Rubisco function, evolution, and engineering

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin Benson Bassham (CBB) cycle is the dominant carbon fixation pathway on earth, driving >99.5% of the ~120 billion tons of carbon that are "fixed" as sugar, by plants, algae and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast and it is a bottleneck in flux through the pathway. This presents a paradox - why hasn't rubisco evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs, and that rubisco variants have been optimized for their native physiological environment. Here we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and, thereby, promote plant growth

Minimal cobalt metabolism in the marine cyanobacterium Prochlorococcus.

Despite very low concentrations of cobalt in marine waters, cyanobacteria in the genus Prochlorococcus retain the genetic machinery for the synthesis and use of cobalt-bearing cofactors (cobalamins) in their genomes. We explore cobalt metabolism in a Prochlorococcus isolate from the equatorial Pacific Ocean (strain MIT9215) through a series of growth experiments under iron- and cobalt-limiting conditions. Metal uptake rates, quantitative proteomic measurements of cobalamin-dependent enzymes, and theoretical calculations all indicate that Prochlorococcus MIT9215 can sustain growth with less than 50 cobalt atoms per cell, ∼100-fold lower than minimum iron requirements for these cells (∼5,100 atoms per cell). Quantitative descriptions of Prochlorococcus cobalt limitation are used to interpret the cobalt distribution in the equatorial Pacific Ocean, where surface concentrations are among the lowest measured globally but Prochlorococcus biomass is high. A low minimum cobalt quota ensures that other nutrients, notably iron, will be exhausted before cobalt can be fully depleted, helping to explain the persistence of cobalt-dependent metabolism in marine cyanobacteria

The Bioinformatics Virtual Coordination Network: An open-source and interactive learning environment

Lockdowns and “stay-at-home” orders, starting in March 2020, shuttered bench and field dependent research across the world as a consequence of the global COVID-19 pandemic. The pandemic continues to have an impact on research progress and career development, especially for graduate students and early career researchers, as strict social distance limitations stifle ongoing research and impede in-person educational programs. The goal of the Bioinformatics Virtual Coordination Network (BVCN) was to reduce some of these impacts by helping research biologists learn new skills and initiate computational projects as alternative ways to carry out their research. The BVCN was founded in April 2020, at the peak of initial shutdowns, by an international group of early-career microbiology researchers with expertise in bioinformatics and computational biology. The BVCN instructors identified several foundational bioinformatic topics and organized hands-on tutorials through cloud-based platforms that had minimal hardware requirements (in order to maximize accessibility) such as RStudio Cloud and MyBinder. The major topics included the Unix terminal interface, R and Python programming languages, amplicon analysis, metagenomics, functional protein annotation, transcriptome analysis, network science, and population genetics and comparative genomics. The BVCN was structured as an open-access resource with a central hub providing access to all lesson content and hands-on tutorials (https://biovcnet.github.io/). As laboratories reopened and participants returned to previous commitments, the BVCN evolved: while the platform continues to enable “a la carte” lessons for learning computational skills, new and ongoing collaborative projects were initiated among instructors and participants, including a virtual, open-access bioinformatics conference in June 2021. In this manuscript we discuss the history, successes, and challenges of the BVCN initiative, highlighting how the lessons learned and strategies implemented may be applicable to the development and planning of future courses, workshops, and training programs

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria

Borgs are giant genetic elements with potential to expand metabolic capacity

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as ‘Borgs’. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.ISSN:0028-0836ISSN:1476-468

Borgs are giant genetic elements with potential to expand metabolic capacity

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as 'Borgs'. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance

Patterns of Gene Content and Co-occurrence Constrain the Evolutionary Path toward Animal Association in Candidate Phyla Radiation Bacteria

ABSTRACT Candidate Phyla Radiation (CPR) bacteria are small, likely episymbiotic organisms found across Earth’s ecosystems. Despite their prevalence, the distribution of CPR lineages across habitats and the genomic signatures of transitions among these habitats remain unclear. Here, we expand the genome inventory for Absconditabacteria (SR1), Gracilibacteria, and Saccharibacteria (TM7), CPR bacteria known to occur in both animal-associated and environmental microbiomes, and investigate variation in gene content with habitat of origin. By overlaying phylogeny with habitat information, we show that bacteria from these three lineages have undergone multiple transitions from environmental habitats into animal microbiomes. Based on co-occurrence analyses of hundreds of metagenomes, we extend the prior suggestion that certain Saccharibacteria have broad bacterial host ranges and constrain possible host relationships for Absconditabacteria and Gracilibacteria. Full-proteome analyses show that animal-associated Saccharibacteria have smaller gene repertoires than their environmental counterparts and are enriched in numerous protein families, including those likely functioning in amino acid metabolism, phage defense, and detoxification of peroxide. In contrast, some freshwater Saccharibacteria encode a putative rhodopsin. For protein families exhibiting the clearest patterns of differential habitat distribution, we compared protein and species phylogenies to estimate the incidence of lateral gene transfer and genomic loss occurring over the species tree. These analyses suggest that habitat transitions were likely not accompanied by large transfer or loss events but rather were associated with continuous proteome remodeling. Thus, we speculate that CPR habitat transitions were driven largely by availability of suitable host taxa and were reinforced by acquisition and loss of some capacities. IMPORTANCE Studying the genetic differences between related microorganisms from different environment types can indicate factors associated with their movement among habitats. This is particularly interesting for bacteria from the Candidate Phyla Radiation because their minimal metabolic capabilities require associations with microbial hosts. We found that shifts of Absconditabacteria, Gracilibacteria, and Saccharibacteria between environmental ecosystems and mammalian mouths/guts probably did not involve major episodes of gene gain and loss; rather, gradual genomic change likely followed habitat migration. The results inform our understanding of how little-known microorganisms establish in the human microbiota where they may ultimately impact health

Asgard archaea modulate potential methanogenesis substrates in wetland soil

The roles of Asgard archaea in eukaryogenesis and marine biogeochemical cycles are well studied, yet their contributions in soil ecosystems are unknown. Of particular interest are Asgard archaeal contributions to methane cycling in wetland soils. To investigate this, we reconstructed two complete genomes for soil-associated Atabeyarchaeia, a new Asgard lineage, and the first complete genome of Freyarchaeia, and defined their metabolism in situ. Metatranscriptomics highlights high expression of [NiFe]-hydrogenases, pyruvate oxidation and carbon fixation via the Wood-Ljungdahl pathway genes. Also highly expressed are genes encoding enzymes for amino acid metabolism, anaerobic aldehyde oxidation, hydrogen peroxide detoxification and glycerol and carbohydrate breakdown to acetate and formate. Overall, soil-associated Asgard archaea are predicted to be non-methanogenic acetogens, likely impacting reservoirs of substrates for methane production in terrestrial ecosystems