Biology subjects

Silberg, J. J.

Publications and source records attributed to Silberg, J. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Storing >1 byte of information in 16S ribosomal RNA using orthogonal trans-splicing ribozymes

TEXT ABSTRACTCatalytic-RNA (cat-RNA) expressed from mobile DNA can record cellular events, such as the uptake of plasmids via horizontal gene transfer, by splicing a barcode onto 16S ribosomal RNA (rRNA) - a system termed RNA addressable modification (RAM). However, scaling RAM to record multiple simultaneous biological events requires large numbers of orthogonal cat-RNA whose signals reflect the biological features under investigation rather than variability arising from the barcode sequence. Here, we explore how to design orthogonal cat-RNA to record information about multiple plasmid-encoded traits in parallel. We show that cat-RNA having tRNA-derived barcodes with sequence variation in the anticodon stem-loop present greater signal consistency within Escherichia coli than mRNA-derived barcodes. When orthogonal cat-RNA designs harboring tRNA-derived barcodes were evaluated in Vibrio natriegens and Pseudomonas putida, increased variance was observed compared with Escherichia coli. Nevertheless, the signal consistency was sufficient to use these orthogonal cat-RNAs to report on the relative activities of four promoters and two origins of replication by sequencing barcoded-rRNA derived from the three organisms. These results show how RAM can be multiplexed to report on mobile DNA features in microbial communities and illustrate the importance of accounting for variability in RNA outputs when designing and interpreting multiplexed RNA barcoding data. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/738544v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f6d62aorg.highwire.dtl.DTLVardef@6696c6org.highwire.dtl.DTLVardef@134aeb4org.highwire.dtl.DTLVardef@1b9ceeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Biocontainment attenuation of mobile DNA host range in a wastewater microbiome

Biocontainment systems designed to attenuate the spread of mobile DNA are challenging to evaluate within microbiomes of engineered environments. To better understand how toxin-based biocontainment systems affect horizontal gene transfer (HGT) in a microbiome, we evaluated the host range of pairs of plasmids using orthogonal catalytic RNA (cat-RNA) that amend distinct barcodes to 16S rRNA following HGT. We show that mobilizable (5 kb) and self-mobilizable (60 kb) plasmids, which use the same RP4 transfer machinery but different origins of replication, overlap in their host range when conjugated in parallel into a wastewater community, with 127 of the 143 amplicon sequence variants (ASVs) presenting barcoding signals from both plasmids (89%). We also find that mobilizable plasmids with or without the Escherichia coli CcdB toxin overlap in host range in a wastewater community. Among the two most abundant orders, CcdB attenuated the barcoding signal in Aeromonadales more consistently than Enterobacteriales, which have F plasmids containing the CcdB-CcdA toxin-antitoxin system used for biocontainment. Also, CcdB decreased the abundance of the mobilizable plasmid by >100-fold and yielded mutations in 85% of the reads. Together, these findings reveal how pairs of plasmids expressing orthogonal cat-RNA can be used to monitor the effects of plasmid-encoded traits on mobile DNA persistence following HGT. They also highlight challenges when using biocontainment systems containing genes related to those found in the microbiomes targeted for engineering.

synthetic biology↗

bioRxiv2026

Predicting optimal growth temperatures of bacteria using learned structural information from a single protein

Temperature is a fundamental determinant of bacterial physiology and ecology. Optimal growth temperature (OGT) is highly variable across species, contributing to differences in where and when species are most likely to thrive. Although the OGTs for most bacteria remain unknown, the increasing availability of genomes from uncultivated and cultivated taxa has made it advantageous to build genomic, cultivation-independent models to infer OGT. However, pre-existing genomic models often lack the generalizability and mechanistic grounding required for robust inferences of OGT. We propose a novel framework for predicting bacterial OGT which uses learned protein structural signatures of thermal adaptation. We hypothesize that biophysical tradeoffs which dictate enzymatic functions across variable temperatures provide a more robust empirical basis for OGT prediction than broad genomic features. Our OGT-predicting model, ROSEATE, is based on a single gene, adenylate kinase (ADK), that encodes for a ubiquitous enzyme essential for energy homeostasis. ROSEATE uses high-dimensional latent space encoding via MSA Transformer, a protein language model which embeds ADKs in a manner which preserves biophysical information about embedded proteins. We show that the accuracy of the ROSEATE model is on par with other genome-based models, has a high degree of phylogenetic generalizability, and the ESM embeddings effectively capture key temperature-adaptive enzyme characteristics derived from AlphaFold structures. Because ROSEATE is based on analyses of a single ubiquitous protein, it can be used with metagenomic data to infer the community-level variation in bacterial OGTs. We demonstrate this feature of ROSEATE by reconstructing ADK sequences from over 500 environmental and host-associated metagenomes, successfully distinguishing community-wide thermal preferences across diverse habitats, from polar oceans to mammalian guts. By transitioning from genomic proxies to informationally dense protein structural features, this work provides an efficient, interpretable tool for predicting bacterial OGTs across taxa and whole communities. Author SummaryThe temperature preferences of bacteria are key to determining where species are most likely to grow and how bacterial communities may respond to changes in temperature regimes. Unfortunately, the optimal growth temperatures of most bacteria, including a broad diversity of bacteria found in many host-associated and environmental systems, currently remain unknown as many bacterial species cannot be grown or studied in a laboratory. While we now have genomic data for many bacteria, using these data to infer optimal temperatures for bacterial growth has remained a persistent challenge. We developed and validated a novel approach to predict bacterial temperature preferences. When heated, proteins often unfold, becoming nonfunctional. To adapt to warmer environments, organisms evolve more stable proteins which resist denaturing at high temperatures. Instead of analyzing a bacterias entire genome, our approach uses a protein language model to quantify stability-enhancing changes in a single protein found across all bacteria. We found that this single-protein approach can be used to effectively predict the optimal growth temperatures of individual bacterial species and even whole bacterial communities. By changing how we use genomic information to predict temperature preferences, our framework provides a scalable blueprint for predicting other important bacterial traits from protein structure information.

bioinformatics↗

bioRxiv2025

Effects of matric versus osmotic potential changes on Variovorax beijingensis transcription

Soil microbes must continuously adapt to changes in water availability, which dynamically fluctuates with weather and irrigation, and these adaptations are closely linked to soil CO2 emissions. Soil water potential, which regulates microbe-available water, is controlled by both osmotic and matric potential, which both increase as soils dry. While both parameters can independently increase water potential, the genetic mechanisms underlying microbial responses to both are unknown, with potentially different mechanisms available for soil microbes to respond to these hydrologic parameters. To explore microbial responses to matric versus osmotic potential shifts, we evaluated the growth and transcription of Variovorax beijingensis in soils and liquid cultures of varying water potential. We find this microbe respires in dilute minimal medium (-240 {+/-}104 kPa), in liquid medium supplemented with sucrose (-1323 {+/-}20.8 kPa), and in a pair of matrices that span a similar range of pressures (-183 {+/-}55 and -1393 kPa {+/-}200 kPa). We show that the global gene expression patterns vary significantly across all four conditions, even when the matric potential and osmotic pressure are set to similar values. However, the direction of gene expression changes correlated for 68% of the transcripts arising from an increase in osmotic pressure within liquid medium and an increase in matric potential within the different soils. While a large overlap was observed in the Variovorax transcriptional response to shifts in both osmotic and matric potential, the responses were not identical, with matric potential shifts leading to 2.55-fold more genes exhibiting differential expression. IMPORTANCEIt remains hard to establish how changes in soil water properties affect microbial behaviors that regulate soil health, and the energy with which soil water is held is likely a holistic control on at least some of those microbial behaviors. This energy is controlled by parameters associated with soil saltiness (osmotic potential) and texture (matric potential), which both alter bioavailable water by contributing to total soil water potential. To investigate how the global transcriptional profile of a soil microbe changes when the microbe-available water is altered either by changing soil texture or by changing osmolyte concentrations, we varied osmotic and matric potential individually and performed RNA sequencing. We observe differences in the transcriptome across all conditions analyzed. A larger number of genes are differentially expressed as matric potential increases; however, many of the transcripts differentially expressed as osmotic pressure increases covary with those observed as the matrix potential increases.

systems biology↗

bioRxiv2025

Cross-order detection of bacteriophage transduction in communities using ribosomal RNA barcoding

Bacteriophages (phages) facilitate gene transfer and microbial evolution in all ecosystems and have applications as tools for engineering microbiomes and as antimicrobials. Historic efforts to map phage hosts, such as plaque assays, are limited to culturable bacteria, are low throughput, and are hard to apply in environmentally-relevant contexts. To overcome these limitations, a synthetic ribozyme that stores information about DNA uptake in 16S ribosomal RNA (rRNA) was used to identify phage-host interactions by integrating the ribozyme into phage-plasmid P1 and performing targeted 16S rRNA sequencing following transduction. Experiments in synthetic and wastewater communities revealed Aeromonadales as a novel P1 host order and transduction of P1 into pathogens. Host range varied across phagemids with different origins of replication and phage particles with different tail fibers. This work shows how autonomous barcoding can be used in phages to identify the molecular controls on their host range in communities.

synthetic biology↗

bioRxiv2024

Ribozyme-mediated gene-fragment complementation for non-destructive reporting of DNA transfer within soil

Enzymes that produce volatile metabolites can be coded into genetic circuits to report non-disruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halides. We split the mht gene into two non-functional fragments and attached these to a pair of splicing ribozyme fragments. While the individual mht-ribozyme fragments did not produce methyl halides when transcribed alone in Escherichia coli, co-expression resulted in a spliced transcript that translated the MHT reporter. When cells containing one mht-ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second mht-ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

synthetic biology↗

bioRxiv2024

Gender, interdisciplinary graduate training, and confidence working in teams

Teamwork is recognized as critical to solving complex societal challenges related to energy, health, and sustainability. With graduate education, students often gain teamwork experience through a problem-focused approach where they are brought into existing collaborations to pursue research that is focused on studying questions that have already been identified. Here, we describe an interdisciplinary educational program where graduate students were tasked with leading team formation, problem identification, and research formulation. This "team-first" training approach used a two-year curriculum to bring together students enrolled in diverse engineering and science graduate programs and provided students with a pedagogical understanding of interdisciplinarity, nurtured the development of student communication skills across disciplines, fostered student-led team formation and idea development, and empowered students to forge new connections between research groups. Assessment of three cohorts immediately following curriculum completion (n = 36) revealed significant gains in confidence in teamwork (p < 0.001) when compared to a control group of academic peers (n = 74). These gains varied across demographic groups, with women in science, technology, engineering, and mathematics presenting the strongest gains. This finding illustrates the importance of exploring how interdisciplinary team curricula in graduate school could support overcoming the gender gap in confidence. SignificancePedagogical models for graduate education often neglect the importance of teamwork training. Here, we describe an interdisciplinary training program that was developed to bring together doctoral students from diverse science, technology, engineering, and mathematics disciplines for a two-year curriculum that focused on teamwork training through student-led team formation, problem identification, and research formulation. Following program participation, we measured participant confidence in teamwork relative to a peer group. Our findings reveal gains with confidence in teamwork, with women presenting the strongest increases without negative effects on other groups. This pedagogical approach represents a strategy to close gender gaps in professional role confidence while complementing the benefits of traditional disciplinary training approaches.

scientific communication and education↗

bioRxiv2023

The biochemical impact of extracting an embedded adenylate kinase domain using circular permutation

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, and mass spectrometry suggested that the new termini within the AMP binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Long duration environmental biosensing by recording analyte detection in DNA using recombinase memory

Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater. Alternative reporter outputs are needed that stably record the presence of analytes. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12- homoserine lactone) over 8 days ([~]70 generations) following analyte exposure. These biosensors use analyte sensing to trigger the expression of a recombinase which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using qPCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterized Pseudomonas isolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration and in situ investigation of microbial signaling, community shifts, and gene transfer beyond the reach of traditional environmental biosensors. IMPORTANCELiving microbial sensors can monitor chemicals and biomolecules in the environment in real-time, but they remain limited in their ability to function on the week, month, and year timescales. To determine if environmental microbes can be programmed to record the detection of analytes over longer timescales, we evaluated whether the sensing of a microbial signaling molecule could be recorded through a DNA rearrangement. We show that off-the-shelf DNA memory is suboptimal for long-duration information storage, use iterative design to enable robust functioning over more than a week, and demonstrate DNA memory in an uncharacterized wastewater Pseudomonas isolate. Memory biosensors will be useful for monitoring the role of quorum sensing in wastewater biofilm formation, and variations of this design are expected to enable studies of ecological processes in situ that are currently challenging to monitor using real-time biosensors and analytical instruments.

synthetic biology↗

bioRxiv2023

Information storage across a microbial community using universal RNA memory

Biological recorders can code information in DNA, but they remain challenging to apply in complex microbial communities. To program microbiome information storage, a synthetic catalytic RNA (cat-RNA) was used to write information in ribosomal RNA (rRNA) about gene transfer host range. By reading out native and modified rRNA using amplicon sequencing, we find that 140 out of 279 wastewater microbial community members from twenty taxonomic orders participate in conjugation and observe differences in information storage across amplicon sequence variants. Twenty of the variants were only observed in modified rRNA amplicons, illustrating information storage sensitivity. This autonomous and reversible RNA-addressable memory (RAM) will enable biosurveillance and microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials. One-Sentence SummaryRibosomal RNA sequencing detects cellular events recorded across a wastewater microbial community using synthetic biology.

synthetic biology↗

bioRxiv2023

Microbial sensor variation across biogeochemical conditions in the terrestrial deep subsurface

Microbes can be found in abundance many kilometers underground. While microbial metabolic capabilities have been examined across different geochemical settings, it remains unclear how changes in subsurface niches affect microbial needs to sense and respond to their environment. To address this question, we examined how two component systems (TCS) vary across metagenomes in the Deep Mine Microbial Observatory (DeMMO). TCSs were found at all six subsurface sites, the service water control, and the surface site, with an average of 0.88 sensor histidine kinases (HKs) per 100 genes across all sites. Abundance was greater in subsurface fracture fluids compared with surface-derived fluids, and candidate phyla radiation (CPR) bacteria presented the lowest HK frequencies. Measures of microbial diversity, such as the Shannon diversity index, revealed that HK abundance is inversely correlated with microbial diversity (r2 = 0.81). Among the geochemical parameters measured, HK frequency correlated the strongest with variance in dissolved organic carbon (DOC) (r2 = 0.82). Taken together, these results implicate the abiotic and biotic properties of an ecological niche as drivers of sensor needs, and they suggest that microbes in environments with large fluctuations in organic nutrients (e.g., lacustrine, terrestrial, and coastal ecosystems) may require greater TCS diversity than ecosystems with low nutrients (e.g., open ocean). IMPORTANCEThe ability to detect environmental conditions is a fundamental property of all life forms. However, organisms do not maintain the same environmental sensing abilities during evolution. To better understand the controls on microbial sensor abundance, which remain poorly understood, we evaluated how two-component sensor systems evolved within the deep Earth across sampling sites where abiotic and biotic properties vary. We quantify the relative abundances of sensor proteins and find that sensor systems remain abundant in microbial consortia as depth below the Earths surface increases. We also observe correlations between sensor system abundances and abiotic (dissolved organic carbon variation) and biotic (consortia diversity) properties across the DeMMO sites. These results suggest that multiple environmental properties drive sensor protein evolution and diversification and highlight the importance of studying metagenomic and geochemical data in parallel to understand the drivers of microbial sensor evolution.

microbiology↗

bioRxiv2022

Determinants of multiheme cytochrome extracellular electron transfer uncovered by systematic peptide insertion

The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate MtrA binding to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). EET efficiency was found to be inversely correlated with insertion proximity to the heme prosthetic groups. Mutants with decreased EET also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III)-respiring conditions correlated with anode reduction. The peptide-insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for regulating EET.

synthetic biology↗

bioRxiv2022

Non-destructive chemical sensing within bulk soil using 1000 biosensors per gram of matrix

Gene expression can be monitored in hard-to-image environmental materials using gas-reporting biosensors, but these outputs have only been applied in autoclaved matrices that are hydrated with rich medium. To better understand the compatibility of indicator gas reporting with environmental samples, we evaluated how matrix hydration affects the gas signal of an engineered microbe added to a sieved soil. A gas-reporting microbe presented a gas signal in a forest soil (Alfisol) when hydrated to an environmentally-relevant osmotic pressure. When the gas signal was concentrated prior to analysis, a biosensor titer of 103 cells/gram soil produced a significant signal when soil was supplemented with halides. A signal was also observed without halide amendment, but a higher cell titer (106 cells/gram soil) was required. A sugar-regulated gas biosensor was able to report with a similar level of sensitivity when added to an unsterilized soil matrix, illustrating how gas concentration enables biosensing within a soil containing environmental microbes. These results establish conditions where engineered microbes can report on gene expression in living environmental matrices with minimal perturbation, using biosensor titers that are orders of magnitude lower than the number of cells typically observed in a gram of soil.

synthetic biology↗

bioRxiv2022

Artificial soils reveal individual factor controls on microbial processes

Soil matrix properties influence microbial behaviors that underlie nutrient cycling, greenhouse gas production, and soil formation. However, the dynamic and heterogeneous nature of soils makes it challenging to untangle the effects of different matrix properties on microbial behaviors. To address this challenge, we developed a tunable artificial soil recipe and used these materials to study the abiotic mechanisms driving soil microbial growth and communication. When we used standardized matrices with varying textures to culture gas-reporting biosensors, we found that Gram-negative bacteria grew best in synthetic silt soils, remaining active over a wide range of soil matric potentials, while Gram-positive bacteria preferred sandy soils, sporulating at a low water potentials. Soil texture, mineralogy, and alkalinity all attenuated the bioavailability of an acyl-homoserine lactone (AHL) signaling molecule that controls community-level microbial behaviors. Texture controlled the timing of AHL sensing, while AHL bioavailability was decreased [~]105-fold by mineralogy and [~]103-fold by alkalinity. Finally, we built artificial soils with a range of complexities that converge on the properties of one Mollisol. As artificial soil complexity increased to more closely resemble the Mollisol, microbial behaviors approached those occurring in the natural soil, with the notable exception of organic matter. Properties that behaved additively were soil texture, pH, and mineralogy, while organic matter did not, suggesting that its soil behavior is an emergent property. IMPORTANCEUnderstanding environmental controls on soil microbes is difficult because many abiotic parameters vary simultaneously and uncontrollably when different natural soils are compared, preventing mechanistic determination of any individual soil parameters effect on microbial behaviors. We describe how soil texture, mineralogy, pH, and organic matter content can be varied individually within artificial soils to study their effects on soil microbes. Using microbial biosensors that report by producing a rare indicator gas, we identify soil properties that control microbial growth and attenuate the bioavailability of a diffusible chemical used to control community-level behaviors. We find that artificial soils differentially affect signal bioavailability and the growth of Gram-negative and Gram-positive microbes. We show that some soil properties have additive effects on signal bioavailability, while others exhibit emergent properties. These artificial soils are useful for studying the mechanisms that underlie soil controls on microbial fitness, signaling, and gene transfer.

systems biology↗

bioRxiv2020

A split methyl halide transferase AND gate that reports by synthesizing an indicator gas

It is challenging to detect microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater. To develop a simple approach for monitoring post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann-fold domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that can report on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this protein fragment complementation assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

ABSTRACTHomologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic electron transfer pathway.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Prochlorococcus phage ferredoxin: structural characterization and interactions with cyanobacterial sulfite reductases

Marine cyanobacteria are infected by phage whose genomes encode ferredoxin (Fd) electron carriers. While these Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, it is not clear how the biophysical properties and partner specificities of phage Fds relate to those in photosynthetic organisms. Bioinformatic analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects Prochlorococcus marinus, revealed high similarity to cyanobacterial Fds ([≤]0.5 [A] RMSD). Additionally, pssm2-Fd exhibits a low midpoint reduction potential (-336 mV vs. SHE) similar to other photosynthetic Fds, albeit lower thermostability (Tm = 28{degrees}C) than many Fds. When expressed in an Escherichia coli strain with a sulfite assimilation defect, pssm2-Fd complemented growth when coexpressed with a Prochlorococcus marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterial-encoded oxidoreductases.

biochemistry↗

bioRxiv2019

Family permutation profiling identifies a dynamic protein domain as functionally tolerant to increased conformational entropy

To investigate whether adenylate kinase (AK) homologs differ in their functional tolerance to mutational lesions that alter dynamics, we subjected three homologs having a range of thermostabilities to random circular permutation and evaluated where new protein termini were non-disruptive to activity using a cellular selection and deep mutational scanning. Analysis of the positional tolerance to new termini, which increase local conformational entropy by breaking peptide bonds, showed that bonds were either functionally sensitive to cleavage across all three homologs, differentially sensitive, or uniformly tolerant. The mobile AMP binding domain, which displays the highest calculated contact energies (frustration), presented the greatest tolerance to new termini across all AKs. In contrast, retention of function in the lid and core domains was more dependent upon AK melting temperature. Thus, regions of high energetic frustration tolerated increases in conformational entropy in a manner that was less dependent on thermostability than regions of lower frustration. Our results suggest that family permutation profiling identifies primary structure that has been selected by evolution for high frustration that is critical to enzymatic activity. They also illustrate how deep mutational scanning can be applied to protein homologs in parallel to learn how topology and function govern mutational tolerance.

biochemistry↗