应用HCR-FISH技术研究冷泉沉积物中甲烷厌氧氧化古菌的生存状态

doi:10.3969/j.issn.1001-909X.2025.01.003

摘要/Abstract

摘要：

甲烷厌氧氧化(anaerobic oxidation of methane,AOM)过程是冷泉沉积物中元素循环过程的重要一环。该反应一般由甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)和硫酸盐还原细菌(sulfate-reducing bacteria,SRB)共同完成,两者通常以共生体的方式存在。然而目前尚未获得ANME纯培养菌株,且其缓慢的代谢阻碍了对其代谢特征、协同作用机制的进一步探索和研究。本文通过杂交链反应-荧光原位杂交技术(hybridization chain reaction-fluorescence in situ hybridization,HCR-FISH)并结合16S rRNA基因高通量测序结果,调查了南海Formosa冷泉(简称F冷泉)黑色菌席区域不同深度沉积物中ANME群落组成情况及存在状态。结果显示,ANME-1和ANME-2为F冷泉沉积物中的主要ANME类群,其中,ANME-2和SRB以菌团聚集体的方式存在,而未发现ANME-1类群与细菌成团的情况。这表明ANME-2多与SRB共生进行AOM过程,并且暗示ANME-1与ANME-2存在不同的生理功能和甲烷代谢机制。另外,在所有层位的沉积物样品中,ANME-2/SRB菌团直径主要集中在3~10 μm之间。相关性分析表明,菌团直径分布与沉积物中硫酸盐浓度等环境因子显著相关,指示冷泉环境因子对ANME/SRB菌团生长的影响作用。此外,通过HCR-FISH技术进一步发现,在F冷泉沉积物中存在多个排列整齐、大小均匀的菌团连接形成的菌团簇,这种特殊结构暗示菌团间可能存在着联系或合作关系。本研究揭示了F冷泉不同深度沉积物中ANME类群的存在状态及共生菌团的大小和分布规律,为进一步揭示不同ANME类群在原位冷泉沉积物中的甲烷代谢机制和生态功能提供了基础。

关键词: 冷泉, 沉积物, ANME, SRB, AOM, 微生物共生体, HCR-FISH

Abstract:

The anaerobic oxidation of methane (AOM) is a pivotal component of elemental cycling within cold seep sediments. This process is usually performed by anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which usually exist as symbionts. However, pure cultures of ANME have not yet been obtained, and their slow metabolism hinders further exploration and research into their metabolic characteristics and collaborative mechanisms. In this study, we utilized hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) technology and high-throughput 16S rRNA gene sequencing to investigate the composition and state of ANME communities at different depths of the sediments in the black microbial mat area of the South China Sea Formosa cold seep. The results showed that ANME-1 and ANME-2 were the dominant groups in the sampled Formosa cold seep sediments. Specifically, ANME-2 was found to form consortia with SRB, while no such associations were detected for ANME-1. This observation suggested that ANME-2 and SRB primarily engage in symbiotic AOM processes, highlighting potential differences in physiological roles and methane metabolism pathways between ANME-1 and ANME-2. Furthermore, in sediment samples of all layers, the diameters of ANME-2/SRB consortia were predominantly concentrated between 3-10 μm. Correlation analysis indicated a significant link between the distribution of consortium diameters and environmental factors such as sulfate concentration in the sediment, underscoring the impact of environmental factors on the growth of ANME/SRB consortia. Additionally, using HCR-FISH, we further discovered the presence of multiple consortium clusters in the Formosa cold seep sediment, characterized by orderly connected and uniform-sized consortium, implying possible connections or cooperative relationships among consortia. This study revealed the presence and distribution patterns of ANME groups and sizes of symbiotic microbial consortia in sediment samples from different depths of the Formosa cold seep, laying the foundation for further understanding methane metabolism mechanisms and ecological functions of different ANME groups in situ cold seep sediments.

Key words: cold seep, sediment, ANME, SRB, AOM, microbial consortia, HCR-FISH

中图分类号:

X172
P744.4

何茂雨, 王景, 李思翰, 梁乐文. 应用HCR-FISH技术研究冷泉沉积物中甲烷厌氧氧化古菌的生存状态[J]. 海洋学研究, 2025, 43(1): 22-33.

HE Maoyu, WANG Jing, LI Sihan, LIANG Lewen. Utilizing HCR-FISH to investigate the status of anaerobic methanotrophic archaea in cold seep sediments[J]. Journal of Marine Sciences, 2025, 43(1): 22-33.

图/表 8

表1 本研究中采用的Formosa冷泉沉积物样本信息

Tab.1 Information of sediment samples in Formosa cold seep used in this study

样品编号	沉积物描述	沉积物柱状样深度/cm	样品数量/个
D231-1	还原性沉积物外部对照	14	7
D231-2	还原性沉积物上边缘交界	30	15
D231-3	还原性沉积物中心1	20	10
D231-4	还原性沉积物中心2	18	9
D231-5	还原性沉积物中心下边缘交界	22	11
D231-6	还原性沉积物白色菌席	18	9
合计			61

表2 本研究中使用的HCR-FISH探针序列

Tab.2 HCR-FISH probes used in this study

探针名称		靶向类群	探针序列(5’-3’)	来源
起始探针	ARCH915-H	Archaea	CCGAATACAAAGCATCAACGACTAGAAAAAAGTGCTCCCCCGCCAATTCCT	文献[8]
	EUB338-R	Bacteria	TACGCCCTAAGAATCCGAACCCTATGAAATAGCTGCCTCCCGTAGGAGT	文献[16]
	ANME-1-350-H	ANME-1	CCGAATACAAAGCATCAACGACTAGAAAAAAAGTTTTCGCGCCTGATGC	文献[16]
	ANME-2-538-H	ANME-2	CCGAATACAAAGCATCAACGACTAGAAAAAAGGCTACCACTCGGGCCGC	本研究
	ANME-3-1249-H	ANME-3	CCGAATACAAAGCATCAACGACTAGAAAAAATCGGAGTAGGGACCCATT	本研究
	DSS658-R	Desulfosarcina-Desulfococcus	TACGCCCTAAGAATCCGAACCCTATGAAATATCCACTTCCCTCTCCCAT	本研究
扩增探针	H1		CATAGGGTTCGGATTCTTAGGGCGTAGCAGCATCAATACGCCCTAAGAATCC	文献[21]
	H2		TACGCCCTAAGAATCCGAACCCTATGGGATTCTTAGGGCGTATTGATGCTGC	文献[21]
	R1		TCTAGTCGTTGATGCTTTGTATTCGGCGACAGATAACCGAATACAAAGCATC	文献[21]
	R2		CCGAATACAAAGCATCAACGACTAGAGATGCTTTGTATTCGGTTATCTGTCG	文献[21]

图1 Formosa冷泉沉积物样本中ANME和SRB类群的组成及相关性 (图a表示D231-4站位中ANME类群随深度的变化,图中的“Others”表示除ANME以外的古菌和ASV小于1%的ANME;图b表示D231-4站位中SRB类群随深度的变化,图中的“Others”表示除SRB以外的细菌和ASV小于1%的SRB;图c表示D231站位所有层位样本中ANME和SRB类群的Pearson相关性热图,共使用61个沉积物样品,*表示p<0.05,**表示p<0.01。)

Fig.1 Composition and correlation of ANME and SRB groups in the sediment samples of Formosa cold seep (Figure a illustrates the variation of ANME groups with depth in site D231-4, where “Others” in the figure represents archaea other than ANME and ANME with ASV abundances less than 1%. Figure b depicts the variation of SRB groups with depth in site D231-4, where “Others” denotes bacteria other than SRB and SRB with ASV abundances less than 1%. Figure c presents a Pearson correlation heatmap of ANME and SRB groups across all depth intervals from station D231, based on 61 sediment samples. In the heatmap, * indicates p<0.05, and ** indicates p<0.01.)

图2 Formosa冷泉样本中ANME-SRB菌团的HCR-FISH图像 (图a显示了由古菌和细菌组成的菌团;图b显示了由ANME-2和SRB组成的ANME-SRB菌团;与DSS探针阳性杂交的ANME-SRB菌团中未观察到与靶向ANME-1(图c)或ANME-3(图d)的探针杂交信号。采用寡核苷酸探针DSS658-R和EUB338-R分别靶向SRB类群和细菌,标记为红色;探针ARCH915-H、ANME-1-350-H、ANME-2-538-H和ANME-3-1249-H分别靶向古菌、ANME-1、ANME-2和ANME-3,标记为绿色;DAPI复染用于细胞核染色,显示为蓝色。)

Fig.2 HCR-FISH images of ANME-SRB consortia in the sediment samples of Formosa cold seep (Figure a illustrates microbial consortia composed of archaea and bacteria. Figure b displays ANME-SRB consortia consisting of ANME-2 and sulfate-reducing bacteria (SRB). No hybridization signals were observed for ANME-SRB consortia that were positively hybridized with the DSS probe when probed with ANME-1-targeting (Figure c) or ANME-3-targeting (Figure d) probes. Oligonucleotide probes DSS658-R and EUB338-R were employed to target SRB and bacteria, respectively, and were labeled red. Probes ARCH915-H, ANME-1-350-H, ANME-2-538-H, and ANME-3-1249-H were used to target archaea, ANME-1, ANME-2, and ANME-3, respectively, and were labeled green. DAPI staining was applied for nuclear staining and appeared blue.)

表3 不同深度沉积物中观察到的ANME细胞团数量

Tab.3 The ANME consortia number observed in the sediments at different depths

层位	细胞团数量/个
层位	ANME-1	ANME-2	ANME-3
0~2 cm	0	86	0
4~6 cm	0	71	0
8~10 cm	0	73	0
12~14 cm	0	82	0
16~18 cm	0	78	0
平均	0	78	0

图3 Formosa冷泉样本中菌团大小的组成情况

Fig.3 The composition of the consortia size in the sediment samples of Formosa cold seep

图4 环境因子与菌团大小之间的Pearson相关系数热图

Fig.4 Heatmap of Pearson’s correlation coefficients between environmental factors and consortia sizes

图5 菌团簇的HCR-FISH图像 (菌团与靶向SRB的探针(DSS658-R)阳性杂交,显示为红色; DAPI显示为蓝色。从图a到图c是来自不同z轴的扫描结果。)

Fig.5 The HCR-FISH images of the consortia cluster (The microbial consortia exhibited positive hybridization with the SRB-targeting probe (DSS658-R, red), DAPI staining appeared blue. Figures a to c represent scanning results from different z-axes.)

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