Isolation and Characterization of the Tubular Organelles Induced by Fumarate Reductase Overproduction in Escherichia coli

Elmes, MJ; Scraba, Douglas G.; Weiner, Joël H.

doi:10.1099/00221287-132-6-1429

Cited by 17 publications

(26 citation statements)

References 24 publications

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“…In other examples of E. coli membrane protein overexpression (3,11,43), extended membrane structures form that are enriched in the overexpressed protein.…”

Section: Discussionmentioning

confidence: 99%

“…Characteristic radial dimensions were measured for several micellar structures, including those shown in Fig. 4, and are summarized as averages in angstroms Ϯ standard deviations as follows: radius of hairpin contact (r H ), 52 Ϯ 5 (n ϭ 11); radius at the membrane inner edge (r I ), 245 Ϯ 13 (11); radius at the membrane outer edge (r O ), 300 Ϯ 16 (11); and outermost radius (r P ), 362 Ϯ 32 (3). Estimates of the characteristic dimensions of the receptor, e.g., the end-to-end length, were determined as differences of two radial dimensions, e.g., r P Ϫ r H , on each micellar assembly individually and then averaged.…”

Section: Vol 185 2003 Membrane Assemblies Formed By Tsr 3637mentioning

confidence: 99%

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Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr

et al. 2003

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The serine receptor (Tsr) from Escherichia coli is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways. Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria. In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor. Three types of assemblies were observed: ring-like "micelles" with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and "zippers," which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains. The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length. The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains. Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell.The serine receptor (Tsr), one of four methyl-accepting chemotaxis proteins (MCPs) that span the inner membrane of Escherichia coli, initiates responses and governs adaptation to changes in the serine concentration. MCPs belong to a large class of transducers (21, 46), which sense a variety of environmental cues and are the inputs to sensory pathways that bias cell movement toward favorable environments (12). The chemotaxis pathways belong to the two-component superfamily of signal transduction pathways (17, 42), which are chiefly found in prokaryotes. A two-component pathway consists of a sensor, which is frequently an integral membrane protein possessing kinase activity, and one or more cytoplasmic phosphate-accepting response regulator proteins. The transmembrane sensor-kinases of the chemotaxis pathways are often noncovalent complexes between MCPs (which have no enzyme activity) and two soluble cytoplasmic proteins, namely, an adaptor protein (CheW) and a kinase (CheA) (15, 39).Elucidation of the structure and distribution of receptors in the membrane of the cell is integral to understanding the molecular basis of signaling by the transmembrane sensor (MCP-CheW-CheA) complexes. X-ray structure determination of the soluble domains has clearly defined the dimeric organization of the 60-kDa receptor subunits (19,31,45), and functional studies have helped to elucidate the role of dimer organization in the mechanism of transmembrane signaling (references 32 and 12 and refer...

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“…In other examples of E. coli membrane protein overexpression (3,11,43), extended membrane structures form that are enriched in the overexpressed protein.…”

Section: Discussionmentioning

confidence: 99%

Section: Vol 185 2003 Membrane Assemblies Formed By Tsr 3637mentioning

confidence: 99%

Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr

et al. 2003

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“…This model for the structure of DMSO reductase differs from that of fumarate reductase (10,25) in which the FrdA subunit umbrellas the FrdB subunit, protecting it from iodination and proteolysis (10). Overproduction of DMSO reductase did not induce the biosynthesis of a protein-lipid organelle as was seen with fumarate reductase (12). This may be due to differences in the expression level; fumarate reductase can account for 70 to 90% of the protein in a membrane preparation (10,39), whereas DMSO reductase never exceeds 30% (40).…”

mentioning

confidence: 96%

“…The purified inner membrane vesicles prepared from HB101 or HB101(pDMS159) were adsorbed to hydrophilic carbon films on copper mesh grids and reacted with the specified primary and secondary gold-conjugated antibodies (12).…”

mentioning

confidence: 99%

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli

et al. 1990

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Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S-and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, or DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.Dimethyl sulfoxide (DMSO) reductase of Escherichia coli (5) is a terminal enzyme expressed constitutively under anaerobic conditions and is capable of reducing DMSO and trimethylamine-N-oxide (TMAO). We have reported that the E. coli DMSO reductase is a membrane-bound iron-sulfur molybdoenzyme genetically distinct from the single or dual subunit forms of TMAO reductase (40). The structural genes encoding the enzyme have been cloned, and their DNA sequences have been determined (4, 7). The enzyme has been purified to homogeneity and shown to be composed of three nonidentical subunits: a catalytic subunit (DmsA; 82,600 daltons), an iron-sulfur subunit (DmsB; 23,600 daltons), and a membrane anchor subunit (DmsC; 22,700 daltons).The presence of one constitutive and three inducible forms of TMAO reductase have been demonstrated (32,36,42); however, the significance and precise localization of these multiple forms of TMAO reductase in the energy transduction mechanisms of the cell are not clear. The enzymes responsible for TMAO and DMSO reduction in certain marine organisms and nonphotosynthetic bacteria have been characterized in some detail (2). A single enzyme in Rhodobacter sp. has been found to be responsible for the reduction of both DMSO and TMAO (23). The reductases in these organisms were found to be localized in the periplasmic compartment.

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“…7). The pellet obtained after ultracentifugation (fraction P3; mainly cytoplasmic membranes) contained much less TrkA protein (lane 8), and the soluble protein fraction was almost free of it (lane 10).…”

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confidence: 99%

Physical mapping of the K+ transport trkA gene of Escherichia coli and overproduction of the TrkA protein

Hamann¹,

Bossemeyer²,

Bakker³

1987

J Bacteriol

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The position on the Escherichia coli chromosome of trkA, a gene coding for a membrane protein involved in K+ transport by the constitutive uptake system Trk, was determined. We observed that the gene is transcribed in a clockwise direction and that it is located at 72.4 min on the chromosome in a 1.75-kilobase NruI-EcoRV DNA fragment 1.0 kilobase upstream of rplQ. We localized an additional gene encoding a 17,000-molecular-weight protein of unknown function between the trkA and rplQ genes. A plasmid, pDB3, was constructed in which the transcription of the trkA gene was put under the control of the lambda pL promoter. pDB3-containing cells of a strain, which contained the temperature-sensitive lambda repressor cI857 in the chromosome, overproduced the 53,000-molecular-weight TrkA protein at the nonpermissive temperature to such an extent that TrkA became the major cell protein. From cell fractionation studies, we conclude that the overproduced TrkA protein forms aggregates.

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Isolation and Characterization of the Tubular Organelles Induced by Fumarate Reductase Overproduction in Escherichia coli

Cited by 17 publications

References 24 publications

Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr

Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli

Physical mapping of the K+ transport trkA gene of Escherichia coli and overproduction of the TrkA protein

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