isolation-characterization-cyanobacterial-extracellular

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Wellesley College

Due to a typesetting error, 25 rows were omitted from Table 3 in the original version of this Data Descriptor. These missing rows correspond to the following sample names.

Publisher Correction: Marine microbial metagenomes sampled across space and time.

Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.

Charting the Complexity of the Marine Microbiome through Single-Cell Genomics.

As the smallest and most abundant primary producer in the oceans, the cyanobacterium  is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for  over the years; all have been unsuccessful to date, despite some success with their close relative,  . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into  cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

Toward a genetic system in the marine cyanobacterium .

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus  was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally.

Deep-sea microbes as tools to refine the rules of innate immune pattern recognition.

Small subunit rRNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly used primers with >300 million rRNA gene sequences retrieved from globally distributed marine metagenomes. The best-performing primers compared to 16S rRNA of bacteria and archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering cyanobacterial and chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA specific; 82%)-demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median  = 0.98,  = 272), indicating amplicons can produce equally accurate community composition data compared with shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly completely universal primer set that could accurately quantify biogeochemically important taxa in ecosystems ranging from the deep sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data.  PCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes. Our results demonstrate that primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is theoretically possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides, and present a software pipeline that can guide rational design of primers for any environment with available meta'omic data.

Evaluating and Improving Small Subunit rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys.

The picocyanobacteria  and  are found throughout the ocean's euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this region, depriving them of light for extended periods of time. Here, we demonstrate that members of these genera can adapt to tolerate repeated periods of light energy deprivation. Strains kept in the dark for 3 d and then returned to the light initially required 18-26 d to resume growth, but after multiple rounds of dark exposure they began to regrow after only 1-2 d. This dark-tolerant phenotype was stable and heritable; some cultures retained the trait for over 132 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of  NATL2A, indicating that an epigenetic change is likely responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation-one potential mechanism mediating epigenetic inheritance in bacteria-occurs in . LC-MS/MS analysis showed that while DNA methylations, including 6 mA and 5 mC, are found in some other  strains, there were no methylations detected in either the parental or dark-tolerant NATL2A strains. These findings suggest that  utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation, and highlights phenotypic heterogeneity as an additional dimension of  diversity.

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation.

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species.

Article	Year
Publisher Correction: Marine microbial metagenomes sampled across space and time. Scientific data\| PubMed ID: 31113983	2019
Charting the Complexity of the Marine Microbiome through Single-Cell Genomics. Cell\| PubMed ID: 31835036	2019
Toward a genetic system in the marine cyanobacterium . Access microbiology\| PubMed ID: 33005871	2020
Deep-sea microbes as tools to refine the rules of innate immune pattern recognition. Science immunology\| PubMed ID: 33712473	2021
Evaluating and Improving Small Subunit rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys. mSystems\| PubMed ID: 34060911	2021
Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation. Limnology and oceanography\| PubMed ID: 34690365	2021
Prochlorococcus extracellular vesicles: molecular composition and adsorption to diverse microbes. Environmental microbiology\| PubMed ID: 34766712	2021

Steven J. Biller

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