EmrE is an
- Publication Date:
- NSF-PAR ID:
- Journal Name:
- Applied and environmental microbiology
- Sponsoring Org:
- National Science Foundation
More Like this
EmrE is an
Escherichia colimultidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (p K a) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of p K avalues for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent p K avalues and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E.more ». Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent p K avalues reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.
The biological membrane is an efficient barrier against water-soluble substances. Solute transporters circumvent this membrane barrier by transporting water-soluble solutes across the membrane to the other sides. These transport proteins are thus required for all living organisms. Microorganisms, such as bacteria, effectively exploit solute transporters to acquire useful nutrients for growth or to expel substances that are inhibitory to their growth. Overall, there are distinct types of related solute transporters that are grouped into families or superfamilies. Of these various transporters, the major facilitator superfamily (MFS) represents a very large and constantly growing group and are driven by solute- and ion-gradients, making them passive and secondary active transporters, respectively. Members of the major facilitator superfamily transport an extreme variety of structurally different substrates such as antimicrobial agents, amino acids, sugars, intermediary metabolites, ions, and other small molecules. Importantly, bacteria, especially pathogenic ones, have evolved multidrug efflux pumps which belong to the major facilitator superfamily. Furthermore, members of this important superfamily share similar primary sequences in the form of highly conserved sequence motifs that confer useful functional properties during transport. The transporters of the superfamily also share similarities in secondary structures, such as possessing 12- or 14-membrane spanning α-helices and themore »
Fine scale transitions of the microbiota and metabolome along the gastrointestinal tract of herbivorous fishesAbstract Background Gut microorganisms aid in the digestion of food by providing exogenous metabolic pathways to break down organic compounds. An integration of longitudinal microbial and chemical data is necessary to illuminate how gut microorganisms supplement the energetic and nutritional requirements of animals. Although mammalian gut systems are well-studied in this capacity, the role of microbes in the breakdown and utilization of recalcitrant marine macroalgae in herbivorous fish is relatively understudied and an emerging priority for bioproduct extraction. Here we use a comprehensive survey of the marine herbivorous fish gut microbial ecosystem via parallel 16S rRNA gene amplicon profiling (microbiota) and untargeted tandem mass spectrometry (metabolomes) to demonstrate consistent transitions among 8 gut subsections across five fish of the genus of Kyphosus . Results Integration of microbial phylogenetic and chemical diversity data reveals that microbial communities and metabolomes covaried and differentiated continuously from stomach to hindgut, with the midgut containing multiple distinct and previously uncharacterized microenvironments and a distinct hindgut community dominated by obligate anaerobes. This differentiation was driven primarily by anaerobic gut endosymbionts of the classes Bacteroidia and Clostridia changing in concert with bile acids, small peptides, and phospholipids: bile acid deconjugation associated with early midgut microbiota, small peptidemore »
Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar HeidelbergGarrido, Daniel (Ed.)ABSTRACT The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S . Heidelberg strain using various routes of inoculation and determined if S . Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S . Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S . Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoirmore »
The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.