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. 2003 Jan;69(1):24-32.
doi: 10.1128/AEM.69.1.24-32.2003.

Induction of sucrose utilization genes from Bifidobacterium lactis by sucrose and raffinose

Marla I Trindade  1 , Valerie R Abratt, Sharon J Reid
Affiliations

Induction of sucrose utilization genes from Bifidobacterium lactis by sucrose and raffinose

Marla I Trindade et al. Appl Environ Microbiol. 2003 Jan.

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.

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Figures

FIG. 1.
FIG. 1.
Growth curves for B. lactis grown in media containing of each of the various sugars used at a concentration of 1% (wt/vol). An overnight culture of B. lactis in BYG medium was diluted 10−2 into the relevant media, and the growth was monitored by measuring the optical density at 600 nm (OD600 nm) at the times indicated. Symbols: ⋄, glucose; ▪, sucrose; ▵, glucose plus sucrose; •, raffinose; ○, oligofructose.
FIG. 2.
FIG. 2.
Genetic organization of the scr genes of the B. lactis sucrose utilization system. Transcriptional polarities are indicated by arrows. The thick and thin lines represent insert and vector, respectively. Plasmids pSuc1 and pSuc2 originated from the Sau3A gene bank constructed in the BglII site of the vector pEcoR251. Plasmid pSuc3 was constructed from pSuc1 and pSuc2, and pΔST8 and pΔReg2 were subcloned from pSuc3 into pEcoR251. The sucrase activity conferred on E. coli by the constructs was measured in cell extracts and was expressed in micromoles of reducing sugar per minute per milligram of protein. Assays were performed in duplicate, and standard deviations are indicated in parentheses. Sizes (in kilobases) are indicated after the restriction enzyme abbreviations. B, BamHI; Bg/S, BglII/Sau3A; H, HindIII; P, PstI; PvI, PvuI; Sa, SalI; Sm, SmaI; St, StuI.
FIG. 3.
FIG. 3.
Multiple-sequence alignment of ScrR of B. lactis (BlscrR) and other sucrose regulators, including those of S. mutans (SmscrR) (accession no. Q54430), L. lactis (LlsacR) (accession no. Q04939), and P. pentosaceus (PpscrR) (accession no. P43472). Identical amino acids are indicated by asterisks, and similar amino acids are indicated by dots. The helix-turn-helix motif (enclosed in boxes) is conserved in all these proteins, which belong to the GalR-LacI family of regulators.
FIG. 4.
FIG. 4.
Mapping of the transcription start site of the B. lactis scrP gene by primer extension analysis. (A) Primer extension products obtained by using RNA extracted from B. lactis grown in BY medium containing sucrose. (B) DNA sequencing fluorogram corresponding to the region analyzed. The Cy5-labeled SP primer was used for both the primer extension and sequencing reactions. (C) Nucleotide sequence of the promoter regions preceding the scrP and scrR genes. Putative promoters (−35 and −10 regions) and ribosome-binding sites (SD) are indicated by boldface type and underlining. The transcriptional start site (TS) of scrP, as determined by primer extension analysis, is indicated by boldface type. The arrows indicate the directions of the direct and indirect repeat sequences. The indirect repeat is a perfect palindrome which may act as an operator site for a GalR-LacI family regulatory protein.
FIG. 5.
FIG. 5.
RNA slot blot analysis of scrP, scrT, and scrR mRNA in mid-logarithmic-phase cells of B. lactis grown in different carbon sources. (A) Gene-specific mRNA and 16S rRNA signals detected by slot blot analysis. (B) mRNA levels expressed as ratios of the gene-specific hybridization signal to the 16S rRNA hybridization signal. Experiments were performed in duplicate, and standard deviations are indicated in parentheses. GS, glucose plus sucrose; R, raffinose; OF, oligofructose; S, sucrose; G, glucose; ND, no gene-specific signal detected.
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