Multiprotein System Research Articles

Self-lysis microbial consortia for predictable multi-proteins assembly

The scope of bioengineering is expanding from the design of single strain to the microbial communities, allowing for the division-of-labor in synthesizing the multi-protein systems. Predicting the composition of the final product during the biomanufacturing process, however, can be difficult. Consortia-based manufacturing has the potential to boost production efficiency, but this benefit primarily holds in the upstream. The current format of downstream process heavily relies on the centralized facility, and is not economical and flexible to address the demands in small-scale. Here, we present a concise and manageable platform to enable the multi-protein system assembly. We engineer a self-lysis microbial consortium, where each strain lyses autonomously at high densities and produces a single protein component. The product fraction can be precisely tuned by varying the inoculation ratio. Utilizing this platform, we assemble a classical 34-component PURE (protein synthesis using recombinant elements) system. We have further optimized the downstream process of the biomanufacturing by incorporating the porous structure of polymeric materials. The encapsulated autolysis consortium can produce and release the proteins while maintaining the cell factories enclosed in the materials by exporting the multi-protein system for collection. Our research provides a novel approach to the flexible and controllable production of multi-protein systems, opening up new possibilities for pathway assembly and portable biomanufacturing.

Metal regulation of Mycobacterium tuberculosis SufB intein splicing at the host-pathogen crossroad.

Intein sequences self-excise from precursor proteins to generate functional proteins in various organisms. Thus, regulation of intein splicing at the host-pathogen interface can determine the fate of infection by controlling generation of essential proteins in microbes. For instance, Mycobacterium tuberculosis (Mtu) SufB intein splicing is crucial for the functionality of SUF complex. This multiprotein system is the sole pathway for [Fe-S] cluster biogenesis in mycobacteria during oxidative stress and Fe starvation. Although metal toxicity and metal starvation are components of host immunity, correlation of metal stress to Mtu SufB intein splicing is missing till date. Current study examines the splicing and N-terminal cleavage reactions of Mtu SufB precursor protein in presence of micronutrient metal ions like Zn+2, Cu+2, and Fe+3/+2. A known intein splicing inhibitor Pt+4 was also tested to support its proposed role as an anti-TB agent. Mtu SufB precursor protein exhibited significant attenuation of splicing and N-terminal cleavage reactions across different concentration ranges for Pt+4, Cu+2, Zn+2, while Fe+3 interaction resulted in precursor accumulation. UV-Vis spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), Tryptophan fluorescence assay, and dynamic light scattering (DLS) techniques analyzed metal-protein interaction. Mutagenesis experiments and Ellman's assay identified plausible metal co-ordination sites within Mtu SufB protein. Analyzing the metal effect on Mtu SufB splicing may provide elemental information about the fate of mycobacterial infection, and a probable mechanism to attenuate intracellular survival of Mtu. Current research hints at the host regulatory mechanism on SufB splicing in its native environment and a likely target for developing next-generation anti-TB drugs.

Comprehensive identification and hydrophobic analysis of key proteins affecting foam capacity and stability during the evolution of egg white foam

Each protein in a multiprotein system assumed a different role at the colloidal interface. To determine exactly which proteins played a key role in the evolution of egg white foam, label-free quantitative proteomics techniques were employed to detect key proteins associated with egg white foam properties. There were 35 (14 up-regulated and 21 down-regulated) and 25 (13 up-regulated and 12 down-regulated) key proteins related to egg white foam capacity and stability, respectively. The hydrophobicity prediction results revealed that the hydrophobicity of the amino acid sites, the grand average of hydropathicity (GRAVY) of the whole molecule and the spatial arrangement of the hydrophobic domain all influenced the air-water interface properties of these key proteins. Among the high abundance proteins in egg white, ovalbumin was identified as a key protein associated with superior foam capacity, and ovostatin, although a trace protein, could not be ignored for the role it assumed in foam formation. Meanwhile, ovomucoid and lysozyme could result in excellent foam stability. This study was integral to the ensuing discussion of the air-water interface behavior of single or multiple key protein systems. • Key proteins related to egg white foam properties were identified using proteomics. • Ovalbumin and ovostatin gave egg white their excellent foam capacity. • Ovomucoid and lysozyme were closely related to foam stability. • The distribution of hydrophobic domains greatly affected the interfacial activity.

Protein interactome mapping of Porphyromonas gingivalis provides insights into the formation of the PorQ-Z complex of the type IX secretion system.

Porphyromonas gingivalis is an anaerobic Gram-negative human oral pathogen highly associated with the more severe forms of periodontal disease. Porphyromonas gingivalis utilises the type IX secretion system (T9SS) to transport ∼30 cargo proteins, including multiple virulence factors, to the cell surface. The T9SS is a multiprotein system consisting of at least 20 proteins, and recently, we characterised the protein interactome of these components. Similar to the T9SS, almost all biological processes are mediated through protein-protein interactions (PPIs). Therefore, mapping PPIs is important to understand the biological functions of many proteins in P. gingivalis. Herein, we provide native migration profiles of over 1000 P. gingivalis proteins. Using the T9SS, we demonstrate that our dataset is a useful resource for identifying novel protein interactions. Using this dataset and further analysis of T9SS P. gingivalis mutants, we discover new mechanistic insights into the formation of the PorQ-Z complex of the T9SS. This dataset is a valuable resource for studies of P. gingivalis.

Immunoregulation of the decidualization program: focus on the endoplasmic reticulum stress.

Decidualization denotes the reprogramming of endometrial stromal cells that includes the secretion of different mediators like cytokines, chemokines, and the selective recruitment of immune cells. This physiological process involves changes in the secretome of the endometrial stromal cells leading to the production of immunomodulatory factors. The increased amount of protein secretion is associated with a physiological endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR), allowing the expansion of ER and the machinery to assist the protein folding. Notably, the signaling pathways involved in the ER stress and the UPR are interconnected with the onset of a sterile inflammatory response, as well as with angiogenesis. Both of these processes have a key role in decidualization and placentation, therefore, alterations in them could lead to pregnancy complications. In this review, we will discuss how the induction of ER stress and the UPR processes that accompanies the decidualization are associated with embryo implantation and whether they might condition pregnancy outcome. The ER stress activates/triggers sensing proteins which, among others, induces kinase/RNAse-TXNIP expression, activating the NLRP3 inflammasome. This multiprotein system allows caspase-1 activation, which catalyzes the cleavage of the inactive IL-1β proform toward the mature secretory form, with pro-implantatory effects. However, the sterile inflammatory response should be later controlled in favor of a tolerogenic microenvironment to sustain pregnancy. In accordance, alterations of the ER stress and UPR processes can be reflected in recurrent implantation failures (RIF), recurrent pregnancy loss (RPL), or complications associated with deficient placentation, such as preeclampsia (PE).

The Tol-Pal system is required for peptidoglycan-cleaving enzymes to complete bacterial cell division

Tol-Pal is a multiprotein system present in the envelope of Gram-negative bacteria. Inactivation of this widely conserved machinery compromises the outer membrane (OM) layer of these organisms, resulting in hypersensitivity to many antibiotics. Mutants in the tol-pal locus fail to complete division and form cell chains. This phenotype along with the localization of Tol-Pal components to the cytokinetic ring in Escherichia coli has led to the proposal that the primary function of the system is to promote OM constriction during division. Accordingly, a poorly constricted OM is believed to link the cell chains formed upon Tol-Pal inactivation. However, we show here that cell chains of E. coli tol-pal mutants are connected by an incompletely processed peptidoglycan (PG) layer. Genetic suppressors of this defect were isolated and found to overproduce OM lipoproteins capable of cleaving the glycan strands of PG. Among the factors promoting cell separation in mutant cells was a protein of previously unknown function (YddW), which we have identified as a divisome-localized glycosyl hydrolase that cleaves peptide-free PG glycans. Overall, our results indicate that the cell chaining defect of Tol-Pal mutants cannot simply be interpreted as a defect in OM constriction. Rather, the complex also appears to be required for the activity of several OM-localized enzymes with cell wall remodeling activity. Thus, the Tol-Pal system may play a more general role in coordinating OM invagination with PG remodeling at the division site than previously appreciated.

Towards capturing cellular complexity: combining encapsulation and macromolecular crowding in a reverse micelle.

Confinement and macromolecular crowding are acknowledged to be fundamental for the understanding of biomolecular systems. This computational study combines these two phenomena by investigating a multi-protein system encapsulated in a reverse micelle. Contemporary controversies regarding force field accuracy with respect to hydration in such systems were addressed by scaling the non-bonded, non-charged interaction of water with the surfaces, i.e. protein and surfactant (λ-scaling). While based on extensive atomistic simulations, our analysis is of mesoscopic nature with a focus on dielectric properties since recent experimental studies have gained insight on protein mutual orientation via dielectric permittivity. We find two dielectric mechanisms, one allowing for parallel and orthogonal protein dipole alignment while the other leads to preferred anti-parallel alignment. Which mechanism actually occurs is decided by the degree of hydration of both the proteins and the capsule interface. We raise the question whether the findings for reverse micelles can be transferred to biological cells. The embedding in the highly polar outside medium plays an important role in the answer.

Synthetic microbial consortia enable rapid assembly of pure translation machinery.

Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome

The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol ε DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1-Tof1-Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication.

Regulation of Renal Electrolyte Transport by WNK and SPAK-OSR1 Kinases.

The discovery of four genes responsible for pseudohypoaldosteronism type II, or familial hyperkalemic hypertension, which features arterial hypertension with hyperkalemia and metabolic acidosis, unmasked a complex multiprotein system that regulates electrolyte transport in the distal nephron. Two of these genes encode the serine-threonine kinases WNK1 and WNK4. The other two genes [kelch-like 3 (KLHL3) and cullin 3 (CUL3)] form a RING-type E3-ubiquitin ligase complex that modulates WNK1 and WNK4 abundance. WNKs regulate the activity of the Na(+):Cl(-) cotransporter (NCC), the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), and other transport pathways. Interestingly, the modulation of NCC occurs via the phosphorylation by WNKs of other serine-threonine kinases known as SPAK-OSR1. In contrast, the process of regulating the channels is independent of SPAK-OSR1. We present a review of the remarkable advances in this area in the past 10 years.

A Discontinuous Potential Model for Protein-Protein Interactions.

Protein-protein interactions play an important role in many biologic and industrial processes. In this work, we develop a two-bead-per-residue model that enables us to account for protein-protein interactions in a multi-protein system using discontinuous molecular dynamics simulations. This model deploys discontinuous potentials to describe the non-bonded interactions and virtual bonds to keep proteins in their native state. The geometric and energetic parameters are derived from the potentials of mean force between sidechain-sidechain, sidechain-backbone, and backbone-backbone pairs. The energetic parameters are scaled with the aim of matching the second virial coefficient of lysozyme reported in experiment. We also investigate the performance of several bond-building strategies.

Interregional Coevolution Analysis Revealing Functional and Structural Interrelatedness between Different Genomic Regions in Human Mastadenovirus D.

Human mastadenovirus D (HAdV-D) is exceptionally rich in type among the seven human adenovirus species. This feature is attributed to frequent intertypic recombination events that have reshuffled orthologous genomic regions between different HAdV-D types. However, this trend appears to be paradoxical, as it has been demonstrated that the replacement of some of the interacting proteins for a specific function with other orthologues causes malfunction, indicating that intertypic recombination events may be deleterious. In order to understand why the paradoxical trend has been possible in HAdV-D evolution, we conducted an interregional coevolution analysis between different genomic regions of 45 different HAdV-D types and found that ca. 70% of the genome has coevolved, even though these are fragmented into several pieces via short intertypic recombination hot spot regions. Since it is statistically and biologically unlikely that all of the coevolving fragments have synchronously recombined between different genomes, it is probable that these regions have stayed in their original genomes during evolution as a platform for frequent intertypic recombination events in limited regions. It is also unlikely that the same genomic regions have remained almost untouched during frequent recombination events, independently, in all different types, by chance. In addition, the coevolving regions contain the coding regions of physically interacting proteins for important functions. Therefore, the coevolution of these regions should be attributed at least in part to natural selection due to common biological constraints operating on all types, including protein-protein interactions for essential functions. Our results predict additional unknown protein interactions. Human mastadenovirus D, an exceptionally type-rich human adenovirus species and causative agent of different diseases in a wide variety of tissues, including that of ocular region and digestive tract, as well as an opportunistic infection in immunocompromised patients, is known to have highly diverged through frequent intertypic recombination events; however, it has also been demonstrated that the replacement of a component protein of a multiprotein system with a homologous protein causes malfunction. The present study solved this apparent paradox by looking at which genomic parts have coevolved using a newly developed method. The results revealed that intertypic recombination events have occurred in limited genomic regions and been avoided in the genomic regions encoding proteins that physically interact for a given function. This approach detects purifying selection against recombination events causing the replacement of partial components of multiprotein systems and therefore predicts physical and functional interactions between different proteins and/or genomic elements.

Revisiting the NaCl cotransporter regulation by with-no-lysine kinases.

The renal thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is the salt transporter in the distal convoluted tubule. Its activity is fundamental for defining blood pressure levels. Decreased NCC activity is associated with salt-remediable arterial hypotension with hypokalemia (Gitelman disease), while increased activity results in salt-sensitive arterial hypertension with hyperkalemia (pseudohypoaldosteronism type II; PHAII). The discovery of four different genes causing PHAII revealed a complex multiprotein system that regulates the activity of NCC. Two genes encode for with-no-lysine (K) kinases WNK1 and WNK4, while two encode for kelch-like 3 (KLHL3) and cullin 3 (CUL3) proteins that form a RING type E3 ubiquitin ligase complex. Extensive research has shown that WNK1 and WNK4 are the targets for the KLHL3-CUL3 complex and that WNKs modulate the activity of NCC by means of intermediary Ste20-type kinases known as SPAK or OSR1. The understanding of the effect of WNKs on NCC is a complex issue, but recent evidence discussed in this review suggests that we could be reaching the end of the dark ages regarding this matter.

Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system

The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS–PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS–PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition.

Thermoresponsive copolymer decorated surface enables controlling the adsorption of a target protein in plasma.

The control of protein/surface interactions by external stimuli is often required in bioapplications such as bioseparation and biosensors. Although regulation of protein adsorption has been achieved on the surfaces modified with stimuli-responsive polymers, controlled protein adsorption is still challenging for a target protein in a multiprotein system. The present study developed a concept of surface design for the controlled adsorption of a specific protein from plasma by combining a thermoresponsive polymer with an affinity ligand on the surface. In this regard, a polyurethane (PU) surface was modified with the copolymer of N-isopropylacrylamide (NIPAAm) and a ε-lysine-containing monomer (LysMA). ε-Lysine is a specific ligand for plasminogen that was used as the model "target protein" in this study. The PU-P(NIPAAm-co-Lys) surfaces exhibited distinct thermoresponsivity of plasminogen adsorption from plasma with a larger quantity adsorbed at 37 °C than at 23 °C. By contrast, the surfaces showed a low level of adsorption for other plasma proteins at both temperatures. In addition, plasminogen adsorbed on a PU-P(NIPAAm-co-Lys) surface could be partly desorbed by lowering the temperature, and the activity of plasminogen adsorbed was well preserved. We believe that the concept developed in this study can be extended to other proteins by combining PNIPAAm and specific ligands with affinities for the proteins of interest.

Adsorption and Release Behaviors of Vascular Endothelial Growth Factor on Porous Hydroxyapatite Ceramic Under Competitive Conditions

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, ChinaVascular endothelial growth factor plays a positive role in the vascularization of bone grafts afterimplantation. In the present study, we constructed a multi-protein system and investigated itsadsorption and release on porous hydroxyapatite ceramic under competitive conditions, and wealso evaluated its bioactivity by cells proliferation experiments after it released from the ceramic.The results showed that vascular endothelial growth factor always had a high adsorption on poroushydroxyapatite ceramic, whether having the inhibition of other serum proteins or not. When incu-bated in the cell culture medium containing 10% fetal calf serum, the pre-loaded vascular endothelialgrowth factor showed a slow and sustained release from the ceramic. After 7 days of incubation,its accumulative release rate was only about 50%. The high adsorption and slow release of vas-cular endothelial growth factor on porous hydroxyapatite ceramic should be ascribed to the strongelectrostatic attraction between them as well as the abundant nano-sized micropores present onthe ceramic surface. The cell culture experiments con“rmed that the released vascular endothelialgrowth factor from porous hydroxyapatite ceramic could promote the proliferation of human umbilicalvein endothelial cells signi“cantly, indicating that its bioactivity was still maintained after desorb-ing from the ceramic. As thus, it could be inferred that porous hydroxyapatite ceramic may adsorband accumulate the endogenous vascular endothelial growth factor at the early implantation stage,which then releases from the ceramic surface gradually and takes effect on the ingrown vascularendothelial cells, leading to the rapid vascularization of the implant.

Subcellular location of the coupling protein TrwB and the role of its transmembrane domain

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.

Biophysical and computational fragment-based approaches to targeting protein–protein interactions: applications in structure-guided drug discovery

Drug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein-protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids - so-called hotspots - may contribute the majority of interaction-free energy. The challenges posed by protein-protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein-protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods - nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry - to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein-protein interactions and have therapeutic value.

Evolution of Conjugation and Type IV Secretion Systems

Genetic exchange by conjugation is responsible for the spread of resistance, virulence, and social traits among prokaryotes. Recent works unraveled the functioning of the underlying type IV secretion systems (T4SS) and its distribution and recruitment for other biological processes (exaptation), notably pathogenesis. We analyzed the phylogeny of key conjugation proteins to infer the evolutionary history of conjugation and T4SS. We show that single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) conjugation, while both based on a key AAA+ ATPase, diverged before the last common ancestor of bacteria. The two key ATPases of ssDNA conjugation are monophyletic, having diverged at an early stage from dsDNA translocases. Our data suggest that ssDNA conjugation arose first in diderm bacteria, possibly Proteobacteria, and then spread to other bacterial phyla, including bacterial monoderms and Archaea. Identifiable T4SS fall within the eight monophyletic groups, determined by both taxonomy and structure of the cell envelope. Transfer to monoderms might have occurred only once, but followed diverse adaptive paths. Remarkably, some Firmicutes developed a new conjugation system based on an atypical relaxase and an ATPase derived from a dsDNA translocase. The observed evolutionary rates and patterns of presence/absence of specific T4SS proteins show that conjugation systems are often and independently exapted for other functions. This work brings a natural basis for the classification of all kinds of conjugative systems, thus tackling a problem that is growing as fast as genomic databases. Our analysis provides the first global picture of the evolution of conjugation and shows how a self-transferrable complex multiprotein system has adapted to different taxa and often been recruited by the host. As conjugation systems became specific to certain clades and cell envelopes, they may have biased the rate and direction of gene transfer by conjugation within prokaryotes.

Sided Functions of an Arginine–Agmatine Antiporter Oriented in Liposomes

The arginine-dependent extreme acid resistance system helps enteric bacteria survive the harsh gastric environment. At the center of this multiprotein system is an arginine-agmatine antiporter, AdiC. To maintain cytoplasmic pH, AdiC imports arginine and exports its decarboxylated product, agmatine, resulting in a net extrusion of one "virtual proton" in each turnover. The random orientation of AdiC in reconstituted liposomes throws up an obstacle to quantifying its transport mechanism. To overcome this problem, we introduced a mutation, S26C, near the substrate-binding site. This mutant exhibits substrate recognition and pH-dependent activity similar to those of the wild-type protein but loses function completely upon reaction with thiol reagents. The membrane-impermeant MTSES reagent can then be used as a cleanly sided inhibitor to silence those S26C-AdiC proteins whose extracellular portion projects from the external side of the liposome. Alternatively, the membrane-permeant MTSEA and membrane-impermeant reducing reagent, TCEP, can be used together to inhibit proteins in the opposite orientation. This approach allows steady-state kinetic analysis of AdiC in a sided fashion. Arginine and agmatine have similar Michaelis-Menten parameters for both sides of the protein, while the extracellular side selects arginine over argininamide, a mimic of the carboxylate-protonated form of arginine, more effectively than does the cytoplasmic side. Moreover, the two sides of AdiC have different pH sensitivities. AdiC activity increases to a plateau at pH 4 as the extracellular side is acidified, while the cytoplasmic side shows an optimal pH of 5.5, with further acidification inhibiting transport. This oriented system allows more precise analysis of AdiC-mediated substrate transport than has been previously available and permits comparison to the situation experienced by the bacterial membrane under acid stress.