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W Goebel

Publications and source records attributed to W Goebel.

At least 145 records · Page 8Linked to original sources
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↗
PubMed1990

Characterization of a Listeria monocytogenes-specific protein capable of inducing delayed hypersensitivity in Listeria-immune mice.

Recovery of the host after infection by the intracellular pathogen Listeria monocytogenes is dependent on cell-mediated immunity. Little is known of the nature of listerial antigens that induce cell-mediated responses in the infected host. In this study we report on the identification and cloning of an Escherichia coli recombinant encoding a listerial antigen, designated ImaA, capable of eliciting a specific delayed-type hypersensitivity response in Listeria-immune mice. Nucleotide sequencing of the Listeria DNA insert in plasmid pLM10 showed that the ImaA gene product consisted of 170 amino acids with a molecular weight of 17,994. The predicted amino acid sequence suggests that the protein is localized to the bacterial plasma membrane or cell wall. The ImaA gene was unique to the pathogenic species L. monocytogenes and Listeria ivanovii; it was not present in any other species of the genus Listeria.

Amino Acid Sequence↗
PubMed1990

Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes.

Listeriolysin is a virulence factor that appears to be necessary for the intracellular survival of Listeria monocytogenes. As shown in this investigation, listeriolysin is produced in only small amounts by clinical isolates of L. monocytogenes belonging to the serogroup 1/2a, but its synthesis can be induced by heat shock and to a lesser extent by oxidative stress. In addition to about 15 heat shock proteins that appear to be common to L. monocytogenes and Listeria species that are nonpathogenic for humans, at least five heat shock proteins are specifically coinduced with listeriolysin in all L. monocytogenes strains under heat shock conditions but not in the other Listeria species. One type of L. monocytogenes mutant blocked in the expression of listeriolysin failed to synthesize several of these specific heat shock proteins.

Bacterial Toxins↗
PubMed1990

Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes, and basophils and from polymorphonuclear granulocytes.

We investigated the role of Escherichia coli expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains differed in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature. The various bacterial strains differed in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene B4 generation and reduced w-oxidation of leukotriene B4. Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E. coli.

Adhesins, Escherichia coli↗
PubMed1990

The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes.

The gene of Listeria monocytogenes that encodes a major extracellular protein (p60) was cloned in Escherichia coli. The gene was designated iap, as p60 was previously shown to represent an invasion-associated protein (M. Kuhn and W. Goebel, Infect. Immun. 57:55-61, 1989). The recombinant E. coli clone expressed p60, as shown by immunoblotting. The complete nucleotide sequence of iap was determined. The deduced amino acid sequence of p60 (484 amino acids) contains a putative N-terminal signal sequence of 27 amino acids and an extended repeat region consisting of 19 threonine-asparagine units. Hybridization with the entire iap gene revealed the presence of homologous sequences in most other Listeria species. In contrast, a 400-base-pair internal iap probe which contained the whole repeat region hybridized only with genomic DNA from L. monocytogenes. Four oligonucleotides previously described as specific probes for the detection of L. monocytogenes (A. R. Datta, B. A. Wentz, D. Shook, and M. W. Trucksess, Appl. Environ. Microbiol. 54:2933-2937, 1988) were shown to be part of the iap gene.

Amino Acid Sequence↗
PubMed1990

Translocation and compartmentalization of Escherichia coli hemolysin (HlyA).

Hemolysin plasmids were constructed with mutations in hlyB, hlyD, or both transport genes. The localization of hemolysin activity and HlyA protein in these mutants was analyzed by biochemical and immunological methods. It was found that mutants defective in hlyB accumulated internal hemolysin, part of which was associated with the inner membrane and was degraded in the late logarithmic growth phase. In an HlyB+ HlyD- mutant, hemolysin was predominantly localized in the membrane compartment. Labeling of these Escherichia coli cells with anti-HlyA antibody indicated that part of HlyA, presumably the C-terminal end but not the pore-forming domains, was already transported to the cellular surface. This finding suggests that HlyB is able to recognize the C-terminal signal of the HlyA protein and to initiate its translocation across the membranes.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b.

In culture supernatants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as listeriolysin O (LLO). In the case of L. ivanovii a second major supernatant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supernatants of L. ivanovii a sphingomyelinase and a lecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-terminal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.

Amino Acid Sequence↗
PubMed1989

Mutations affecting hemolysin production in Listeria monocytogenes located outside the listeriolysin gene.

We have investigated the molecular basis of spontaneous mutations leading to non-hemolytic and avirulent variants of the Listeria monocytogenes serotype 1/2a strain NCTC 7973 using Southern hybridization to DNA fragments that harbor the listeriolysin gene (hlyA) and adjacent regions cloned from a L. monocytogenes serotype 1/2a strain. The analysis of such non-hemolytic variants revealed the presence of a deletion of 300 base pairs, located 1.6 kb upstream of an otherwise intact listeriolysin gene. The importance of regions upstream of the hlyA gene in controlling the expression of the listeriolysin gene was further emphasized by the detection of a transposon-derived nonhemolytic mutant in which the transposon had inserted approximately 200 bp upstream of the listeriolysin gene. We conclude that at least two elements, contained within a region encompassing 1.6 kb of sequences upstream of the hlyA gene, may be required for expression of the listeriolysin gene.

Bacterial Toxins↗
PubMed1989

Synthesis of listeriolysin in Listeria monocytogenes under heat shock conditions.

Listeriolysin, produced by all virulent Listeria monocytogenes isolates, is an essential virulence factor which appears to be necessary for the intracellular survival of these bacteria. It has been postulated that the intracellular environment imposes stress conditions similar to heat shock on invading bacteria. We show here that listeriolysin was still very efficiently synthesized in one Listeria monocytogenes strain even intracellularly and induced under heat shock conditions in another L. monocytogenes strain. Listeriolysin appears to be the only major extracellular protein synthesized under heat shock conditions; all other heat shock proteins remain cell associated.

Bacterial Toxins↗