Induction of Sucrose Utilization Genes from Bifidobacterium lactis by Sucrose and Raffinose

Abstract

The probiotic organism Bifidobacterium lactis was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of B. lactis was identified by complementation of a gene library in Escherichia coli. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The scrP gene was expressed constitutively from its own promoter in E. coli grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with scrP performed by using RNA isolated from B. lactis identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In B. lactis, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the scrP, scrR, and scrT genes in B. lactis indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous E. coli indicated that ScrR functions as a positive regulator.Bifidobacteria are gram-positive anaerobes that occur in large numbers in the intestines of humans and other animals (44). Of the several hundred species of bacteria that colonize the large intestine, bifidobacteria are generally considered to be health promoting and beneficial (2, 23). Because of these benefits, considerable research is being directed at promoting the growth of bifidobacteria in the large intestine. One method is to introduce more bifidobacteria into the bowel by the consumption of fermented milk products containing the bacteria. Another approach is to use prebiotics, which are defined as nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or the activity of one or a limited number of colonic bacteria (14). A variety of fructose-containing oligosaccharides, including inulin and oligofructose, have been reported to stimulate the growth of bifidobacteria (15, 16). Other nondigestible oligosaccharides, including raffinose and stachyose, as well as the oligosaccharides containing xylose, galactose, and maltose, have also been shown to have similar effects (35). Bifidobacterium lactis has been used industrially in fermented foods, such as yoghurt, cheese, beverages, sausages, infant formulas, and cereals. It is also available in natural remedies and supplemented tablets. B. lactis has been reported to be useful for supporting and balancing a healthy intestinal flora, for reducing the incidence of diarrhea and intestinal infections, for promoting the absorption of nutrients, for supporting the immune system, fo...

Bifidobacterium longum Requires a Fructokinase (Frk; ATP: d -Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

“…Bioinformatic analyses complement biochemical data reporting the purification and characterization of Bifidobacterium sp. enzymes that are capable of hydrolyzing various glycosidic bonds from plant-derived carbohydrates and are often inducible by the released monomeric molecules (15,22,29,33,41,48,56,58,63). Moreover, several studies conducted on fructose-containing polymers as potential selective substrates for colonic bacteria have provided evidence that bifidobacteria are able to ferment these carbohydrates, in particular the short linear chains of ␤-2-1-linked fructosyl units (7,24,30,36,37).…”

Desenvolvimento de buttermilk probiótico

“…lactis, anteriormente classificada como Bifidobacterium lactis 15 , foi escolhido para esta pesquisa por ser empregado industrialmente como probiótico em iogurtes, leites fermentados, queijos, bebidas, molhos, suplementos dietéticos, fórmulas infantis e cereais há mais de dez anos 16,25 .…”

Carbohydrate metabolism in Bifidobacteria

“…Several studies have shown that sucrose phosphorylase from B. adolescentis DSM20083 [144], B. animalis subsp. lactis [132] and B. longum subsp. longum [54] can hydrolyze sucrose in the presence of free phosphate with the release of glucose-1-phosphate and fructose molecules, thus bypassing the ATP-requiring step of the glucose phosphorylation/hexokinase reaction in preparation for entry into the ''Bifid shunt'' (Fig.…”

“…However, in just a few cases the precise action of the LacI-type regulator has been investigated in bifidobacteria [97,132]. The RbsR protein, which controls ribose utilization through the regulation of the rbs operon of B. breve UCC2003, is presumed to act as an effective transcriptional repressor in the absence of the effector molecule, D-ribose, thereby preventing transcription [97].…”