Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media

Pulschen, André Arashiro; Bendia, Amanda Gonçalves; Fricker, Ashwana D.; Pellizari, Vivian H.; Galante, Douglas; Rodrigues, Fábio

doi:10.3389/fmicb.2017.01346

Cited by 72 publications

(58 citation statements)

References 84 publications

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“…The significantly lower cell counts as determined by the plate count method are possibly because some members of the hemolymph microorganisms failed to grow on the 2216E medium. This could be probably caused by unfavorable energy or nutrient supplementation (20), the absence of certain growth signals (31), insufficient incubation time (32), inhibition by bacteriocin-producing microorganisms (33), or dependence on other microorganisms (34). Moreover, some slow-growing or oligotrophic microorganisms in the hemolymph may form minicolonies which could not be observed with the naked eye on the agar plate (35).…”

Section: Discussionmentioning

confidence: 99%

Hemolymph Microbiomes of Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method

Zhang

Sun

Zhang

et al. 2018

Appl Environ Microbiol

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Symbiotic microorganisms have been found in the hemolymph (blood) of many aquatic invertebrates, such as crabs, shrimps and oysters. Hemolymph is a critical site in host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies using selective media (e.g., TCBS, 2216E, and LB) for enumerating and isolating microbial cells. However, doubts remain about the "true" representation of the microbial abundance and diversity of symbiotic microorganisms in hemolymph, particularly for uncultivable microorganisms which are believed to be more abundant than the cultured. To explore this, we developed a culture-independent cell extraction method for separating microbial cells from the hemolymph of three aquatic invertebrates (, , and) involving filtration through a 5-μm mesh filter membrane (the Filtration Method). A combination of the Filtration Method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. More than 2.6 × 10 cells/mL of microbial cells dominated by and, and, and and, were detected in the hemolymph of ,, and , respectively. A parallel study for investigating the hemolymph microbiome by comparing the Filtration Method and a culture-dependent method (the Plate Count Method), showed significantly higher microbial abundances (between 26 and 369-folds difference; P<0.05) and less biased community structures of the Filtration Method than those of the Plate Count Method. Furthermore, hemolymph of the three invertebrates harbored many potential pathogens, including ,, and Finally, the Filtration Method provides a solution that improves understanding of the metabolic functions of uncultivable hemolymph microorganisms (e.g., metagenomics) devoid of host hemocytes contamination. Microorganisms are found in invertebrates' hemolymph, a critical site in host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies. However, doubts remain about the "true" representation of hemolymph microbiome. This study developed a culture-independent cell extraction method that could separate microbial cells from the hemolymph of three aquatic invertebrates (, , and) based on filtration through a 5-μm mesh filter membrane (the Filtration Method). A combination of the Filtration Method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. Our results demonstrate that the hemolymph of aquatic invertebrates harbors a much higher microbial abundance and distinct microbial community composition than previously estimated. Furthermore, this work provides a less biased solution for studying the metabolic functions of uncultivable hemolymph microbiota devoid of host hemocytes contamination.

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Section: Discussionmentioning

confidence: 99%

Hemolymph Microbiomes of Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method

Zhang

Sun

Zhang

et al. 2018

Appl Environ Microbiol

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“…In Antarctic soils, the percentage of yet-uncultured genera is expected to be much higher [14] than the 82% described for general soils [15]. A considerable fraction of such uncultured bacteria corresponds to members of the NP-rich Actinobacteria [16,17] and Cyanobacteria [18,19] phyla.King George Island is one of the largest ice-free and biodiverse areas in maritime Antarctica [20]. It harbors a high density of scientific stations, particularly in Maxwell Bay [21], where most of terrestrial biology research takes place.…”

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Diversity of Bacterial Biosynthetic Genes in Maritime Antarctica

et al. 2020

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Bacterial natural products (NPs) are still a major source of new drug leads. Polyketides (PKs) and non-ribosomal peptides (NRP) are two pharmaceutically important families of NPs and recent studies have revealed Antarctica to harbor endemic polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, likely to be involved in the production of novel metabolites. Despite this, the diversity of secondary metabolites genes in Antarctica is still poorly explored. In this study, a computational bioprospection approach was employed to study the diversity and identity of PKS and NRPS genes to one of the most biodiverse areas in maritime Antarctica-Maxwell Bay. Amplicon sequencing of soil samples targeting ketosynthase (KS) and adenylation (AD) domains of PKS and NRPS genes, respectively, revealed abundant and unexplored chemical diversity in this peninsula. About 20% of AD domain sequences were only distantly related to characterized biosynthetic genes. Several PKS and NRPS genes were found to be closely associated to recently described metabolites including those from uncultured and candidate phyla. The combination of new approaches in computational biology and new culture-dependent and -independent strategies is thus critical for the recovery of the potential novel chemistry encoded in Antarctica microorganisms. target the highly conserved ketosynthase (KS) and adenylation (AD) domains of PKS and NRPS genes, respectively. A growing number of studies have employed the PCR-based sequence tag approach to uncover PKS and NRPS gene diversity across different environmental microbiomes [7][8][9].Extreme environments, such as Antarctica, have the potential to reveal novel molecules since they are still a poorly explored source of chemical scaffolds and novel chemical diversity [10,11]. As such, metabarcoding approaches to study PKS and NRPS diversity in Antarctic ecosystems represent a simple and effective strategy to understand the biosynthetic potential of a large number of sampling sites [7]. A couple of such studies have been carried out very recently in Antarctica [12,13] revealing that soils at different Antarctica locations harbor multiple endemic PKS and NRPS genes, likely to be involved in the synthesis of new chemical diversity. In Antarctic soils, the percentage of yet-uncultured genera is expected to be much higher [14] than the 82% described for general soils [15]. A considerable fraction of such uncultured bacteria corresponds to members of the NP-rich Actinobacteria [16,17] and Cyanobacteria [18,19] phyla.King George Island is one of the largest ice-free and biodiverse areas in maritime Antarctica [20]. It harbors a high density of scientific stations, particularly in Maxwell Bay [21], where most of terrestrial biology research takes place. Although bacterial diversity studies have been conducted in this area [22,23], the diversity of bacterial biosynthetic genes has not been a target of study. Here, we perform a detailed study on soil samples from Maxwell Bay, with the main goal of assessing the...

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“…Suplementar o meio de cultivo com ácido nalidíxico reduz o crescimento de bactérias não filamentosas e favorece a seleção de linhagens produtoras de compostos bioativos (CHU; KHOO;CHEAH, 2017;DEMAIN, 2013;BALTZ, 2007). A utilização dos usuais meios de cultivo, em baixas concentrações possibilita o isolamento de grupos estritamente oligotróficos e reduz a competição por espaço, decorrente ao crescimento de grupos bacterianos de crescimento rápido (PULSCHEN et al, 2017;WANG et al, 2014).…”

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“…., 2017), como por exemplo o Continente Antártico. O artigo "Isolation of uncultured bacteria from Antarctica using long incubation periods and low nutrition media"(PULSCHEN et al, 2017) demonstra claramente o potencial da região à descoberta de novos grupos bacterianos (familia, gênero e espécie).Streptomyces alni, equivalente a 28 diferenças nucleotídicas (dif/nt). Streptomyces alni NRRL B-24611 T foi descrita em 2009, através dos esforços de prospecção entre o grupo de pesquisa da Universidade de Beijing e o laboratório farmacêutico Zhejiang Hisun.…”

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Actinobactérias da Antártica produtoras de compostos anticâncer

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Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media

Cited by 72 publications

References 84 publications

Hemolymph Microbiomes of Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method

Hemolymph Microbiomes of Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method

Diversity of Bacterial Biosynthetic Genes in Maritime Antarctica

Actinobactérias da Antártica produtoras de compostos anticâncer

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