Nucleotide sequence, expression and transcriptional analysis of the Bifidobacterium longum MB 219 lacZ gene

The gene encoding beta-galactosidase was isolated by functional complementa tion of Escherichia coli Front Bifidobacterium longum MB219, which exhibite d the highest activity among ten Bifidobacterium strains tested of the spec ies B. longum, B. breve, B. adolescentis, B. indicum. B. animalis and B, cu niculi. The nucleotide sequence of the 5.0-kb fragment conferring the posit ive beta-galactosidase phenotype to E. coli revealed the presence of a lacZ -type gene encoding a 1023-amino-acid protein that was preceded by a riboso me binding site. A sequence showing 72% identity with the proline tRNA of B acillus subtilis and a gene probably encoding the DNA-3-methyladenine glyco sydase I were located downstream from the lacZ gene, after a gap of 30-50 u nsequenced base pairs. By primer-extension analysis, the transcription star t site of the lacZ gene was mapped 65 nt upstream from the start codon, and it enabled identification of the -10 region of the putative promoter. The nucleotide sequence of lacZ and its deduced amino acid sequence were compar ed with those of beta-galactosidase genes and enzymes from other microorgan isms. High similarity was demonstrated be tween the B. longum beta-galactos idase and its counterparts in Lactobacillus delbruckii subsp. bulgaricus, S treptococcus salivarius subsp. thermophilus, E. coli, Clostridium acetobuty licum, Leuconostoc lactis, Klebsiella pneumoniae and Kluyveromyces marxianu s var. lactis, all belonging to the LacZ family. The B. longum MB219 lacZ g ene was cloned in Bifidobacterium and its expression was observed in strain s with otherwise low levels of endogenous activity. The expression increase d by factors of 1.5-50 and enabled those strains that do not grow on lactos e to use this sugar as sole carbon source.