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S Falkow

Publications and source records attributed to S Falkow.

At least 235 records · Page 13Linked to original sources
PubMed1983

Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli.

A chromosomal hemolysin determinant was isolated in the form of recombinant plasmid pSF4000 from a human urinary tract isolate. Escherichia coli J96. A restriction endonuclease fragment map of pSF4000 was constructed. The ampicilin resistance transposon Tn/ was used as a site-specific mutagen in conjunction with in vitro derived deletions to localize the hemolysin determinant to a 7.0-kilobase region of pSF4000. Seventeen hemolytic E. coli isolates from human urinary tract infections were found to share similar DNA sequences with J96 hemolysin sequences, using a hemolysin-specific restriction endonuclease fragment as a hybridization probe. In all 17 hemolytic E. coli strains the hemolysin gene sequences were localized to the bacterial chromosome. The hemolysin-specific gene sequences can be found only in hemolytic strains by colony blot hybridization, suggesting that in the evolutionary sense the hemolysin-specific genetic sequences have only recently been introduced into the E. coli chromosome. Evidence is also presented that in the regions neighboring the J96 chromosomal hemolysin there are additional unique DNA sequences not commonly found in other E. coli isolates.

Base Sequence↗
PubMed1983

Tn5-induced mutations affecting virulence factors of Bordetella pertussis.

Transposon Tn5 was used to isolate mutants of Bordetella pertussis. Strains with Tn5 insertions were screened for loss of virulence-associated factors, including filamentous hemagglutinin, hemolysin, and pertussis toxin. Several mutants deficient for hemolysin production were obtained. All produced dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin, but were found to vary with respect to adenylate cyclase production. One hemolysin mutant had no detectable adenylate cyclase activity; others had 0.6% or 16% wild-type activity, whereas a fourth seemed to be unaffected in terms of adenylate cyclase activity. Mutants deficient in the ability to hemagglutinate sheep erythrocytes were also isolated. These mutants either failed to synthesize or produced reduced amounts of three protein species of 200,000, 130,000, and 100,000 daltons, all of which reacted with antiserum to filamentous hemagglutinin. Pertussis toxin mutants were identified by screening culture supernatants for failure to induce a clustered growth pattern in Chinese hamster ovary cells, and identification was confirmed by the standard histamine-sensitizing assay in mice. These mutants will be useful to determine the relative contribution of each virulence factor to pathogenicity as well as to determine the identity of the antigens important in protective immunity.

Adenylyl Cyclases↗
PubMed1983

Transposon insertion and subsequent donor formation promoted by Tn501 in Bordetella pertussis.

The mercuric chloride resistance transposon, Tn501, was introduced into Bordetella pertussis by using the chimeric plasmid pUW942, which is unable to replicate in this species. Tn501 insertions which conferred a thiamine requirement were the predominant insertion class. In many cases, the mercuric chloride-resistant transconjugants were also resistant to the other plasmid markers, but failure to detect plasmid DNA in these isolates indicated that integration of the entire plasmid into the chromosome had occurred. One such insertion was further characterized. Southern hybridization with a Tn501-specific probe indicated that chromosomal DNA from one strain containing the integrated plasmid had two copies of Tn501 and an intervening copy of the plasmid associated with the chromosome. The presence of the plasmid was unstable, and derivatives which had lost all of the plasmid markers except mercuric chloride resistance were obtained. These strains had a single copy of Tn501 and had lost all of the rest of the plasmid-specific sequences. Strains containing the plasmid in the integrated state could act as genetic donors and mobilize chromosomal genes.

Bordetella pertussis↗
PubMed1983

Characterization of conjugative plasmid EDP208.

EDP208 is a conjugative plasmid belonging to incompatibility group IncF0 lac, A restriction endonuclease map of this plasmid was constructed using five restriction enzymes: BamHI, HindIII, PvuI, SstI, and XhoI. On the basis of these mapping studies, the plasmid was found to be 90 kilobases in length. Clones were constructed from four large HindIII fragments of plasmid EDP208. One fragment, HindIII-20.5, was found to contain the lac genes and the origin of vegetative replication (oriV). Another fragment, HindIII-27.5, was found to contain all of the genes necessary for sex pilus formation, but it was nontransmissible. However, when used to complement a plasmid carrying an adjacent fragment, HindIII-23, the transfer of the latter occurred, suggesting that HindIII-23 contains the origin of transfer (oriT). The further localization of genes concerned with pilus biosynthesis was achieved by transposon mutagenesis. Six EDP208::Tn1 and thirty-seven EDP208::Tn5 mutants were isolated on the basis of their resistance to f1, a filamentous phage which adheres to intact pilus tips. The positions of the inserted transposons were determined on the restriction map and a 16.5-kilobase region was found to be required for pilus synthesis.

Conjugation, Genetic↗
PubMed1983

Plasmid association and nucleotide sequence relationships of two genes encoding heat-stable enterotoxin production in Escherichia coli H-10407.

Plasmid DNA from enterotoxigenic Escherichia coli strains H-10407 and H-10407-P was examined for nucleotide sequence homology to two E. coli genes encoding infant mouse-active heat-stable enterotoxins (ST). A 62-megadalton plasmid of strain H-10407 contained sequences homologous to the gene encoding a toxin designated STIb, previously isolated from a human isolate of E. coli. A 42-megadalton plasmid of strains H-10407 and H-10407-P contained sequences homologous to the gene encoding a toxin designated STIa, previously isolated from bovine and porcine isolates of E. coli.

Bacterial Toxins↗
PubMed1983

Target cell specificity of wild-type E. coli and mutants and clones with genetically defined adhesins.

The species, individual and tissue specificity of bacterial binding reactions was studied using wild-type E. coli strains from diarrhoea or urinary tract infection, and derivatives with genetically manipulated adhesins. E. coli J96 and GR12 were isolated from the urine of patients with acute pyelonephritis; E. coli strains expressing the CFAI and II antigens from the stools of patients with diarrhoea. E. coli J96, GR12 and CFAI induced mannose-resistant agglutination of human erythrocytes; E. coli J96 and GR12 in addition carried mannose-sensitive adhesins. Mutants of GR12 with either or both of these adhesins were obtained through chemical mutagenesis. Cloning of 6-8 mdal fragments of chromosomal DNA from J96 into E. coli K12 resulted in expression of pili and binding properties in the previously non-piliated and non-binding strain. Bacterial binding was registered to target cells from different human tissues; small intestinal brush borders, uroepithelial and buccal cells and erythrocytes and was compared between species using rabbit intestinal brush borders, mouse bladder cells and guinea pig erythrocytes. Individual variation was illustrated by agglutination of human P1 erythrocytes and those of blood group p lacking the globoseries glycolipid receptors. Specific recognition of globoseries glycolipid receptors was defined as capacity to agglutinate guinea pig erythrocytes after but not before coating with globotetraosylceramide. Binding specific for mannose-containing receptors was diagnosed by mannose-reversible agglutination of guinea pig erythrocytes. The binding pattern of the wild-type strains was related to the site of infection, i.e. the CFAI and II strains bound to small intestinal brush borders and the pyelonephritogenic E. coli to uroepithelial cells, but not vice versa. The mutants and clones retained the binding properties of the parent/donor both in degree and specificity of binding. Strains with adhesins specific for globoseries glycolipids attached to human uroepithelia, mouse uroepithelial and buccal cells. Within the group of strains with mannose-sensitive adhesins heterogeneity was observed. Strains sharing ability to agglutinate guinea pig erythrocytes to a mannose-reversible manner bound or did not bind to human buccal cells, to human uroepithelial cells and agglutinated or did not agglutinate human erythrocytes. The results demonstrate the usefulness of genetic technology in the study of bacterial binding reactions. The role of pili as adhesins is discussed.

Adhesins, Escherichia coli↗
PubMed1983

Bacterial plasmids--an overview.

Resistance to antimicrobial substances and various factors contributing to pathogenicity are only some of the bacterial traits that can be determined by plasmid genes. Not all plasmids are involved with resistance, nor is all resistance to antimicrobials plasmid-mediated. Nevertheless, the impact of R-plasmids on the choice of antimicrobial therapy is substantial, especially in hospital and when dealing with certain community pathogens, such as Haemophilus influenzae. The discovery of transposition elements has helped us to understand the rapidity with which certain resistance determinants develop and become disseminated among diverse microbial species. Similarly, not all determinants of microbial pathogenicity are plasmid encoded and the genes for many toxins, cellular attachment pili, iron sequestration systems, etc. are found on the bacterial chromosomes. In Yersinia, pathogenicity is even more complex in that plasmid determinants play an essential role, but only in concert with chromosomal genes. The particularly significant features of such pathogenicity-determined plasmid-mediated genes is not that they are found on extrachromosomal elements, but that they may possess greater genetic mobility. Another consequence of the presence of virulence genes on bacterial plasmids is that it makes them easier to study by the newer techniques of microbial genetics and molecular biology.

Anti-Bacterial Agents↗
PubMed1982

Isolation of enterotoxin structural gene deletion mutations in Vibrio cholerae induced by two mutagenic vibriophages.

Phenotypically nontoxinogenic mutants of Vibrio cholerae were isolated after infection with either of two mutagenic vibriophages, VcAI and VcA2ctsl. DNA isolated from these mutants was analyzed for toxin gene sequences by the Southern blotting method with 32P-labeled probes derived from the cloned A and B subunit genes for the heat-labile enterotoxin of Escherichia coli, designated LT. Several of the mutant isolates were shown by this method to have lost all sequences hybridizing to the LT probes, indicating that these clones contain deletion mutations that removed the structural gene(s) for cholera toxin. The mutants were prototrophic and grew normally, in vitro, demonstrating that the toxin is not essential for the growth and viability of V. cholerae. Moreover, the toxin gene deletion mutants multiplied well in vivo in ligated rabbit intestine. Because of these growth properties and the stability of deletion mutations, these strains are promising candidates for testing as live oral vaccine strains for protection against cholera.

Bacteriophages↗
PubMed1982

Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity.

The biological significance of bacterial extracellular proteases that specifically cleave human IgA1 is unknown. We have prepared a gene bank of gonococcal chromosomal DNA in Escherichia coli K-12 using a cosmid cloning system. Among these clones, we have identified and characterized an E. coli strain that elaborates an extracellular endopeptidase that is indistinguishable from gonococcal IgA1 protease in its substrate specificity and action on human IgA1. Analysis of recombinant plasmids and examination of plasmid-specific peptides in minicells have shown that the IgA1 protease activity in E. coli is associated with expression of a Mr 140,000 peptide. We have isolated IgA1 protease-deficient mutants of Neisseria gonorrhoeae by reintroduction of physically defined deletions of the cloned gene into the gonococcal chromosome by transformation.

Chromosome Mapping↗
PubMed1982

Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes.

The applicability of examining clinical specimens with a DNA hybridization technique for genes encoding enterotoxins was examined using enterotoxigenic Escherichia coli (ETEC) that produced both heat-labile toxin (LT) and heat-stable toxin (ST) (24 isolates), ETEC that produced LT only (17 isolates), and ETEC that produced ST only (22 isolates) from Thailand. ETEC was identified with Y-l adrenal cell and suckling mouse assays. All were homologous with radiolabeled fragments of DNA encoding LT or ST of porcine origin (ST-P) or of human origin (ST-H). Strains of ETEC that produced ST only from rural Thailand were homologous with the ST-H probe only, whereas strains isolated in Bangkok were homologous with the ST-H probe, the ST-P probe, or both probes. The hybridization technique detected ETEC in all stool samples of patients with diarrhea from whom ETEC was isolated and in ETEC-inoculated water containing other species of bacteria. The DNA hybridization assay is useful for characterizing and identifying environmental sources of ETEC.

Adult↗
PubMed1982

Common R-plasmids in Staphylococcus aureus and Staphylococcus epidermidis during a nosocomial Staphylococcus aureus outbreak.

During a 7-month period in 1978 to 1979, 31 patients and personnel at a Kentucky hospital were colonized or infected with a Staphylococcus aureus strain resistant to clindamycin, erythromycin, gentamicin, methicillin, penicillin, and tetracycline. S. epidermidis with similar antibiotic resistance patterns had been isolated in this hospital in the year before the S. aureus outbreak. A 32-megadalton R-plasmid, pUW3626, mediating resistance to penicillin and gentamicin, was present in these isolates and in coisolated S. epidermidis from the same outbreak. By colony hybridization, pUW3626 was homologous to gentamicin R-plasmids from staphylococci isolated in other geographic areas. Our studies suggest that the emergency of antibiotic resistance in S. Aureus may result from genetic transfer from S. epidermidis as well as from the interhospital spread of resistant staphylococci.

Anti-Bacterial Agents↗
PubMed1982

Characterization of ampicillin resistance plasmids from Haemophilus ducreyi.

Seven strains of Haemophilus ducreyi from diverse geographic origins were analyzed for their plasmid content. All strains were multiply resistant, but only resistance to ampicillin was transferred to Escherichia coli by transformation. The H. ducreyi plasmids encoding for ampicillin resistance were 7.4, 5.7, and 3.6 megadaltons and encoded for part or all of TnA, and ampicillin transposon. The relatedness of these plasmids was examined by restriction endonuclease digestion and DNA-DNA homology with isolated DNA fragments from TnA.

Ampicillin↗
PubMed1982

Genetics of hemolysin of Escherichia coli.

The expression of alpha-hemolysin is a property frequently associated with Escherichia coli extraintestinal infections. We have examined the genetic basis for hemolysin expression by an E. coli strain isolated from a human urinary tract infection. The genes necessary for hemolysin synthesis were found to be chromosomal and to map near the ilv gene cluster. Isogenic hly+ and hly derivatives were also prepared and tested for virulence in the chicken embryo model system. Hemolysin was found to be necessary but not in itself sufficient for E. coli virulence in this in vitro model.

Animals↗
PubMed1982

Plasmid transfer to Bordetella pertussis: conjugation and transformation.

Plasmids of the P and W incompatibility groups were introduced into Bordetella pertussis by conjugation. Plasmid DNA isolated from B. pertussis could be reintroduced by transformation. DNA isolated from Escherichia coli could not be introduced into B. pertussis by transformation if this DNA contained HindIII restriction sites. We have demonstrated that HindIII sites are modified by B. pertussis. Plasmids of the FI and FII incompatibility groups could not be introduced into B. pertussis by conjugation, and nonconjugative plasmids of the ColE1 and Q incompatibility groups could not be introduced by transformation. Our ability to introduce plasmids in the laboratory suggests that the apparent lack of plasmids in natural isolates of B. pertussis is not due to an inability to act as a plasmid recipient.

Bordetella pertussis↗
PubMed1982

Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast.

Enterotoxigenic strains of Vibrio cholerae O-1, biotype El Tor, isolated from a case of cholera in Texas in 1973, an outbreak of cholera in Louisiana in 1978, and Louisiana sewage samples in 1980 and 1981 were analyzed for their genetic similarities. Chromosomal DNA was isolated from each strain, digested with restriction endonuclease, and analyzed by the Southern blot technique. A radioactive probe consisting of Escherichia coli heat-labile enterotoxin DNA detected cholera toxin gene sequences in these strains and demonstrated that the toxin gene sequence, if not the entire chromosomal DNA, is identical in these strains and distinctly different from other strains of V. cholerae isolated throughout the world. In addition, two strains of enterotoxigenic V. cholerae non-O-1 isolated from clinical cases, were analyzed and found to possess cholera toxin genes which differed in the DNA sequence from the V. cholerae O-1 strains. We concluded that a single strain of enterotoxigenic V. cholerae O-1 is resident in the U.S. Gulf Coast and that a second reservoir of cholera toxin genes exists in V. cholerae non-O-1 strains in Louisiana.

Cholera↗