Danielle Margarita

Polymerase chain reaction with the 30-kb circular plasmid of Borrelia burgdorferi B31 as a target for detection of the Lyme borreliosis agents in cerebrospinal fluid

The polymerase chain reaction (PCR) was developed for use in the detection of Borrelia burgdorferi sensu lato, the Lyme disease agent. A 333-bp fragment of the 30-kbp circular plasmid from Borrelia burgdorferi B31 was amplified and PCR products were analysed by DNA-DNA hybridization. Sensitivity was enhanced by addition of a carrier to the samples before treatment and enabled detection of as few as 1 to 10 bacteria. Specific products were obtained only with the Lyme disease agents, but not with other spirochetes or unrelated bacteria. B. burgdorferi sensu lato was detected in cerebrospinal fluid (CSF) from 11 out of 45 patients with confirmed Lyme neuroborreliosis. In a prospective study, 20 out of 315 CSF samples from potential patients were PCR-positive. Forty uninfected patients were PCR-negative.

First isolation of bacteriophages for a spirochaete: potential genetic tools for Leptospira.

Three bacteriophages of the saprophytic aquicole bacterium Leptospira biflexa were isolated from sewage waters from the outskirts of Paris, France. These phages do not infect representative strains of the pathogenic species Leptospira interrogans, and their host range is restricted to serovar patoc of the saprophytic species. The phages were found to be lytic and no lysogenic state could be demonstrated. Electron micrographs showed that the phages were morphologically identical and had polyhedral heads and contractile tails. Their genomes were sensitive to restriction enzymes and consisted of double-stranded DNA. Pulsed-field agarose gel electrophoresis indicated that their genomes were linear: 60 kb for LE1 and 50 kb for LE3 and LE4.

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132 628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation. metH was repressed tenfold, presumably indirectly, by L-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.

Cloning of genes required for amino acid biosynthesis from Leptospira interrogans serovar icterohaemorrhagiae

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.

Fine structure analysis of the threonine operon in Escherichia coli K-12

SummaryA fine structure analysis of the threonine operon in Escherichia coli K-12 was performed by deletion mapping. Lambda transducing bacteriophages carrying various parts of the threonine operon were isolated from strains in which the lacZ gene was fused to a thr gene. We tested for recombination between deletions of the threonine promotor extending into the threonine operon, carried by the phage, and bacterial thr auxotrophs. The relative order of thrO (operator) mutations was established. We propose that an operator region is located between a promoter region and the structural genes. Mutations leading to the desensitization of the aspartokinase I-homoserine dehydrogenase I towards threonine were localized in two different regions of the thrA gene.

Construction and physical mapping of plasmids containing the metJBLF gene cluster of E. coli K12

SummaryIn vitro recombination techniques were used to clone the E. coli metJBLF gene cluster in a plasmid vector. Several chimeric plasmids were obtained, analyzed by restriction mapping and characterized genetically. The combined results establish that the met gene cluster is contained on an approximately 5.6 kilobase segment of bacterial DNA with metL between metB and metF. The origin of metL was localized precisely by its DNA sequence and its transcription direction was established.

Characterization of a defective ? bacteriophage transducing the threonine operon of Escherichia coli K12

SummaryPhage λdthrc carries the entire threonine operon of E. coli K12 and can complement all known thrA, thrB and thC mutations. Threonine plus isoleucine repress the threonine operon carried by the phage in either the vegetative or the prophage form. Density gradient analysis of λdthrc demonstrates that this phage is defective and less dense than the parental phage λcI857 S7. Marker rescue experiments indicate that the bacterial threonine operon replaces λ genes coding for late functions of the phage. However, λdthrc retains all the genes necessary for λ head biosynthesis. In vitro complementation between λ tails and λ heads produced by λdthrc has been demonstrated.

Immunochemical comparison of a bifunctional enzyme, aspartokinase II-homoserine dehydrogenase II, and its two proteolytic fragments

Abstract We report here a comparison between immunochemical properties of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II of E.coli K12 and of its two isolated proteolytic fragments. Both fragments, one inactive and one endowed with homoserine dehydrogenase activity, react with antibodies raised against the native enzyme. Some of the antibodies elicited against the dehydrogenase fragment can recognize regions of this fragment which are not exposed in the entire enzyme. The immunochemical results are used to discuss a simple model in which this bifunctional enzyme is folded up in two domains. The organization of aspartokinase II-homoserine dehydrogenase II is compared to that of another bifunctional enzyme aspartokinase I-homoserine dehydrogenase I with which it shares some sequence homology.