High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain.

A method is described for mutagenesis of Pseudomonas fluorescens strains by electroporation with the transposon delivery vector pUT/mini-Tn5 Km. The transposition process was shown to be optimal at 12.5 kV cm-1 for a pulse time (Bowen and Koslak, 1992) of about 4 ms. The Pseudomonas fluorescens L6.5 target strain exhibited maximal electrocompetence when harvested at the middle of the exponential growth phase. As many as 7.7 10(5) mutants per picomole of delivery vector (7.5 kb) could be obtained, and these kanamycin-resistant mutants were shown to have lost the pUT plasmid. By external calibration with plasmids of increasing size (from 11.5 to 60.1 kb), the efficiency of the transformation process was evaluated to be approximately 1.31 x 10(8) transformants per picomole of delivery vector. Efficiency of the transposition process was 0.58%. This rapid method was used to tag for the cloning three independent chromosomal loci responsible for the Alk+ phenotype of Pseudomonas fluorescens L6.5 strain.

Cloning and sequencing of the gene encoding the AmpC beta-lactamase of Morganella morganii.

The chromosomal beta-lactamase gene of a clinical isolate of Morganella morganii was cloned in Escherichia coli and sequenced. The beta-lactamase had a pI of 7.4 and conferred a typical AmpC susceptibility pattern. The insert obtained was found to encode a protein of 379 amino acids. Its deduced amino acid sequence revealed it to be a class C beta-lactamase: 39-56% identity with chromosomal AmpC beta-lactamases of Serratia marcescens, Yersinia enterocolitica, Citrobacter freundii, Enterobacter cloacae and Escherichia coli; and 37-56% identity with plasmid-mediated beta-lactamases (MOX-1, CMY-1, FOX-1, ACT-1, LAT-1, BIL-1 and CMY-2). The ampC gene was linked to a gene only part of which (450 bp) was cloned homologous to the regulatory ampR genes of chromosomal class C beta-lactamases.

A beta-lactamase-overproducing strain of Alcaligenes denitrificans subsp. xylosoxydans isolated from a case of meningitis.

A resistant (R) clinical isolate of Alcaligenes denitrificans subsp. xylosoxydans was recovered from CSF during treatment including piperacillin. This variant selected in vivo, and a second variant obtained in vitro from the initially susceptible (S) strain, both exhibited resistance to penicillins (ticarcillin, piperacillin) and cephalosporins, but remained susceptible to latamoxef and imipenem. Clavulanate (2 mg/L) restored the susceptibility of the two R-variants to penicillins. A beta-lactamase of pI 9.5 was detected in both S and R strains, but overproduction was observed only in the in-vivo and in-vitro R-variants. This inducible beta-lactamase hydrolysed benzylpenicillin, cephalothin and cephaloridine efficiently, but amoxycillin, ticarcillin and cefoperazone were only moderate substrates. The enzyme was inhibited by clavulanate, cloxacillin and imipenem (IC50 between 3 and 9 mM), but not by aztreonam and chloride ions (1 mM). Resistance to beta-lactams was not transferable by conjugation to Escherichia coli or Pseudomonas aeruginosa, and DNA agarose gel electrophoresis indicated that no plasmid was present in the isolates. Restriction patterns of chromosomal DNA isolated from the S and R isolates were similar after digestion by NotI and HindIII.

[Role of a free-living amoeba from water, Acanthamoaba castellani, in the transport of naked or enveloped animal viruses]. / Rôle d'une amibe libre de l'eau, Acanthamoeba castellanii, dans le transport de virus animaux nus ou enveloppés.

The trophozoïts of Acanthamoeba castellanii are unable to adsorb poliovirus or vesicular stomatitis Virus. After encystment in medium containing respectively 5.4 x 10(8) and 3 x 10(9) P.F.U./ml cysts did not contain Viruses. These data do not agree with a current hypothesis by which water's free Amoeba could carry animal Viruses.

[Detection and localization of a ribonuclease activity associated with vaccinia virus]. / Mise en évidence et localisation d'une activité ribonucléase associée au virus de la vaccine.

A ribonuclease associated with vaccinia virus can be detected when reduced concentrations of nucleotides are used for an in vitro RNA synthesis assay. The non-viral origin of this ribonuclease may be inferred from its external location and from its variable activity on different purified virus stocks. The detection of this ribonuclease activity on purified virus grown without foetal Calf serum may suggest that this enzyme is of cellular origin.

[A new method permitting the quantitative determination of virus present in surface waters]. / Nouvelle méthode permettant la détermination quantitative des virus présents dans les eaux de surface.

With a 34% efficiency, not related to the amount of viruses, a simple and inexpensive apparatus allows quantitative recovery of enteroviruses from river-water.