Dataset Information

Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation.

ABSTRACT: The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages.IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities-the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.

SUBMITTER: Bartelme RP

PROVIDER: S-EPMC6992367 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation.

Bartelme Ryan P RP Custer Joy M JM Dupont Christopher L CL Espinoza Josh L JL Torralba Manolito M Khalili Banafshe B Carini Paul P

mSphere 20200129 1

The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized tha ...[more]

PMID: 31996418

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Project description:Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3-20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

Project description:Cultivated bacterioplankton representatives from diverse lineages and locations are essential for microbiology, but the large majority of taxa either remain uncultivated or lack isolates from diverse geographic locales. We paired large-scale dilution-to-extinction (DTE) cultivation with microbial community analysis and modeling to expand the phylogenetic and geographic diversity of cultivated bacterioplankton and to evaluate DTE cultivation success. Here, we report results from 17 DTE experiments totaling 7,820 individual incubations over 3 years, yielding 328 repeatably transferable isolates. Comparison of isolates to microbial community data for source waters indicated that we successfully isolated 5% of the observed bacterioplankton community throughout the study; 43% and 26% of our isolates matched operational taxonomic units and amplicon single-nucleotide variants, respectively, within the top 50 most abundant taxa. Isolates included those from previously uncultivated clades such as SAR11 LD12 and Actinobacteria acIV, as well as geographically novel members from other ecologically important groups like SAR11 subclade IIIa, SAR116, and others, providing isolates in eight putatively new genera and seven putatively new species. Using a newly developed DTE cultivation model, we evaluated taxon viability by comparing relative abundance with cultivation success. The model (i) revealed the minimum attempts required for successful isolation of taxa amenable to growth on our media and (ii) identified possible subpopulation viability variation in abundant taxa such as SAR11 that likely impacts cultivation success. By incorporating viability in experimental design, we can now statistically constrain the effort necessary for successful cultivation of specific taxa on a defined medium.IMPORTANCE Even before the coining of the term "great plate count anomaly" in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the "most wanted" bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress toward answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

Project description:Shallow-sea hydrothermal systems experience continuous fluctuations of physicochemical conditions due to seawater influx which generates variable habitats, affecting the phylogenetic composition and metabolic potential of microbial communities. Until recently, studies of submarine hydrothermal communities have focused primarily on chemolithoautotrophic organisms, however, there have been limited studies on heterotrophic bacteria. Here, fluorescence in situ hybridization, high throughput 16S rRNA gene amplicon sequencing, and functional metagenomes were used to assess microbial communities from the shallow-sea hydrothermal system off Kueishantao Island, Taiwan. The results showed that the shallow-sea hydrothermal system harbored not only autotrophic bacteria but abundant heterotrophic bacteria. The potential for marker genes sulfur oxidation and carbon fixation were detected in the metagenome datasets, suggesting a role for sulfur and carbon cycling in the shallow-sea hydrothermal system. Furthermore, the presence of diverse genes that encode transporters, glycoside hydrolases, and peptidase indicates the genetic potential for heterotrophic utilization of organic substrates. A total of 408 cultivable heterotrophic bacteria were isolated, in which the taxonomic families typically associated with oligotrophy, copiotrophy, and phototrophy were frequently found. The cultivation-independent and -dependent analyses performed herein show that Alphaproteobacteria and Gammaproteobacteria represent the dominant heterotrophs in the investigated shallow-sea hydrothermal system. Genomic and physiological characterization of a novel strain P5 obtained in this study, belonging to the genus Rhodovulum within Alphaproteobacteria, provides an example of heterotrophic bacteria with major functional capacity presented in the metagenome datasets. Collectively, in addition to autotrophic bacteria, the shallow-sea hydrothermal system also harbors many heterotrophic bacteria with versatile genetic potential to adapt to the unique environmental conditions.

Dataset Information

Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation.

Publications

Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation.

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