Genetic tool development in marine protists: Emerging model organisms for experimental cell biology

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Bidirectional redox cycling of phenazine-1-carboxylic acid by Citrobacter portucalensis MBL drives increased nitrate reduction

Phenazines are secreted metabolites that microbes use in diverse ways, from quorum sensing to antimicrobial warfare to energy conservation. Phenazines are able to contribute to these activities due to their redox activity. The physiological consequences of cellular phenazine reduction have been extensively studied, but the counterpart phenazine oxidation has been largely overlooked. Phenazine-1-carboxylic acid (PCA) is common in the environment and readily reduced by its producers. Here, we describe its anaerobic oxidation by Citrobacter portucalensis strain MBL, which was isolated from topsoil in Falmouth, MA, and which does not produce phenazines itself. This activity depends on the availability of a suitable terminal electron acceptor, specifically nitrate or fumarate. When C. portucalensis MBL is provided reduced PCA and either nitrate or fumarate, it continuously oxidizes the PCA. We compared this terminal electron acceptor-dependent PCA-oxidizing activity of C. portucalensis MBL to that of several other γ-proteobacteria with varying capacities to respire nitrate and/or fumarate. We found that PCA oxidation by these strains in a fumarate-or nitrate-dependent manner is decoupled from growth and correlated with their possession of the fumarate or periplasmic nitrate reductases, respectively. We infer that bacterial PCA oxidation is widespread and genetically determined. Notably, reduced PCA enhances the rate of nitrate reduction to nitrite by C. portucalensis MBL beyond the stoichiometric prediction, which we attribute to C. portucalensis MBL’s ability to also reduce oxidized PCA, thereby catalyzing a complete PCA redox cycle. This bidirectionality highlights the versatility of PCA as a biological redox agent

The Discovery and Biological Mechanisms of a Widespread Phenazine's Oxidation

During the 2017 Microbial Diversity course at the Marine Biological Laboratory in Woods Hole, MA, Scott Saunders and Yinon Bar-On started enrichment cultures in hopes of dis-covering biological oxidation of phenazine-1-carboxylic acid (PCA). I took these enrich-ment cultures and described their PCA oxidation activity. From one of the mixed cultures, I isolated a bacterial strain that recapitulated the behavior of the enrichment. I identified it as a strain of Citrobacter portucalensis via a whole-genome analysis and called the strain "MBL" in reference to the Marine Biological Laboratory. Using a combination of analytical chemistry, quantitative fluorescence measurements, and genetic engineering, I showed that C. portucalensis MBL couples PCA oxidation to each mode of anaerobic respiration it employs with nitrate, fumarate, dimethyl sulfoxide (DMSO), and trimethylamine-N-oxide (TMAO) as terminal electron acceptors (TEAs). I further found that most of the PCA oxidation activi-ty depends on electron flux through the quinone/quinol pool but can be driven by certain terminal reductase complexes when no quinones are available, particularly in the case of ni-trate reductases. Every bacterial strain I tested catalyzed PCA oxidation when provided the appropriate TEA. My described mechanism for bacterial PCA oxidation is generalizable and implies that this previously undocumented phenomenon should occur wherever PCA is produced in rhizosphere environments.</p

Genetic tool development in marine protists: Emerging model organisms for experimental cell biology

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Draft Genome Sequence of the Redox-Active Enteric Bacterium Citrobacter portucalensis Strain MBL

We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid

Draft Genome Sequence of the Redox-Active Enteric Bacterium Citrobacter portucalensis Strain MBL

We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

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Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways