sodium-bisulfite and Chromosome-Deletion

Tn554 is a high-frequency, site-specific transposable element of Staphylococcus aureus which has integrative properties resembling those of temperate bacteriophages. Tn554 inserts at a unique chromosomal location, designated att554. att554 contains a core hexanucleotide sequence, 5'-GATGTA-3' (nucleotides numbered -3 to +3). Most of the time (greater than 99%) insertion occurs immediately 3' to this sequence; the resulting orientation of Tn554 to att554 is designated as the (+) orientation. Infrequent insertions immediately 5' to the core sequence result in the opposite, or (-) orientation. Mutational analysis of a cloned att554 site indicates that deletions extending from the left and ending at -15 or from the right ending between +8 and +12 reduced attachment site efficiency. Plasmids with deletions extending closer to the insertion site, although still retaining the core sequence from -3 to +3, were totally inactive. Tn554 insertions into partially active att554 sites retained normal site- and orientation-specificity with respect to att554, but they frequently contained abnormal sequences at the junction of att554 and the 3' end of Tn554. These data indicate that att554 contains a short nucleotide sequence essential for transposition and flanking sequences that greatly increase the frequency of recombination.

Topics: Base Sequence; Chromosome Deletion; Chromosomes, Bacterial; DNA Transposable Elements; Genes, Bacterial; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Restriction Mapping; Staphylococcus aureus; Sulfites

The analysis of the deletion derivative of pBS359 obtained as a result of sodium bisulphite mutagenesis and of recombinant derivatives pBS361-pBS363 permitted to map genes of the broad-host-range pBS222 plasmid which participate in replication, maintenance and mobilization. These genes are localized within the coordinates 0.2 to 2.5 kb in the region including a unique HindIII restriction site on the pBS222 physical map. Possible participation of the in vitro synthesized polypeptides in providing functions of cosmopolitanism and mobilization is being considered. Putative molecular-genetic structure of pBS222 and the presence of active recombination points are discussed, as well as the merits of the employed method for obtaining derivatives. The derivatives obtained and recombinant plasmids belong to the smallest plasmids which may be inherited in various gram-negative bacteria.

Topics: Bacteria; Chromosome Deletion; Chromosome Mapping; Cloning, Molecular; DNA Replication; DNA Restriction Enzymes; DNA, Bacterial; Genes, Bacterial; R Factors; Recombination, Genetic; Sulfites

Nine single C-to-T transitions were introduced into the -26 to -12 region of the Klebsiella pneumoniae nifL promoter by bisulphite mutagenesis of M13 heteroduplexes containing a 15 nucleotide single-stranded loop. Mutant promoter fragments were inserted into translational lac fusion vectors to utilise beta-galactosidase activity as a measure of promoter efficiency. Mutations in invariant nucleotides found in the consensus sequence for nif promoters gave a strong 'down' promoter phenotype with respect to transcriptional activation. Mutations in semi-conserved residues had a much weaker down phenotype, whereas a mutation which increased homology to the consensus sequence enhanced promoter strength. One mutant showed increased activation by ntrC and decreased activation by nifA.

Topics: Bacterial Proteins; Base Sequence; Chromosome Deletion; Escherichia coli; Genes; Genes, Bacterial; Klebsiella pneumoniae; Mutagens; Mutation; Promoter Regions, Genetic; Sulfites