Isolation of selective microorganisms from hot spring microbial mats using co-culturing strategy: a perspective of microbial interaction

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Microorganisms are microscopic ‘little wonder’ with the widest living range and the most abundant biological diversity on the Earth. They are important material basis for human survival and important source for biotechnology innovation. However, due to the limited knowledge on the majority of uncultivated microbial species, the “Great Plate Count Anomaly” [1] is still a puzzle, and hitherto uncultured microorganisms are vividly imaged as microbial “dark matters” [2]. We, however, do understand that these “dark matters” play unprecedented roles in carbon and nitrogen cycling, in novel natural products chemistry and in maintaining the balance of the environment. Culturing and effectively describing the vast majority of microorganisms have always been a challenging task in microbiological research, as biologists often say, “To really know them, you have to grow them” [3].

In our new study, a total of 26 hot spring microbial mat (HSMM) samples were collected from Yunnan and Tibet hot springs. Relying on the 16S rRNA gene high-throughput sequencing data, a co-occurrence network analysis was performed to understand the interactions between the microbial communities. A targeted selection for member of the phylum Chloroflexi-Thermus­ indicated genera Chloroflexus (13.9%), Thermus (11.05%) and Roseiflexus (8.44%) occupy the peripheral nodes, while members with low abundance formed the key nodes. Among these key nodes, genera Tepidimonas, Geobacillus, Meiothermus and Sphingomonas have been previously isolated. After a preliminary screening test, strain Tepidimonas SYSU G00190W (190W) was confirmed as a growth promoting strain that particularly activate the growth of tested Chloroflexi strains. Therefore, spent culture medium (SCM) based on 190W was prepared to selectively target the isolation and cultivation of Chloroflexi (Fig. 1). The medium was found to be particularly useful for isolation of many previously uncultivated Chloroflexi, and the newly isolated strains include putativenovel isolates related to genera Roseiflexus and Chloroflexus while two strain formed distinct lineages within the phylum Chloroflexi.

Fig. 1. Overview of the sampling sites and the workflow for the network-directed isolation procedure from HSMMs. (a) Location of the sampling sites; (b) Representative samples (Y1, Y2 and Y3 from Yunnan, and A96 from Tibet hot springs) that are used for isolation; (c) Workflow use to predict key-node taxa and screening for growth-promoting strains. SCM plate was effective in the isolation of targeted microorganisms from the HSMMs.

As the growth-promotion on Chloroflexi spp. was observed with SCM, we hypothesized that Tepidimonas sp. may have strong ability to synthesize and excrete extracellular metabolites that facilitate the growth of Chloroflexi strains. Subsequently, a non-targeted extracellular metabolomes analysis was conducted on 190W. 83 low molecular weight organic substances (LMWOS) were detected among the metabolites that are significantly accumulated during its growth, Representative LMWOS was further tested on the newly isolated strains, result showed that pantothenic acid and 3-indoleacetic acid facilitate growth of all the tested strains, imidazole acetic acid and deoxycytidine showed enhance growth to Chloroflexus strains while putrescine improved the growth of Roseoflexus strains, Further, uracil, cytosine and adenine facilitate the growth of other Chloroflexi .

Our work provides strong experimental evidence that key node bacteria is critical for rapid and easy cultivation of hard and/or slow-growing strains from HSMMs in the laboratory condition. Our study further provides an idea for cultivating of unculturable microorganisms in other environments.

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The research team of Prof. Wen-Jun Li at School of Life Sciences of Sun Yat-Sen University have been focusing on the extremophiles and their ecology for more than twenty years. Details of our previous researches could be found with following information.

Insights into ecological roles and evolution of methyl coenzyme M reductase containing hot spring Archaea.

Genomic inference of the metabolism and evolution of the archaeal phylum Aigarchaeota

Discovery of Druggability-Improved Analogues by Investigation of LL-D49194α1 Biosynthetic Pathway.

Discovery and Biosynthesis of Atrovimycin, an Antitubercular and Antifungal Cyclodepsipeptide Featu-ring Vicinal-dihydroxylated Cinnamic Acyl Chain.

Update on the classification of higher ranks in the phylum Actinobacteria.

[1] Staley, J. T. & Konopka, A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Ann. Rev. Microbiol. 39, 321-346 (1985)

[2] Lok, C. Mining the microbial dark matter. Nature 522, 270-273 (2015).

[3] Charnock C, Tunsjø H, Hjeltnes B. To really know them you have to grow them. Bioingeniøren 8, 18-23 ( 2017).

Wen-Jun Li

Professor, Sun Yat-Sen University