Denice C. Bay

Diversity and evolution of the small multidrug resistance protein family

BackgroundMembers of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins.ResultsA thorough annotation of unidentified/putative SMR sequences was performed placing sequences into each of the three SMR protein subclass designations, namely small multidrug proteins (SMP), suppressor of groEL mutations (SUG), and paired small multidrug resistance (PSMR) using protein alignments and phylogenetic analysis. Examination of SMR subclass distribution within Bacteria and Archaea taxa identified specific Bacterial classes that uniquely encode for particular SMR subclass members. The extent of selective pressure acting upon each SMR subclass was determined by calculating the rate of synonymous to non-synonymous nucleotide substitutions using Syn-SCAN analysis. SUG and SMP subclasses are maintained under moderate selection pressure in comparison to integron and plasmid encoded SMR homologues. Conversely, PSMR sequences are maintained under lower levels of selection pressure, where one of the two PSMR pairs diverges in sequence more rapidly than the other. SMR genomic loci surveys identified potential SMR efflux substrates based on its gene association to putative operons that encode for genes regulating amino acid biogenesis and QAC-like metabolites. SMR subclass protein transmembrane domain alignments to Bacterial/Archaeal transporters (BAT), DMT, and MFS sequences supports SMR participation in multidrug transport evolution by identifying common TM domains.ConclusionBased on this study, PSMR sequences originated recently within both SUG and SMP clades through gene duplication events and it appears that SMR members may be evolving towards specific metabolite transport.

Small multidrug resistance protein EmrE reduces host pH and osmotic tolerance to metabolic quaternary cation osmoprotectants.

The small multidrug resistance (SMR) transporter protein EmrE in Escherichia coli is known to confer resistance to toxic antiseptics classified as quaternary cation compounds (QCCs). Naturally derived QCCs synthesized during metabolic activities often act as osmoprotectants, such as betaine and choline, and participate in osmotic homoestasis. The goal of this study was to determine if EmrE proteins transport biological QCC-based osmoprotectants. Plasmid-encoded copies of E. coli emrE and the inactive variant emrE-E14C (emrE with the E → C change at position 14) were expressed in various E. coli strains grown in either rich or minimal media at various pHs (5 to 9) and under hypersaline (0.5 to 1.0 M NaCl and KCl) conditions to identify changes in growth phenotypes induced by osmoprotectant transport. The results demonstrated that emrE expression reduced pH tolerance of E. coli strains at or above neutral pH and when grown in hypersaline media at or above NaCl or KCl concentrations of 0.75 M. Hypersaline growth conditions were used to screen QCC osmoprotectants betaine, choline, l-carnitine, l-lysine, l-proline, and l-arginine. The study identified that betaine and choline are natural QCC substrates of EmrE.

Outer membrane protein OmpW participates with small multidrug resistance protein member EmrE in quaternary cationic compound efflux.

In Escherichia coli, the small multidrug resistance (SMR) transporter protein EmrE confers host resistance to a broad range of toxic quaternary cation compounds (QCC) via proton motive force in the plasma membrane. Biologically produced QCC also act as EmrE osmoprotectant substrates within the cell and participate in host pH regulation and osmotic tolerance. Although E. coli EmrE is one of the most well-characterized SMR members, it is unclear how the substrates it transports into the periplasm escape across the outer membrane (OM) in Gram-negative bacteria. We tested the hypothesis that E. coli EmrE relies on an unidentified OM protein (OMP) to complete the extracellular release of its QCC. Eleven OMP candidates were screened using an alkaline phenotypic growth assay to identify OMP involvement in EmrE-mediated QCC efflux. E. coli single-gene deletion strains were transformed with plasmid-carried copies of emrE to detect reduced-growth and rescued-growth phenotypes under alkaline conditions. Among the 11 candidates, only the ΔompW strain showed rescued alkaline growth tolerance when transformed with pEmrE, supporting the corresponding proteins involvement in EmrE osmoprotectant efflux. Coexpression of plasmids carrying the ompW and emrE genes transformed into the E. coli ΔompW and ΔemrE strains demonstrated a functional complementation restoring the original alkaline loss-of-growth phenotype. Methyl viologen drug resistance assays of pEmrE and pOmpW plasmid-complemented E. coli ΔompW and wild-type strains found higher host drug resistance than with other plasmid combinations. This study confirms our hypothesis that the porin OmpW participates in the efflux of EmrE-specific substrates across the OM.

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent.

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane alpha-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (</=1muM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (>/=100nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.

Visualizing a multidrug resistance protein, EmrE, with major bacterial lipids using Brewster angle microscopy

Understanding lipid-protein interactions to enhance our knowledge of membrane architecture is a critical step in the development of novel therapeutic measures to respond to the drastic rise of drug resistant microorganisms. Escherichia coli contains a small archetypal inner membrane multidrug resistance protein, EmrE, that must multimerize to be functional but this multimerization is difficult to demonstrate in vivo. We studied three major E. coli lipids (phosphatidylethanolamine, phosphatidylglycerol and cardiolipin) that varied in head group structure, acyl chain length and saturation. These were investigated both in the presence and absence of EmrE to determine which lipid(s) EmrE influenced most strongly. Langmuir monolayers and Brewster angle microscopy demonstrated that varying each head group, acyl chain length and saturation contributed to differences in membrane packing and affected lipid-protein associations. Long unsaturated anionic lipids were influenced most strongly by EmrE. Shorter acyl chains initiated string-like formations of EmrE clusters, whereas longer chains contributed to enhance protein clustering. Longer partially unsaturated acyl chains in phosphatidylglycerol showed a significant surface pressure decrease in the presence of the protein, indicating that the monolayer was destabilized. Interestingly, longer unsaturated chains of cardiolipin formed the most stable monolayer in the presence of EmrE. These studies indicate cardiolipin acyl chains that hydrophobically match protein helical lengths stabilize EmrE structural forms.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host.

Spectroscopic analysis of small multidrug resistance protein EmrE in the presence of various quaternary cation compounds

Escherichia coli EmrE protein is the archetypical member of the small multidrug resistance protein family in bacteria and confers host resistance to a wide assortment of toxic quaternary cation compounds by secondary active efflux. This protein can form a variety of multimers under various membrane mimetic conditions, and the consensus of most biochemical and biophysical studies indicate that the active form is a dimer. The purpose of this study is to characterize the conformation of organically extracted detergent solubilized EmrE protein known to predominate as monomer yet demonstrates ligand binding ability. Active site EmrE-E14 replacements were also examined as functionally inactive controls for this study. EmrE was solubilized in detergents, sodium dodecyl sulfate (SDS) and dodecyl maltoside (DDM), and protein conformation was examined in the presence of four known quaternary cation compound (QCC) substrates, tetraphenyl phosphonium (TPP), methyl viologen, cetylpyridinium, and ethidium. SDS-Tricine PAGE analysis of both detergent solubilized proteins revealed that DDM-EmrE preparations enhanced the formation of dimer (and in some cases trimer) forms in the presence of all four QCC above 25 QCC:1 EmrE molar ratios. Examination of EmrE and its active site variant tertiary structures in DDM by circular dichroism spectropolarimetry, intrinsic Trp fluorescence quenching and second order derivative ultraviolet absorbance revealed that the variant fails to bind TPP but interacts with all other compounds. The results of this study show that monomeric detergent solubilized EmrE is capable of forming multimeric complexes that are enhanced by chemically diverse QCCs.

'Come into the fold': A comparative analysis of bacterial redox enzyme maturation protein members of the NarJ subfamily.

Redox enzyme maturation proteins (REMPs) are system-specific chaperones required for the maturation of complex iron sulfur molybdoenzymes that are important for anaerobic respiration in bacteria. Although they perform similar biological roles, REMPs are strikingly different in terms of sequence, structure, systems biology, and type of terminal electron acceptor that it supports for growth. Here we critically dissect current knowledge pertaining to REMPs of the nitrate reductase delta superfamily, specifically recognized in Escherichia coli to include NarJ, NarW, TorD, DmsD, and YcdY, also referred to as the NarJ REMP subfamily. We show that NarJ subfamily members share sequence homology and similar structural features as revealed by alignments performed on structurally characterized REMPs. We include an updated phylogenetic analysis of subfamily members, justifying their classification in this subfamily. The structural and functional roles of each member are presented herein and these discussions suggest that although NarJ subfamily members are related in sequence and structure, each member demonstrates remarkable uniqueness, validating the concept of system-specific chaperones.

Spectroscopic analysis of the intrinsic chromophores within small multidrug resistance protein SugE

Small multidrug resistance (SMR) protein family member, SugE, is an integral inner membrane protein that confers host resistance to antiseptic quaternary cation compounds (QCC). SugE studies generally focus on its resistance to limited substrates in comparison to SMR protein EmrE. This study examines the conformational characteristics of SugE protein in two detergents, sodium dodecyl sulphate (SDS) and dodecyl maltoside (DDM), commonly used to study SMR proteins. The influence of cetylpyridinium (CTP) and cetrimide (CET) using SugE aromatic residues (4W, 2Y, 1F) as intrinsic spectroscopic probes was also determined. Organically extracted detergent solubilized Escherichia coli SugE protein was examined by SDS-Tricine PAGE and various spectroscopic techniques. SDS-Tricine PAGE analysis of SugE in either detergent demonstrates the protein predominates as a monomer but also dimerizes in SDS. Far-UV region circular dichroism (CD) analysis determined that the overall α-helix content SugE in SDS and DDM was almost identical and unaltered by QCC. Near-UV region CD, fluorescence, and second-derivative ultraviolet absorption (SDUV) indicated that only DDM-SugE promoted hydrophobic environments for its Trp and Tyr residues that were perturbed by QCC addition. This study identified that only the tertiary structure of SugE protein in DDM is altered by QCC.

The cmbT gene encodes a novel major facilitator multidrug resistance transporter in Lactococcus lactis.

Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in this study, it can be concluded that CmbT is a novel major facilitator multidrug resistance transporter candidate in L. lactis, with a possible signaling role in sulfur metabolism.