Structure-function correlation of outer membrane protein porin from Paracoccus denitrificans

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.     

The preparation of tightly coupled membrane vesicles from Paracoccus denitrificans

Nonequilibrium sedimentation of membrane vesicles from Paracoccus denitrificans through Ficoll gradients results in a separation into two fractions. The fraction which pellets through dense Ficoll is uncoupled. The second fraction, retarded by dense Ficoll, shows both improved concentrative transport activity and greater uncoupler stimulation of respiration as compared to the original vesicle preparation.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

The twin-arginine translocation pathway is necessary for correct membrane insertion of the Rieske Fe/S protein in Legionella pneumophila

The twin-arginine translocation (Tat) pathway translocates folded proteins across the cytoplasmic membrane. Proteins transported through this secretion system typically carry two arginine residues in their signal peptide that is cleaved off during translocation. Recently, we demonstrated the presence of the Tat pathway in Legionella pneumophila Philadelphia-1 and the Rieske Fe/S protein PetA was one of the predicted Tat substrates. Because we observed that the signal peptide of PetA is not processed and that this protein is still membrane associated in the tat mutants, correct membrane insertion was assayed using a trypsin sensitivity assay. We conclude that the Tat pathway is necessary for correct membrane insertion of L. pneumophila PetA.     

Tracking the unfolding and refolding pathways of outer membrane protein porin from Paracoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.     

The function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite. Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50 = 15 microM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.     

A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase

A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.     

Cytochrome c-550 mediates electron transfer from inducible periplasmic c-type cytochromes to the cytoplasmic membrane of Paracoccus denitrificans

Electron transfer from periplasmic cytochromes c to the membrane-bound respiratory chain has been studied with the isolated cytochromes and membrane preparations from Paracoccus denitrificans. When reduced cytochromes were incubated with spheroplasts only the constitutive cytochrome c-550 was rapidly oxidized. The inducible cytochromes c-551i and c-553i were not oxidized at appreciable rates. Cytochrome c-550 was able to mediate the transfer of electrons from either cytochrome c-551i or cytochrome c-553i to the membrane preparation.     

Removal of Paracoccus denitrificans outer membrane material by sodium chloride

The effects of water washing and NaCl treatment on the cell surface of P. denitrificans were studied. Both treatments caused a release of material from cells. Chemical studies showed that NaCl treatment released material containing components characteristic of outer membrane. This treatment also increased the susceptibility of the organism to lysozyme. Scanning electron microscopy was used to monitor the effects of water washing and NaCl treatment on the cell surface. Both treatments were shown to alter the appearance of the cell surface. The disruptive effects of these procedures were found to be dependent upon the age of the culture.Non-Standard Abbreviations WW water wash - SE saline extract     

Iron-regulated outer membrane proteins and non-siderophore-mediated iron acquisition by Paracoccus denitrificans

Abstract Deprivation of Paracoccus denitrificans of iron in sodium molybdate-containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non-sodium molybdate-containing medium, where iron deprivation had little effect on growth rate. Five high M _r outer membrane proteins and catechol production were induced in iron-deprived cultures. The fifth protein, M _r 72 000, was produced later than the others. Growth of iron-deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation-induced protein production, and led to production of an M _r 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the M _r 23 000 and high M _r proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, 'low affinity' iron uptake pathway.     

The twin-arginine leader-binding protein,DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase

The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria. Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits. Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase. In this work, the targeting of DmsD within Escherichia coli was investigated. Using cell fractionation and Western blot analysis, DmsD is found to be associated with the inner membrane of wild-type E. coli and a dmsABC mutant E. coli under anaerobic conditions. In contrast, DmsD is predominantly found in the cytoplasmic fraction of a Delta tatABCDE strain, which suggests that DmsD interacts with the membrane-associated Tat complex. Under aerobic conditions DmsD was also found primarily in the cytoplasmic fraction of wild-type E. coli, suggesting that physiological conditions have a significant effect upon the targeting of DmsD to the inner membrane. Size exclusion chromatography data and membrane washing studies indicate that DmsD is interacting tightly with an integral membrane protein and not with the lipid component of the E. coli inner membrane. Additional investigation into the nature of this interaction revealed that the TatB and TatC subunits of the translocase are important for the interaction of DmsD with the E. coli inner membrane.     

Iron-dependent production of a heat-modifiable, 23,000-Mr outer membrane protein in Paracoccus denitrificans.

Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.     

The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans

There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of Gram-negative bacteria. It has been reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity. We have demonstrated that a major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of Paracoccus denitrificans. The degree of stimulation produced by melittin is dependent upon (i) the nature of the respiratory substrates, (ii) the pH, (iii) the presence of Mg2+, (iv) the melittin: membrane protein ratio, and (v) the growth history of the cells from which the membrane vesicles were derived. Melittin-induced enhancement of TMPD:ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of nonpermeant substrate to the interior of the vesicle. The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane thereby abolishing respiratory control. Collectively these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography. These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans containing aminotransferase activity

Summary To establish an efficient production method for l-phenylalanine, the production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans pFPr-1 containing aminotransferase activity was investigated. By using intact cells, 0.74M l-phenylalanine was produced from 0.8M phenylpyruvate (conversion yield, 92.5%). Moreover, by using immobilized cells with -carrageenan, when the space velocity was 0.1 h^-1 at 30°C, 0.135 M l-phenylalanine was produced from 0.15 M phenylpyruvate (conversion yield, 90%). The half-life of the l-phenylalanine-forming activity of the column was estimated to be about 30 days at 30°C.     

The reversibility of active sulphate transport in membrane vesicles of Paracoccus denitrificans.

An uncoupler-sensitive active transport of sulphate into membrane vesicles prepared from the plasma membrane of Paracoccus denitrificans (previously Micrococcus denitrificans) can be driven by respiration or by a trans-membrane pH gradient (alkaline inside) generated by the addition either of KCL ( in the presence of nigericin) or of NH4CL. Valinomycin does not substitute for nigericin. Respiration-driven transport is observed in right-side-out vesicles but not in inside-out vesicles, whereas transport driven by the addition of KCL (in the presence of nigericin) or of NH4CL is observed in both types of membrane vesicle. The active transport of sulphate into these vesicles is shown to be carrier-mediated by its sensitivity to thiol-group reagents. It is proposed that the sulphate carrier in the plasma membrane of P. denitrificans operates by a mechanism of electroneutral proton symport, and is capable of actively transporting sulphate in either direction across the plasma membrane, but that in whole cells respiration-driven proton expulsion drives the accumulative uptake of sulphate.