Biology subjects

Savage, D. F.

Publications and source records attributed to Savage, D. F..

28 records · Page 2Linked to original sources

bioRxiv2022

Evolution of Carbon Isotope Fractionation in Cyanobacteria

The history of Earths carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. We tested the intersection of environment and evolution by measuring both biomass ({varepsilon}p) and enzymatic ({varepsilon}Rubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to >>1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger {varepsilon}p values than WT, despite having a much smaller {varepsilon}Rubisco (17.23 {+/-} 0.61{per thousand} vs. 25.18 {+/-} 0.31{per thousand} respectively). Measuring both enzymatic and biomass fractionation revealed a surprising result--ANC {varepsilon}p exceeded ANC {varepsilon}Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. However, these models were corrected by accounting for cyanobacterial physiology, notably the CO2 concentrating mechanism (CCM). Our model suggested that additional fractionating processes like powered inorganic carbon uptake systems contribute to {varepsilon}p, and this effect is exacerbated in ANC. Understanding the evolution of rubisco and the CCM is therefore critical for interpreting the carbon isotope record. Large fluctuations in that record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2. Significance StatementEarth scientists rely on chemical fossils like the carbon isotope record to derive ancient atmospheric CO2 concentrations, but interpretation of this record is calibrated using modern organisms. We tested this assumption by measuring the carbon isotope fractionation of a reconstructed ancestral rubisco enzyme (>1 billion years old) in vivo and in vitro. Our results contradicted prevailing models of carbon flow in Cyanobacteria, but our data could be rationalized if light-driven uptake of CO2 is taken into account. Our study showed that the carbon isotope record tracks both the evolution of photosynthesis physiology as well as changes in atmospheric CO2, highlighting the value of considering both evolution and physiology for comparative biological approaches to understanding Earths history.

evolutionary biology↗

bioRxiv2022

Trajectories for the evolution of bacterial CO2-concentrating mechanisms

Cyanobacteria rely on CO2 concentrating mechanisms (CCMs) that depend on {approx}15 genes to produce two protein complexes: an inorganic carbon (Ci) transporter and a 100+ nm carboxysome compartment that encapsulates rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting CCM components prohibit growth in todays atmosphere (0.04% CO2), indicating that CCMs evolved to cope with declining environmental CO2. Indeed, geochemical data and models indicate that atmospheric CO2 has been generally decreasing from high concentrations over the last {approx}3.5 billion years. We used a synthetic reconstitution of a bacterial CCM in E. coli to study the co-evolution of CCMs with atmospheric CO2. We constructed strains expressing putative ancestors of modern CCMs -- strains lacking one or more CCM components -- and evaluated their growth in a variety of CO2 concentrations. Partial forms expressing CA or Ci uptake genes grew better than controls in intermediate CO2 levels ({approx}1%); we observed similar phenotypes in genetic studies of two autotrophic bacteria, H. neapolitanus and C. necator. To explain how partial CCMs improve growth, we advance a model of co-limitation of autotrophic growth by CO2 and HCO3-, as both are required to produce biomass. Our model and results delineated a viable trajectory for bacterial CCM evolution where decreasing atmospheric CO2 induces an HCO3- deficiency that is alleviated by acquisition of CAs or Ci uptake genes, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity. SignificanceThe greenhouse gas content of the ancient atmosphere is estimated using models and measurements of geochemical proxies. Some inferred high ancient CO2 levels using models of biological CO2 fixation to interpret the C isotopes found in preserved organic matter. Others argued that elevated CO2 could reconcile a faint young Sun with evidence for liquid water on Earth. We took a complementary "synthetic biological" approach to understanding the composition of the ancient atmosphere by studying present-day bacteria engineered to resemble ancient autotrophs. By showing that it is simpler to rationalize the emergence of modern bacterial autotrophs if CO2 was once high, these investigations provided independent evidence for the view that CO2 concentrations were significantly elevated in the atmosphere of early Earth.

microbiology↗

bioRxiv2022

Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution

Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individuals and measure their physical and chemical properties. To overcome this, we previously developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces which counteract Brownian motion, thus enabling extended observation. Here, we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitor the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Further, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL traps ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/484789v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1fccfacorg.highwire.dtl.DTLVardef@a639b6org.highwire.dtl.DTLVardef@116fff6org.highwire.dtl.DTLVardef@fe95fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Discovery of a carbonic anhydrase-Rubisco supercomplex within the alpha-carboxysome

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation - Rubisco and carbonic anhydrase - and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the -carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the -carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into -carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 [A] single-particle cryo-electron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCAs binding site overlaps with that of CsoS2 but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme, but also as a central hub for mediating assembly through protein interactions.

biochemistry↗

bioRxiv2021

Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression

Cas13 is a family of unique RNA-targeting CRISPR-Cas effectors, making it an appealing tool for probing and perturbing RNA function. However only a few Cas13 homologs have been shown to mediate robust RNA targeting in human cells, suggesting that unknown elements may be limiting their efficacy. Furthermore, many Cas13 enzymes show high degrees of toxicity upon targeting and have not been shown to mediate specific knockdown in other cell types such as E. coli. Here, we show that catalytically inactive Cas13 enzymes can be repurposed for efficient translational repression in bacteria with no associated growth defects. To achieve this advance, we carried out a directed evolution screen to engineer functional Cas13a variants, and identified a number of stabilizing mutations, which enabled efficient post transcriptional knockdown of gene expression. In vitro characterization of the resulting engineered Lbu Cas13a mutant, termed eLbu, revealed both stabilization and altered cleavage kinetics. Finally, we show that eLbu can be used for efficient exon skipping in human cells. This work represents the first demonstration of targeted translational repression in E. coli using a CRISPR enzyme, as well as the first directed evolution of a Cas13 enzyme. Such a platform could allow for engineering other aspects of this protein family to obtain more robust RNA targeting tools.

bioengineering↗

bioRxiv2021

The encapsulin from Thermatoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based organelles found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a [~]25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model T. maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryoEM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the T. maritima encapsulin, the decameric cargo protein with 5-fold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier--it also plays a significant role in the structure and function of the cargo enzyme.

biochemistry↗

bioRxiv2020

A nanocompartment containing the peroxidase DypB contributes to defense against oxidative stress in M. tuberculosis

Encapsulin nanocompartments are an emerging class of prokaryotic protein-based organelles consisting of an encapsulin protein shell that encloses a protein cargo1. Genes encoding nanocompartments are widespread in bacteria and archaea, and recent works have characterized the biochemical function of several cargo enzymes2. However, the importance of these organelles to host physiology is poorly understood. Here, we report that the human pathogen Mycobacterium tuberculosis (Mtb) produces a nanocompartment that contains the dye-decolorizing peroxidase DypB. We show that this nanocompartment is important for the ability of Mtb to resist oxidative stress in low pH environments, including during infection of host cells and upon treatment with a clinically relevant antibiotic. Our findings are the first to implicate a nanocompartment in bacterial pathogenesis and reveal a new mechanism that Mtb uses to combat oxidative stress.

microbiology↗

bioRxiv2019

α-carboxysome formation is mediated by the multivalent and disordered protein CsoS2

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism, feeding high concentrations of CO2 to the enzyme Rubisco for fixation. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the -carboxysome, there is little molecular insight into protein-protein interactions which drive the assembly process. Here we show that the N-terminus of CsoS2, an intrinsically disordered protein found in the -carboxysome, possesses a repeated peptide sequence that binds Rubisco. X-ray structural analysis of the peptide bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate CsoS2 acts as an interaction hub to condense Rubisco and enable efficient -carboxysome formation.

biochemistry↗

bioRxiv2018

Genome-wide screening reveals a novel class of carbonic anhydrase-like inorganic carbon transporters in chemoautotrophic bacteria

Many bacterial autotrophs rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, including a 200+ MDa protein organelle called the carboxysome, a systematic screen of CCM components has not been carried out. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the {gamma}-proteobacterium H. neapolitanus. Our screen revealed an operon encoding a domain of unknown function (PFAM:PF10070) and putative cation transporter subunit (PFAM:PF00361) is critical for CCM function. These two proteins, which we name DabA and DabB for \"DABs accumulate bicarbonate,\" function as a heterodimeric, energy-coupled inorganic carbon pump in E. coli. Furthermore, DabA has distant homology to a {beta}-carbonic anhydrase and binds a zinc ion necessary for activity. Based on these results, we propose that DABs function as vectorial CAs coupled to cation gradients and serve as inorganic carbon pumps throughout prokaryotic phyla.

molecular biology↗

bioRxiv2018

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

synthetic biology↗