A non-amplified FISH protocol to identify simultaneously different bacterial groups attached to eukaryotic phytoplankton

Cruz-López, Ricardo; Maske, Helmut

doi:10.1007/s10811-014-0379-2

Cited by 6 publications

(9 citation statements)

References 39 publications

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“…All in situ hybridizations were performed as described in Cruz-López and Maske (2014) . In brief, before hybridization, bacterial cells were partially digested with 400,000 U ml –1 lysozyme (Sigma, L6876) dissolved in buffer containing 100 mM Tris-HCl, 50 mM EDTA, pH 8.0 for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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The Vitamin B1 and B12 Required by the Marine Dinoflagellate Lingulodinium polyedrum Can be Provided by its Associated Bacterial Community in Culture

Cruz-López

Maske

2016

Front. Microbiol.

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In this study we established the B1 and B12 vitamin requirement of the dinoflagellate Lingulodinium polyedrum and the vitamin supply by its associated bacterial community. In previous field studies the B1 and B12 demand of this species was suggested but not experimentally verified. When the axenic vitamin un-supplemented culture (B-ns) of L. polyedrum was inoculated with a coastal bacterial community, the dinoflagellate’s vitamin growth limitation was overcome, reaching the same growth rates as the culture growing in vitamin B1B7B12-supplemented (B-s) medium. Measured B12 concentrations in the B-s and B-ns cultures were both higher than typical coastal concentrations and B12 in the B-s culture was higher than in the B-ns culture. In both B-s and B-ns cultures, the probability of dinoflagellate cells having bacteria attached to the cell surface was similar and in both cultures an average of six bacteria were attached to each dinoflagellate cell. In the B-ns culture the free bacterial community showed significantly higher cell abundance suggesting that unattached bacteria supplied the vitamins. The fluorescence in situ hybridization (FISH) protocol allowed the quantification and identification of three bacterial groups in the same samples of the free and attached epibiotic bacteria for both treatments. The relative composition of these groups was not significantly different and was dominated by Alphaproteobacteria (>89%). To complement the FISH counts, 16S rDNA sequencing targeting the V3–V4 regions was performed using Illumina-MiSeq technology. For both vitamin amendments, the dominant group found was Alphaproteobacteria similar to FISH, but the percentage of Alphaproteobacteria varied between 50 and 95%. Alphaproteobacteria were mainly represented by Marivita sp., a member of the Roseobacter clade, followed by the Gammaproteobacterium Marinobacter flavimaris. Our results show that L. polyedrum is a B1 and B12 auxotroph, and acquire both vitamins from the associated bacterial community in sufficient quantity to sustain the maximum growth rate.

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Section: Methodsmentioning

confidence: 99%

“…The images were processed in ImageJ ( Schneider et al, 2012 ). For each spectral channel a summary image was composed by selecting the pixels of maximum intensity within the stack, and using the ‘Find Edges’ and ‘Despeckle’ functions to reduce images noise ( Cruz-López and Maske, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

The Vitamin B1 and B12 Required by the Marine Dinoflagellate Lingulodinium polyedrum Can be Provided by its Associated Bacterial Community in Culture

Cruz-López

Maske

2016

Front. Microbiol.

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“…This observation was further confirmed by direct addition of purified VF into the L. polyedrum culture which growth increased 1.5 times more than those without VF. These results imply that the presence of the siderophore vibrioferrin clearly influences L. polyedrum growth, but that other factors such as vitamin B12 (Cruz-Lopez and Maske, 2015) provided by bacteria may also be important in promoting this apparent bacterial-algal mutualism.…”

Section: Discussionmentioning

confidence: 83%

Iron and Harmful Algae Blooms: Potential Algal-Bacterial Mutualism Between Lingulodinium polyedrum and Marinobacter algicola

2018

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Phytoplankton blooms can cause acute effects on marine ecosystems either due to their production of endogenous toxins or due to their enormous biomass leading to major impacts on local economies and public health. Despite years of effort, the causes of harmful algal blooms (HAB) are still not fully understood. Our hypothesis is that bacteria that produce photoactive siderophores may provide a bioavailable form of iron to commensally associated phytoplankton, which could in turn affect algal growth and bloom dynamics. Here we report a laboratory-based study of binary cultures of the dinoflagellate Lingulodinium polyedrum, a major HAB species, with Marinobacter algicola DG893, a phytoplankton-associated bacterium that produces the photoactive siderophore vibrioferrin. Comparing binary cultures of L. polyedrum with both the wild type and the vibrioferrin minus mutant of M. algicola shows that bacteria are necessary to promote dinoflagellate growth and that this growth promotion effect is at least partially related to the ability of the bacterium to supply bioavailable iron via the siderophore vibrioferrin. These results support the notion of a carbon for iron mutualism in some bacterial-algal interactions.

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“…Fluorescence in situ hybridization (FISH) based methods reveal spatial distribution of interacting partners, for instance showing stratification and co-aggregation patterns in biofilms [53] or bacterial groups attached to phytoplankton host [54]. In addition to resolving spatial structure, imaging, for example, based on fluorescent dyes, can be used to assess general metabolic state of community members [55,56 ].…”

Section: Isotope Labeling For Tracing Community-scale Pathwaysmentioning

confidence: 99%

“…Microscopic detection of these structures can be a powerful tool in identifying interacting microorganisms. Illustrative is an example of nanotubes formed by cross-feeding Escherichia coli auxotrophs [51] or variety of formations in acid mine drainage community, such as cytoplasmic bridges, pili, and 'synaps like connections' [52].Fluorescence in situ hybridization (FISH) based methods reveal spatial distribution of interacting partners, for instance showing stratification and co-aggregation patterns in biofilms [53] or bacterial groups attached to phytoplankton host [54]. In addition to resolving spatial structure, imaging, for example, based on fluorescent dyes, can be used to assess general metabolic state of community members [55,56 ].…”

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confidence: 99%

Metabolic interactions in microbial communities: untangling the Gordian knot

Ponomarova

Patil

2015

Current Opinion in Microbiology

229

177

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Metabolic exchanges are ubiquitous in microbial communities. However, detecting metabolite cross-feedings is difficult due to their intrinsically dynamic nature and the complexity of communities. Thus, while exhaustive description of metabolic networks operating in natural systems is a task for the future, the battle of today is divided between detailed characterizations of small, reduced complexity microbial consortia, and focusing on particular metabolic aspects of natural ecosystems. Detecting metabolic interactions requires methodological blend able to capture species identity, dependencies and the nature of exchanged metabolites. Multiple combinations of diverse techniques, from metagenomics to imaging mass spectrometry, offer solutions to this challenge, each combination being tailored to the community at hand.

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A non-amplified FISH protocol to identify simultaneously different bacterial groups attached to eukaryotic phytoplankton

Cited by 6 publications

References 39 publications

The Vitamin B1 and B12 Required by the Marine Dinoflagellate Lingulodinium polyedrum Can be Provided by its Associated Bacterial Community in Culture

The Vitamin B1 and B12 Required by the Marine Dinoflagellate Lingulodinium polyedrum Can be Provided by its Associated Bacterial Community in Culture

Iron and Harmful Algae Blooms: Potential Algal-Bacterial Mutualism Between Lingulodinium polyedrum and Marinobacter algicola

Metabolic interactions in microbial communities: untangling the Gordian knot

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