CN106047755A - 一株空间克氏库克菌株lct‑h5 - Google Patents

一株空间克氏库克菌株lct‑h5 Download PDF

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CN106047755A
CN106047755A CN201610455942.8A CN201610455942A CN106047755A CN 106047755 A CN106047755 A CN 106047755A CN 201610455942 A CN201610455942 A CN 201610455942A CN 106047755 A CN106047755 A CN 106047755A
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lct
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刘长庭
张学林
潘磊
姜学革
方向群
郭英华
王俊峰
周宏�
徐绸
刘岩
余昳
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Chinese PLA General Hospital
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Abstract

本发明涉及一株从“神舟九号”返回舱中分离得到下行菌株空间克氏考克氏菌株LCT‑H5，为革兰氏阳性球菌，以四联状排列为主，亦有成对、散在排列，可利用葡萄糖，蕈糖、甘露醇、蔗糖等多种碳源。对该菌进行16S rRNA测序，鉴定其为克氏考克氏菌属成员。对该菌进行了全基因组测序，发现该菌基因组大小为2.4 Mbp，包括1991个编码序列和101个RNA基因，COG数据库比对共注释到22组COG功能分类。此专利对研究密闭飞行器内微生物种类分布等提供了一定帮助。

Description

一株空间克氏库克菌株LCT-H5
技术领域
本发明涉及一株空间环境下分离的空间克氏考克氏菌株LCT-H5及其生物学特性、全基因组学测序。
背景技术
从登月成功到未来的火星探索，人类探索外太空的活动不仅越来越频繁，其范围也越来越大。微生物是自然环境中的一部分，很多细菌也存在于人类皮肤、口腔、鼻咽部和胃肠道等部位。有研究发现在模拟微重力条件下培养的鼠伤寒沙门氏菌对小鼠感染的毒力会增强，而且航天员的免疫力在空间环境下有不同程度的下降，容易导致机会性感染。目前，在空间站中已检测出包括肺炎克雷伯氏菌、金黄色葡萄球菌、铜绿假单胞菌、大肠杆菌、沙雷氏菌、蜡状芽孢杆菌和肠球菌等多种细菌。因此，对这些细菌进行深入研究有助于了解空间环境对这些细菌的影响极其致病力和耐药性的改变机制，为载人航天提供医学保障。
早期对许多环境微生物的认识仅来自于对16S rRNA的研究，一些重要的遗传信息很难获得。现在越来越多的新的培养和分子生物学方法，例如基因组学、蛋白质组学、宏基因组学等，被用来研究新获得微生物的潜在特征和功能等。同时随着测序技术的更新换代和高通量测序技术的出现，细菌基因组的测序成本和所需时间已大幅度降低。
发明内容
本发明提供的菌株空间克氏考克氏菌株LCT-H5为“神舟九号”返回舱中分离得到下行菌株。本发明提供的空间克氏考克氏菌株LCT-H5菌株，其保藏号为CGMCC No.12573。
菌株LCT-H5可在MH固体平板上形成稍凸起的圆形、湿润、黄色的菌落，专性需氧，为革兰氏阳性球菌。可利用多种碳源，包括葡萄糖，蕈糖、甘露醇、蔗糖等。对头孢唑啉、头孢曲松、环丙沙星、诺氟沙星、阿米卡星、庆大霉素等均敏感。
所述菌株LCT-H5，进行全基因组测序并进行分析，发现该菌基因组大小为2.4Mbp，包括1991个编码序列和101个RNA基因，COG数据库比对共注释到22组COG功能分类(表1)。
表1 菌株LCT-H5基因特征
Attribute Value % of totala
Size (bp) 2409488
G+C content (bp) 1730012 71.8
Coding region (bp) 2127578 88.3
Total genes 2092 100
RNA genes 101 4.83
Protein-coding genes 1991 95.17
Genes in paralog clusters 39 1.86
Genes assigned to COGs
1 or more conserved domains 992 47.42
2 or more conserved domains 112 5.35
3 or more conserved domains 27 1.29
4 or more conserved domains 16 0.76
Genes with signal peptides 72 3.44
Genes with transmembrane helices 550 26.29
Paralogous groups 17 0.81
附图说明
图1菌株LCT-H5 的MH 平板培养。
图2菌株LCT-H5的16S rRNA基因序列NJ系统发育树。
图3菌株LCT-H5的革兰氏染色。
图4菌株LCT-H5的电子显微镜观察。
图5-6 菌株LCT-H5的基因组测序、组装及注释。
具体实施方式
下述实施实例中所用的实验方法，如无特殊说明，均为常规方法。
下述实施实例中所用的材料、试剂，如无特殊说明，均可从商业途径获得。
实施实例一：菌株LCT-H5的培养。
菌株LCT-H5为 “神舟九号”飞船的返回舱内分离得到。菌株分别于37℃培养箱或摇床中进行静置或振荡培养，液体培养基为脑心浸出液培养基(BHI，Oxiod)，配方为 37 g/L BHI；固体培养基为MH 平板（图1）。
实施实例二：菌株LCT-H5菌种鉴定及系统发育树构建。
菌株LCT-H5接种至BHI液体培养基，37℃，180 rpm过夜培养，6000 rpm离心5 min收集菌体，弃上清。利用Qiagen公司的基因组DNA 提取试剂盒(Code No. 51304，详细步骤见产品说明书) 提取收集的菌体的基因组DNA。以LCT-H5的基因组DNA为模板，利用细菌16srRNA通用测序引物27F（5’- AGAGTTTGATCCTGGCTCAG-3’）和1492R（5’-TACGGCTACCTTGTTACGACTT-3’）扩增其16s rRNA片段并测序，得到其碱基序列。用 CLUSTALW（http://www.ebi.ac.uk/Tools/msa/clustalw2/）进行多序列比对，并用 MEGA 5.0构建NJ 系统发育树（图2）。
实施实例三：菌株LCT-H5的革兰氏染色。
采用革兰氏染色试剂盒（Code No. G1060, Solarbio）对LCT-H5菌体进行染色。BHI液体培养基过夜培养菌体，将菌液滴于载玻片中央，在酒精灯上略加温，使之迅速干燥。结晶紫、沙黄染色液对干燥菌体一次染色脱色，详细步骤见产品说明书。染色完成并晾干后，置于光学显微镜下观察染色情况（图3）。
实施实例四：菌株LCT-H5的扫描电子显微镜观察。
在扫描电子显微镜（SEM）下观察菌体形态。首先用2.5%戊二醛、PBS缓冲液以及1%锇酸清洗菌体，乙醇梯度脱水并乙酸异戊酯置换，样品干燥后做喷金处理，处理好的样品置于FEI Quanta 200型扫描电镜下观察（图4）。
实施实例五：菌株LCT-H5的基因组测序、组装及注释。
基因组DNA样品委托美吉生物有限公司进行全基因组序列测定、基因预测、基因功能注释、非编码RNA预测等工作，序列分析工作委托美吉生物有限公司和上海瀚宇生物科技有限公司共同完成。基因组DNA 提取后进行质量鉴定，在浓度和纯度达到测序要求后进行全基因组测序。利用Covaris进行基因组DNA片段化，构建插入片段约300-500 bp 的基因组测序文库，通过桥式PCR扩增得到DNA簇，然后采用Illumina Hiseq2000测序技术完成该菌株的基因组扫描测序，获得约1 Gb 的原始测序数据。Illumina HiSeq2000 测序得到的原始图像数据经过Base Calling 转化为序列数据，结果以FASTQ 文件格式来存储，包含测序read 的序列信息以及测序质量信息；原始数据经过预处理去除接头、引物及低质量数据后，利用SOAPdenovo （http://soa p.genomics.org.cn/）拼接软件对优化序列进行多个Kmer 参数的拼接，得到最优的组装结果，并运用GapCloser 软件对组装结果进行局部内洞填充和碱基校正；分别利用RNAmmer 和tRNAscan-SE 软件对基因组中包含的rRNA 和tRNA进行预测；利用Glimmer 3.0（http://www.cbcb.umd.edu/software/glimmer/）软件进行基因预测；利用各种数据库进行基因功能注释和分类（图5、6）。
[0025]
关于LCT-H5菌株的保藏说明
A. 菌种的保藏单位名称和地址
名称：中国微生物菌种保藏管理委员会普通微生物中心
地址：北京市朝阳区北辰西路1号院3号 中国科学院微生物研究所
B. 交机构保藏的日期
2016年6月1日
C. 保藏机构给予的保藏号
CGMCC No.12573
D. 分类命名
克氏考克氏菌 Kocuria kristinae。

Claims (3)

1.一株从“神舟九号”飞船返回舱中分离得到下行菌株克氏考克氏菌株LCT-H5菌株，其保藏号为CGMCC No.12573。
2.根据权利要求1所述的一株从“神舟九号”返回舱中分离得到的下行菌株克氏考克氏菌株LCT-H5，其特征在于：为革兰氏阳性球菌，以四联状排列为主，亦有成对、散在排列，可利用葡萄糖，蕈糖、甘露醇、蔗糖等多种碳源。
3.根据权利要求1所述的一株从“神舟九号”返回舱中分离得到下行菌株克氏考克氏菌株LCT-H5菌株，进行全基因组测序并进行分析。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111621444A (zh) * 2019-12-31 2020-09-04 华南理工大学 一株提高酿造酱油风味品质的克氏库克菌及其应用

Citations (2)

* Cited by examiner, † Cited by third party
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WO2006029632A2 (en) * 2004-09-17 2006-03-23 Chr.Hansen A/S Chimeric phage-derived particles, methods for their production and use
CN102827771A (zh) * 2012-08-30 2012-12-19 东北农业大学 一种腐乳专用生产发酵剂及其制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006029632A2 (en) * 2004-09-17 2006-03-23 Chr.Hansen A/S Chimeric phage-derived particles, methods for their production and use
CN102827771A (zh) * 2012-08-30 2012-12-19 东北农业大学 一种腐乳专用生产发酵剂及其制备方法

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伍蕊等: "克氏库克菌血流感染1例并文献复习", 《中国感染与化疗杂志》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111621444A (zh) * 2019-12-31 2020-09-04 华南理工大学 一株提高酿造酱油风味品质的克氏库克菌及其应用
CN111621444B (zh) * 2019-12-31 2022-03-01 华南理工大学 一株提高酿造酱油风味品质的克氏库克菌及其应用

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