Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA

Recent crystal structures of the multidrug ATP‐binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208′) in the intracellular domains of its two half‐transporters, close to the nucleotide‐binding domains (NBDs), which are in close proximity of each other in the outward‐facing state but not in the inward‐facing state. Using intermolecular cysteine crosslinking between E208C and E208C′ in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide‐free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half‐transporters like a nucleotide‐free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

YddG from Escherichia coli promotes export of aromatic amino acids

The inner membrane protein YddG of Escherichia coli is a homologue of the known amino acid exporters RhtA and YdeD. It was found that the yddG gene overexpression conferred resistance upon E. coli cells to the inhibiting concentrations of l-phenylalanine and aromatic amino acid analogues, dl-p-fluorophenylalanine, dl-o-fluorophenylalanine and dl-5-fluorotryptophan. In addition, yddG overexpression enhanced the production of l-phenylalanine, l-tyrosine or l-tryptophan by the respective E. coli-producing strains. On the other hand, the inactivation of yddG decreased the aromatic amino acid accumulation by these strains. The cells of the E. colil-phenylalanine-producing strain containing overexpressed yddG accumulated less l-phenylalanine inside and exported the amino acid at a higher rate than the cells of the isogenic strain containing wild-type yddG. Taken together, these results indicate that YddG functions as an aromatic amino acid exporter.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

Molecular basis of bacterial resistance to chloramphenicol and florfenicol

Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance--even in the absence of a selective pressure imposed by the use of Cm or Ff.     

Two polyurethanases PueA and PueB are major extracellular lipases partly secreted by the mediation of their cognate ABC exporter AprDEF in Pseudomonas protegens Pf-5

Two polyurethanases PueA and PueB from Pseudomonas protegens Pf-5 have been reported to have hydrolytic activity against synthetic p-nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf-5), two heterologous co-expression strains and their three control strains in lipase-free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf-5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf-5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf-5 and BL21(DE3).     

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P_trc) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P_trc-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.     

Enhanced L-cysteine production by overexpressing potential L-cysteine exporter genes in an L-cysteine-producing recombinant strain of Corynebacterium glutamicum

ABSTRACT

We identified L-cysteine exporter candidates of Corynebacterium glutamicum and investigated the effect of overexpression of the potential L-cysteine exporter genes on L-cysteine production in a recombinant strain of C. glutamicum. Overexpression of NCgl2566 and NCgl0580 resulted in enhanced L-cysteine production in an L-cysteine-producing recombinant strain of C. glutamicum.     

A novel variant of mouse MATE-1 H+/organic cation antiporter with a long hydrophobic tail

Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H⁺-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters.