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Journal of Tropical Oceanography >

2022 , Vol. 41 >Issue 1: 52 - 61

DOI: https://doi.org/10.11978/2021018

Marine Biology

Draft genome sequence and comparative genome analysis of Alliroseovarius sp. Z3

  • LIU Wei , 1, 2 ,
  • GUO Haipeng , 1, 2 ,
  • DONG Pengsheng 1, 2 ,
  • YAN Mengchen 1, 2 ,
  • ZHANG Demin 1, 2
Expand
  • 1. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo 315800, China
  • 2. School of Marine Sciences, Ningbo University, Ningbo 315800, China
GUO Haipeng. email:

Copy editor: YAO Yantao

Received date: 2021-02-13

  Revised date: 2021-04-08

  Online published: 2021-04-12

Supported by

National Natural Science Foundation of China(31672658)

General Research Project of Zhejiang Education Department, China(Y201839299)

Agricultural Major Project of Ningbo, China(2017C110001)

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Abstract

Alliroseovarius sp. Z3 was isolated from guts of Litopenaeus vannamei. In the study, Illumina Hiseq sequencing platform was used for genome sequencing; then, the genome was assembled, annotated by software SOAPdenovo etc., and compared with other five similar type strains. Genome annotation results showed that Z3 genome consists of 3525503 bp with a guanine-cytosine (GC) content of 59.4%, and 3509 protein-coding genes. The results of 16S rRNA sequence alignment, average nucleotide identity (ANI), DNA-DNA hybridization (DDH) and collinearity analysis showed that strain Z3 had the highest 16S rRNA similarity with Aliiroseovarius crassostreae CV919-312T, at 98.20%, and had the highest DDH and ANI values with Aliiroseovarius sediminilitoris DSM 29439T, at 26.80% and 84.95%, respectively. It was considered to be a new species of Alliroseovarius, and was named Aliiroseovarius sp. Z3. Comparative genomic analysis displayed that Aliiroseovarius sp. Z3 shared 2005 core orthologous gene clusters with other five similar type strains, and Z3 had 413 specific genes that are related to carbohydrate transport and metabolism, amino acid transport and metabolism, replication, recombination and repair, etc. After functional annotations, we found that Z3 possesses a complete denitrification pathway, indicating that Z3 can utilize nitrate and nitrite in the environment. These results related to the genome features and comparative genome of strain Z3 not only enrich the gene source of Aliiroseovarius genus, but also provide a molecular basis for studying its characteristics of denitrification.

Key words： shrimp gut; Aliiroseovarius sp. Z3; phylogenetic analysis; denitrification; comparative genome analysis

Cite this article

LIU Wei , GUO Haipeng , DONG Pengsheng , YAN Mengchen , ZHANG Demin . Draft genome sequence and comparative genome analysis of Alliroseovarius sp. Z3[J]. Journal of Tropical Oceanography, 2022 , 41(1) : 52 -61 . DOI: 10.11978/2021018

红杆菌科(Rhodobacteracea)细菌中的玫瑰杆菌分支(Roseobacter clade)是海洋环境(如海水、沉积物、藻类、无脊椎动物、脊椎动物、生物膜和高盐微生物垫)中最丰富的异养菌之一。据统计, 沿海浮游细菌群落的20%和海水混合层浮游细菌群落的15%都由玫瑰杆菌组成(Buchan et al, 2005; Choi et al, 2006; Kent et al, 2018; Sharpe et al, 2020)。玫瑰杆菌已被证实具有广泛的生理生化特征, 如可以通过分解并释放碳、硫和其他化合物而参与海洋生物化学循环(Mou et al, 2008; Reisch et al, 2011)。此外, 玫瑰杆菌分支细菌在水产养殖中也很常见, 它是虾肠道和养殖水体中含量最丰富的细菌之一(Guo et al, 2020; Huang et al, 2020), 并且部分玫瑰杆菌具有反硝化作用(Pujalte et al, 2014), 可以降低养殖水体中的硝酸盐、亚硝酸盐含量。
别样玫瑰变色杆菌属(Aliiroseovarius)细菌是玫瑰杆菌分支的成员, 为革兰氏阴性菌, 细胞呈杆状, 单极生长, 部分具有运动性(除Aliiroseovarius marinus)。其中, A. crassostreae CV919-312T(过去被分类为Roseovarius crassostreae)为别样玫瑰变色杆菌的模式种, 由Boettcher等(1999)从牡蛎中首次分离获得, 已被证实为牡蛎的病原菌, 可以导致幼年牡蛎患病(Kessner, 2015)。该属菌株还可从近海沉积物(A. sediminilitoris)(Park et al, 2013)、海水(A. marinus、A. pelagivivens)(Jung et al, 2011; Park et al, 2015)、海鞘(A. halocynthiae)(Kim et al, 2012)等生境分离出来。目前对该属菌株的研究大多都集中在分离纯化、新种鉴定和生理生化特征等方面(Boettcher et al, 2005; Park et al, 2013), 而对该属细菌的基因组信息和生态功能等研究报道则较少。
本研究通过对从对虾肠道中分离出的一株Aliiroseovarius属细菌的基因组进行测序和注释, 探究该物种的遗传进化特征。同时利用比较基因组学技术, 确定其直系同源基因及特有基因集群, 从基因组学层面解释该菌株的环境适应性、脱氮能力等, 为开发和利用玫瑰杆菌资源提供理论支撑。

1 材料与方法

1.1 DNA提取及基因组测序

菌株Z3为本课题组从健康凡纳滨对虾(Litopenaeus vannamei)肠道中分离获得(活体取样), 在海洋肉汤2216(MB, Difco)培养基中37℃恒温扩增培养18h后, 12000rpm离心5min收集细胞。依据DNA试剂盒(Bacteria DNA kit, OMEGA, USA)的说明书提取其基因组DNA, 并用NanoDrop ND-1000分光光度计(NanoDrop Technologies, Wilmington, DE, USA)对获得的DNA进行质量把控, 采用Illumina HiSeq PE150平台完成基因组草图测序(上海美吉生物医药科技有限公司, 上海, 中国)。

1.2 基因组装及注释

将从Illumina HiSeq平台测序获得的数据经CASAVA软件(Masella et al, 2012)碱基识别分析转化为原始测序序列后过滤处理, 去除包含Adapter的序列以及低质量数据, 得到有效数据后用SOAPdenovo v2.04软件(Luo et al, 2012)完成序列组装。再使用CISA软件(http://sb.nhri.org.tw/CISA/en/CISA)进行整合, 对组装结果进行补洞和优化。采用Glimmer v3.02软件(http://www.cbcb.umd.edu/software/glimmer/)对Z3基因组进行基因结构及蛋白基因的预测。将预测到的编码蛋白质的基因(Coding sequence, CDS)分别与直系同源基因簇数据库(Cluster of Orthologous Groups of proteins, COG)(Natale et al, 2000)、京都基因和基因组百科全书数据库(Kyoto Encyclopedia of Genes and Genomes, KEGG)(Kanehisa et al, 2000)进行比对, 获得蛋白功能注释。

1.3 系统发育及同源基因分析

提取基因组中的16S rRNA基因全长序列, 利用EzBioCloud(https://www.ezbiocloud.net/)和NCBI(https://blast.ncbi.nlm.nih.gov/Blast.cgi)数据库的核苷酸BLAST工具对16S rRNA基因全长序列进行比对, 下载相似度最高的14株模式菌株序列, 使用Mega X软件(Kumar et al, 2018)基于最大似然法(1000bootstrap)构建系统发育树。根据16S rRNA基因全长序列比对结果, 从EzBioCloud数据库中下载与Z3基因序列相似度最高的5个Aliiroseovarius属模式菌株以及6个其他玫瑰杆菌属模式菌株的基因组序列及相关信息。5株Aliiroseovarius属模式菌分为别: A. crassostreae CV919-312T(GCA_0013077 65.1), A. sediminilitoris DSM 29439T(GCA_9001099 55.1), A. marinus A6024T(GCA_004360145.1), A. halocynthiae MA1-10T(GCA_007004645.1)和A. pelagivivens GYSW-22T(GCA_900302485.1)。根据前期Glimmer预测的基因结构, 利用Orthofinder (Emms et al, 2019)筛选12株菌之间的同源基因。基于Orthofinder输出的单拷贝直系同源基因, 首先使用Muscle(Edgar, 2004)进行多序列比对, 而后使用Gbolcks(Castresana, 2000)提取保守序列(参数: -b4=5, -b5=h), 再将保守序列合并后用Raxml (Stamatakis, 2014)绘制系统发育树(参数:-f=a, -x= 123, -p=123, -n=1000, -m=PROTGAMMAAU TO)。为了进一步确定Z3的分类学地位, 本研究还进行了ANI(Average nucleotide identity)(http://jspecies.ribohost.com/jspeciesws/) (Lee et al, 2016)、DDH(DNA-DNA hybridization)(https://ggdc.dsmz.de) (Meier-Kolthoff et al, 2013)和共线性分析(Mauve软件)(Darling et al, 2004)。最后, 从Orthofinder输出的序列中提取5株Aliiroseovarius属菌株和Z3的核心基因及Z3的特异性基因, 用CD-hit(Li et al, 2006)(参数: -c=0.9, -n=5)去冗余后, 分别用COG和KEGG数据库进行功能注释。

2 结果与分析

2.1 基因组装与注释

菌株Z3经测序组装后, 得到11个基因组的框架(Scaffolds), 它们的长度范围为181350~ 528243bp。Z3的基因组大小为3525503bp, 平均GC含量为59.4%, 包含3509个预测的CDS, 编码基因总长度3221889bp, 占整个基因组的91.39%。将Z3的基因组序列提交到NCBI的GenBank数据库中(登录号为JAFMZK000000000)进行比对后, 再将CDS与COG数据库比对, 对CDS进行同源基因注释分类, 将基因划分为24类, 以英文大写字母B—Z代表每一类的代码(图1)。其中, 参与氨基酸的运输与代谢(E, Amino acid transport and metabolism)的蛋白质最多, 共有399个; 其次为一般功能预测(R, General function prediction)、转录(K, Transcription)以及翻译、核糖体结构与生物合成(J, Translation, ribosomal structure and biosynthesis), 基因数量分别为320、286和265个。通过与KEGG数据库比对, 从Z3的基因组中找到代谢通路相关的基因2070个(图2), 共分为37个类型, 可归为细胞过程(Cellular processes)、环境信息处理(Environmental information processing)、基因信息处理(Genetic information processing)、人类疾病(Human diseases)和代谢(Metabolism)5个大类。其中, 代谢大类的氨基酸代谢(Amino acid metabolism)、碳水化合物代谢(Carbohydrate metabolism)和环境信息处理大类的膜运输(Membrane transport)部分被注释到较多的基因, 分别为249、234和222个。
图1 别样玫瑰变色杆菌Z3基因组的COG功能分类

Fig. 1 Gene distribution based on Cluster of Orthologous Groups of proteins (COG) of Aliiroseovarius sp. Z3

图2 别样玫瑰变色杆菌Z3基因组的KEGG代谢通路

Fig. 2 Genetic Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways of Aliiroseovarius sp. Z3

2.2 系统发育分析

从Z3基因组中提取完整的16S rRNA基因全长序列进行比对并构建系统发育树。序列比对结果表明, 菌株Z3与Aliiroseovarius属中两个种的模式株A. crassostreae CV919-312T和A. sediminilitoris DSM 29439T的16S rRNA基因相似度最高, 分别为98.20%和98.06%。基于16S rRNA基因构建的系统发育树结果如图3所示, 菌株Z3与A. crassostreae CV919-312T聚在一枝。为了进一步鉴定Z3, 选取与Z3 16S rRNA基因相似度最高的5个Aliiroseovarius属模式株的基因组以及其他6株相近属模式株的基因组, 提取单拷贝直系同源基因构建系统发育树(图4), 结果发现Aliiroseovarius sp. Z3与A. sediminilitoris DSM 29439T的亲缘关系最近, 被分到同一枝。比较分析Z3与5株Aliiroseovarius属模式株基因组间的ANI和DDH, 结果发现6株菌两两之间的ANI值范围为83.61%~86.50%(表1), 其中Z3与其他5个种模式株的ANI值为84.95%~84.33%, 明显小于同种之间的ANI界定值95%(Kim et al, 2014), 表明菌株Z3可能为Aliiroseovarius属的一个新种。此外, Z3与A. sediminilitoris DSM 29439T的DDH值最高为26.8%(表2), 远低于同种分类阈值(70%)(Richter et al, 2009), 进一步说明菌株Z3为Aliiroseovarius属的新种, 将其命名为Aliiroseovarius sp. Z3。
图3 基于最大似然法构建的别样玫瑰变色杆菌Z3 16S rRNA基因的系统发育树

Fig. 3 The phylogenetic tree of Aliiroseovarius sp. Z3 16S rRNA gene based on the maximum likelihood method

图4 基于单拷贝直系同源基因构建的系统发育树

Fig. 4 The phylogenetic tree based on the single copy orthologous gene families

表1 6株别样玫瑰变色杆菌属细菌平均核苷酸同一性的值

Tab. 1 Average nucleotide identity (ANI) values of six Aliiroseovarius spp.

Aliiroseovarius sp. Z3 A. crassostreae CV919-312T A. halocynthiae MA1-10T A. marinus A6024T A. pelagivivens GYSW-22T A. sediminilitoris DSM 29439T
Aliiroseovarius sp. Z3 * 84.51% 84.33% 84.87% 84.49% 84.95%
A. crassostreae CV919-312T 84.51% * 84.82% 84.95% 85.04% 83.92%
A. halocynthiae MA1-10T 84.33% 84.81% * 85.82% 85.52% 83.69%
A. marinus A6024T 84.87% 84.94% 85.83% * 86.50% 83.62%
A. pelagivivens GYSW-22T 84.51% 85.02% 85.52% 86.50% * 83.63%
A. sediminilitoris DSM 29439T 84.95% 83.92% 83.69% 83.61% 83.65% *
表2 别样玫瑰变色杆菌Z3与其他5株模式株DNA- DNA杂交的值

Tab. 2 DNA-DNA hybridization (DDH) values between Aliiroseovarius sp. Z3 and other five Aliiroseovarius spp.

Aliiroseovarius sp. Z3
A. crassostreae CV919-312T 20.90%
A. sediminilitoris DSM 29439T 26.80%
A. marinus A6024T 20.60%
A. halocynthiae MA1-10T 20.30%
A. pelagivivens GYSW-22T 20.30%

2.3 比较基因组学分析

2.3.1 6株别样玫瑰变色杆菌的基因组基本特征

Z3与参考菌株的基因组基本特征如表3所示, 它们在基因组大小、GC含量等方面十分相近, 基因组大小在3.13~3.73Mb之间, GC含量介于57.1%~59.9%, 表明Z3符合Aliiroseovarius属细菌基因组的基本特征。
表3 Z3和与其近缘的5株别样玫瑰变色杆菌属模式株的基因组基本特征

Tab. 3 Genome features summary of Z3 and other five similar type strains from Aliiroseovarius genus

名称 大小/Mb GC含量/% 编码蛋白 分离来源
Aliiroseovarius sp. Z3 3.53 59.4 3509 中国浙江宁波: 凡纳滨对虾肠道
A. crassostreae CV919-312T 3.73 58.4 3693 美国缅因州达马里斯科塔河: 患病牡蛎
A. sediminilitoris DSM 29439T 3.41 58.7 3323 中国南部吉野岛: 近海沉积物
A. marinus A6024T 3.13 59.9 3027 中国山东日照: 海水
A. halocynthiae MA1-10T 3.39 57.1 3349 南海: 海鞘
A. pelagivivens GYSW-22T 3.33 58.1 3218 韩国巨济岛: 海水

2.3.2 基因组共线性分析

通过Mauve软件分别对Z3和其他5株别样玫瑰变色杆菌的基因组进行分析比对, 结果如图5所示。由图可看出Z3与A. sediminilitoris DSM 29439T之间的共线性最好, 但Z3与参考菌株的基因组之间均存在或多或少的插入、缺失、易位和倒位等基因重排现象, 这可能归结于它们为适应不同生存环境而产生了基因变化。
图5 菌株Z3与其他5个别样玫瑰变色杆菌Aliiroseovarius crassostreae CV919-312T(a)、Aliiroseovarius sediminilitoris DSM 29439T(b)、Aliiroseovarius halocynthiae MA1-10T(c)、Aliiroseovarius marinus A6024T(d)和Aliiroseovarius pelagivivens GYSW-22T(e)的共线性分析

图中数值表示基因片段大小

Fig. 5 Synteny blocks between Aliiroseovarius sp. Z3 and other five Aliiroseovarius spp., Aliiroseovarius. crassostreae CV919-312T (a), Aliiroseovarius sediminilitoris DSM 29439T (b), Aliiroseovarius. halocynthiae MA1-10T (c), Aliiroseovarius marinus A6024T (d), and Aliiroseovarius pelagivivens GYSW-22T (e). The value in the figure indicates the size of the gene fragment

2.3.3 核心基因和泛基因组分析

核心基因组(Core genome)是指同时出现在所有被分析的基因组中的一系列同源基因的合集, 副基因组(Accessory genome)被定义为基因组的特异性基因和部分共享基因的合集, 泛基因组(Pan genome)是指所有基因组的全部基因的合集(Inglin et al, 2018; Zeb et al, 2020), 核心基因组和泛基因组常被用以评价相似的物种内的基因多样性(Zhang et al, 2018), 本文中核心基因仅指6个菌株间的直系同源核心基因。核心基因和泛基因组分析结果表明, Aliiroseovarius sp. Z3与其他5株别样玫瑰变色杆菌的基因组共有5044个直系同源基因, 其中核心基因有2005个(39.75%)(图6)。同时, 各菌株存在的特有基因数分别为548个(A. crassostreae CV919-312T)、274个(A. halocynthiae MA1-10T)、117个(A. marinus A6024T)、145个(A. pelagivivens GYSW-22T)、182个(A. sediminilitoris DSM 29439T)和259个(Aliiroseovarius sp. Z3), 说明这6株菌具有较高同源性的同时也存在一些进化上的差异。1013个直系同源核心基因经COG注释后被分到19个类别中(表4), 其中一些高度保守的生物过程被注释到较多基因, 如氨基酸的运输与代谢(E, Amino acid transport and metabolism)包含305个基因, 无机离子转运与代谢(P, Inorganic ion transport and metabolism)包含235个基因, 能量产生与转化(C, Energy production and conversion)包含149个基因。为了更全面地了解Z3基因组中的418个特有基因, 本文对其进行了COG和KEGG功能注释(表4)。结果显示, 其中198个基因被注释到18个COG功能类别中, 如有15个基因被注释到碳水化合物转运与代谢(G, Carbohydrate transport and metabolism)类别, 12个基因被注释为与无机离子转运和代谢(P, Inorganic ion transport and metabolism)相关。
图6 6株别样玫瑰变色杆菌的直系同源基因和特异性基因簇的数量

Fig. 6 The numbers of orthologous and unique gene clusters among six Aliiroseovarius spp.

表4 基于COG数据库比对的6株别样玫瑰变色杆菌属细菌的直系同源核心基因和Z3的特异性基因

Tab. 4 The number of core orthologous gene clusters of six Aliiroseovarius spp. and specific 418 genes in the genome of Aliiroseovarius sp. Z3 based on COG categories analysis

COG分类 核心基因/个 Z3的特异性基因/个
未分类(Unassigned) 992 220
能量产生与转换(C) 149 13
细胞周期调控、细胞分裂、染色体分区(D) 9 2
氨基酸转运与代谢(E) 305 14
核苷酸转运与代谢(F) 22 2
碳水化合物的转运与代谢(G) 139 15
辅酶转运与代谢(H) 97 6
脂类转运与代谢(I) 78 6
翻译、核糖体结构和生物合成(J) 30 3
转录(K) 217 12
复制、重组和修复(L) 31 12
细胞壁/膜/包体生物合成(M) 76 10
细胞运动(N) 2 0
翻译后修饰、蛋白质周转和分子伴侣(O) 46 8
无机离子转运与代谢(P) 235 12
次生代谢物的生物合成、转运和分解代谢(Q) 48 2
功能未知(S) 184 68
信号转导机制(T) 110 4
细胞内运输、分泌和囊泡运输(U) 17 3
防御机制(V) 44 6

2.3.4 反硝化代谢通路基因分析

将Aliiroseovarius sp. Z3的CDS与KEGG数据库比对, 结果发现Z3拥有大量与碳(C)、氮(N)、磷(P)、硫(S)代谢相关的基因。值得注意是, 其具有完整的反硝化途径(Denitrification)的基因(图7), 如硝酸还原酶(napA)、亚硝酸还原酶(nirS)、一氧化氮还原酶(norB、norC)和一氧化二氮还原酶(nosZ), 说明Z3可以进行完整的反硝化作用。其近缘细菌A. crassostreae CV919-312T也同样被报道过具有反硝化作用(Boettcher et al, 2005)。但6株别样玫瑰变色杆菌的核心基因组的KEGG注释结果显示它们都仅具有硝酸还原酶(napA、narG), 即均只可将硝酸盐还原为亚硝酸盐, 无反硝化作用的相关基因(图7), 且除A. crassostreae CV919- 312T外的其余4株别样玫瑰变色杆菌也未见具有反硝化能力的报道。
图7 反硝化代谢途径相关基因

虚线框表示Z3拥有的基因; 数值表示氮元素的化合价态

Fig. 7 The gene related to denitrification pathway. The dashed box indicates that Z3 has the gene, and the number indicates the chemical valence of the nitrogen element

3 讨论

全基因组测序技术由于可以在分子水平上系统地研究菌株的遗传进化信息、与宿主的互作机制、致病机理、鉴定分类等, 目前已经被广泛应用于微生物的鉴定与分析中(Yu et al, 2017)。本研究从凡纳滨对虾肠道中分离获得一株具有潜在脱氮功能的Aliiroseovarius属细菌, 并对其进行了基因组草图测序和比较基因组学分析。通过16S rRNA基因全长序列比对发现, 该菌株与模式菌株A. crassostreae CV919-312T具有最高的相似度为98.2%, 通过构建单拷贝同源基因系统发育树发现其与A. sediminilitoris DSM 29439T聚类最近, 说明菌株Z3为Aliiroseovarius属细菌。为了进一步鉴定该菌株, 通过基因组比对分析发现菌株Z3与模式菌株A. sediminilitoris DSM 29439T具有最高的ANI值为84.95%, 介于6株Aliiroseovarius属细菌两两比对的ANI值之间, 说明其可能为该属的新种。此外, DDH比对发现, 菌株Z3与其他5株亲缘关系较近的菌株的DDH值低于新种鉴定阈值(Richter et al, 2009), 进一步说明该菌株为Aliiroseovarius属的新种, 故将其命名为Aliiroseovarius sp. Z3。
硝酸盐和亚硝酸盐是养殖水体的主要污染物之一(Xing et al, 2016), 长期暴露于高水平的硝酸盐、亚硝酸盐环境中, 可能会引起氧化应激, 影响水生动物的抗氧化系统(Camargo et al, 2005; Sun et al, 2014)。反硝化作用可以使硝酸盐还原为亚硝酸盐再到氮气, 是水体最主要的脱氮过程, 对水体的氮元素平衡和富营养化控制有重要意义(刘志迎 等, 2019)。菌株Z3基因组具有编码完整的反硝化途径的硝酸还原酶(napA)、亚硝酸还原酶(nirS)、一氧化氮还原酶(norB、norC)、一氧化二氮还原酶(nosZ)等基因, 表明Z3具有将硝酸盐转化为氮的潜力, 有助于改善养殖水体的水质(Philippot, 2002; Graham et al, 2010), 这与A. crassostreae CV919-312T具有的反硝化作用相一致(Pujalte et al, 2014)。此外, 有研究表明反硝化作用与生物膜形成息息相关, 如Abdelhamed等(2021)发现nasA、norB和nosZ在细菌形成生物膜时高度表达。本研究发现Z3基因组中具有编码生物膜形成和群体效应相关的基因, 说明菌株Z3可以通过形成生物膜发挥反硝化作用。本文通过对菌株Z3进行基因组测序及比较基因组分析, 不仅探究了它的分子机理和作用机制, 且丰富了Aliiroseovarius属细菌的研究, 为玫瑰杆菌分支细菌的多样性研究提供了参考。

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