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W Lubitz

Publications and source records attributed to W Lubitz.

At least 91 records · Page 5Linked to original sources
PubMed1997

Proline 21, a residue within the alpha-helical domain of phiX174 lysis protein E, is required for its function in Escherichia coli.

PhiX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis-trans isomerization of proline residues within alpha-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded alpha-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli. Oligomerization of protein P21G-StrpA was not disturbed.

Amino Acid Sequence↗
PubMed1997

Characterization of Natronobacterium magadii phage phi Ch1, a unique archaeal phage containing DNA and RNA.

A novel archaeal bacteriophage, phi Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii(L13) was used to demonstrate infectivity of phage phi Ch1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that phi Ch1 is a temperate phage. The phage morphology resembles other members of Myoviridae-infecting Halobacterium species. In solution below 2M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that phi Ch1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80-700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and phi Ch1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage phi Ch1 is the first virus described for the genus natronobacterium, and the first phage containing DNA and RNA in mature phage particles.

Archaea↗
PubMed1997

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

ATP-Binding Cassette Transporters↗
PubMed1997

Molecular characterization of the Bacillus stearothermophilus PV72 S-layer gene sbsB induced by oxidative stress.

S-layer protein variation from a hexagonally ordered (SbsA; 130 kDa) to a obliquely ordered (SbsB; 98 kDa) protein in Bacillus stearothermophilus PV72 is mediated by an increased oxygen supply. To elucidate the molecular basis of S-layer protein variation in B. stearothermophilus PV72, the sbsB gene, coding for the 98-kDa protein, was cloned by means of inverse PCR technology and sequenced. The sbsB coding region cloned in pUC18 was expressed in Escherichia coli, without its own regulatory upstream sequences but with its putative transcriptional terminator. The reading frame of sbsB (2,760 nucleotides) is predicted to encode a protein of 920 amino acids, including the signal sequence. Amino acid sequence comparison of SbsA and SbsB did not reveal any significant homology. The expression of sbsB in E. coli resulted in an accumulation of SbsB self-assembly products in the cytoplasm.

Amino Acid Sequence↗
PubMed1997

Bacterial ghosts as multifunctional vaccine particles.

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 variety of bacteria including Escherichia coli. Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, and Helicobacter pylori. Such ghosts are used as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In recombinant ghosts, foreign proteins can be inserted into the inner membrane prior to E-mediated lysis via specific N-, or C-, or N- and C-terminal anchor sequences. The export of proteins into the periplasmic space or the expression of recombinant S-layer proteins vastly extents the capacity of ghosts or recombinant ghosts as carriers of foreign epitopes or proteins. Oral, aerogenic or parenteral applications of (recombinant) ghosts in experimental animals 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 used as vaccines or as carriers of relevant antigens. The inserted target antigens into the inner membrane or into S-layer proteins are not limited in size.

Adjuvants, Immunologic↗
PubMed1997

Vaccination by aerosols: modulation of clearance mechanisms in the lung.

Inhalation and deposition within the the airways are the initial steps before pathogens of the respiratory tract are able to adhere and colonize their host. Once the microorganisms are deposited in the lung lining fluids they do not remain at the location where they first came in contact with the mucous membranes. It is long known that lung clearance mechanisms translocate all deposited particles. At least, most of them are swallowed and cleared via the gastrointestinal tract. Aerosol vaccination with inactivated or (recombinant) live bacteria has been shown to be an efficient way to induce local protection against lung diseases. It can be assumed that the local concentration of the vaccine and the deposition pattern of the vaccine within the lung limit the strength of a local or systemic immune response. The local concentration of airborne bacterial antigen necessary to initiate a mucosal immunity in the respiratory tract is known for a very few microorganisms. Bacterial survival, infectivity, deposition, and persistence characteristics have to be defined when aerosols are included in vaccination experiments.

Aerosols↗
PubMed1996

Site-directed mutations affecting the spectroscopic characteristics and midpoint potential of the primary donor in photosystem I.

Photosystem I is a member of the iron-sulfur center or type I reaction centers. The primary electron donor in photosystem I is a chlorophyll a dimer termed P700. The biophysical properties of P700 are well understood, but the protein environment that gives it such unique properties is unknown. We have characterized site-directed mutants of the photosystem I reaction center protein PsaB and identified an amino acid, His-656, that interacts closely with one of the P700 chlorophylls. Mutation of His-656 to Asn or Ser increases the oxidation midpoint potential of P700/P700+. by 40 mV. The P700/P700+. optical difference spectra show the appearance of a new bleaching band at 667 nm. Electron nuclear double resonance spectroscopy indicates a significant increase in the hyperfine coupling corresponding to methyl protons at position 12 of the spin carrying chlorophyll a of P700+. The implication of these results to current structural models of the photosystem I reaction center is discussed.

Amino Acid Sequence↗
PubMed1996

Shift of the special pair redox potential: electrostatic energy computations of mutants of the reaction center from Rhodobacter sphaeroides.

Shifts of the special pair redox potential of the photosynthetic reaction center of Rhodobacter sphaeroides are considered for several point mutations [Lin. X., Murchison, H. A., Nagarijan, V., Parson, W. W., Allen, J. P., & Williams, J. C. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269] in the neighborhood of the special pair. The shifts are calculated from electrostatic energies by solving Poisson's equation for energy-minimized structures of the reaction center. Different conditions for the evaluation of the electrostatic energy are probed. To test the influence of the hydrogen bonding at the acetyl groups of the special pair, the orientation and torsion potential of the acetyl groups are varied. The calculated shifts of the midpoint potential of double and triple mutants can approximately be obtained from the corresponding shifts of the single point mutations. The calculated shifts agree with the measured values for all single and double mutants considered. However, a clear decision between different acetyl group conformations was only possible for the mutants HF(L168) and HF(L168) + LH(L131) where the calculated shifts of the redox potential agree with experiments only if the acetyl oxygen atom at DM points toward the Mg2+ ion of DL. This is corroborated by computations of the interaction energy of the acetyl group at DM, which adopts a lower value in the wild-type reaction center if its oxygen atom is bonded to the Mg2+ ion of DL.

Bacteriochlorophylls↗
PubMed1996

Classification and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics.

Most of the bacterial species are still unknown. Consequently, our knowledge about bacterial ecology is poor and expectations about specialized species with novel enzymatic functions or new products are high. Thus, bacterial identification is a growing field of interest within microbiology. In this review, suitability of developments for identification based on miniaturized biochemical and physiological investigations of bacteria are evaluated. Special emphasis is given to chemotaxonomic methods such as analysis of quinone system, fatty acid profiles, polar lipid patterns, polyamine patterns, whole cell sugars, peptidoglycan diaminoacids, as well as analytical fingerprinting methods and cellular protein patterning. 16S rDNA sequencing introduced to investigate the phylogenetic relationships of bacteria, nucleic acids hybridization techniques and G + C content determination are discussed as well as restriction fragment length polymorphism (RFLP), macrorestriction analysis and random amplified polymorphic DNA (RAPD). The importance of the different approaches in classification and identification of bacteria according to phylogenetic relationships are demonstrated on selected examples.

Bacteria↗
PubMed1996

Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides.

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.

Amino Acid Sequence↗
PubMed1996

Bacterial ghosts: non-living candidate vaccines.

Expression of cloned PhiX174 gene E in bacteria results in lysis of bacteria. It is unique among phage lysis systems as it introduces a transmembrane tunnel structure through the cell envelope complex of Gram-negative bacteria. The resulting bacterial ghosts have intact envelope structures devoid of cytoplasmic contents. E-mediated lysis has been achieved in a variety of Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Klebsiella pneumoniae, and Actinobacillus pleuropneumoniae. Such ghosts, derived from human or animal pathogens, have been proposed as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In 'recombinant ghosts', foreign proteins (e.g., viral proteins) are inserted into the inner membrane via specific N-, or C-, or N- and C-terminal anchor sequences prior to lysis. Relevant advantages of (recombinant) bacterial ghosts as immunogens include: (i) inactivation procedures that denature relevant immunogenic determinants are not employed in the production of ghosts used as vaccines or as carriers of relevant antigens; (ii) the recombinant proteins are inserted into a highly immune stimulatory environment; (iii) there is no size limitation of the foreign protein moieties: multiple antigenic determinants can be presented simultaneously; (iv) bacterial ghosts can be produced inexpensively in large quantities; (v) (recombinant) ghosts are stable for long periods of time and do not require the cold chain storage system. Intraperitoneal, subcutaneous or intramuscular applications of recombinant ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and viral components. Initial aerosol vaccinations of swine with ghosts from Actinobacillus pleuropneumoniae showed that protective immunity can be established by this route of application and that the well-preserved surface structures of ghosts obtained by E-mediated lysis are able to target the mucosal immune system.

Animals↗
PubMed1996

Induction of protective immunity by aerosol or oral application of candidate vaccines in a dose-controlled pig aerosol infection model.

In order to outline basic concepts for the design of a bacterial aerosol infection model, the development of a pig model with Actinobacillus pleuropneumoniae is described. First, reproducibility of aerosol parameters should be maintained by optimizing generating and sampling conditions. Survival rates of the chosen strain must be predictable. Secondly, inhalation conditions for the recipients have to be standardized to enable the determination of deposition sites and the dose administered. Subsequently, dose-response relationship should be evaluated to find a suitable challenge dose. Furthermore, it seems necessary to establish methods to obtain local specimens for determination of the local immune responses. The present study demonstrates that after aerosol challenge pigs were completely protected after inhalation and partially protected after oral application of A. pleuropneumoniae vaccines and describes techniques to administer bacteria in a dose-dependent, viable way. Using the infection model several stages of the disease from acute pleuropneumonia to chronic infection can be induced for research purposes.

Actinobacillus Infections↗
PubMed1996

Heterologous expression and self-assembly of the S-layer protein SbsA of Bacillus stearothermophilus in Escherichia coli.

The cell surface of Bacillus stearothermophilus PV72 is covered by a regular surface layer (S-layer) composed of single species of protein, SbsA, with a molecular weight of 130,000. Recently, the sequence of the corresponding gene (sbsA) has been determined. The SbsA coding region including the signal sequence was cloned as a polymerase chain reaction (PCR) product into a low-copy-number vector under the transcriptional control of the lambda pL promoter. Expression of sbsA was shown to be thermally inducible from the resulting vector pBK4 in a strain of Escherichia coli expressing the lambda cl857 from the chromosome. As shown by ultrathin sectioning of whole cells and immunogold labelling using SbsA- specific antibodies, expression of sbsA in E. coli led to accumulation of sheet-like self-assembling products of the protein in the cytoplasm. No SbsA protein was detected either in the periplasm or in the supernatant fractions. Long-term expression of sbsA from pBK4, including in the late stationary phase, did not lead to degradation of SbsA.

Bacterial Outer Membrane Proteins↗
PubMed1996

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings.

Bacteria↗
PubMed1995

Study of heme Fe(III) ligated by OH- in cytochrome b-559 and its low temperature photochemistry in intact chloroplasts.

EPR properties of Cyt b-559 have been investigated in intact chloroplasts that are functionally competent in O2 evolution and in CO2 fixation. After chemical oxidation of Cyt b-559 by 10 mM 2,3-dicyano, 4,5-dichloro-p-benzoquinone (DDQ) the major part of Cyt b-559 is found to be present in the high spin Fe(III) form. Only a small fraction of low spin heme Fe(III) (less than 5%) was formed by chemical or light-induced oxidation. This fraction increased during aging of intact chloroplasts. A comparison with the EPR signal of Fe(III) in myoglobin (Mb) reveals that the structure of the high spin signal in intact chloroplasts is indicative for the presence of an axial OH- ligand at the heme Fe(III). This type of ligation comprised a considerable part (approximately 40%) of the total Cyt b-559 content. Removal of the Mn-cluster caused a change of the EPR parameters of OH- ligation. When in intact chloroplasts the heme Fe is chemically oxidized to Fe(III) ligated by OH-, this OH- ligation disappeared after a subsequent illumination at 80K by red light. Upon illumination at 140K this disappearance was accompanied by the formation of a high spin Fe(III) that is not ligated by OH-. These results are discussed in terms of removal of OH- from Fe(III) caused by structural changes or photooxidation at a complex of Cyt b-559 that could possibly also comprise the Mn-cluster. This photooxidation is assumed to be accompanied by the formation of a bound OH. radical. The possibility is discussed that this process is related to photosynthetic water oxidation.

Benzoquinones↗
PubMed1995

ENDOR studies of the primary donor cation radical in mutant reaction centers of Rhodobacter sphaeroides with altered hydrogen-bond interactions.

The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer. The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7226-7230; Schenck, C. C., Gaul, D., Steffen M., Boxer S. G., McDowell L., Kirmaier C., & Holten D. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp 229-238, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

Cations↗
PubMed1995

EPR and ENDOR investigation of the primary electron acceptor radical anion QA.- in iron-depleted photosystem II membrane fragments.

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

Anions↗