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

Abstract

The gene encoding β-galactosidase was isolated by functional complementation of Escherichia coli from Bifidobacterium longum MB219, which exhibited the highest activity among ten Bifidobacterium strains tested of the species B. longum, B. breve, B. adolescentis, B. indicum, B. animalis and B. cuniculi. The nucleotide sequence of the 5.0-kb fragment conferring the positive β-galactosidase phenotype to E. coli revealed the presence of a lacZ-type gene encoding a 1023-amino-acid protein that was preceded by a ribosome binding site. A sequence showing 72% identity with the proline tRNA of Bacillus subtilis and a gene probably encoding the DNA-3-methyladenine glycosydase I were located downstream from the lacZ gene, after a gap of 30-50 unsequenced base pairs. By primer-extension analysis, the transcription start 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 compared with those of β-galactosidase genes and enzymes from other microorganisms. High similarity was demonstrated between the B. longum β-galactosidase and its counterparts in Lactobacillus delbruckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, E. coli, Clostridium acetobutylicum, Leuconostoc lactis, Klebsiella pneumoniae and Kluyveromyces marxianus var. lactis, all belonging to the LacZ family. The B. longum MB219 lacZ gene was cloned in Bifidobacterium and its expression was observed in strains with otherwise low levels of endogenous activity. The expression increased by factors of 1.5 - 50 and enabled those strains that do not grow on lactose to use this sugar as sole carbon source.