Network-directed efficient isolation of previously uncultivated Chloroflexi and related bacteria in hot spring microbial mats

Abstract

The perplexity of the complex multispecies community interactions is one of the many reasons why majority of the microorganisms are still uncultivated. We analyzed the entire co-occurrence networks between the OTUs of Tibet and Yunnan hot spring samples, and found that less abundant OTUs such as genus Tepidimonas (relative abundant <1%) had high-degree centricity (key nodes), while dominant OTUs particularly genus Chloroflexus (relative abundant, 13.9%) formed the peripheral vertexes. A preliminary growth-promotion assay determined that Tepidimonas sp. strain SYSU G00190W enhanced the growth of Chloroflexus sp. SYSU G00190R. Exploiting this result, an ameliorated isolation medium containing 10% spent-culture supernatant of Tepidimonas sp. strain SYSU G00190W was prepared for targeted isolation of Chloroflexi in the Tibet and Yunnan hot spring samples. 16S rRNA gene fingerprinting characterized majority of the colonies isolated from these media as previously uncultivated Chloroflexi, of which 36 are potential novel species (16S rRNA sequence identity <98.5%). Metabolomes studies indicated that the spent-culture supernatant comprises several low-molecular-weight organic substrates that can be utilized as potential nutrients for the growth of these bacteria. These findings suggested that limited knowledge on the interaction of microbes provide threshold to traditional isolation method.

From ecophysiology to cultivation methodology: filling the knowledge gap between uncultured and cultured microbes

“…The bacterial diversity profile is highly diverse in marine biofilms, and consists of several unclassified groups, including members of the classes Solibacteres, Acidobacteria, and Blastocatellia (Zhang et al 2019). Moreover, > 70% of the bacterial community in hot spring microbial mats belong to not-yetcultured microbial groups (Xian et al 2020). Similarly, uncultured microbial communities are dominant in biofilms associated with most of other unique and underexplored environments, such as deserts, acid mine drainages, human guts (Amin et al 2020;Lear et al 2009;Lozupone et al 2012;Madinger et al 2016), or even in the largely explored soil ecosystem (Lindahl 1996) where they aggregate and adhere to any available surface.…”

“…3). To date, we have finished the description of at least 500 novel bacterial and archaeal taxa, most of which were recovered by improving isolation procedures, including pre-treatment conditions, media optimization, and designing new isolation methods (Asem et al 2018;Qin et al 2009;Xian et al 2020). These novel microbial taxa consisted of one new class, 23 new orders/sub-orders, 29 new families, and over 80 new genera.…”

“…Moreover, certain strains could not cope with the simulated environment during subsequent cultivation, and thus lose their viability. In recent years, development and advancement of sequencing technology have helped devise new and advanced culturomics approaches (Cross et al 2019;Lagier et al 2016;Xian et al 2020). Despite their limitations, these new developments are expected to provide access to many novel previously uncultivated bacteria, and to divulge their metabolic pathways as well as ecological roles.…”

Diversity, metabolism and cultivation of archaea in mangrove ecosystems

“…Archaea involved in sulfate reduction, such as Bathyarchaeota and Thermoprofundales, could be a target for isolation (Pan et al 2020;Zhou et al 2019). Furthermore, co-occurrence networks are used to analyze potential interactions and seek "key node" microbes in a microbial communities (Xian et al 2020). Co-cultures of Rice Cluster I with propionate-oxidizing H 2 -producing bacterium and Prometheoarchaeum syntrophicum MK-D1 with Methanogenium are two successful examples for microbial interaction (Imachi et al 2020;Sakai et al 2008).…”

Network And Meta-Omics Reveal The Key Degrader And Cooperation Patterns In An Efficient 1,4-Dioxane-Degrading Microbial Community

“…Statistically signi cant associations between different nodes in the resulting network can be the guidance of identifying probable cooperative bacteria. Recent studies have proved the feasibility of predictions based on networks with the isolation of previously uncultivated bacteria [10] and cooperative bacteria for pollutant degradation [11].…”