TY - JOUR A1 - Bender, L. A1 - Ott, M. A1 - Debes, A. A1 - Rdest, U. A1 - Heesemann, J. A1 - Hacker, Jörg T1 - Distribution, expression and long range mapping of legiolysin gene (lly) specific DNA sequences in Legionellae N2 - The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal Uy-specific DNA probe was used in Southern hybridizations for the detection of Uy-specific DNA in the genomes of legioneUae and other gram-negative pathogenic bacteria. Under conditi9ns of high stringency, tlie Uy DNA probe specifically reacted with DNA fragments fr9m L. pneumophiüz isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bact~ria of other genera. The Uy genewas mapped by pulsed-field gel electrophoresis to the respective genomic Notl fragments of Legionelltz isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59744 ER - TY - JOUR A1 - Bender, L. A1 - Ott, M. A1 - Marre, R. A1 - Hacker, Jörg T1 - Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE) N2 - Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of Not I cleaved genomic DNA. The genome of L pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five Not I fragments. Two virulent derivatives. derived from L pneumophila Philadelphia I. which were obtained by prolonged passage on artificial cuhure media, did not differ from their isogenic virulent strain according the Not I fragment pattern. By summing the lengths of the Notl fragments, the genome size of L. pneumophila Philadelphia I was calculated as approximately 3.9 Mb. Environmental L pneumophila strains exhibited different Not I pattems, as did Legionella strains not belongi'ng to the species pneumophila. The usefulness of DNA long range mapping of Legionella ssp. with Notl for epidemiology and evaluation of their evolutionary rela· tionships is discussed. KW - Infektionsbiologie KW - Legionella ssp. KW - Genome analysis KW - Orthogonal field attenuation gel electrophoresis Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59657 ER - TY - JOUR A1 - Blum, G. A1 - Ott, M. A1 - Cross, A. A1 - Hacker, Jörg T1 - Virulence determinants of Escherichia coli O6 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques N2 - A total of 16 Escherichia coli 06 strains isolated from cases of extraintestinal infections were analysed for the genetic presence and phenotypic expression of fimbrial adhesins ( P, S/FIC, type I), aerobactin and hemolysin. ln addition restriction fragment length polymorphisms (RFLPs) of Xbal-cleaved genomic DNA of seven selected strains, separated by orthogonal field alternation gel electrophoresis {OFAGE) were determined and virulence-associated DNA probes were used for Southern hybridization studies of the Xbal-cleaved genomic DNAs. The virulence characteristics and hybridization patterns obtained differed between the various isolates. ln three isolates hemolysin genes and P fimbrial determinants were located on the same Xbal fragments. Furthermore, multiple copies of FIC determinants (foc) could be detected in two strains. Our data show that the new technique of pulse field electrophoresis tagether with Southern hybridization represents a powerful tool for the genetic analysis of pathogenic bacteria. KW - Infektionsbiologie KW - E. coli serotype 06 KW - extraintestinal isolates KW - virulence factors KW - gene probes KW - DNA lang range mapping KW - epidemiology Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59717 ER - TY - JOUR A1 - Hacker, Jörg A1 - Bender, L. A1 - Ott, M. A1 - Wingeder, J. A1 - Lund, B. A1 - Marre, R. A1 - Goebel, W. T1 - Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vivo and in vitro in various extraintestinal Escherichia coli isolates N2 - No abstract available KW - Infektionsbiologie KW - P-fimbriae KW - hemolysin KW - genomic deletions KW - extraintestinal E. coli KW - virulence modulation Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59608 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Hof, H. T1 - Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic E. coli by using a wild-type gene fusion N2 - No abstract available KW - Infektionsbiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59874 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Ludwig, B. A1 - Rdest, U. T1 - Intracellular survival and expression of virulence determinants of Legionella pneumophila N2 - Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as weil as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human.embryo lung fibro:blastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further sturlies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella. Zusammenfassung: Intrazelluläres Überleben N2 - Intrazelluläres Überleben und Expression der Virulenzdeterminanten vonLegionella pneumophila. L. pneumophila, der Erreger der Legionärskrankheit, hat die Fähigkeit, sowohl in Makrophagen als auch in Protozoen zu leben und sich dort zu vermehret;t. Legionellen inhibieren die Fusion von Phagosom und Lysosom und hemmen die Ansäuerung des Phagosoms. Mit Hilfe von zwei unterschiedlichen Zellkultur-Systemen konnte gezeigt werden, daß Legionella-Stämme ihre Virulenz nach Laborpassage verlieren. Um die Mechanismen zu studieren, die für das intrazelluläre Überleben von Legionellen verantwortlich sind, haben wir eine Genbank des Legionella pneumophila-Stammes Philadelphia I in Escherichia coli K-12angelegt. Mit Hilfe der Cosmid-Klonierungstechnik war es möglich, fünf putative Virulenzfaktoren zu klonieren. Zwei von diesen Faktoren haben hämolytische Eigenschaften und drei sind Membran-assoziierte Proteine mit Molekulargewichten von 19, 26 und 60 kilodalton. Eines der hämolytischen Proteine, das Legiolysin, lysiert spezifisch humane Erythrozyten. Das zweite Hämolysin zeigt zusätzlich proteolytische Eigenschaften und schädigt sowohl Vero- als auch CHO-Zellen. Weitere Studien sind notwendig, um die Rolle der klonierten Proteine in der Pathogenese von Legionella exakt zu bestimmen. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59681 ER - TY - JOUR A1 - Hacker, Jörg A1 - Ott, M. A1 - Schmidt, G. A1 - Hull, R. A1 - Goebel, W. T1 - Molecular cloning of the F8 fimbrial antigen from Escherichia coli N2 - The genetic determinant coding for the Pspecific F8 fimbriae was cloned from · the chromosome of the Escherichia coli wild-type strain 2980 (018: K5: H5: FlC, F8). The F8 determinant was further subcloned into the Pstl site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned Counterpart was demonstrated. The cloned F8 fimbriäe and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepi thellal cells. The cloned F8 determinant was weil expressed in a variety of host strains. KW - Infektionsbiologie KW - Escherichia coli KW - antigen KW - F8 fimbriae KW - gene cloning Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59391 ER - TY - JOUR A1 - Krallmann-Wenzel, U. A1 - Ott, M. A1 - Hacker, Jörg A1 - Schmidt, G T1 - Chromosomal mapping of genes encoding mannose-sensitive (type I) and mannose-resistant F8(P) fimbriae of Escherichia coli O18:K5:H5 N2 - DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pi/) exist in a single copy on the chromosome of E. coli 018:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and Jeu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomallinkage map. KW - Infektionsbiologie KW - Escherichia coli KW - F1 KW - F8 fimbriae KW - Gene cloning KW - Gene mapping Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59545 ER - TY - JOUR A1 - Ott, M. A1 - Bender, L. A1 - Blum, G. A1 - Schmittroth, M. A1 - Achtmann, M. A1 - Tschäpe, H. A1 - Hacker, Jörg T1 - Virulence patterns and long range mapping of extraintestinal Escherichia coli K1, K5 and K100 isolates: Use of pulse field gel electrophoresis N2 - A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes Kl, KS, and KlOO from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae fpap] and P-related sequences fprs], S fimbriae [s/a)/FlC fimbriae [foc], and type I fimbriae lfim]), aerobactin (aer), and hemolysin (hly). The expression of corresponding virulence factors was also tested. Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships. DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them. Different isolates of the same serotype orten expressed different virulence patterns. The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype. Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of .precise molecular epidemiology. KW - Infektionsbiologie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59738 ER - TY - JOUR A1 - Ott, M. A1 - Bender, L. A1 - Chirinos, E. A1 - Ehret, W. A1 - Hacker, Jörg T1 - Phenotype versus genotype of the 19 kd peptido-glycan associated protein of Legionella (PplA) among Legionellae and other gram-negative bacteria N2 - The protein PpiA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. lt exhibits distinct sequence homology to Iipoproteins of Haemophilus influenzae and E. coli. A ppiA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but alt other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpiA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpiA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not alt, the Legionella species analysed here. KW - Infektionsbiologie KW - Legionellae KW - peptido-glycan associated protein KW - ppl KW - Southern hybridization KW - stringency KW - polymerase chain reaction (PCR) Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59768 ER -