Identification, cloning, sequencing, and characterization of a proposed lactacin B enhancer protein and partial characterization of the Lactobacillus delbrueckii ssp. lactis ATCC 4797 genome by shotgun cloning and random sequencing

Lactacin B is a hydrophobic bacteriocin produced by Lactobacillus acidophilus N2 and is bactericidal against closely related species. In addition to other Lactobacilli, lactacin B is also inhibitory against the food related pathogen Listeria monocytogenes ; making lactacin B a good candidate for food preservation studies.; A lactacin B cell wall associated enhancer protein was purified from L. delbrueckii. The N-terminal region of the purified enhancer was sequenced and used to generate an oligonucleotide (57-mer) for further genetic analysis. The corresponding oligo was used for inverse PCR experiments in search for the genetic determinant encoding the lactacin B enhancer. Inverse PCR produced a 317 bp fragment that hybridized to the degenerate oligonucleotide designed from the corresponding purified enhancer protein sequence. Using the enhancer specific oligonucleotide generated from inverse PCR, 6,250 clones were screened of which four tested positive for the enhancer sequence. Clone RU43e9 produced a 4,623 bp insert that strongly hybridized to the enhancer oligo. Sequencing of clone RU43e9 revealed a partial operon containing three key glycolytic enzymes involved in glycolysis. The triosephosphate isomerase or gene three in the operon contained an open reading frame of 763 bp which encodes a protein of 27,976 Da in size. In addition, the N-terminal region of this open reading frame showed 98% homology to the purified N-terminal region of the lactacin B enhancer protein. Clone RU43e9 produced a 31,000 Da protein corresponding to the lactacin B enhancer. Considering the 6X His tag, which adds an additional 3,000 Da to the fusion protein, clone RU43e9 produced a 28,000 Da protein.; In addition to the lactacin B enhancer gene, an attempt was made to partially characterize the genome of L. delbrueckii by shotgun cloning and random sequencing. A total of 2,688 recombinant plasmids were extracted for sequencing analysis. Sequencing was carried out by performing the forward and reverse sequencing reads on each of the 2,688 clones. Each sequencing run gave an average of 300--350 bp, and the reaction success was roughly 80--85%. Thus far, a total of 1,561,728 bp of sequenced DNA from the L. delbrueckii genome has been completed. Considering overlapping regions or clones that contain similar inserts, we have approximately 30--40% of the entire chromosome completed.; This genetic analysis will provide several physical markers for further characterizing the entire L. delbrueckii genome. In addition to physical markers, this experimental approach will provide an overview of codon usage and capacity, G+C content, frequency of repetitive DNA, presence of transposons and or IS elements, and conserved or unique homologous genes between other species of Gram positive and Gram-negative bacteria. (Abstract shortened by UMI.)...