Membrane Orientation Research Articles

Heliorhodopsin Evolution Is Driven by Photosensory Promiscuity in Monoderms.

ABSTRACTRhodopsins are light-activated proteins displaying an enormous versatility of function as cation/anion pumps or sensing environmental stimuli and are widely distributed across all domains of life. Even with wide sequence divergence and uncertain evolutionary linkages between microbial (type 1) and animal (type 2) rhodopsins, the membrane orientation of the core structural scaffold of both was presumed universal. This was recently amended through the discovery of heliorhodopsins (HeRs; type 3), that, in contrast to known rhodopsins, display an inverted membrane topology and yet retain similarities in sequence, structure, and the light-activated response. While no ion-pumping activity has been demonstrated for HeRs and multiple crystal structures are available, fundamental questions regarding their cellular and ecological function or even their taxonomic distribution remain unresolved. Here, we investigated HeR function and distribution using genomic/metagenomic data with protein domain fusions, contextual genomic information, and gene coexpression analysis with strand-specific metatranscriptomics. We bring to resolution the debated monoderm/diderm occurrence patterns and show that HeRs are restricted to monoderms. Moreover, we provide compelling evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla. In addition, we also describe two novel putative signal-transducing domains fused to some HeRs. We posit that HeRs likely function as generalized light-dependent switches involved in the mitigation of light-induced oxidative stress and metabolic circuitry regulation. Their role as sensory rhodopsins is corroborated by their photocycle dynamics and their presence/function in monoderms is likely connected to the higher sensitivity of these organisms to light-induced damage.IMPORTANCE Heliorhodopsins are enigmatic, novel rhodopsins with a membrane orientation that is opposite to all known rhodopsins. However, their cellular and ecological functions are unknown, and even their taxonomic distribution remains a subject of debate. We provide evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla boundaries. In support of this, we also identify two novel putative signal transducing domains in HeRs that are fused with them. We also observe linkages of HeRs to genes involved in mitigation of light-induced oxidative stress and increased carbon and nitrogen metabolism. Finally, we synthesize these findings into a framework that connects HeRs with the cellular response to light in monoderms, activating light-induced oxidative stress defenses along with carbon/nitrogen metabolic circuitries. These findings are consistent with the evolutionary, taxonomic, structural, and genomic data available so far.

Fabrication of MIL-96 nanosheets and relevant c-oriented ultrathin membrane through solvent optimization

Concurrent manipulation of orientation and thickness of MOF membranes has become a reliable approach for improving their separation performances. In terms of oriented seeded growth, oriented deposition of MOF nanosheets during seeding process and sealing all intergranular gaps with no discernible increase in membrane thickness during secondary growth have become indispensable. In this study, we pioneered the fabrication of uniform hexagonal-shaped, 20–40 nm thick MIL-96 nanosheets through solvent composition optimization (VH2O/VFA = 1/2). Subsequently, spin coating was employed for c-oriented deposition of uniform MIL-96 seed layer, during which the addition of PVP in the suspension was found to be critical for obtaining qualified seed layer. Finally, well-intergrown, highly c-oriented ultrathin MIL-96 membrane was prepared upon employing NMF as the solvent during secondary growth. Gas permeation results implied that prepared MIL-96 membrane exhibited superior gas separation performance compared with that of a-&b-oriented counterpart.

Phonon resonant effect in silicon membranes with different crystallographic orientations

Engineering low-frequency phonon transport in nanostructures with the phonon resonant mechanism has become an important research direction. On the basis of non-equilibrium molecular dynamics simulations, the thermal transport in pristine and resonant Si-membranes bounded with {100}, {110} and {111} facets is investigated. It is found that the creation of surfaces can introduce anisotropic thermal transport due to the lattice symmetry breaking. Besides, ballistic phonon transport is found in pristine membranes with lengths up to 500 nm at low-frequencies with a critical frequency mainly dependent on the crystallographic orientation. Moreover, although surface resonances can dramatically reduce the thermal conductivity of all membranes, the resonant effect strongly relies on membrane orientation. Among the three studied membrane orientations, the resonant effect is maximized in the {111}-membrane, where the thermal conductivity is tuned from the largest one to the smallest one among the three membrane types by resonant pillars. The large thermal conductivity reduction in the {111}-membranes by resonances originated from the reduced spectral heat flux between 3 and 12 THz. Furthermore, the resonant coupling strength can be tuned by the interface vacancy between resonant pillars and the base material, which can enhance phonon transport at an intermediate frequency range. Our work provides further insights on thermal transport engineering by phonon resonances and could be useful for thermal conductivity engineering with surface orientations and resonances.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

PH-Sensitive liposomes with embedded ampholytic derivatives of cholan-24-oic acid

Stimulus-sensitive liposomes have been prepared from zwitterionic dioleoylphosphocholine and ampholytic molecular switches with carboxylic anionic groups and the triazole or isobutylamino cationic ones attached to the opposite ends of the steroid core. When the pH of outer solution was altered from slightly alkaline to slightly acidic, the switches changed their orientation in the liposomal membrane, which induced temporal defects formation and the release of a drug model load. The low-toxic pH-sensitive isobutylamino derivative–dioleoylphosphocholine liposomes demonstrated fast cargo release.

On the Order and Orientation in Liquid Crystalline Polymer Membranes for Gas Separation.

To prevent greenhouse emissions into the atmosphere, separations like CO2/CH4 and CO2/N2 from natural gas, biogas, and flue gasses are crucial. Polymer membranes gained a key role in gas separations over the past decades, but these polymers are often not organized at a molecular level, which results in a trade-off between permeability and selectivity. In this work, the effect of molecular order and orientation in liquid crystals (LCs) polymer membranes for gas permeation is demonstrated. Using the self-assembly of polymerizable LCs to prepare membranes ensures control over the supramolecular organization and alignment of the building blocks at a molecular level. Robust freestanding LC membranes were fabricated that have various, distinct morphologies (isotropic, nematic cybotactic, and smectic C) and alignment (planar and homeotropic), while using the same chemical composition. Single gas permeation data show that the permeability decreases with increasing molecular order while the ideal gas selectivity of He and CO2 over N2 increases tremendously (36-fold for He/N2 and 21-fold for CO2/N2) when going from randomly ordered to the highly ordered smectic C morphology. The calculated diffusion coefficients showed a 10-fold decrease when going from randomly ordered membranes to ordered smectic C membranes. It is proposed that with increasing molecular order, the free volume elements in the membrane become smaller, which hinders gasses with larger kinetic diameters (Ar, N2) more than gasses with smaller kinetic diameters (He, CO2), inducing selectivity. Comparison of gas sorption and permeation performances of planar and homeotropic aligned smectic C membranes shows the effect of molecular orientation by a 3-fold decrease of the diffusion coefficient of homeotropic aligned smectic C membranes resulting in a diminished gas permeation and increased ideal gas selectivities. These results strongly highlight the importance of molecular order and orientation in LC polymer membranes for gas separation.

The Sigma-1 receptor is an ER-localized type II membrane protein

The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.

Forward osmosis concentration of high viscous polysaccharides of Dendrobium officinale: Process optimisation and membrane fouling analysis

SummaryPolysaccharides of Dendrobium officinale (DOP) need to be dehydrated and concentrated after extraction for further application. They are usually concentrated by thermal evaporation which consumes great energy. However, high viscosity of DOP makes the concentration more difficult even using non‐thermal membrane technologies such as nanofiltration (NF) or reverse osmosis (RO). In this study, effects of process conditions, such as membrane orientations, draw solutions and their concentrations, and flowrate on forward osmosis (FO) concentration of viscous DOP were studied. Active layer to feed solution mode, cross flowrate at 240 mL min−1, and draw solution of 3 m NaCl have been found as the optimal conditions. Foulants on the membrane surface with loose structure could be easily cleaned and removed by hydraulic flushing. DOP concentrated by FO achieved almost 1.3 times at the same time compared with that in NF and RO. DOP could be further concentrated for 1.5 folds at longtime without significant decrease in water flux. In addition, slight reverse solutes in FO process could reduce the viscosity of high viscous DOP, which was good for concentration. Accordingly, FO is a potential technology for concentration of high viscous polysaccharides such as DOP.

Fabrication of highly (110)-Oriented ZIF-8 membrane at low temperature using nanosheet seed layer

Preferred orientation represents a main concern for microstructure control and performance optimization of MOF membranes. To date, it remains a challenging task to precisely control preferred orientation of ZIF-8 membrane. Herein, we prepared highly (110)-oriented ZIF-8 membrane by oriented secondary growth. Among various elements, preparation of uniform ZIF-8 nanosheet seeds through thermal conversion of identically shaped ZIF-L precursors, spin-coating-assisted oriented deposition of (110)-oriented ZIF-8 seed layer, and controlled in-plane secondary growth at low temperature were found crucial for obtaining well-intergrown (110)-oriented ZIF-8 membrane whose H2/CO2 ideal selectivity exceeded H2/N2 and H2/CH4 under ambient conditions, owing to preferential CO2 adsorption capacity of not only PEI but also ZIF-8 nanosheets compared with their bulk counterparts. Our research highlighted the significance of preferred orientation regulation in tailoring the gas permeation behavior of MOF membranes.

Brine reject dilution with treated wastewater for indirect desalination

The forward osmosis (FO) process was suggested as a pretreatment to a multi-stage flashing (MSF) plant to reduce the environmental impact of brine discharge and the chemicals used. Yet, there is no study investigating the performance of the FO process pretreatment to the MSF plant using tertiary sewage effluent (TSE) as a feed solution. Combining MSF brine with the TSE generates a considerable permeation flux, reducing the membrane area and capital cost. This study evaluated the performance of the FO process for indirect desalination of the MSF brine, considering membrane fouling, cleaning, required membrane area and the specific power consumption. The FO process used a thin-film composite (TFC) membrane to dilute the brine reject from the MSF plant by the TSE and hence converting waste solutions into a feasible water resource. A considerable high water flux (±35 L/m2h) was generated and slightly decreased throughout each experiment's 4 cycles. An enhancement in the water permeability was observed in the FO tests with a prefiltration of the brine reject and the wastewater with 20 μm and an osmotic backwash cleaning of the used membrane. The prefiltration of the draw and feed solutions was effective in minimizing the impact of fouling. Maximum power consumption of 0.007 kWh/m³ was consumed in the forward osmosis process without prefiltration and decreased to 0.006 kWh/m³ in the FO process. The proposed FO system successfully diluted the brine reject’ divalent ions, reducing their concentration to 43% in some cases. Depending on the FO membrane orientation, the TSE feed solution resulted in a 276%–473% reduction in the number of FO elements required in the FO process compared to the seawater feed solution.

Production of cooling water by Ti3C2Tx MXene interlayered forward osmosis membranes for post-combustion CO2 capture system

Forward osmosis (FO) technology has the potential to replace the trim cooler and to supply cooling water for a post-combustion CO2 capture system. Herein, a high-performance MXene (Ti3C2Tx) interlayered FO membrane was fabricated. Thanks to the ability of MXene to adjust the structures of substrate and polyamide, the MXene interlayered FO (MXene-FO) membrane showed improved permeability and selectivity in a monoethanolamine (MEA) draw solution. The performance of MXene-FO membrane was investigated at different membrane orientations and temperatures with amino acid/salt (AAS) solutions (taurine/KOH and β-alanine/KOH) as draw solutions. Because the osmotic pressures provided by AAS solutions (104.4 ± 0.3 bar for 3 mol/L taurine/KOH and 153.7 ± 0.2 bar for 5 mol/L β-alanine/KOH solutions) are higher than that of 5 mol/L MEA solution (93.0 ± 0.1 bar), the MXene-FO membrane exhibited higher water fluxes in AAS solutions. Besides, the good performance of the MXene-FO membrane was shown with the presence of small specific amino acid fluxes and specific KOH fluxes. To investigate the feasibility of using saline water as the feed solution, 3.5 wt% NaCl solution was applied and the results indicated that the MXene-FO membrane maintained stable water fluxes and small specific solute fluxes. This work demonstrates the potential of nanomaterials interlayered FO membranes for cooling water production in a post-combustion CO2 capture system.

Nitrate modulation of Bacillus sp. biofilm components: a proposed model for sustainable bioremediation.

The presence of different pollutants in wastewater hinder microbial growth, compromise enzymatic activity or compete for electrons required for bioremediation pathway. Therefore, there is a need to use a single microorganism that is capable of tolerating different toxic compounds and can perform simultaneous bioremediation. In the present study, nitrate reducing bacteria capable of decolorizing azo dye was identified as Bacillus subtillis sp. DN using protein profiling, morphological and biochemical tests X-ray diffraction pattern, Raman spectroscopy and cyclic voltammetry confirm that the bacterium under study possesses membrane-bound nitrate reductase and that is capable of direct electron transfer. The addition of nitrate concentrations (0-50mM) resulted in increased biofilm formation with variable exopolysaccharides, protein, and eDNA. Fourier Transform Infrared spectrum revealed the presence of a biopolymer at high nitrate concentrations. Effective capacitance and conductivity of the cells grown in different nitrate concentrations suggest changes in the relative position of polar groups, their relative orientation and permeability of cell membrane as detected by dielectric spectroscopy. The increase in biofilm shifted the removal of the azo dye from biodegradation to bioadsorption. Our results indicate that nitrate modulates biofilm components. Bacillus sp. DN granular biofilm can be used for simultaneous nitrate and azo dye removal from wastewater.

Tryptophan, an Amino-Acid Endowed with Unique Properties and Its Many Roles in Membrane Proteins

Tryptophan is an aromatic amino acid with unique physico-chemical properties. It is often encountered in membrane proteins, especially at the level of the water/bilayer interface. It plays a role in membrane protein stabilization, anchoring and orientation in lipid bilayers. It has a hydrophobic character but can also engage in many types of interactions, such as π–cation or hydrogen bonds. In this review, we give an overview of the role of tryptophan in membrane proteins and a more detailed description of the underlying noncovalent interactions it can engage in with membrane partners.

Regulating the Orientation of Hydrogen-Bonded Organic Framework Membranes Based on Substrate Modification

Regulating the Orientation of Hydrogen-Bonded Organic Framework Membranes Based on Substrate Modification

Microbial Rhodopsins: The Last Two Decades.

Microbial rhodopsins are diverse photoreceptive proteins containing a retinal chromophore and are found in all domains of cellular life and are even encoded in genomes of viruses. These rhodopsins make up two families: type 1 rhodopsins and the recently discovered heliorhodopsins. These families have seven transmembrane helices with similar structures but opposing membrane orientation. Microbial rhodopsins participate in a portfolio of light-driven energy and sensory transduction processes. In this review we present data collected over the last two decades about these rhodopsins and describe their diversity, functions, and biological and ecological roles.

Preparation and Evaluation of Hemoglobin-Bovine Serum Albumin Nanoparticles with Red Blood Cell Membrane Directional Coating

To prepare hemoglobin-bovine serum albumin nanoparticles with red blood cell membrane directional coating (RBC-Hb/BSA-NP) and to evaluate their physicochemical properties and long circulation capability. The bovine serum albumin nanoparticles modified with human hemoglobin (Hb/BSA-NP) were prepared by the solvent evaporation method. Then, the red blood cell (RBC) membrane was coated on the surface of Hb/BSA-NP by applying physical extrusion. The particle size, zeta potential and morphology of the resulting RBC-Hb/BSA-NP were characterized. The optimal amount of RBC membrane was determined by evaluating the integrity of the RBC membrane coating on RBC-Hb/BSA-NP, while the orientation of the RBC membrane was determined by measuring the content of sialic acid content on the surface of the RBC-Hb/BSA-NP. The anti-phagocytosis ability against macrophage of the RBC-Hb/BSA-NP was evaluated with fluorescence microscope and flow cytometry in vitro , and the long circulation capability of the nanoparticles was assessed through pharmacokinetic experiment in vivo. The RBC-Hb/BSA-NP showed an average size of (127.7±3.5) nm, a zeta potential of (-17.1±0.28) mV, a clear core-shell structure and good stability within 72 h. The RBC membrane extracted from 0.8 mL of whole blood can completely coat 1 mL of Hb/BSA-NP ( ρ BSA=10 mg/mL). The content of sialic acid on the RBC-Hb/BSA-NP was comparable to that of natural red blood cells without significant change, indicating the correct orientation of RBC membranes on the surface of the nanoparticles. Compared with regular BSA nanoparticles, RBC-Hb/BSA-NP could significantly reduce the uptake in macrophages, and it's circulation time in vivo was greatly prolonged. RBC-Hb/BSA-NP was successfully constructed, and it was shown that the delivery system had prolonged circulation time.

Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

In situ construction of an immobilized b-oriented titanium silicalite spherical molecular sieve membrane

Oriented molecular sieve membranes prepared on the surface of a specifically shaped substrate have been applied in the separation and catalysis process, but there is no report on the directional growth of molecular sieve membranes on the surface of spherical substrates. Here, a simple in situ crystallization method was employed to successfully prepare a single layer of b-oriented continuous titanium silicalite-1 (TS-1) molecular sieve membrane on the surface of 2-mm alumina beads. First, a titania intermediate layer was loaded on the surface of beads by the dipping method, and the surface was then modified with the TBOT modification solution. The ratio of the precursor solution was n(TEOS):n(TBOT):n(TPAOH):n(H2O) = 1:0.010:0.15:40 and was crystallized at 180 °C for 72 h to prepare the membrane. The content of the templating agent and water affected the size of TS-1 crystal grains and membrane crosslinking—both in turn influenced the continuity and orientation of the molecular sieve membrane. Spherical membrane catalyst was applied to catalyze the epoxidation of chloropropene in a fixed bed reactor, and the conversion of hydrogen peroxide and selectivity of epichlorohydrin attained 87.4% and 93.2%, respectively. This work provides a foundation for the design of novel TS-1 immobilized catalyst.

Inside-Out-Oriented Cell Membrane Biomimetic Magnetic Nanoparticles for High-Performance Drug Lead Discovery.

Biomimetic cell membrane-coated nanoparticles have been broadly applied because of their superior biochemical properties. The right-side-out cell membrane coating manner provides nanoparticles with an immune-evasive stealth function in vivo. However, this acts as a drag for drug discovery when the drug targets are the intracellular domain of transmembrane receptors. Herein, inside-out-oriented cell membrane-coated nanoparticles were prepared for screening tyrosine kinase inhibitors, which specifically interacted with the intracellular kinase domain of the epidermal growth factor receptor. Biotinylated human lung adenocarcinoma epithelial cell membranes specifically interacted with streptavidin-immobilized Fe3O4 magnetic nanoparticles and then formed inside-out-oriented cell membrane-coated magnetic nanoparticles (IOCMMNPs). The cell membrane orientation of the IOCMMNPs was successfully confirmed by immunogold electron microscopy, fluorescently labeled confocal microscopy, sialic acid quantification assay, and the adsorption capacity assay. Moreover, IOCMMNPs possessed satisfactory binding capacity, selectivity, and high sensitivity (limit of detection = 0.4 × 10-3 μg mL-1). Ultimately, IOCMMNPs successfully targeted two main compounds from Strychnos nux-vomica whose potential antitumor activities were further validated by pharmacological studies. The application of the inside-out cell membrane coating strategy further enhances the drug screening efficiency and broadens the insight and methodologies for drug lead discovery. This inside-out cell membrane coating concept also provides a method for the future development of engineered cell membrane-coated nanotechnology.

Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC

Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an Nin-Cin orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.