Kinetics of electron transfer between the tetrahemic cytochrome and the special pair in isolated reaction centers of Roseobacter denitrificans

We have analyzed the rate of electron transfer between the tetrahemic cytochrome and the primary electron donor in isolated reaction centers of Roseobacter denitrificans as a function of the ambient redox potential. Three different phases are observed: a slow phase (half-time > ms), and two fast phases with half-times of 5 µs and 380 ns. The slow phase is present at high redox potential, it corresponds to the kinetics of charge recombination between the photo-oxidized primary electron acceptor P⁺ and the reduced primary acceptor (Q _A ^– ). The 5 µs phase titrates with the reduction of the highest potential heme (HP₁). This phase corresponds to the electron transfer between heme HP₁ and P⁺. At redox potentials where the second high potential heme HP₂ becomes reduced, the 5 µs phase disappears and is replaced by the 380 ns phase, which is therefore related to the electron transfer between the high potential heme HP₂ and P⁺. To explain the large difference in the rate of oxidation of HP₁ and HP₂ we propose a tentative model where the heme HP₂ is closest to P.     

Interaction of Porin from Yersinia pseudotuberculosis with Different Structural Forms of Endogenous Lipopolysaccharide

The interaction of Yersinia pseudotuberculosis porin solubilized in deoxycholate with the S- and R-forms of endogenous lipopolysaccharide (LPS) was studied by the quenching of intrinsic protein fluorescence. The samples of S-LPS differed both in the length of O-specific polysaccharide (n = 1 and 4) and in the acylation degree of the 3-hydroxytetradecanoic acid residues of the lipid A moiety (12-66%). R-LPS (12%) binding to porin was found to occur with positive cooperativity on two integrated structural regions of the R-LPS macromolecule, namely, core oligosaccharide and lipid A. The mode of porin interaction with low-acylated S-LPSs (15 or 20%) coincided with a model involving three types of binding sites. The shape of Scatchard curves of binding indicates that a complex formation between porin and low-acylated S-LPS is cooperative at low and moderate ligand concentration, whereas at near-saturating LPS concentrations porin binds to LPS independently on two types of binding sites. The O-specific polysaccharide chain in the S-LPS macromolecule increases the affinity of its interaction with porin in comparison with R-LPS–porin binding. A significant increase (to 66%) in the degree of S-LPS acylation substantially changed its porin-binding character: the process becomes anti-cooperative with lowered affinity. Thus, the features of LPS–porin interaction significantly depend on the conformational changes in the LPS molecule due to expanding of its hydrophobic region.     

Outer membrane porin protein of Campylobacter jejuni

Abstract Protein e, a 43-kDa protein from the outer membrane of Campylobacter jejuni UA580, was purified and reconstituted into lipid bilayer membranes. It was shown to form small channels with a single channel conductance of 8.82 nS in 1M KCl. Zero current potential measurements demonstrated that the channel was approx. 10-fold selective for K⁺ over Cl⁻ ions. A porin with a similar single channel conductance was observed in fractions from the outer membrane of Campylobacter fetus UA60.     

Purification,characterization, crystallization,and preliminary X-ray results from Paracoccus denitrificans porin

The porin from Paracoccus denitrificans ATCC 13543 was purified and crystallized. Two crystal forms were obtained from porin solutions with β-d-octylglucopyra-noside as detergent. Crystals of form I belong to the monoclinic spacegroup C2 with unit cell dimensions a = 112.2 Å, b = 193.8 Å, c = 100.5 Å and β = 129.2°. There is 1 trimer per asymmetric unit. Crystals of form II are triclinic with α = 89.7 Å, b = 98.8 Å, c = 112.5 Å, b = 112.5Å, β = 101.8°, γ = 106.7° (2 trimers per asymmetric unit). Both crystal forms diffract to 3 Å. © 1995 Wiley-Liss, Inc.     

The Benefit of a Roseobacter Species on the Survival of Scallop Larvae

A marine strain (BS107), identified as a Roseobacter species, was antagonistic to Vibrio species on agar plates. Results suggested that the inhibitory effect was displayed only in the presence of another bacterium. Quantification of the antibacterial activity showed that 48-hour-coculture supernatants from BS107 and another bacterial strain (V. anguillarum 408) reached the highest titers of bacterial inhibition. The antibacterial substance was also liberated when supernatants from V. anguillarum 408 were added to pure cultures of the inhibition-productive bacterium. The presence of a proteinaceous molecule may induce BS107 to display the inhibitory effect. The antibacterial substance was sensitive to trypsin (8000 U/ml) and stable at 100°C. Cell extracts of the isolate BS107 (10⁶ cells/ml) significantly enhanced scallop larval survival, thus being beneficial to the rearing process. Received December 8, 1997; accepted July 15, 1998.     

Cation-selective Pathway of OmpF Porin Revealed by Anomalous X-ray Diffraction

The OmpF porin from the Escherichia coli outer membrane folds into a trimer of β-barrels, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the β-barrel pore to form a narrow “constriction zone”. A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the β-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P6₃ space group and two different data sets were obtained at 2.6 Å and 3.0 Å resolution with K⁺ and Rb⁺, respectively. The Rb⁺-soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 Å and cation positions were determined. A PEG molecule was observed in the pore region for both the K⁺ and Rb⁺-soaked crystals, where it interacts with loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.     

A Structural Analysis of the Regularly Arranged Porin on the Outer Membrane of Campylobacter jejuni Based on Correlation Averaging

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer-assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4±0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 ±0.1 nm.     

Characterization of the Photosynthetic Apparatus and Proteome of Roseobacter denitrificans

The phototrophic capacity of aerobic anoxygenic phototrophic bacteria endows them with a selective advantage over other heterotrophic bacteria in the oligotrophic ocean. Here, we reported the phototrophic features and proteome of an aerobic phototrophic bacterium Roseobacter denitrificans under starvation stress. The fluorescence induction and relaxation measurements suggested that the photosynthetic capacity in R. denitrificans was preserved but was lower than in the photoautotrophic bacterium Rhodobacter sphaeroides. The existence of light-harvesting complexes (LH1 and LH2) and the reaction center (RC) in the native membrane were demonstrated through atomic force microscopy image analysis as direct evidence of their phototrophy. The homology-based LH1–RC complex structure was proposed in which RC was the Rb. sphaeroides homolog structure surrounded by the LH1. Moreover, the protein expression profiles of cells in the stationary phase under heterotrophic and mixotrophic conditions show that light enhanced or activated some proteins such as carbon monoxide dehydrogenase and NifU to cope with the low levels of amino acids and carbon sources under starvation conditions.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.     

Identification of two voltage-dependent anion channel-like protein sequences conserved in Kinetoplastida

The eukaryotic porin superfamily consists of two families, voltage-dependent anion channel (VDAC) and Tom40, which are both located in the mitochondrial outer membrane. In Trypanosoma brucei, only a single member of the VDAC family has been described. We report the detection of two additional eukaryotic porin-like sequences in T. brucei. By bioinformatic means, we classify both as putative VDAC isoforms.     

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.     

Effects of various culture environments on expression of major outer membrane proteins from Porphyromonas gingivalis

We examined the effects of various culture environments on major outer membrane proteins from Porphyromonas gingivalis ATCC 33277. Major outer membrane protein patterns on gel electrophoresis showed little difference over the culturable range of osmolarity and pH. With elevated temperature or prolonged culture, the intensities of the gingipain bands decreased; however, bands of RagA, RagB and the putative porins were relatively stable. Similar results were observed with several different culture media. Although the precise functions of RagA, RagB and the putative porins are unknown, these factors may be strongly related to the initiation and progression of adult periodontitis.     

Unusual Constriction Zones in the Major Porins OmpU and OmpT from Vibrio cholerae

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Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)₂ with the catalytic (BchY/BchZ)₂ protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF₄⁻. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)₂. A second [4Fe-4S] cluster was identified on (BchY/BchZ)₂. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.     

Physicochemical Characterization of the Reassembled Dimer of an Integral Membrane Protein OmpF Porin

The in vitro reassembled species of OmpF porin, which was renatured from its denatured monomer using n-octyl-β-D-glucopyranoside, was characterized by low-angle laser light scattering photometry, circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering measurements. The light scattering measurement reconfirmed that the reassembled species was the dimer of the protein. Circular dichroism spectra of the reassembled dimer showed a native-like β-structure. A small-angle X-ray scattering measurement indicated that the size of the reassembled dimer was nearly equal to that of the native trimer under the present experimental conditions. In a thermal denaturation experiment followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the reassembled dimer was less stable than the native trimer.     

Involvement of Porin N,N-dicyclohexylcarbodiimide-Reactive Domain in Hexokinase Binding to the Outer Mitochondrial Membrane

The proportion of hexokinase that is bound to the outer mitochondrial membrane is tissue specific and metabolically regulated. This study examined the role of the N,N-dicyclohexylcarbodiimide-binding domain of mitochondrial porin in binding to hexokinase I. Selective proteolytic cleavage of porin protein was performed and peptides were assayed for their, effect on hexokinase I binding to isolated mitochondria. Specificity of DCCD-reactive domain binding to hexokinase I was demonstrated by competition of the peptides for porin binding sites on hexokinase as well as by blockage hexokinase binding by N,N-dicyclohexylcarbodiimide. One of the peptides, designated as 5 kDa (the smallest of the porin peptides, which contains a DCCD-reactive site), totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme. These experiments demonstrate that there exists a direct and specific interaction between the DCCD-reactive domain of VDAC and hexokinase I. The peptides were further characterized with respect to their effects on certain functional properties of hexokinase I. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. To evaluate further the outer mitochondrial membranes role in the hexokinase binding, insertion of VDAC was examined using isolated rat mitochondria. Pre-incubation of mitochondria with purified porin strongly increases hexokinase I binding to rat liver mitochondria. Collectively, the results imply that the high hexokinase-binding capability of porin-enriched mitochondria was due to a quantitative difference in binding sites.     

In vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: Cloning of an ompR 2 mutation in Escherichia coli K-12

Abstract We have devised a simple in vivo scheme for moving chromosomal mutations onto multicopy plasmids in Escherichia coli K-12. A plasmid clone of the relevant wild-type gene is first integrated into the chromosome of a PolA⁻ strain carrying the desired mutation. The plasmid cointegrate formed is then resolved by P1 transduction to a PolA⁺ host. A certain fraction of these transductants will have the mutant allele on the plasmid. Employing this scheme we cloned an ompR 2 mutation onto a multicopy plasmid. To show that the plasmid actually contained the ompR 2 mutation, this allele was introduced back into the chromosome by the gene replacement technique of Gutterson and Koshland [1] and shown to be indistinguishable from the original ompR 2 by genetic mapping and phenotype.     

Membrane protein dynamics versus environment: simulations of OmpA in a micelle and in a bilayer

The bacterial outer membrane protein OmpA is one of the few membrane proteins whose structure has been solved both by X-ray crystallography and by NMR. Crystals were obtained in the presence of detergent, and the NMR structure is of the protein in a detergent micelle. We have used 10 ns duration molecular dynamics simulations to compare the behaviour of OmpA in a detergent micelle and in a phospholipid bilayer. The dynamic fluctuations of the protein structure seem to be ca 1.5 times greater in the micelle environment than in the lipid bilayer. There are subtle differences between the nature of OmpA-detergent and OmpA-lipid interactions. As a consequence of the enhanced flexibility of the OmpA protein in the micellar environment, side-chain torsion angle changes are such as to lead to formation of a continuous pore through the centre of the OmpA molecule. This may explain the experimentally observed channel formation by OmpA.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide