Biomedical subjects

W Lubitz

Publications and source records attributed to W Lubitz.

At least 109 records · Page 6Linked to original sources

PubMed1995

Evaluation of the E. coli ribosomal rrnB P1 promoter and phage-derived lysis genes for the use in a biological containment system: a concept study.

A concept study devised for the development of a biological containment system has been conducted. We show that the lysis genes of different phage origin function in a variety of bacteria. They may therefore be suited for conditional suicide cassettes. Moreover, we tested whether the Escherichia coli rrnB P1 promoter could function as an environmentally responsive element sensing poor growth conditions expected after an accidental release of E. coli production strains from a bioreactor. Mimicking poor nutrient conditions by production of the alarmone guanosine tetraphosphate (ppGpp) with a plasmid encoded ppGpp synthetase I, the rrnB P1 promoter activity was completely turned off. These experiments suggested that the rrnB P1 promoter may be used as an efficient biosensor for altered growth conditions. A concept for a conditional suicide system employing the rrnB P1 promoter and phage-derived lysis genes as key components is discussed.

Amino Acid Sequence↗

PubMed1995

Site-directed mutagenesis of conserved histidines in the helix VIII domain of PsaB impairs assembly of the photosystem I reaction center without altering spectroscopic characteristics of P700.

The chloroplast psaB gene encodes one of the polypeptides of the photosystem I reaction center heterodimer that coordinates the electron transfer components P700, A0, and A1. Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordinate the P700 reaction center chlorophyll(s) and the initial electron acceptor, A0. Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosystem I reaction center biogenesis and function. It is demonstrated that these histidine residues are essential for stable accumulation of the photosystem I reaction center. Protein pulse-labeling shows that changing the histidine residues impairs a post-translational step in reaction center assembly. Photosystem I complexes from the mutants have been characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P700+. In all cases we determine that spectroscopic characteristics of P700+ remain unchanged. The implications of these results to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.

Amino Acid Sequence↗

PubMed1995

Electronic structure of Q-A in reaction centers from Rhodobacter sphaeroides. I. Electron paramagnetic resonance in single crystals.

The magnitude and orientation of the electronic g-tensor of the primary electron acceptor quinone radical anion, Q-A, has been determined in single crystals of zinc-substituted reaction centers of Rhodobacter sphaeroides R-26 at 275 K and at 80 K. To obtain high spectral resolution, EPR experiments were performed at 35 GHz and the native ubiquinone-10 (UQ10) in the reaction center was replaced by fully deuterated UQ10. The principal values and the direction cosines of the g-tensor axes with respect to the crystal axes a, b, c were determined. Freezing of the single crystals resulted in only minor changes in magnitude and orientation of the g-tensor. The orientation of Q-A as determined by the g-tensor axes deviates only by a few degrees (< or = 8 degrees) from the orientation of the neutral QA obtained from an average of four different x-ray structures of Rb. sphaeroides reaction centers. This deviation lies within the accuracy of the x-ray structure determinations. The g-tensor values measured in single crystals agree well with those in frozen solutions. Variations in g-values between Q-A, Q-B, and UQ10 radical ion in frozen solutions were observed and attributed to different environments.

Anions↗

PubMed1995

Two-stage model for integration of the lysis protein E of phi X174 into the cell envelope of Escherichia coli.

As a tool for determining the topology of the small, 91-amino acid phi X174 lysis protein E within the envelope complex of Escherichia coli, a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used. The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations. At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E. coli.

Bacteriolysis↗

PubMed1995

Oral immunization of pigs with viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces pulmonary and systemic antibodies and protects against homologous aerosol challenge.

A dose-defined aerosol infection of pigs was used to study the immunogenic and protective potentials of oral immunization with dead or live Actinobacillus pleuropneumoniae serotype 9 reference strain CVI 13261 against an aerogenic challenge. Pigs were vaccinated with a single dose of 10(11) CFU of viable (n = 8) or inactivated (n = 8) A. pleuropneumoniae given orally in a gelatin capsule. After 3 weeks, vaccinated pigs and nonvaccinated controls were challenged aerogenically with a dose of 10(8) CFU of A. pleuropneumoniae CVI 13261. The protective efficacy of oral immunization was evaluated by clinical and postmortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples for assessment of local antibodies. Isotype-specific antibody responses in sera and in bronchoalveolar lavage fluids were determined by enzyme-linked immunosorbent assays based on whole-cell antigen. Oral immunization did not induce clinical side effects. After aerosol challenge, two animals of both vaccinated groups (25% in each case) showed a moderate fever for 2 days, whereas all four pigs (100%) of the nonvaccinated control group developed severe fever. In contrast to the controls, which developed severe pleuropneumonia, the vaccinated pigs had only mild pulmonary lesions. Three weeks after challenge, 13 of 16 vaccinated pigs (81%) were found to be free of pathomorphological changes of the lungs. From two of these pigs immunized with live bacteria we were able to reisolate A. pleuropneumoniae. A significant systemic and pulmonary increase in the concentrations of immunoglobulin A (IgA), IgM, and IgG antibodies reactive with A. pleuropneumoniae was detectable after aerosol challenge in both vaccinated groups. Immunization with viable bacteria was found to induce significantly higher concentrations of each Ig isotype in bronchoalveolar lavage fluids and sera than immunization with inactivated A. pleuropneumoniae. These serological findings were not reflected in the reduction in clinical disease after challenge in comparison to the case for the pigs vaccinated with inactivated bacteria. We concluded that a single oral administration of A. pleuropneumoniae provides partial clinical protection against aerosol challenge infection in the respiratory tract.

Actinobacillus Infections↗

PubMed1995

High-level poly(beta-hydroxybutyrate) production in recombinant Escherichia coli in sugar-free, complex medium.

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50-80% PHB/cell dry mass when the cells are grown in Luria-Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria-Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the beta-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter-operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria-Bertani medium without glucose supplement.

Acetyl-CoA C-Acyltransferase↗

PubMed1994

Comparative study of reaction centers from photosynthetic purple bacteria: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Reaction centers (RCs) from four species of purple bacteria, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and the recently discovered bacterium Rhodospirillum centenum, have been characterized by optical spectroscopy [Wang, S., Lin, X., Woodbury, N. W., & Allen, J. P. (1994) Photosynth. Res. (submitted for publication)] and magnetic resonance spectroscopy. All RCs contain a bacteriochlorophyll (BChl) a dimer as the primary donor. For Rb. sphaeroides and Rs. rubrum the donor QY optical band is at approximately 865 nm, compared to approximately 850 nm for Rb. capsulatus and Rs. centenum. The primary donor in the RCs can be converted between these two forms by the addition or removal of charged detergents. The electronic structure of the cation radical of the primary electron donor P+. was investigated in these species using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and electron nuclear triple resonance (TRIPLE) spectroscopy. The EPR line widths of P+. vary significantly and the ENDOR and Special TRIPLE spectra reveal drastic differences in the spin density distribution of the dimer for the different species. Reaction centers from Rb. sphaeroides and Rs. rubrum have a slightly asymmetric spin density distribution over the two halves of the dimer. The respective ratios are 2:1 and 1.6:1 in favor of the L-half of the BChl a dimer. In contrast, the spectra of P+. in reaction centers from Rb. capsulatus and Rs. centenum show an almost complete localization of the unpaired electron on the L-half of the dimer (ratio approximately 5:1).(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Spin Resonance Spectroscopy↗

PubMed1994

Sequence analysis of the sbsA gene encoding the 130-kDa surface-layer protein of Bacillus stearothermophilus strain PV72.

Bacillus stearothermophilus (Bs) contains a surface-layer (S-layer) protein (SbsA), which forms a hexagonal array on the cell wall. In order to understand the structural/functional relationship of SbsA from Bs PV72, the entire nucleotide (nt) sequence of the sbsA gene was determined from three overlapping fragments. The 3'-end was cloned and expressed in Escherichia coli, whereas the 5'-region was amplified from the genome of Bs PV72 by the polymerase chain reaction using two overlapping fragments. The open reading frame (3684 nt) of sbsA is predicted to encode a protein of 1228 amino acids (aa). The SbsA is synthesized with a leader sequence of 30 aa. The predicted SbsA aa profile was similar to most other sequenced S-layer proteins, containing more acidic than basic aa (pI 5.1) and a very low amount of sulfur-containing aa. Based on aa sequence data, SbsA has weak homology of with the S-layer proteins from B. sphaericus, Rickettsia rickettsii, B. brevis HPD31 and B. brevis 47 (OWP).

Amino Acid Sequence↗

PubMed1994

Expression of the Alcaligenes eutrophus phbA gene in Escherichia coli using a positive selection vector based on phage Lambda lysis genes.

A new positive selection vector, pGS23, based on the Lambda lysis cassette has been designed for efficient expression of homologous and heterologous genes in Escherichia coli. The plasmid permits controlled expression of a gene of interest under transcriptional control of the lac promoter with translation initiation of coding sequences directed by the phage T7 gene 10 ribosome binding site. The application of the vector system was tested for high level expression of the heterologous phbA gene of Alcaligenes eutrophus in E. coli.

Acetyl-CoA C-Acyltransferase↗

PubMed1994

Production of Vibrio cholerae ghosts (VCG) by expression of a cloned phage lysis gene: potential for vaccine development.

The protein E-specific lysis mechanism of the Escherichia coli-specific bacteriophage PhiX174 was employed to produce Vibrio cholerae ghosts (VCG). VCG consist of both rounded and collapsed cells that have lost their cytoplasmic contents through an E-specific hole in the cell envelope. These ghosts are proposed as non-living material for immunization against cholera. A specific membrane anchor sequence was used to insert the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) fusion protein into the cell envelope of V. cholerae. The identity of the expression products was confirmed by Western blot analysis employing an RT-specific monoclonal antibody. HIV-1 RT was chosen as a model for the purpose of evaluating heterologous gene expression in V. cholerae and the carrier potential of VCG. Intraperitoneal immunization of mice was used to evaluate the immunogenic potential of VCG. Preliminary results showed significant seroconversions to intact whole-cell vibrio antigens in mice immunized with VCG or a heat-killed whole-cell vibrio preparation.

Animals↗

PubMed1994

Immunogenicity of Vibrio cholerae ghosts following intraperitoneal immunization of mice.

The immunogenic potential of Vibrio cholerae ghosts (VCG) in comparison with heat-killed whole-cell vibrios (WCV) was evaluated after intraperitoneal immunization of adult mice. Swiss white mice received four doses of VCG or WCV intraperitoneally, consisting of 500 micrograms of lyophilized material in 200 microliters of phosphate-buffered saline (PBS), pH 7.4. The control group received 200 microliters of PBS. Serum samples were collected from all mice on the day of immunization and on days 14, 24, 35 and 62 postimmunization. Sera were examined for vibriocidal antibodies by the microtitre and tube-dilution methods and Vibrio-specific serum IgG antibodies were assessed by ELISA. IgG antibodies to intact WCV were detected in sera from all animals immunized with VCG or WCV. The response was specific and of high magnitude. Significantly higher antibody responses were obtained when sera from both VCG- and WCV-immunized mice were titrated against VCG. The immunogenicity of VCG in evoking serum IgG responses was higher than that of WCV. However, the immunogenicity of the two antigen preparations was comparable in terms of seroconversion for vibriocidal antibodies. These results demonstrate that VCG administered intraperitoneally evoke Vibrio-specific serum IgG responses as well as vibriocidal antibody activity in mice.

Animals↗

PubMed1993

Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300.

Expression of the cloned PhiX174 gene E causes lysis of the Gram-negative bacterium Escherichia coli, which led to the proposal that a two-membrane system is necessary for the protein E lysis function. Gene E was cloned in an E. coli/Bacillus subtilis shuttle vector and expressed in the Gram-positive bacterium Staphylococcus carnosus TM300. Regulated gene E expression had a lethal effect on S. carnosus; however, no lysis was detected, lending support to the hypothesis.

Bacteriophage phi X 174↗

PubMed1993

Lambda kil-mediated lysis requires the phage context.

The lambda kil gene has been shown to be responsible for premature lysis effected by addition of chloramphenicol between 15 and 20 min after thermal induction of a lambda prophage. Here, we localized the kil reading frame. The kil gene, represented by lambda orf47, overlaps genes cIII and gam. Expression of the plasmid-borne kil gene resulted in growth arrest, a reduction of colony-forming units and filament formation. However, kil-mediated cell lysis could not be triggered by chloramphenicol when the plasmid borne kil gene was expressed, suggesting that kil-induced cell lysis requires the phage context.

Bacteriophage lambda↗

PubMed1993

The missing link in phage lysis of gram-positive bacteria: gene 14 of Bacillus subtilis phage phi 29 encodes the functional homolog of lambda S protein.

In most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane. The lesion is brought about by a second phage-encoded lysis function. For the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium. Thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive. Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli. Production of protein 14 in E. coli resulted in cell death, whereas production of protein 14 concomitantly with the phi 29 lysozyme or unrelated murein-degrading enzymes led to lysis, suggesting that membrane-bound protein 14 induces a nonspecific lesion in the cytoplasmic membrane.

Amino Acid Sequence↗

PubMed1992

Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli.

Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.

Adenosine Triphosphatases↗

PubMed1990

A bifunctional vector system for controlled expression and subsequent release of the cloned gene product by phi X174 lysis protein-E.

A new bifunctional Escherichia coli cloning vector, pSB50, is presented. The plasmid allows controlled expression of a gene of interest under control of the lac promoter and the subsequent release of the cloned product by the use of bacteriophage phi X174 lysis protein-E, the gene of which is under control of the phage Lambda pL promoter. To ensure optimal repression of the Lambda pL promoter and the lac promoter in plasmid pSB50, E. coli strain UB89-1 was constructed which carries a chromosomal copy of the lambda cl857 repressor allele and the laclq1 allele, respectively. Here, we employ the E-based lysis system to release human prourokinase to the culture medium.

Bacteriophages↗

PubMed1990

Translational efficiency of phi X174 lysis gene E is unaffected by upstream translation of the overlapping gene D reading frame.

The lysis gene E of bacteriophage phi X174 is entirely embedded in gene D. Expression studies of genes D and E in Escherichia coli minicells and lysis times obtained in the presence or absence of D translation showed that the simultaneous expression of gene D does not affect protein E production. Thus, unlike other overlapping gene pairs, gene E expression is independent from the upstream translation of gene D. lacZ fusion studies and primer extension inhibition analysis (toeprinting) revealed an intrinsically weak E ribosome-binding site, which seems to be the major factor determining the low expression rate of the gene and thus proper scheduling of cell lysis.

Amino Acid Sequence↗

PubMed1990

Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E.

Biochemical and genetic studies have suggested that a transmembrane tunnel structure penetrating the inner and outer membranes is formed during the lytic action of bacteriophage phi X174 protein E. In this study we directly visualized the lysis tunnel by using high-magnification scanning and transmission electron microscopy.

Bacteriophage phi X 174↗