Biomedical subjects

W Lubitz

Publications and source records attributed to W Lubitz.

At least 19 recordsLinked to original sources

PubMed2000

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Balstochloris viridis. Influence of mutations at position M208.

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (phi(*-)A) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BCh1), B(A), and the BPh, phiA, that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of (phi(*-)A at 160 K indicates that two distinct states of phi(*-)A are present in the wild type and the mutant YF(M208). Based on a comparison with phi(*-)A in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of phiA. Only one phi(*-)A state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state phi(*-)AQ(*-)A is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-302]. The corresponding absorbance changes in the BCh1 Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of phiA changes its orientation. It is concluded that this distinct structural relaxation of phiA can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.

Cold Temperature↗

PubMed2000

Intramuscular immunization with genetically inactivated (ghosts) Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state.

Bacterial ghosts are empty cell envelopes achieved by the expression of a cloned bacteriophage lysis gene and, unlike classical bacterins, suffer no denaturing steps during their production. These properties may lead to a superior presentation of surface antigens to the immune system. Currently available porcine Actinobacillus pleuropneumoniae vaccines afford only minimal protection by decreasing mortality but not morbidity. Pigs which survive infection can still be carriers of the pathogen, so a herd once infected remains infected. Carrier pigs harbour A. pleuropneumoniae in their nasal cavities, in their tonsils, or within lung lesions. A dose-defined nose-only aerosol infection model for pigs was used to study the immunogenic and protective potential of systemic immunization with ghosts made from A. pleuropneumoniae serotype 9 reference strain CVI 13261 against an homologous aerogenous challenge. Pigs were vaccinated twice intramuscularly with a dose of 5x10(9) CFU ghosts (GVPs) or formalin-inactivated A. pleuropneumoniae bacterins (BVPs). After 2 weeks vaccinated pigs and non-vaccinated placebo controls (PCs) were challenged with a dose of 10(9) CFU by aerosol. The protective efficacy of immunization was evaluated by clinical, bacteriological, serological and post-mortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples (BALF) for assessment of local antibodies. Isotype-specific antibody responses in serum and BALF were determined by ELISAs based on whole-cell antigen. Immunization with ghosts did not cause clinical side-effects. After aerosol challenge PCs developed fever and pleuropneumonia. GVPs or BVPs were found to be fully protected against clinical disease or lung lesions in both vaccination groups, whereas colonization of the respiratory tract with A. pleuropneumoniae was only prevented in GVPs. Specific immunoglobins against A. pleuropneumoniae were not detectable in BALF after immunization. A significant systemic increase of IgM, IgA, IgG(Fc'), or IgG(H+L) antibodies reactive with A. pleuropneumoniae was measured in GVPs and BVPs when compared to the non-exposed controls. BVPs reached higher titers of IgG(Fc') and IgG(H+L) than GVPs. However, prevention of carrier state in GVPs coincided with a significant increase of serum IgA when compared to BVPs. These results suggest that immunization with ghosts, that bias antibody populations specific to non-denaturated surface antigens, may be more efficacious in protecting pigs against colonization and infection than bacterins.

Actinobacillus Infections↗

PubMed2000

Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

The Ni-A and the Ni-B forms of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F have been studied in single crystals by continuous wave and pulsed EPR spectroscopy at different temperatures (280 K, 80 K, and 10 K). For the first time, the orientation of the g-tensor axes with respect to the recently published atomic structure of the active site at 1.8 A resolution was elucidated for Ni-A and Ni-B. The determined g-tensors have a similar orientation. The configuration of the electronic ground state is proposed to be Ni(III) 3d1/z 2 for Ni-A and Ni-B. The gz principal axis is close to the Ni-S(Cys549) direction; the gx and the gy axes are approximately along the Ni-S(Cys546) and Ni-S(Cys81) bonds, respectively. It is proposed that the difference between the Ni-A and Ni-B states lies in a protonation of the bridging ligand between the Ni and the Fe.

Binding Sites↗

PubMed2000

The archaeal halophilic virus-encoded Dam-like methyltransferase M. phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli.

The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M. Dam in DNA mismatch repair.

Amino Acid Sequence↗

PubMed2000

Heterodimeric versus homodimeric structure of the primary electron donor in Rhodobacter sphaeroides reaction centers genetically modified at position M202.

Using light-induced Fourier-transform infrared (FTIR) difference spectroscopy of the photo-oxidation of the primary donor (P) in chromatophores from Rhodobacter sphaeroides, we examined a series of site-directed mutants with His M202 changed to Gly, Ser, Cys, Asn or Glu in order to assess the ability of these side chains to ligate the Mg atom of one of the two bacteriochlorophylls (BChl) constituting P. In the P+QA-/PQA FTIR difference spectra of the mutants HG(M202), HS(M202), HC(M202) and HN(M202), the presence of a specific electronic transition at approximately 2650-2750 cm-1 as well as of associated vibrational (phase-phonon) bands at approximately 1560, 1480 and 1290 cm-1 demonstrate that these mutants contain a BChl/BChl homodimer like that in native reaction centers with the charge on P+ shared between the two coupled BChl. In contrast, the absence of all of these bands in HE(M202) shows that this mutant contains a BChl/bacteriopheophytin heterodimer with the charge localized on the single BChl, as previously determined for the mutant HL(M202). Furthermore, the spectra of the heterodimers HE(M202) and HL(M202) are very similar in the 4000-1200 cm-1 IR range. Perturbations of the 10a-ester and 9-keto carbonyl modes for both the P and P+ states are observed in the homodimer mutants reflecting slight variations in the conformation and/or in position of P. These perturbations are likely to be due to a repositioning of the dimer in the new protein cavity generated by the mutation.

Dimerization↗

PubMed2000

Conversion of solvent evaporation residues from the AB- (acetone - butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters.

In a bioconversion study based on utilisation of by-products from the AB- (acetone - butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents. The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp. G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate-(PHA-)accumulation up to 40% per dry weight. A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs. Concentrations of 1-butanol, which is known for its membrane damaging properties in microorganisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth. No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate. Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process. The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.

Acetone↗

PubMed2000

Conversion of solvent evaporation residues from the AB- (acetone-butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters.

In a bioconversion study based on utilisation of by-products from the AB- (acetone-butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents. The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp. G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate- (PHA-)accumulation up to 40% per dry weight. A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs. Concentrations of 1-butanol, which is known for its membrane damaging properties in micro-organisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth. No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate. Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process. The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.

1-Butanol↗

PubMed1999

Bacterial cell envelopes (ghosts) but not S-layers activate human endothelial cells (HUVECs) through sCD14 and LBP mechanism.

Bacterial cell-envelopes (called ghosts) and surface layers (S-layers) are discussed to be used as vaccines and/or adjuvants, consequently it is necessary to find out which immunomodulatory mediators are induced in human cells. The present work focuses on the effects of ghosts (Escherichia coli O26:B6), S-layers (Bacillus stearothermophilus) in comparison with LPS and antibiotic-inactivated whole bacteria (E. coli O26:B6) on human umbilical vein endothelial cells (HUVEC) with regard to the release of interleukin 6 (IL-6) and the expression of surface E-selectin and the role of lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and serum for this activation. Endothelial cells responded to ghosts, whole bacteria and LPS with IL-6 release up to 15000 pg/ml and surface E-selectin expression, while in contrast the response to S-layers with IL-6 release up to 500 pg/ml was very weak. Compared to LPS, 10-100-fold higher concentrations of bacterial ghosts and whole bacteria were required to induce the cytokine synthesis and E-selectin expression. IL-6 release and E-selectin expression of HUVECs were reduced in the absence of serum and equivalent to unstimulated samples. We have also studied the role of CD14 and LBP for the activation of endothelial cells using antiCD14 and antiLBP antibodies (Ab). AntiCD14 and antiLBP Ab both inhibited IL-6 release and E-selectin expression in a dose dependent manner after stimulation with ghosts, whole bacteria and LPS but had no effect on S-layers stimulated cells. AntiCD14 Ab inhibited more effectively than antiLBP Ab. These findings suggest that bacterial ghosts but not S-layers activate HUVECs through sCD14 and LBP dependent mechanisms.

Acute-Phase Proteins↗

PubMed1999

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.

2,6-Dichloroindophenol↗

PubMed1999

Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis.

The primary quinone acceptor radical anion Q(A)(-)(*) (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe(2+) is replaced by diamagnetic Zn(2+). The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridisan exchange rate of the iron of approximately 50% +/- 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)(-)(*) in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ(4)(-)(*)) dissolved in 2-propanol identifies Q(A)(-)(*) as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)(-)(*) in the RC as compared with MQ(4)(-)(*) in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)(-)(*) in the ZnRCs, two (14)N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(delta)((1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)(-)(*) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P(960)(+) (*)Q(A)(-)(*) in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339].

Anions↗

PubMed1999

Bacterial ghosts as drug carrier and targeting vehicles.

A novel system for the packaging of drugs as well as vaccines is presented. Bacterial ghosts are intact, non-denatured bacterial envelopes that are created by lysis of bacteria through the expression of cloned phage PhiX174 gene E. Inhibition of induced E-mediated lysis by MgSO(4), harvesting of cells by centrifugation, and resuspension in low-ionic-strength buffers leads to rapid, violent lysis and results in empty bacterial envelopes with large (approximately 1 microm in diameter) openings. The construction of plasmid pAV1, which encodes a streptavidin fusion protein with an N-terminal membrane anchor sequence, allows the loading of the inner side of the cytoplasmic membrane with streptavidin. The functionality and efficacy of binding of even large biotinylated compounds in such streptavidin ghosts (SA-ghosts) was assessed using the enzyme alkaline phosphatase. The successful binding of biotinylated fluorescent dextran, as well as fluorescent DNA complexed with biotinylated polylysine, was demonstrated microscopically. The display by bacterial ghosts of morphological and antigenic surface structures of their living counterparts permits their attachment to target tissues such as the mucosal surfaces of the gastrointestinal and respiratory tract, and their uptake by phagocytes and M cells. In consequence, SA-ghosts are proposed as drug carriers for site-specific drug delivery.

Alkaline Phosphatase↗

PubMed1999

Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses.

Aerosol immunization is a safe way to induce complete protection against pleuropneumonia in pigs caused by the lung pathogenic bacterium Actinobacillus pleuropneumoniae. In order to determine the local immune responses of vaccinees in concomitant with protection, lung lining fluid before and 3 weeks after immunization from pigs immunized three times with aerosols of either genetically inactivated ghosts which represent whole cell envelope preparations, or irradiated bacteria were examined following an homologous aerosol challenge. Specific antibody isotypes in the bronchoalveolar lavage were assayed by whole cell ELISAs. Total and relative numbers of cells including lymphocyte subsets were determined. In both vaccinated groups a net influx of plasma cells and lymphocytes, as well as a significant increase of specific IgG occurred. Concurrently, the CD4+/CD8+ ratio was found to increase after aerosol immunization. The lymphocyte subsets of IgG+ and IgA+ cells were found significantly higher in the group immunized with irradiated bacteria when compared to pigs immunized with bacterial ghosts. The latter group showed a significant increase of IgA, IgM, and a net influx of lymphoid blasts and granulocytes in the bronchoalveolar lining fluid. Although differences between the local immune responses of both immunized groups occurred, a significant increase of specific IgG and a net influx of plasma cells and lymphocytes were found to be associated with complete protection against a homologous aerosol challenge infection.

Actinobacillus Infections↗

PubMed1999

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Adjuvants, Immunologic↗

PubMed1999

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.

Animals↗

PubMed1999

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli.

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.

Bacteriolysis↗

PubMed1999

New strategies for combination vaccines based on the extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Adjuvants, Immunologic↗

PubMed1999

Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis.

In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.

Bacillus subtilis↗

PubMed1999

Determination of the distance between Y(Z)ox* and QA-* in photosystem II by pulsed EPR spectroscopy on light-induced radical pairs.

Out-of-phase electron spin echo envelope modulation (ESEEM) spectroscopy was used to determine the distances within two consecutive radical pair states initiated by a laser flash in photosystem II membrane fragments at pH 11. The distance between the spin density centers of the primary electron donor cation radical, P680+*, and the reduced plastoquinone acceptor, QA-*, has been found to be 27.7+/-0.7 A in agreement with previous results. Near room temperature and at high pH, P680+* is reduced by Y(Z), a redox active tyrosine residue, on a sub-microsecond timescale. As a consequence, the subsequent radical pair state, Y(Z)ox*-QA-*, could be investigated after almost complete reduction of P680+* by Y(Z). The determined dipolar electronic spin-spin coupling within the radical pair Y(Z)ox*QA-* corresponds to a distance of 34+/-1 A between the two molecules.

Electron Spin Resonance Spectroscopy↗