Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria

Rygaard, Anita Mac; Thøgersen, Mariane Schmidt; Nielsen, Kristian Fog; Gram, Lone; Bentzon-Tilia, Mikkel

doi:10.1128/aem.00243-17

Cited by 39 publications

(43 citation statements)

References 59 publications

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“…Molecular surveys have shown that sponges host rich microbial communities (40, 41); however, only a minor component of this richness has been cultivated via the conventional cultivation method (32). Extensive cultivation efforts have made use of several alternative techniques to isolate previously uncultured sponge-associated bacteria, including adding the sponge extracts or its skeleton to the media (42, 43), using oligotrophic media (36), adding antibiotics to inhibit fast-growing bacteria (44), using alternative gelling agent (45), and using in situ diffusion devices (21, 22) and floating filters (42). These alternative approaches yielded an increased novelty of sponge isolates and improved cultivability rates up to 14% in some cases (42).…”

Section: Discussionmentioning

confidence: 99%

Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Jung

Machida

Nakao

et al. 2019

Preprint

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Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situcultivation) is an effective approach. In the present study, we appliedin situcultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method’s efficiencies with those of the conventional method. Then, we attempted to clarify the key and unknown mechanism ofin situcultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring “growth initiation factor” for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated viain situcultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived fromin situand SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of thein situstrains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on thein situstrains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).ImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situcultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.

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Section: Discussionmentioning

confidence: 99%

Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Jung

Machida

Nakao

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Media were solidified using 0.75% (w/v) gellan gum (GELRITE, Carl Roth, Karlsruhe, DE), since agar has been shown before to impair the cultivability of environmental bacteria (Narihiro and Kamagata, 2013;Tanaka et al, 2014;Rygaard et al, 2017). Before autoclaving the pH was set to between 8 and 8.5, to achieve a final pH of approximately 8.…”

Section: (A) Platesmentioning

confidence: 99%

Cultivation of Bacteria From Aplysina aerophoba: Effects of Oxygen and Nutrient Gradients

et al. 2020

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Sponge-associated bacteria possess biotechnologically interesting properties but as yet have largely evaded cultivation. Thus, "omics"-based information on the ecology and functional potential of sponge symbionts is awaiting its integration into the design of innovative cultivation approaches. To cultivate bacteria derived from the marine sponge Aplysina aerophoba, nine novel media formulations were created based on the predicted genomic potential of the prevalent sponge symbiont lineage Poribacteria. In addition, to maintain potential microbial metabolic interactions in vitro, a Liquid-Solid cultivation approach and a Winogradsky-column approach were applied. The vast majority of microorganisms in the inoculum appeared viable after cryopreservation of sponge specimen as determined by selective propidium monoazide DNA modification of membrane-compromised cells, however, only 2% of the initial prokaryotic diversity could be recovered through cultivation. In total, 256 OTUs encompassing seven prokaryotic phyla were cultivated. The diversity of the cultivated community was influenced by the addition of the antibiotic aeroplysinin-1 as well as by medium dilution, rather than carbon source. Furthermore, the Winogradsky-column approach reproducibly enriched distinct communities at different column depths, amongst which were numerous Clostridia and OTUs that could not be assigned to a known phylum. While some bacterial taxa such as Pseudovibrio and Ruegeria were recovered from nearly all applied cultivation conditions, others such as Bacteroidetes were specific to certain medium types. Predominant sponge-associated prokaryotic taxa remained uncultured, nonetheless, alternative cultivation approaches applied here enriched for previously uncultivated microbes.

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“…Only about 1% of marine bacteria can be easily studied using standard laboratory procedures [ 53 ]. This is a major drawback for the understanding of how those microorganisms interact.…”

Section: Examples Of Wto Network Using the Wto R Packagementioning

confidence: 99%

wTO: an R package for computing weighted topological overlap and a consensus network with integrated visualization tool

et al. 2018

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BackgroundNetwork analyses, such as of gene co-expression networks, metabolic networks and ecological networks have become a central approach for the systems-level study of biological data. Several software packages exist for generating and analyzing such networks, either from correlation scores or the absolute value of a transformed score called weighted topological overlap (wTO). However, since gene regulatory processes can up- or down-regulate genes, it is of great interest to explicitly consider both positive and negative correlations when constructing a gene co-expression network.ResultsHere, we present an R package for calculating the weighted topological overlap (wTO), that, in contrast to existing packages, explicitly addresses the sign of the wTO values, and is thus especially valuable for the analysis of gene regulatory networks. The package includes the calculation of p-values (raw and adjusted) for each pairwise gene score. Our package also allows the calculation of networks from time series (without replicates). Since networks from independent datasets (biological repeats or related studies) are not the same due to technical and biological noise in the data, we additionally, incorporated a novel method for calculating a consensus network (CN) from two or more networks into our R package. To graphically inspect the resulting networks, the R package contains a visualization tool, which allows for the direct network manipulation and access of node and link information. When testing the package on a standard laptop computer, we can conduct all calculations for systems of more than 20,000 genes in under two hours. We compare our new wTO package to state of art packages and demonstrate the application of the wTO and CN functions using 3 independently derived datasets from healthy human pre-frontal cortex samples. To showcase an example for the time series application we utilized a metagenomics data set.ConclusionIn this work, we developed a software package that allows the computation of wTO networks, CNs and a visualization tool in the R statistical environment. It is publicly available on CRAN repositories under the GPL −2 Open Source License (https://cran.r-project.org/web/packages/wTO/).

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Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria

Cited by 39 publications

References 59 publications

Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Cultivation of Bacteria From Aplysina aerophoba: Effects of Oxygen and Nutrient Gradients

wTO: an R package for computing weighted topological overlap and a consensus network with integrated visualization tool

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