Proton translocation coupled to dimethyl sulfoxide reduction in anaerobically grown Escherichia coli HB101.

Proton translocation coupled to dimethyl sulfoxide (DMSO) reduction was examined in Escherichia coli HB101 grown anaerobically on glycerol and DMSO. Rapid acidification of the medium was observed when an anaerobic suspension of cells, preincubated with glycerol, was pulsed with DMSO, methionine sulfoxide, nitrate, or trimethylamine N-oxide. The DMSO-induced acidification was sensitive to the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (60 microM) and was inhibited by the quinone analog 2-n-heptyl-4-hydroxy-quinoline-N-oxide (5.6 microM). Neither sodium azide nor potassium cyanide inhibited the DMSO response. An apparent----H+/2e- ratio of 2.9 was obtained for DMSO reduction with glycerol as the reductant. Formate and H2(g), but not lactate, could serve as alternate electron donors for DMSO reduction. Cells grown anaerobically on glycerol and fumarate displayed a similar response to pulses of DMSO, methionine sulfoxide, nitrate, and trimethylamine N-oxide with either glycerol or H2(g) as the electron donor. However, fumarate pulses did not result in acidification of the suspension medium. Proton translocation coupled to DMSO reduction was also demonstrated in membrane vesicles by fluorescence quenching. The addition of DMSO to hydrogen-saturated everted membrane vesicles resulted in a carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone-sensitive fluorescence quenching of quinacrine dihydrochloride. The data indicate that reduction of DMSO by E. coli is catalyzed by an anaerobic electron transport chain, resulting in the formation of a proton motive force.     

H+/e- stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells.

Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H+/e- stoichiometry being close to 2.5 and 1.5, respectively. In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O2 and to 0 for DMSO. These data suggest that (i) the H+/e- stoichiometry for E. coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12.

Two membrane-bound hydrogenase isoenzymes present in Escherichia coli during anaerobic growth have been resolved. The isoenzymes are immunologically and electrophoretically distinct. The physically more abundant isoenzyme (hydrogenase 1) contains a subunit of Mr 64,000 and is not released from the membrane by exposure to either trypsin or pancreatin. The second isoenzyme (hydrogenase 2) apparently contributes the greater part of the membrane-bound hydrogen:benzyl viologen oxidoreductase activity and exists in two electrophoretic forms revealed by nondenaturing polyacrylamide gel analysis. This isoenzyme is irreversibly inactivated at alkaline pH and gives rise to an active, soluble derivative when the membrane-bound enzyme is exposed to either trypsin or pancreatin. Both hydrogenase isoenzymes contain nickel.     

Dimethyl sulfoxide reductase of Escherichia coli: an investigation of function and assembly by use of in vivo complementation.

Dimethyl sulfoxide (DMSO) reductase of Escherichia coli is a membrane-bound, terminal anaerobic electron transfer enzyme composed of three nonidentical subunits. The DmsAB subunits are hydrophilic and are localized on the cytoplasmic side of the plasma membrane. DmsC is the membrane-intrinsic polypeptide, proposed to anchor the extrinsic subunits. We have constructed a number of strains lacking portions of the chromosomal dmsABC operon. These mutant strains failed to grow anaerobically on glycerol minimal medium with DMSO as the sole terminal oxidant but exhibited normal growth with nitrate, fumarate, and trimethylamine N-oxide, indicating that DMSO reductase is solely responsible for growth on DMSO. In vivo complementation of the mutant with plasmids carrying various dms genes, singly or in combination, revealed that the expression of all three subunits is essential to restore anaerobic growth. Expression of the DmsAB subunits without DmsC results in accumulation of the catalytically active dimer in the cytoplasm. The dimer is thermolabile and catalyzes the reduction of various substrates in the presence of artificial electron donors. Dimethylnaphthoquinol (an analog of the physiological electron donor menaquinone) was oxidized only by the holoenzyme. These results suggest that the membrane-intrinsic subunit is necessary for anchoring, stability, and electron transport. The C-terminal region of DmsB appears to interact with the anchor peptide and facilitates the membrane assembly of the catalytic dimer.     

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.     

Proton translocation coupled to nitrite reduction in anaerobically grown Escherichia coli

Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli. Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells. This extrusion was sensitive to a proton conductor, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite. Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H+-ATPase. These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H+-ATPase. From the results obtained by the measurement of proton extrusion in nitrite reductase-deficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E. coli.     

Membrane cytochromes of Escherichia coli grown aerobically and anaerobically with nitrate

Redox titration has been coupled to spectroscopic techniques, enzyme fractionation, and the use of mutants to examine the cytochrome composition of the membranes from cells grown aerobically and anaerobically with nitrate. A combination of techniques was found to be necessary to resolve the cytochromes. At least six b-type cytochromes were present. Besides cytochromes bfdh and bnr, components of the formate dehydrogenase-nitrate reductase pathway, cytochromes b556, b555, b562, and o, characteristic of aerobic respiratory pathways, were present. The midpoint oxidation-reduction potentials of the aerobic b-type cytochromes suggested that the sequence of electron transfer is: cytochrome b556 leads to b555 leads to b562 leads to O2.     

Dimethyl sulfoxide reductase is not required for trimethylamine N-oxide reduction in Escherichia chcoli

Deletion mutants of Escherichia coli lacking dimethyl sulfoxide (DMSO) reductase activity and consequently unable to utilize DMSO as an electron acceptor for anaerobic growth have been isolated. These mutants retained the ability to use trimethylamine N-oxide (TMAO) as an electron acceptor and the TMAO reductase activity was found to be unaltered. Heating the cell-free extract of the wild-type strain at 70 degrees C for 15 min selectively inactivated the DMSO reductase activity while the TMAO reductase activity remained unchanged for at least 1 h.     

Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli

Proton translocation, coupled to formate oxidation and hydrogen evolution, was studied in anaerobically grown fermenting Escherichia coli JW136 carrying hydrogenase 1 (hya) and hydrogenase 2 (hyb) double deletions. Rapid acidification of the medium by EDTA-treated anaerobic suspension of the whole cells or its alkalization by inverted membranes was observed in response to application of formate. The formate-dependent proton translocation and 2H(+)-K(+) exchange coupled to H(2) evolution were sensitive to the uncoupler, carbonylcyanide-m-chlorophenylhydrazone, and to copper ions, inhibitors of hydrogenases. No pH changes were observed in a suspension of formate-pulsed aerobically grown ("respiring") cells. The apparent H(+)/formate ratio of 1.3 was obtained in cells oxidizing formate. The 2H(+)-K(+) exchange of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes does take place in JW136 cell suspension. Hydrogen formation from formate by cell suspensions of E. coli JW136 resulted in the formation of a membrane potential (Deltapsi) across the cytoplasmic membrane of -130 mV (inside negative). This was abolished in the presence of copper ions, although they had little effect on the value of Deltapsi generated by E. coli under respiration. We conclude that the hydrogen production by hydrogenase 3 is coupled to formate-dependent proton pumping that regulates 2H(+)-K(+) exchange in fermenting bacteria.     

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

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, 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.     

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.     

Subcellular distribution of alkalophilic Bacillus penicillinase produced by Escherichia coli HB101 carrying pEAP31

Summary The extracellular production of alkalophilic Bacillus penicillinase by Escherichia coli HB101 carrying pEAP31 was dependent on the cultivation temperature. Extracellular production occurred only above 26°C. The penicillinase produced by the organism grown at lower temperatures accumulated in the periplasm of the cells. At high temperature, the penicillinase accumulated transiently in the periplasm and then was released gradually from the cells. The penicillinase that accumulated in the periplasm of the organism grown at low temperature could also be released by shifting to a high temperature.     

A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide

It is known that Escherichia coli methionine mutants can grow on both enantiomers of methionine sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E. coli that can reduce each of these enantiomers to methionine (met). Previous studies have identified two members of the methionine sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated. One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met. However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E. coli extracts is very low. A new member of the Msr family (fRMsr) has been identified in E. coli extracts that reduces free met-R-(o) to met. Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E. coli.     

High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli.

The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine. The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter. Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein. The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin. Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity.