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W Goebel

Publications and source records attributed to W Goebel.

At least 91 records · Page 5Linked to original sources
PubMed1992

Structural and functional properties of the p60 proteins from different Listeria species.

The major extracellular protein p60 of Listeria monocytogenes seems to be required for this microorganism's adherence to and invasion of 3T6 mouse fibroblasts but not for adherence to human epithelial Caco-2 cells. Western blot analysis with polyclonal antibodies against p60 of L. monocytogenes indicated the presence of cross-reacting proteins in the culture supernatants of all Listeria species. Protein p60 of L. monocytogenes could restore adhesion of the L. monocytogenes mutant RIII (impaired in the synthesis of p60) to mouse fibroblasts more efficiently than that of Listeria grayi. The amino acid sequences of the p60-related proteins of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi indicated highly conserved regions of about 120 amino acids at both the N-terminal and the C-terminal ends. The middle portions of these proteins, consisting of about 240 amino acids, varied considerably. These parts include the repeat domain consisting of repetitions of Thr (T) and Asn (N) which was present only, albeit in different arrangements, in the p60 proteins of L. monocytogenes and L. innocua. The p60-related proteins of L. grayi, L. ivanovii, L. seeligeri, and L. welshimeri each contained an insertion of 54 amino acids which was absent in the p60 proteins of L. monocytogenes and L. innocua.

Amino Acid Sequence↗
PubMed1992

Microbial strategies to prevent oxygen-dependent killing by phagocytes.

Microorganisms which are taken up by professional phagocytic cells of a host organism (e.g., by macrophages and polymorphonuclear leukocytes) encounter a series of antimicrobial events including confrontation with toxic oxygen species, derived mainly from the superoxide radical produced by phagocytic NADPH oxidase after uptake of the microorganism. Many microbes are susceptible to the oxygen-dependent phagocytic stress and are efficiently killed. The strategies of some microorganisms to bypass an encounter with the phagocytes' reactive oxygen species, and biochemical systems contributing to the microbes' resistance to killing by reactive oxygen species are outlined.

Animals↗
PubMed1991

Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme.

During an investigation into the substrate specificity and processing of subtilisin Carlsberg from Bacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97-101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, Mr = 42 kDa) was not processed to the mature form (Mr = 30 kDa) and was not released into the medium by a protease-deficient B. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants from B. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0-3 h after onset of stationary phase) of a processing intermediate (P38c, Mr = 38 kDa) of this protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.

Bacillus↗
PubMed1991

Mutations affecting pore formation by haemolysin from Escherichia coli.

By introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9-37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI-DIII) located between amino acids 238-410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498-830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670-830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed.

Amino Acid Sequence↗
PubMed1991

Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA.

A plasmid integration technique was developed for insertional inactivation of chromosomal Listeria monocytogenes genes. A Listeria-Escherichia coli shuttle vector (pLSV1) was constructed which carried the temperature-sensitive gram-positive replication origin from plasmid pTV32(Ts). An internal fragment of the listeriolysin gene (lisA) was cloned into pLSV1 to create pLSV2. In L. monocytogenes pLSV2 transformants, plasmid pLSV2 integrated into the L. monocytogenes chromosome at a frequency of 2 x 10(-3) via lisA homology and these cells could be selected at 42 degrees C using a plasmid-encoded erythromycin resistance. Plasmid integration resulted in disruption of the lisA gene, production of a truncated, immunologically cross-reactive listeriolysin protein and loss of the hemolytic phenotype. An improved Listeria protoplast transformation method is also described which facilitates genetic manipulation of Listeria species.

Bacterial Toxins↗
PubMed1991

Studies on the pathogenicity of Listeria monocytogenes.

The characterization of mutants of Listeria monocytogenes with reduced virulence properties is described. Reduction in the amount of the extracellular protein p60 (encoded by the ipa gene) leads to cell filaments with impaired invasiveness. Mutants which cannot synthesize listeriolysin are still invasive but unable to survive within phagocytic cells. One type of listeriolysin-negative mutants is defective in the synthesis of a positive regulatory element PrfA which coordinately regulates the listeriolysin gene (lisA) together with several other genes, including those for a phosphatidylinositol-specific phospholipase and a metalloprotease.

Antigens, Bacterial↗
PubMed1991

Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene.

The entire nucleotide sequence of an open reading frame located immediately downstream of the listeriolysin gene from a virulent Listeria monocytogenes serotype 1/2a strain was determined. The product of the open reading frame was 510 amino acids with a predicted molecular weight of 57,400. The deduced amino acid sequence of this open reading frame is highly similar to that of a family of secreted metalloproteases produced by various members of the genus Bacillus, of which thermolysin is the prototype. Immunoblots performed with specific antisera raised against thermolysin from Bacillus stearothermophilus allowed the detection of a 60-kDa polypeptide, corresponding to the pro-form of the protease, in culture supernatants of L. monocytogenes strains. In maxicell experiments, Escherichia coli recombinants harboring this open reading frame also specifically directed production of a 60-kDa protein. Protease activity was low to undetectable in both Listeria strains and E. coli recombinants. This is due to lack of processing of the inactive pro-form of the protease to its mature active form in both species. We have designated this gene mpl for metalloprotease of L. monocytogenes. The gene was present only in pathogenic L. monocytogenes strains, in which it was physically linked to the listeriolysin gene.

Amino Acid Sequence↗
PubMed1991

Characterization of monoclonal antibodies against alpha-hemolysin of Escherichia coli.

Monoclonal antibodies (MAbs) were raised against native and denatured alpha-hemolysin (HlyA) of Escherichia coli. Binding of the MAbs to native, denatured, and erythrocyte-complexed active wild-type hemolysin and mutant derivatives was tested. All 15 MAbs analyzed bound to native hemolysin, even when the toxin was complexed with human erythrocytes. While some MAbs were unable to bind to a specific native mutant hemolysin, others could not even bind to mutant hemolysin carrying deletions remote from their actual binding sites. A rough determination of the binding sites of 15 MAbs on HlyA was performed by Western immunoblot analysis using CNBr fragments of HlyA and mutant hemolysin proteins. Interestingly, the binding sites of the MAbs against native hemolysin seem to be more randomly distributed on HlyA than are those of MAbs against denatured hemolysin. Three MAbs inhibited the hemolytic activity significantly. Two of these MAbs bound to the hydrophobic region, and the other one bound to the repeat domain of HlyA. The use of synthetic peptides from these regions allowed determination of the linear epitopes for two of these MAbs.

Animals↗
PubMed1991

Expression of the iap gene coding for protein p60 of Listeria monocytogenes is controlled on the posttranscriptional level.

Expression of the iap gene of Listeria monocytogenes encoding a major extracellular protein (p60) was analyzed. Different start sites for transcription of the iap gene were identified by primer extension analysis in L. monocytogenes and in a recombinant Escherichia coli clone. The mutant RIII of L. monocytogenes represents a member of the frequently occurring L. monocytogenes R mutants, which form cell chains and produce greatly reduced amounts of p60. However, the concentrations of iap-specific mRNA were similar in mutant RIII and the wild-type strain. The introduction of additional copies of the iap gene from wild-type L. monocytogenes led to an equal increase of iap mRNA in both strains, but overexpression of protein p60 was only observed in the wild-type strain. The nucleotide sequences of both iap genes and their 5' noncoding regions were identical in all parts that are essential for efficient transcription of the iap gene, translation of the iap-specific mRNA, and transport of the p60 protein. These data suggest that the expression of the iap gene in L. monocytogenes is controlled on the posttranscriptional level by a specific factor that is defective in mutant RIII.

Amino Acid Sequence↗
PubMed1991

Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases.

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene.

Amino Acid Sequence↗
PubMed1991

Cloning and expression in Escherichia coli of a gene encoding superoxide dismutase from Listeria ivanovii.

A chromosomal DNA fragment from the gram-positive bacterium Listeria ivanovii (ATCC 19119) encoding a superoxide dismutase (SOD) gene has been cloned in Escherichia coli QC779 (sodAsodB) using the plasmid vector pTZ19R. The DNA fragment inserted into the plasmid showed high structural instability in E. coli QC779 (recA+), but turned out to be a stable 1.95 kbp DNA fragment when transformed into E. coli DH5 alpha (recA-). The gene is expressed in both of these E. coli strains at high levels. Preliminary studies showed that the activity of the recombinant SOD within E. coli DH5 alpha was up to 13-times the combined activity of both E. coli SODs. The recombinant SOD forms active hybrid SODs with both E. coli SODs in vivo.

Bacterial Proteins↗
PubMed1990

Cloning of the resistant EcoRII recognition site of phage T7 into an EcoRII-sensitive plasmid makes the site susceptible to the restriction enzyme.

The recognition sequence 5'-CC(A/T)GG for EcoRII in the bacteriophage T7 genome is refractory to this restriction endonuclease, despite not bearing the specific (protective) methylation. Following the integration of this site as part of a 219 bp fragment (in which the recognition sequence is flanked by about 100 bp of T7 origin) into the EcoRII-sensitive vector pUC18, the T7 site becomes susceptible to cleavage, too. The same is true of recombinant pBR322 plasmids containing the T7-derived recognition site. The results show that the flanking sequences are not immediately responsible for the refractory behaviour of EcoRII sites and are in agreement with data according to which EcoRII requires the coordinated presence of at least two recognition sites in its DNA substrate.

Cloning, Molecular↗
PubMed1990

Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins.

We studied the efficiency of the pHly152-derived haemolysin transport system using PhoA-HlyA fusion proteins and different constructs which provide HlyB/HlyD in trans. The optimal C-terminal HlyA signal consists of the last 60 amino acids. Longer stretches of HlyA do not improve the transport efficiency of PhoA-HlyA fusion proteins. The introduction of deletions and/or replacements in the 60 amino acid HlyA signal domain revealed at least three functional regions with different degrees of specificity. Amino acids 1-21 (numbered from the N-terminal part of the 60 amino acid HlyA signal), termed region I, could be replaced by a Pro-containing peptide. The other two regions II and III (amino acids 22-40 and 41-60, respectively) seem to interact directly with the HlyB/HlyD translocator since a PhoA fusion protein which contains either of the two regions was still secreted in a HlyB/HlyD-dependent mode, albeit at low efficiency. An efficient trans-complementing HlyB/HlyD system was only obtained from the pHLy152-encoded hly determinant when the regulatory hlyR element was provided in cis. Secretion of the PhoA-HlyA fusion protein did not interfere with the secretion of HlyA even when the fusion protein was induced to a high level. This suggests that the capacity of the HlyB/HlyD translocation system is high and not normally saturated by its natural HlyA substrate.

Amino Acid Sequence↗
PubMed1990

Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence.

Alkaline phosphatase (AP) is secreted into the medium when the carboxy-terminal 25 amino acids are replaced by the 60 amino acid carboxy-terminal signal peptide (HlyAs) of Escherichia coli haemolysin (HlyA). Secretion of the AP-HlyAs fusion protein is dependent on HlyB and HlyD but independent of SecA and SecY. The efficiency of secretion by HlyB/HlyD is decreased when AP carries its own N-terminal signal peptide. Translocation of this fusion protein into the periplasm is not observed even in the absence of HlyB/HlyD. The failure of the Sec export machinery to transport the latter protein into the periplasm seems to be due in part to the loss of the carboxy-terminal sequence of AP since even AP derivatives which do not carry the HlyA signal peptide but lack the 25 C-terminal amino acids of AP are localized in the membrane but not translocated into the periplasm.

Alkaline Phosphatase↗
PubMed1990

Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vitro and in vivo in various extraintestinal Escherichia coli isolates.

Fimbrial adhesins and hemolysins contribute to pathogenicity of extraintestinal Escherichia coli isolates causing urinary tract infections (UTI), sepsis and new born meningitis (NBM). Using gene cloning techniques and pulse field electrophoresis in combination with Southern hybridizations it was demonstrated that the genetic determinants coding for P and 'P-related' fimbrial adhesins and hemolysins are closely linked on the chromosomes of different pathogenic E. coli wild-type isolates. For two UTI strains, 536 (O6:K15) and J96 (O4:K6), a co-deletion of the linked gene clusters coding for hemolysin and fimbriae was observed. The deleted DNA regions which also comprise flanking DNA sequences were termed 'pathogenicity DNA islands'. Such 'pathogenicity DNA islands' were also detected in the genome of O18:K1 isolates of OMP type 6 but were absent on the chromosomes of O18:K1 strains of OMP type 9. A mutant strain, 536-22 was selected from rat kidneys after intraurethral infection of animals with the wild-type parental strain 536. This particular isolate also shows deletions of 'pathogenicity islands' leading to a non-pathogenic phenotype. It is therefore concluded that excisions of 'pathogenicity islands' from chromosomes of pathogenic E. coli strains are not restricted to the laboratory but also occur in vivo. The generation of deletions may represent a general mechanism of bacterial virulence modulation.

Adhesins, Escherichia coli↗
PubMed1990

Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes.

We have isolated, by molecular cloning and genetic complementation of a listeriolysin-negative mutant, a gene required for the expression of this virulence factor in Listeria monocytogenes. The mutant strain SLCC53, which was nonhemolytic and avirulent, harbored a deletion of 450 base pairs located approximately 1500 base pairs upstream of the listeriolysin gene. No transcripts corresponding to the listeriolysin gene were detected in the mutant. DNA sequencing of this region from the hemolytic strain EGD revealed that the region deleted in the mutant would abrogate expression of a 27-kDa polypeptide. Introduction of a recombinant plasmid expressing this 27-kDa polypeptide restored hemolytic activity to the mutant and increased the hemolytic activity of the wild-type L. monocytogenes strain EGD. We have designated the gene encoding the 27-kDa polypeptide prfA, for positive regulatory factor of listeriolysin (lisA) expression. The prfA gene regulates transcription of the lisA gene positively.

Amino Acid Sequence↗