Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography

Abstract A method for purifying TBP2 from N. meningitidis has been developed using affirnity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver training after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.     

Tetrameric repeat units associated with virulence factor phase variation in Haemophilus also occur in Netsseria spp. and Moraxella catarrhalis

Abstract The tetrameric repeat units 5'-CAAT-3' and 5'-GCAA-3' are associated with phase variable expression of lipopolysaccharide biosynthetic genes in Haemophilus influenzae . Four other tetrameric repeat units have also been reported from H. influenzae strain Rd, 5'-CAAC-3', 5'-GACA-3', 5'-AGCT-3', and 5'-TTTA-3', which are also associated with putative virulence factors. Using oligonucleotide probes corresponding to five tandem copies of each of these tetramers, we have screened three strains of Neisseria meningitidis and one each of Neisseria gonorrhoeae, Neisseria lactamica, Haemophilus parainfluenzae, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiceptica and Moraxella catarrhalis for the presence of these motifs. We have demonstrated the presence of multiple copies of the 5'-GCAA-3' motif in all the Neisseria strains tested, and also the repeated motif 5'-CAAC-3' in M. catarrhalis . We have further demonstrated by Southern blot analysis that the 5'-CAAC-3' repeats detected in M. catarrhalis are probably associated with the same genes as in H. influenzae , but that the 5'-GCAA-3' motifs in N. meningitidis are not. The use of characterised tetrameric DNA sequences as hybridisation probes may prove useful in the identification of novel phase variable virulence determinants in organisms other than H. influenzae .     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.     

Sequence of a gene encoding a (poly ManA) alginate lyase active on Pseudomonas aeruginosa alginate

Abstract To overcome problems associated with Western blotting of denatured proteins, we have used quantitative immunoelectrophoretic techniques to perform functional analysis of the Neisseria gonorrhoeae common antigen. Using these techniques, we show (a) that Neisseria gonorrhoeae expresses an antigen that is cross-reactive with the common antigen of Pseudomonas aeruginosa and Legionella micdadei and with the GroEl-like protein of Chlamydia , and (b) that this N. gonorrhoeae common antigen has lectin-like activity and can be precipitated with three different sugars immobilized on agarose beads: α- d -glucosamine, maltose and fucose.