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W Goebel

Publications and source records attributed to W Goebel.

At least 181 records · Page 10Linked to original sources
PubMed1989

Isolation and characterization of genes coding for proteins involved in the cytolysis by Listeria ivanovii.

We established a library of chromosomal DNA of Listeria ivanovii in the pTZ19R plasmid system, using Escherichia coli DH5 alpha as the host. One recombinant clone reacted strongly with a polyclonal antiserum raised against the listeriolysin O and a second exoprotein (24kDa) of L. ivanovii, which is most probably also involved in cytolytic processes. The recombinant E. coli clone may contain part of the listeriolysin O gene of L. ivanovii.

Blotting, Western↗
PubMed1988

Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli.

A sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant. Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312. HlyR was active in cis but its activity was orientation-dependent. The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element. Stepwise removal of the hlyR sequence from its 5' end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin. A similar observation was made when hlyR was shortened by ExoIII from its 3' end, which suggests that more than one functional region may be present in the hlyR sequence. A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter. The nucleotide sequence of hlyR is rich in A + T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending.

Base Sequence↗
PubMed1988

The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes.

The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+. These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca2+ in haemolysis is presented.

Amino Acid Sequence↗
PubMed1988

Hemolysin from Listeria--biochemistry, genetics and function in pathogenesis.

Thiol-activated hemolysins (listeriolysins) from Listeria monocytogenes (Sv4b) and Listeria ivanovii were purified to homogeneity. The N-terminal amino acid sequences of the 58 kDa listeriolysin of L. ivanovii and of a 24 kDa protein which may represent the CAMP-factor of L. ivanovii were determined. Antibodies raised against the L. ivanovii listeriolysin and anti-streptolysin O antibodies were used in Western blot analyses to detect listeriolysin(s) in virulent and avirulent Listeria strains. It was found that all virulent strains of L. monocytogenes synthesize and secrete listeriolysin (Mr 58-59 kDa), albeit in significantly variable quantities. No protein cross-reaction with anti-listeriolysin antibodies or anti-streptolysin O-antibodies was present in the supernatant of Listeria innocua, Listeria welshimeri, Listeria grayi and Listeria murrayi strains. Furthermore, the avirulent but hemolytic Listeria seeligeri did not cross-react with these antibodies. In a L. monocytogenes (strain EGD) gene bank constructed in Escherichia coli two types of hemolytic clones were identified. The first type carried recombinant plasmids with a common 2.0 kb fragment coding for a 23 kDa protein. This hemolytic activity was not activated by DTT and the 23 kDa protein did not cross react with anti-listeriolysin or anti-streptolysin antibodies. The other type of hemolytic clones was detected by using anti-streptolysin O antibodies to screen the gene bank. Some of these clones synthesized a protein of 61 kDa which cross reacted with anti-streptolysin O (or anti-listeriolysin) antibodies. By transposon Tn916 mutagenesis of L. monocytogenes two types of nonhemolytic mutants were obtained. Type I produced no extracellular protein that cross reacted with anti-listeriolysin (or anti SLO) antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and transcriptional analysis of the aerCaerA region of Aeromonas sobria encoding aerolysin and its regulatory region.

The nucleotide sequence of a 2510 base pair chromosomal fragment containing the aerolysin gene aerA, and its regulatory region aerC, from a clinical isolate of Aeromonas sobria was determined. The aerolysin gene coded for a 54.5 kD polypeptide and had a G + C content of 59%, indicating that it is endogenous to the genus Aeromonas. In contrast, the aerC region was characterized by its high A + T content (61%) and the presence of a core motif, aATAAAa, repeated eight times within 300 base pairs. A 12 base pair repeat, 5'AATAAAACCGGG3', present within this region occurred as a direct repeat 544 base pairs away, within the coding region of aerolysin. RNA polymerase binding studies and S1 mapping allowed the detection of two divergent non-overlapping promoters within aerC. Despite having identical transcriptional start sites in both A. sobria and Escherchia coli, the amount of aerolysin transcript produced in E. coli is 30-40 times less than that found in A. sobria. The signal peptide of preproaerolysin was shown by deletion to be essential for export of the toxin to the external medium. The mature toxin is a hydrophilic protein with no hydrophobic stretches long enough to cross a membrane. A search for similarities to the primary sequence of aerolysin revealed that the toxin may share a functional similarity to haemolysin (hlyA) of E. coli.

Aeromonas↗
PubMed1988

Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes.

The gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen. The only known property of L. monocytogenes which has been shown to be involved in virulence is a hemolysin, listeriolysin (J. L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986; S. Kathariou, P. Metz, H. Hof, and W. Goebel, J. Bacteriol. 169:1291-1297, 1987). Using our previously obtained transposon Tn916-induced hemolysin-negative mutants of L. monocytogenes Sv1/2a (Mackaness strain), we demonstrated that the loss of hemolysin reduced significantly the rate of survival of the bacteria in mouse peritoneal macrophages but did not reduce their uptake. It was further shown that virulent L. monocytogenes strains could invade the mouse embryo fibroblast 3T6 cell line, i.e., mammalian cells which are nonprofessional phagocytes. This uptake was inhibited by cytochalasin B and hence seems to be accomplished by parasite-induced endocytosis. Hemolysin was not essential for this step. Strains of other Listeria species could not efficiently penetrate the 3T6 cells.

Animals↗
PubMed1988

Levels of Listeria monocytogenes hemolysin are not directly proportional to virulence in experimental infections of mice.

The sulfhydryl-activated hemolysin of Listeria monocytogenes has been implicated in the virulence of the bacteria. Although loss of hemolytic activity by means of transposon mutagenesis is accompanied by loss of virulence in the mouse infection model, a direct relationship between in vitro production of hemolysin and virulence was not observed. Noticeable deviations in the extent of hemolysin production appeared to leave virulence unaffected.

Animals↗
PubMed1988

Evidence for two different gas vesicle proteins and genes in Halobacterium halobium.

Most halobacteria produce gas vesicles (GV). The well-characterized species Halobacterium halobium and some GV+ revertants of GV- mutants of H. halobium produce large amounts of GV which have a spindlelike shape. Most other GV+ revertants of H. halobium GV- mutants and other recently characterized halobacterial wild-type strains possess GV with a cylindrical form. The number of intact particles in the latter isolates is only 10 to 30% of that of H. halobium. Analysis of GV envelope proteins (GVPs) by electrophoresis on phenol-acetic acid-urea gels showed that the GVP of the highly efficient GV-producing strains migrated faster than the GVP of the low-GV-producing strains. The relative molecular mass of the GVP was estimated to be 19 kilodaltons (kDa) for high-producing strains (GVP-A) and 20 kDa for low-producing strains (GVP-B). Amino acid sequence analysis of the first 40 amino acids of the N-terminal parts of GVP-A and GVP-B indicated that the two proteins differed in two defined positions. GVP-B, in relation to GVP-A, had Gly-7 and Val-28 always replaced by Ser-7 and Ile-28, respectively. These data suggest that at least two different gvp genes exist in H. halobium NRL. This was directly demonstrated by hybridization experiments with gvp-specific DNA probes. A fragment of plasmid pHH1 and a chromosomal fragment of H. halobium hybridized to the probes. Only a chromosomal fragment hybridized to the same gyp probes when both chromosomal and plasmid DNAs from the low-GV-producing halobacterial wild-type strains SB3 and GN101 were examined. These findings support the assumption that GVP-A is expressed by a pHH1-associated gvp gene and GVP-B by a chromosomal gvp gene.

Amino Acid Sequence↗
PubMed1988

Comparison of four hemolysin-producing organisms (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) for release of inflammatory mediators from various cells.

We investigated the role of various hemolysin-producing strains (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) in induction of inflammatory mediators, e.g., histamine release from rat mast cells as well as the chemiluminescence response and the release of lipoxygenase transformation products from human polymorphonuclear neutrophils. Our data show that the hemolysin-positive bacteria as well as the hemolysin-positive culture supernatants were active in inducing the chemiluminescence response, leukotriene (LTB4 and LTC4) release from human granulocytes, and histamine release from rat mast cells. The degree of leukotriene release was dependent on the hemolysin type and on the expression of hemolysin activity. The E. coli alpha-hemolysin and the aerolysin-producing A. hydrophila were the most potent stimuli whether washed bacteria or bacterial supernatant was used. Bacteria expressing the S. marcescens hemolysin and the listeriolysin were only poor inducers of leukotriene generation. In contrast to leukotriene generation, all hemolysin-positive strains induced nearly the same histamine release in a dose-dependent manner. Our data suggest a potent role for various hemolysins as virulence factors in inducing the release of inflammatory mediators.

Aeromonas↗
PubMed1988

Active and inactive forms of hemolysin (HlyA) from Escherichia coli.

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.

Chromatography↗
PubMed1987

Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of Escherichia coli haemolysin (HlyA).

Fusion of the alkaline phosphatase gene (phoA) which lacks its own signal peptide sequence to the N-terminal region of hlyA, the structural gene for Escherichia coli haemolysin, leads to active alkaline phosphatase (AP). AP activity depends on the length of the N-terminal region of hlyA. An optimum is reached when 100-200 amino acids of HlyA are fused to PhoA but fusion of as little as 13 amino acids of HlyA to PhoA is sufficient to yield appreciable AP activity. When cells are treated with lysozyme most of the AP activity is found associated with the membrane fraction but a substantial amount is also found in the soluble fraction, most of which may represent a periplasmic pool of AP. The soluble portion of AP activity is significantly increased when the cells are disrupted by ultrasonication, which indicates that the fusion proteins are only loosely associated with the membrane and that large parts are already located on the outside of the cytoplasmic membrane. The expected fusion proteins were identified in the soluble and the membrane fractions and their amounts in these fractions correlated well with AP activity.

Alkaline Phosphatase↗
PubMed1987

Mutations affecting activity and transport of haemolysin in Escherichia coli.

Temperature-sensitive mutants that exhibit an altered haemolytic phenotype were isolated from Escherichia coli harbouring the plasmid pHly152. Complementation with recombinant plasmids carrying one of the four hly genes (C, A, B or D) allowed localization of the hly(ts) mutations. A ts mutation in hlyC leads to a pro----leu exchange in amino acid position 53 of HlyC. Two ts mutations in HlyA were found in positions 312 (ser----pro) and 315 (thr----ile). Both amino acid exchanges are located in the same hydrophobic domain of HlyA which extends from amino acids 299 to 327. Two different mutations were introduced by site-specific mutagenesis in this hlyA domain: one by an exchange of ala, val to asp, glu (positions 313, 314) altering the hydrophobicity of this region and another which removes most of this hydrophobic portion. Both mutants have entirely lost the haemolytic activity but the mutant haemolysins are still efficiently transported across both membranes when hlyB and hlyD are provided. Functional HlyC is not required for the transport of the mutant haemolysins. Two site-specific mutations at the N-terminal end of hlyA (one at amino acid position 2 leading to a thr----pro exchange and another deleting ile and thr at positions 4 and 5) also do not affect the transport of the altered haemolysins. The thr----pro exchange enhances the haemolytic activity of the corresponding mutant, whereas the ile, thr deletion exhibits little or no effect on the haemolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Comparison of the genetic determinant coding for the S-fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants.

DNA probes specific for different regions of the S-fimbrial adhesin (sfa) determinant were constructed and hybridized with DNA sequences coding for P (F8 and F13), mannose-sensitive hemagglutinating type 1 (F1A), and F1C fimbriae. While the sfa and F1C DNA determinants exhibited homology along their entire lengths, the P-fimbrial and type 1-fimbrial determinants exhibited homology to regions of the sfa cluster responsible for the control of transcription and, to a minor extent, to regions coding for proteins involved in biogenesis and/or adhesion of the fimbriae and for the N-terminal part of the fimbrillin subunit.

Adhesins, Escherichia coli↗