Lactose metabolism in Lactobacillus bulgaricus: analysis of the primary structure and expression of the genes involved

Abstract

The genes coding for the lactose permease and 0-galactosidase, two proteins involved in the metabolism of lactose by LactobaciUlus bulgaricus, have been cloned, expressed, and found functional in Escherichia coli. The nucleotide sequences of these genes and their flanking regions have been determined, showing the presence of two contiguous open reading frames (ORFs). One of these ORFs codes for the lactose permease gene, and the other codes for the P-galactosidase gene. The lactose permease gene is located in front of the I-galactosidase gene, with 3 bp in the intergenic region. The two genes are probably transcribed as one operon. Primer extension studies have mapped a promoter upstream from the lactose permease gene but not the ,-galactosidase gene. This promoter is similar to those found in E. coli with general characteristics of GC-rich organisms. In addition, the sequences around the promoter contain a significantly higher number of AT base pairs (80%) than does the overall L. bulgaricus genome, which is rich in GC (GC content of 54%). The amino acid sequences obtained from translation of the ORFs are found to be highly homologous (similarity of 75%) to those from Streptococcus thermophilus. The first 460 amino acids of the lactose permease shows homology to the melibiose transport protein of E. coli. Little homology was found between the lactose permease of L. bulgaricus and E. coli, but the residues which are involved in the binding and the transport of lactose are conserved. The carboxy terminus is similar to that of the enzyme Ill of several phosphoenolpyruvate-dependent phosphotransferase systems.Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are dairy lactic acid bacteria that are widely used in the fermentation of milk (25). During their growth in milk, lactose is used as the primary energy source. Two systems for transport and metabolism of lactose are known in lactic acid bacteria: (i) a phosphoenolpyruvate (PEP) lactose phosphotransferase system (PTS) with a phospho-p-galactosidase enzyme (21) and (ii) a lactose permease system with a P-galactosidase. The PEP-PTS system is found in many species, including lactococci (reviewed in references 30 and 31).L. bulgaricus utilizes lactose via the second pathway. Lactose is brought into the cell as the free sugar and cleaved by P-galactosidase. The glucose moiety of the sugar is further metabolized while the galactose moiety is released (8, 23). The lactose operon of Escherichia coli, which also has a lactose permease (lacY) and 3-galactosidase (lacZ), has been extensively studied, and the proteins involved are being characterized (1-4, 11, 13, 24). Not much is known, however, about the system in lactic acid bacteria. Recently, there has been a report on the cloning and sequencing of the ,-galactosidase gene from L. bulgaricus (28). This group found that ,B-galactosidase from L. bulgaricus has an average similarity of 34% to that from E. coli, with stretches of high homology around the regions involved in activit...

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

“…The spacing between the -35 and -10 promoter sequences is 20 nt, and the distance between the ribosomebinding site and the start codon is 8 nt. The -35 sequence (TClJ.lGGIl lTlAT) is identical to that reported for the lactose permease from Lactobacillus bulgaricus (39). The putative ribosome-binding site sequence GGGGG is identical to that reported by Lerch et al (41) for the L-HicDH gene from Lactobacillus confusus and similar to ribosome-binding site sequences tabulated by Alpert and Chassy (3) for lactobacilli.…”

Secondary solute transport in bacteria

“…lactis and the citrate-H+-Na + symporter (CitS) of K. pneumoniae [62], and the melibiose carriers (MelB) of E. coli, S. typhimurium and K. pneumoniae and the galactoside-H + symporters (LacS) of Streptococcus thermophilus and Lactobacillus bulgaricus [54,102,103,104,105].…”

Energy transduction in lactic acid bacteria

“…The genes encoding the lactose (galactoside) transport proteins (LacS) of S. thermophilus and L. hulgaricus have been cloned, characterized and functionally expressed in E. coli [27,28,32,33].…”