GENTANGLE: integrated computational design of gene entanglements.

SUMMARY: The design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased stability. The design of functional overlapping gene pairs is a challenging procedure, and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high-performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user-specified gene pairs. AVAILABILITY AND IMPLEMENTATION: The GENTANGLE source code and its submodules are freely available on GitHub at https://github.com/BiosecSFA/gentangle. The DATANGLE (DATA for genTANGLE) repository contains related data and results and is freely available on GitHub at https://github.com/BiosecSFA/datangle. The GENTANGLE container is freely available on Singularity Cloud Library at https://cloud.sylabs.io/library/khyox/gentangle/gentangle.sif. The GENTANGLE repository wiki (https://github.com/BiosecSFA/gentangle/wiki), website (https://biosecsfa.github.io/gentangle/), and user manual contain detailed instructions on how to use the different components of software and data, including examples and reproducing the results. The code is licensed under the GNU Affero General Public License version 3 (https://www.gnu.org/licenses/agpl.html).

Prolonging genetic circuit stability through adaptive evolution of overlapping genes.

The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more 'benign' mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis.

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by "programming" cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including "monoculture" and "transmitter-receiver" models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the "collective" attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.

Projection Microstereolithographic Microbial Bioprinting for Engineered Biofilms.

Microbes are critical drivers of all ecosystems and many biogeochemical processes, yet little is known about how the three-dimensional (3D) organization of these dynamic organisms contributes to their overall function. To probe how biofilm structure affects microbial activity, we developed a technique for patterning microbes in 3D geometries using projection stereolithography to bioprint microbes within hydrogel architectures. Bacteria were printed and monitored for biomass accumulation, demonstrating postprint viability of cells using this technique. We verified our ability to integrate biological and geometric complexity by fabricating a printed biofilm with two E. coli strains expressing different fluorescence. Finally, we examined the target application of microbial absorption of metal ions to investigate geometric effects on both the metal sequestration efficiency and the uranium sensing capability of patterned engineered Caulobacter crescentus strains. This work represents the first demonstration of the stereolithographic printing of microbials and presents opportunities for future work of engineered biofilms and other complex 3D structured cultures.

Plutonium Redox Transformation in the Presence of Iron, Organic Matter, and Hydroxyl Radicals: Kinetics and Mechanistic Insights.

Plutonium (Pu) redox and complexation processes in the presence of natural organic matter and associated iron can impact the fate and transport of Pu in the environment. We studied the fate of Pu(IV) in the presence of humic acid (HA) and Fe(II) upon reaction with H2O2 that may be generated by photochemical and other reactions. A portion of Pu(IV) was oxidized to Pu(V/VI), which is primarily ascribed to the generation of reactive intermediates from the oxidation of Fe(II) and Fe(II)-HA complexes by H2O2. The kinetics of Pu(IV) oxidation is pH-dependent and can be described by a model that incorporates Pu redox kinetics with published HA-modified Fenton reaction kinetics. At pH 3.5, the presence of HA slowed Pu(IV) oxidation, while at pH 6, HA accelerated Pu(IV) oxidation in the first several hours followed by a reverse process where the oxidized Pu(V/VI) was reduced back to Pu(IV). Analysis of Pu-associated particle size suggests that Pu oxidation state is a major driver in its complexation with HA and formation of colloids and heteroaggregates. Our results revealed the H2O2-driven oxidation of Pu(IV)-HA-Fe(II) colloids with implications to the transient mobilization of Pu(V/VI) in organic-rich redox transition zones.

Influence of Uranium Concentration and pH on U-Phosphate Biomineralization by Caulobacter OR37.

Uranium contamination of soils and groundwater in the United States represents a significant health risk and will require multiple remediation approaches. Microbial phosphatase activity coupled to the addition of an organic P source has recently been studied as a remediation strategy that provides an extended release of inorganic P (Pi) into U-contaminated sites, resulting in the precipitation of meta-autunite minerals. Previous laboratory- and field-based biomineralization studies have investigated environments with relatively high U concentrations (>20 µM). However, most contaminated sites have much lower U concentrations (<2 µM). The Environmental Protection Agency (EPA) limit for U in drinking water is 0.126 µM. Reaching this regulatory limit becomes challenging as U concentrations approach autunite solubility. We studied the precipitation of U(VI)-phosphate minerals by an environmental isolate of Caulobacter sp. (strain OR37) from an Oak Ridge, Tennessee, U-contaminated site. Abiotic U(VI) solubility experiments reveal that U(VI)-phosphate minerals do not form in the presence of excess Pi (500 µM) when U(VI) concentrations are <1 µM and pH is <5. When OR37 cells are reacted under the same conditions with Pi or glycerol-2-phosphate, U(VI)-phosphate mineral formation was observed, along with the formation of intracellular polyphosphate granules. These results show that bacteria provide supersaturated microenvironments needed for U(VI)-phosphate mineralization while hydrolyzing organic P sources. This provides a pathway to lower U concentrations to below EPA limits for drinking water.

Microbe-Encapsulated Silica Gel Biosorbents for Selective Extraction of Scandium from Coal Byproducts.

Scandium (Sc) has great potential for use in aerospace and clean energy applications, but its supply is currently limited by a lack of commercially viable deposits and the environmental burden of its production. In this work, a biosorption-based flow-through process was developed for extraction of Sc from low-grade feedstocks. A microbe-encapsulated silica gel (MESG) biosorbent was synthesized through sol-gel encapsulation of Arthrobacter nicotianae, a bacterium that selectively adsorbs Sc. Microscopic imaging revealed a high cell loading and macroporous structure, which enabled rapid mass transport and adsorption/desorption of metal ions. The biosorbent displayed high Sc selectivity against lanthanides and major base metals, with the exception of Fe(III). Following pH adjustment to remove Fe(III) from an acid leachate prepared from lignite coal, a packed-bed column loaded with the MESG biosorbent exhibited near-complete Sc separation from lanthanides; the column eluate had a Sc enrichment factor of 10.9, with Sc constituting 96.4% of the total rare earth elements. The MESG biosorbent exhibited no significant degradation with regard to both adsorption capacity and physical structure after 10 adsorption/desorption cycles. Overall, our results suggest that the MESG biosorbent offers an effective and green alternative to conventional liquid-liquid extraction for Sc recovery.

The UzcRS two-component system in Caulobacter crescentus integrates regulatory input from diverse auxiliary regulators.

The UzcRS two-component system in Caulobacter crescentus mediates widespread transcriptional activation in response to the metals U, Zn and Cu. Unexpectedly, a screen for mutations that affected the activity of the UzcR-regulated urcA promoter (PurcA ) revealed four previously uncharacterized proteins whose inactivation led to metal-independent induction of PurcA . Using molecular genetics and functional genomics, we find that these auxiliary regulators control PurcA expression by modulating the activity of UzcRS through distinct mechanisms. An ABC transporter with a periplasmic metallo-aminopeptidase domain forms a sensory complex with UzcRS, antagonizing metal dependent stimulation by virtue of its ATPase and peptidase domains. Two MarR-like transcription factors synergistically regulate UzcRS activity by repressing the expression of the membrane proteins UzcY and UzcZ, which stimulate UzcRS activity and enhance its sensitivity to a more environmentally relevant U/Zn/Cu concentration range. Additionally, the membrane protein UzcX, whose expression is positively regulated by UzcR, provides a mechanism of feedback inhibition within the UzcRS circuit. Collectively, these data suggest that UzcRS functions as the core-signaling unit within a multicomponent signal transduction pathway that includes a diverse set of auxiliary regulators, providing further insight into the complexity of signaling networks.

Selective and Efficient Biomacromolecular Extraction of Rare-Earth Elements using Lanmodulin.

Lanmodulin (LanM) is a recently discovered protein that undergoes a large conformational change in response to rare-earth elements (REEs). Here, we use multiple physicochemical methods to demonstrate that LanM is the most selective macromolecule for REEs characterized to date and even outperforms many synthetic chelators. Moreover, LanM exhibits metal-binding properties and structural stability unseen in most other metalloproteins. LanM retains REE binding down to pH ≈ 2.5, and LanM-REE complexes withstand high temperature (up to 95 °C), repeated acid treatments, and up to molar amounts of competing non-REE metal ions (including Mg, Ca, Zn, and Cu), allowing the protein's use in harsh chemical processes. LanM's unrivaled properties were applied to metal extraction from two distinct REE-containing industrial feedstocks covering a broad range of REE and non-REE concentrations, namely, precombustion coal and electronic waste leachates. After only a single all-aqueous step, quantitative and selective recovery of the REEs from all non-REEs initially present (Li, Na, Mg, Ca, Sr, Al, Si, Mn, Fe, Co, Ni, Cu, Zn, and U) was achieved, demonstrating the universal selectivity of LanM for REEs against non-REEs and its potential application even for industrial low-grade sources, which are currently underutilized. Our work indicates that biosourced macromolecules such as LanM may offer a new paradigm for extractive metallurgy and other applications involving f-elements.

System-level analysis of metabolic trade-offs during anaerobic photoheterotrophic growth in Rhodopseudomonas palustris.

BACKGROUND: Living organisms need to allocate their limited resources in a manner that optimizes their overall fitness by simultaneously achieving several different biological objectives. Examination of these biological trade-offs can provide invaluable information regarding the biophysical and biochemical bases behind observed cellular phenotypes. A quantitative knowledge of a cell system's critical objectives is also needed for engineering of cellular metabolism, where there is interest in mitigating the fitness costs that may result from human manipulation. RESULTS: To study metabolism in photoheterotrophs, we developed and validated a genome-scale model of metabolism in Rhodopseudomonas palustris, a metabolically versatile gram-negative purple non-sulfur bacterium capable of growing phototrophically on various carbon sources, including inorganic carbon and aromatic compounds. To quantitatively assess trade-offs among a set of important biological objectives during different metabolic growth modes, we used our new model to conduct an 8-dimensional multi-objective flux analysis of metabolism in R. palustris. Our results revealed that phototrophic metabolism in R. palustris is light-limited under anaerobic conditions, regardless of the available carbon source. Under photoheterotrophic conditions, R. palustris prioritizes the optimization of carbon efficiency, followed by ATP production and biomass production rate, in a Pareto-optimal manner. To achieve maximum carbon fixation, cells appear to divert limited energy resources away from growth and toward CO2 fixation, even in the presence of excess reduced carbon. We also found that to achieve the theoretical maximum rate of biomass production, anaerobic metabolism requires import of additional compounds (such as protons) to serve as electron acceptors. Finally, we found that production of hydrogen gas, of potential interest as a candidate biofuel, lowers the cellular growth rates under all circumstances. CONCLUSIONS: Photoheterotrophic metabolism of R. palustris is primarily regulated by the amount of light it can absorb and not the availability of carbon. However, despite carbon's secondary role as a regulating factor, R. palustris' metabolism strives for maximum carbon efficiency, even when this increased efficiency leads to slightly lower growth rates.

Recovery of Rare Earth Elements from Geothermal Fluids through Bacterial Cell Surface Adsorption.

The increasing demand for rare earth elements (REEs) in the modern economy motivates the development of novel strategies for cost-effective REE recovery from nontraditional feedstocks. We previously engineered E. coli to express lanthanide binding tags on the cell surface, which increased the REE biosorption capacity and selectivity. Here we examined how REE adsorption by the engineered E. coli is affected by various geochemical factors relevant to geothermal fluids, including total dissolved solids (TDS), temperature, pH, and the presence of specific competing metals. REE biosorption is robust to TDS, with high REE recovery efficiency and selectivity observed with TDS as high as 165,000 ppm. Among several metals tested, U, Al, and Pb were found to be the most competitive, causing >25% reduction in REE biosorption when present at concentrations ∼3- to 11-fold higher than the REEs. Optimal REE biosorption occurred between pH 5-6, and sorption capacity was reduced by ∼65% at pH 2. REE recovery efficiency and selectivity increased as a function of temperature up to ∼70 °C due to the thermodynamic properties of metal complexation on the bacterial surface. Together, these data define the optimal and boundary conditions for biosorption and demonstrate its potential utility for selective REE recovery from geofluids.

Microbe Encapsulation for Selective Rare-Earth Recovery from Electronic Waste Leachates.

Rare earth elements (REEs) are indispensable components of many green technologies and of increasing demand globally. However, refining REEs from raw materials using current technologies is energy intensive and enviromentally damaging. Here, we describe the development of a novel biosorption-based flow-through process for selective REE recovery from electronic wastes. An Escherichia coli strain previously engineered to display lanthanide-binding tags on the cell surface was encapsulated within a permeable polyethylene glycol diacrylate (PEGDA) hydrogel at high cell density using an emulsion process. This microbe bead adsorbent contained a homogenous distribution of cells whose surface functional groups remained accessible and effective for selective REE adsorption. The microbe beads were packed into fixed-bed columns, and breakthrough experiments demonstrated effective Nd extraction at a flow velocity of up to 3 m/h at pH 4-6. The microbe bead columns were stable for reuse, retaining 85% of the adsorption capacity after nine consecutive adsorption/desorption cycles. A bench-scale breakthrough curve with a NdFeB magnet leachate revealed a two-bed volume increase in breakthrough points for REEs compared to non-REE impurities and 97% REE purity of the adsorbed fraction upon breakthrough. These results demonstrate that the microbe beads are capable of repeatedly separating REEs from non-REE metals in a column system, paving the way for a biomass-based REE recovery system.

Identification of a U/Zn/Cu responsive global regulatory two-component system in Caulobacter crescentus.

Despite the well-known toxicity of uranium (U) to bacteria, little is known about how cells sense and respond to U. The recent finding of a U-specific stress response in Caulobacter crescentus has provided a foundation for studying the mechanisms of U- perception in bacteria. To gain insight into this process, we used a forward genetic screen to identify the regulatory components governing expression of the urcA promoter (PurcA ) that is strongly induced by U. This approach unearthed a previously uncharacterized two-component system, named UzcRS, which is responsible for U-dependent activation of PurcA . UzcRS is also highly responsive to zinc and copper, revealing a broader specificity than previously thought. Using ChIP-seq, we found that UzcR binds extensively throughout the genome in a metal-dependent manner and recognizes a noncanonical DNA-binding site. Coupling the genome-wide occupancy data with RNA-seq analysis revealed that UzcR is a global regulator of transcription, predominately activating genes encoding proteins that are localized to the cell envelope; these include metallopeptidases, multidrug-resistant efflux (MDR) pumps, TonB-dependent receptors and many proteins of unknown function. Collectively, our data suggest that UzcRS couples the perception of U, Zn and Cu with a novel extracytoplasmic stress response.

Reduction of Plutonium(VI) to (V) by Hydroxamate Compounds at Environmentally Relevant pH.

Natural organic matter is known to influence the mobility of plutonium (Pu) in the environment via complexation and reduction mechanisms. Hydroxamate siderophores have been specifically implicated due to their strong association with Pu. Hydroxamate siderophores can also break down into di and monohydroxamates and may influence the Pu oxidation state, and thereby its mobility. In this study we explored the reactions of Pu(VI) and Pu(V) with a monohydroxamate compound (acetohydroxamic acid, AHA) and a trihydroxamate siderophore desferrioxamine B (DFOB) at an environmentally relevant pH (5.5-8.2). Pu(VI) was instantaneously reduced to Pu(V) upon reaction with AHA. The presence of hydroxylamine was not observed at these pHs; however, AHA was consumed during the reaction. This suggests that the reduction of Pu(VI) to Pu(V) by AHA is facilitated by a direct one electron transfer. Importantly, further reduction to Pu(IV) or Pu(III) was not observed, even with excess AHA. We believe that further reduction of Pu(V) did not occur because Pu(V) does not form a strong complex with hydroxamate compounds at a circum-neutral pH. Experiments performed using desferrioxamine B (DFOB) yielded similar results. Broadly, this suggests that Pu(V) reduction to Pu(IV) in the presence of natural organic matter is not facilitated by hydroxamate functional groups and that other natural organic matter moieties likely play a more prominent role.

Recovery of Rare Earth Elements from Low-Grade Feedstock Leachates Using Engineered Bacteria.

The use of biomass for adsorption of rare earth elements (REEs) has been the subject of many recent investigations. However, REE adsorption by bioengineered systems has been scarcely documented, and rarely tested with complex natural feedstocks. Herein, we engineered E. coli cells for enhanced cell surface-mediated extraction of REEs by functionalizing the OmpA protein with 16 copies of a lanthanide binding tag (LBT). Through biosorption experiments conducted with leachates from metal-mine tailings and rare earth deposits, we show that functionalization of the cell surface with LBT yielded several notable advantages over the nonengineered control. First, the efficiency of REE adsorption from all leachates was enhanced as indicated by a 2-10-fold increase in distribution coefficients for individual REEs. Second, the relative affinity of the cell surface for REEs was increased over all non-REEs except Cu. Third, LBT-display systematically enhanced the affinity of the cell surface for REEs as a function of decreasing atomic radius, providing a means to separate high value heavy REEs from more common light REEs. Together, our results demonstrate that REE biosorption of high efficiency and selectivity from low-grade feedstocks can be achieved by engineering the native bacterial surface.

Interactions of Plutonium with Pseudomonas sp. Strain EPS-1W and Its Extracellular Polymeric Substances.

Safe and effective nuclear waste disposal, as well as accidental radionuclide releases, necessitates our understanding of the fate of radionuclides in the environment, including their interaction with microorganisms. We examined the sorption of Pu(IV) and Pu(V) to Pseudomonas sp. strain EPS-1W, an aerobic bacterium isolated from plutonium (Pu)-contaminated groundwater collected in the United States at the Nevada National Security Site (NNSS) in Nevada. We compared Pu sorption to cells with and without bound extracellular polymeric substances (EPS). Wild-type cells with intact EPS sorbed Pu(V) more effectively than cells with EPS removed. In contrast, cells with and without EPS showed the same sorption affinity for Pu(IV). In vitro experiments with extracted EPS revealed rapid reduction of Pu(V) to Pu(IV). Transmission electron microscopy indicated that 2- to 3-nm nanocrystalline Pu(IV)O2 formed on cells equilibrated with high concentrations of Pu(IV) but not Pu(V). Thus, EPS, while facilitating Pu(V) reduction, inhibit the formation of nanocrystalline Pu(IV) precipitates. IMPORTANCE: Our results indicate that EPS are an effective reductant for Pu(V) and sorbent for Pu(IV) and may impact Pu redox cycling and mobility in the environment. Additionally, the resulting Pu morphology associated with EPS will depend on the concentration and initial Pu oxidation state. While our results are not directly applicable to the Pu transport situation at the NNSS, the results suggest that, in general, stationary microorganisms and biofilms will tend to limit the migration of Pu and provide an important Pu retardation mechanism in the environment. In a broader sense, our results, along with a growing body of literature, highlight the important role of microorganisms as producers of redox-active organic ligands and therefore as modulators of radionuclide redox transformations and complexation in the subsurface.

Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation.

Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior. Caulobacter crescentus is unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFa and RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology to Escherichia coli TolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFa and RsaFb led to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in C. crescentusIMPORTANCE Decreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell, largely due to a lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural systems. As one example of a system evolved to prevent intracellular protein accumulation, our study demonstrates that Caulobacter crescentus has two homologous outer membrane transporter proteins that are involved in S-layer export. This is an interesting case study that demonstrates how bacteria can evolve redundancy to ensure adequate protein export functionality and maintain high cellular fitness. Moreover, we provide evidence that these two outer membrane proteins, although being the closest C. crescentus homologs to TolC in E. coli, do not process TolC functionality in C. crescentus.

Bioadsorption of Rare Earth Elements through Cell Surface Display of Lanthanide Binding Tags.

With the increasing demand for rare earth elements (REEs) in many emerging clean energy technologies, there is an urgent need for the development of new approaches for efficient REE extraction and recovery. As a step toward this goal, we genetically engineered the aerobic bacterium Caulobacter crescentus for REE adsorption through high-density cell surface display of lanthanide binding tags (LBTs) on its S-layer. The LBT-displayed strains exhibited enhanced adsorption of REEs compared to cells lacking LBT, high specificity for REEs, and an adsorption preference for REEs with small atomic radii. Adsorbed Tb(3+) could be effectively recovered using citrate, consistent with thermodynamic speciation calculations that predicted strong complexation of Tb(3+) by citrate. No reduction in Tb(3+) adsorption capacity was observed following citrate elution, enabling consecutive adsorption/desorption cycles. The LBT-displayed strain was effective for extracting REEs from the acid leachate of core samples collected at a prospective rare earth mine. Our collective results demonstrate a rapid, efficient, and reversible process for REE adsorption with potential industrial application for REE enrichment and separation.

Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance.

UNLABELLED: The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE: Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus. In particular, we show that outer membrane transporters RsaFa and RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

Shotgun proteomic analysis unveils survival and detoxification strategies by Caulobacter crescentus during exposure to uranium, chromium, and cadmium.

The ubiquitous bacterium Caulobacter crescentus holds promise to be used in bioremediation applications due to its ability to mineralize U(VI) under aerobic conditions. Here, cell free extracts of C. crescentus grown in the presence of uranyl nitrate [U(VI)], potassium chromate [Cr(VI)], or cadmium sulfate [Cd(II)] were used for label-free proteomic analysis. Proteins involved in two-component signaling and amino acid metabolism were up-regulated in response to all three metals, and proteins involved in aerobic oxidative phosphorylation and chemotaxis were down-regulated under these conditions. Clustering analysis of proteomic enrichment revealed that the three metals also induce distinct patterns of up- or down-regulated expression among different functional classes of proteins. Under U(VI) exposure, a phytase enzyme and an ABC transporter were up-regulated. Heat shock and outer membrane responses were found associated with Cr(VI), while efflux pumps and oxidative stress proteins were up-regulated with Cd(II). Experimental validations were performed on select proteins. We found that a phytase plays a role in U(VI) and Cr(VI) resistance and detoxification and that a Cd(II)-specific transporter confers Cd(II) resistance. Interestingly, analysis of promoter regions in genes associated with differentially expressed proteins suggests that U(VI) exposure affects cell cycle progression.