chemotaxis	33
细菌型鞭毛依赖运动 Bacterial-type flagellum-dependent cell motility	18

图6 菌株77基因组的碳水化合物活性酶注释统计

Fig. 6 Statistics of CAZyme annotation of strain 77's genome

表3 菌株77趋化基因预测分类

Table 3 Predictive classification of chemotactic genes of strain 77

基因类别 Gene classification	基因名称 Gene name	基因数量 Number of genes	基因编号 Gene ID	KEGG数据库基因注释 Gene annotation in KEGG database
mcp	mcp	9	gene0255、gene0975、gene1020、gene2196、gene3307、gene3446、gene4030、gene4485、gene4827	甲基趋化受体蛋白Mcp
che	cheA	1	gene2189	感受器激酶CheA
	cheW	1	gene2190	嘌呤结合趋化蛋白CheW
	cheR	2	gene2192、gene4468	甲基转移酶CheR
	cheB	2	gene2193、gene3711	趋化响应调节蛋白CheB
	cheBR	1	gene4467	CheB/CheR融合蛋白
	cheD	1	gene5023	趋化蛋白CheD
wsp	wspF	1	gene3040	趋化响应调节蛋白WspF
	wspD、wspB	2	gene3042、gene3044	趋化相关蛋白WspD、WspB
	wspC	1	gene3043	甲基转移酶WspC
	wspA	1	gene3045	甲基趋化受体蛋白WspA
mot	motA	1	gene2381	鞭毛马达蛋白MotA
aer	aer	2	gene2219、gene2311	趋氧性受体蛋白Aer
tar	tar	1	gene4676	甲基趋化受体蛋白Ⅱ（天冬氨酸感受器受体）
-	-	5	gene0304、gene2191、gene2214、gene2215、gene2217	未知功能趋化蛋白
-	-	1	gene3125	未知功能趋化蛋白
-	-	1	gene3000	未知功能趋化蛋白

表4 菌株77分泌系统分类

Table 4 Classification in secretion system of strain 77

分泌系统Secretion system	数量Amount
Ⅰ型 Type Ⅰ	1
Ⅱ型 Type Ⅱ	11
Ⅲ型 Type Ⅲ	0
Ⅳ型 Type Ⅳ	2
Ⅴ型 Type Ⅴ	0
Ⅵ型 Type Ⅵ	17

图7 耐药基因预测分类

Fig. 7 Predictive classification of drug resistance genes

图8 菌株77对真菌分泌物及根分泌物成分的趋化响应红色圆点表示化合物或无菌水的点接位置,红色箭头指向菌株77因趋化响应而形成的弧形趋化圈

Fig.8 Chemotactic responses of stain 77 to fungal and root exudates The red dots indicate the inoculation site of the candidate chemotactic compounds or sterile water,and the red arrows point to the arc-shaped chemotactic circle formed by the chemotactic response of strain 77

表5 真菌及根分泌物组分对菌株77 OD600的影响

Table 5 Effects of fungal and root exudate components on OD600 value of strain 77

化合物浓度 Concentration/ （mmol^.L^-1）	真菌或根分泌物 Fungal or root exudates				根分泌物 Root exudate		尖孢镰刀菌分泌物 Exudate of Fusarium oxysporum
化合物浓度 Concentration/ （mmol^.L^-1）	延胡索酸 Fumaric acid	α-酮戊二酸 α -ketoglutaric acid	苹果酸 Malic acid	琥珀酸 Succinic acid	水杨酸 Salicylic acid	对羟基苯乙酸4-Hyd-roxyphenylacetic acid	镰刀菌酸 Fusaric acid
0	0.278±0.002 d	0.334±0.002 bc	0.274±0.004 b	0.285±0.012 c	0.295±0.003 d	0.642±0.008 b	0.293±0.004 c
0.5	0.323±0.003 b	0.344±0.002 ab	0.319±0.005 a	0.315±0.004 a	0.334±0.002 b	0.775±0.003 a	0.340±0.002 a
1	0.351±0.001 a	0.351±0.002 a	0.312±0.002 a	0.313±0.006 ab	0.366±0.002 a	0.760±0.019 a	0.322±0.005 b
2	0.304±0.009 c	0.331±0.001 bc	0.285±0.004 b	0.298±0.011 bc	0.317±0.006 bc	0.049±0.002 c	0.272±0.018 d
4	0.299±0.012 c	0.326±0.004 c	0.272±0.006 bc	0.291±0.002 c	0.307±0.003 cd	0.049±0.003 c	0.111±0.007 e
6	0.288±0.002 d	0.308±0.002 d	0.088±0.011 d	0.243±0.012 d	0.223±0.026 e	0.053±0.002 c	0.055±0.004 f
8	0.063±0.002 e	0.095±0.018 e	0.052±0.002 e	0.094±0.005 e	0.054±0.003 f	0.052±0.003 c	0.054±0.005 f
10	0.054±0.004 e	0.056±0.007 f	0.054±0.003 e	0.058±0.002 f	0.051±0.001 f	0.053±0.001 c	0.053±0.003 f

图9 菌株77对常见抗生素的抗性

Fig. 9 Resistances of strain 77 to common antibiotics

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