Complete genome sequence of Haloterrigena turkmenica type strain (4k)

Haloterrigena turkmenica (Zvyagintseva and Tarasov 1987) Ventosa et al. 1999, comb. nov. is the type species of the genus Haloterrigena in the euryarchaeal family Halobacteriaceae. It is of phylogenetic interest because of the yet unclear position of the genera Haloterrigena and Natrinema within the Halobacteriaceae, which created some taxonomic problems historically. H. turkmenica, was isolated from sulfate saline soil in Turkmenistan, is a relatively fast growing, chemoorganotrophic, carotenoid-containing, extreme halophile, requiring at least 2 M NaCl for growth. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the genus Haloterrigena, but the eighth genome sequence from a member of the family Halobacteriaceae. The 5,440,782 bp genome (including six plasmids) with its 5,287 protein-coding and 63 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Crystal structure of deltarhodopsin‐3 from Haloterrigena thermotolerans

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.     

Investigation of ligand binding to the multidrug resistance protein EmrE by isothermal titration calorimetry

Escherichia coli multidrug resistance protein E (EmrE) is an integral membrane protein spanning the inner membrane of Escherichia coli that is responsible for this organism's resistance to a variety of lipophilic cations such as quaternary ammonium compounds (QACs) and interchelating dyes. EmrE is a 12-kDa protein of four transmembrane helices considered to be functional as a multimer. It is an efflux transporter that can bind and transport cytoplasmic QACs into the periplasm using the energy of the proton gradient across the inner membrane. Isothermal titration calorimetry provides information about the stoichiometry and thermodynamic properties of protein-ligand interactions, and can be used to monitor the binding of QACs to EmrE in different membrane mimetic environments. In this study the ligand binding to EmrE solubilized in dodecyl maltoside, sodium dodecyl sulfate and reconstituted into small unilamellar vesicles is examined by isothermal titration calorimetry. The binding stoichiometry of EmrE to drug was found to be 1:1, demonstrating that oligomerization of EmrE is not necessary for binding to drug. The binding of EmrE to drug was observed with the dissociation constant (K(D)) in the micromolar range for each of the drugs in any of the membrane mimetic environments. Thermodynamic properties demonstrated this interaction to be enthalpy-driven with similar enthalpies of 8-12 kcal/mol for each of the drugs in any of the membrane mimetics.     

Siderophores of halophilic archaea and their chemical characterization

Nine halophilic archaea viz., Halobacterium salinarum, Halobacterium sp.1, Halobacterium sp.2, Halobaculum sp., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. isolated from marine salterns around Bhavnagar coast were screened for siderophore production. Five isolates viz., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. produced siderophores as evidenced by positive reaction in FeCl3 test, CAS assay and CAS agar plate test. Determination of chemical nature of siderophores by chemical assays and bioassays identified them as carboxylates. Quantification of siderophores indicated Halorubrum saccharovorum to be the maximum siderophore producer (2.62 RE mg/ml) and Halococcus saccharolyticus to be the least (1.33 RE mg/ml). The present study is the first report on siderophore production in Indian haloarchaeal strains. Mechanism of iron assimilation in four non-siderophore isolates still needs to be investigated further.     

Precious things come in little packages

The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule. The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments. This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.     

Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein

Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.     

Structural analysis of synthetic peptide fragments from EmrE,a multidrug resistance protein,in a membrane-mimetic environment

EmrE, a multidrug resistance protein from Escherichia coli, renders the bacterium resistant to a variety of cytotoxic drugs by active translocation out of the cell. The 110-residue sequence of EmrE limits the number of structural possibilities that can be envisioned for this membrane protein. Four helix bundle models have been considered [Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1996) J. Biol. Chem. 271, 31044-31048]. The validity of EmrE structural models has been probed experimentally by investigations on overlapping peptides (ranging in length from 19 to 27 residues), derived from the sequence of EmrE. The choice of peptides was made to provide sequences of two complete, predicted transmembrane helices (peptides H1 and H3) and two helix-loop-helix motifs (peptides A and B). Peptide (B) also corresponds to a putative hairpin in a speculative beta-barrel model, with the "Pro-Thr-Gly" segment forming a turn. Structure determination in SDS micelles using NMR indicates peptide H1 to be predominantly helical, with helix boundaries in the micellar environment corroborating predicted helical limits. Peptide A adopts a helix-loop-helix structure in SDS micelles, and peptide B was also largely helical in micellar environments. An analogue peptide, C, in which the central "Pro-Thr-Gly" was replaced by "(D)Pro-Gly" displays local turn conformation at the (D)Pro-Gly segment, but neither a continuous helical stretch nor beta-hairpin formation was observed. This study implies that the constraints of membrane and micellar environments largely direct the structure of transmembrane peptides and proteins and study of judiciously selected peptide fragments can prove useful in the structural elucidation of membrane proteins.     

In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit

EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer. Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer. To identify the functional unit of EmrE, a novel approach was developed. In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 degrees C, a treatment that does not impair transport activity. Oligomer formation is highly specific as judged by several criteria, among them the fact that (35)S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells. Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits. The hetero-oligomers thus formed display a decreased affinity to substrates. In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer. It is concluded that, in EmrE, the oligomer is the functional unit.     

Exploring the binding domain of EmrE, the smallest multidrug transporter

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.     

Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis

All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.     

Functional roles of tyrosine 185 during the bacteriorhodopsin photocycle as revealed by in situ spectroscopic studies

Tyrosine 185 (Y185), one of the aromatic residues within the retinal (Ret) chromophore binding pocket in helix F of bacteriorhodopsin (bR), is highly conserved among the microbial rhodopsin family proteins. Many studies have investigated the functions of Y185, but its underlying mechanism during the bR photocycle remains unclear. To address this research gap, in situ two-dimensional (2D) magic-angle spinning (MAS) solid-state NMR (ssNMR) of specifically labelled bR, combined with light-induced transient absorption change measurements, dynamic light scattering (DLS) measurements, titration analysis and site-directed mutagenesis, was used to elucidate the functional roles of Y185 during the bR photocycle in the native membrane environment. Different interaction modes were identified between Y185 and the Ret chromophore in the dark-adapted (inactive) state and M (active) state, indicating that Y185 may serve as a rotamer switch maintaining the protein dynamics, and plays an important role in the efficient proton-pumping mechanism in the bR purple membrane.     

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.     

Parallel topology of genetically fused EmrE homodimers

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.     

Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved in CRP*-dependent mal gene expression in Escherichia coli.

We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfs1 through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nlp, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfs1) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfs1 encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfs1 gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfs1 could be a new regulatory factor involved in maltose metabolism.     

Growth kinetics of extremely halophilic archaea (family halobacteriaceae) as revealed by arrhenius plots

Members of the family Halobacteriaceae in the domain Archaea are obligate extreme halophiles. They occupy a variety of hypersaline environments, and their cellular biochemistry functions in a nearly saturated salty milieu. Despite extensive study, a detailed analysis of their growth kinetics is missing. To remedy this, Arrhenius plots for 14 type species of the family were generated. These organisms had maximum growth temperatures ranging from 49 to 58 degrees C. Nine of the organisms exhibited a single temperature optimum, while five grew optimally at more than one temperature. Generation times at these optimal temperatures ranged from 1.5 h (Haloterrigena turkmenica) to 3.0 h (Haloarcula vallismortis and Halorubrum saccharovorum). All shared an inflection point at 31 +/- 4 degrees C, and the temperature characteristics for 12 of the 14 type species were nearly parallel. The other two species (Natronomonas pharaonis and Natronorubrum bangense) had significantly different temperature characteristics, suggesting that the physiology of these strains is different. In addition, these data show that the type species for the family Halobacteriaceae share similar growth kinetics and are capable of much faster growth at higher temperatures than those previously reported.     

Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance to a diverse group of lipophilic cations. To examine the multimeric state(s), size-exclusion HPLC and sedimentation velocity experiments were performed with EmrE solubilized in N-dodecyl-beta-d-maltopyranoside (DM) detergent. EmrE was purified from Escherichia coli membranes using organic extraction with a 3:1 chloroform:methanol solvent followed by LH-20 chromatography and the recovered pure protein was re-solubilized in a buffer containing 2% DM. The purified protein was analyzed by SEC-HPLC to estimate the monodispersity and to determine the amount of bound detergent. The results show that EmrE is homogeneous in DM with a Stokes radius of 3.6nm compatible with that of a monomer. Sedimentation velocity experiments indicated that the EmrE preparation was monodisperse and supports the fact that the organic extracted protein solubilized in DM is monomeric. This monomeric form of the protein analyzed here is also shown to bind substrate in the micromolar range.     

Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family.

In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis. H. pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities). The H. pylori ftsH product was overexpressed in E. coli and reacted immunologically with an anti-E. coli FtsH serum (T. Tomoyasu, K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H. Niki, S. Hiraga, and T. Ogura, J. Bacteriol. 175:1352-1357, 1993). FtsH was also shown to be present in the membrane fraction of H. pylori, suggesting that it is membrane bound. Disruption of the ftsH gene led to the loss of viability of H. pylori, demonstrating that this gene is essential for cell growth. Overproduction of both H. pylori FtsH and E. coli FtsH together tremendously reduced the growth rate of the E. coli host cells, whereas the growth of the E. coli cells carrying the wild-type E. coli ftsH operon on the chromosome was not significantly affected by overproduction of H. pylori FtsH itself. This result suggests that the abnormal growth of cells results from interaction between H. pylori FtsH and E. coli FtsH.     

Exploring the role of a unique carboxyl residue in EmrE by mass spectrometry

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.