The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine

Abstract

The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTS^Lac) EIICB and EIIA domains, respectively, and lacG for the phospho-β-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTS^Lac are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG abolishes the growth of L. casei in both disaccharides and analysis of LacG activity showed a high selectivity toward phosphorylated compounds, suggesting that LacG is necessary for the hydrolysis of the intracellular phosphorylated lactose and LacNAc. L. casei (lacAB) strain deficient in galactose-6P isomerase showed a growth rate in lactose (0.0293 ± 0.0014 h^-1) and in LacNAc (0.0307 ± 0.0009 h^-1) significantly lower than the wild-type (0.1010 ± 0.0006 h^-1 and 0.0522 ± 0.0005 h^-1, respectively), indicating that their galactose moiety is catabolized through the tagatose-6P pathway. Transcriptional analysis showed induction levels of the lac genes ranged from 130 to 320-fold in LacNAc and from 100 to 200-fold in lactose, compared to cells growing in glucose.

Figure 1

(a) Genetic organization of the lac operon in Lactobacillus casei strain BL23. Hairpin loops indicate rho-independent transcriptional terminators. cre, catabolite responsive element; RAT, ribonucleic antiterminator; LacT is a transcriptional antiterminator; EIICB and EIIA, domains of the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTS^Lac); LacG, phospho-β-galactosidase. (b) Schematic presentation of the pathways for N-acetyllactosamine (LacNAc) and lactose (Lac) transport and metabolism in L. casei BL23. GlcNAc: N-acetylglucosamine; Fru, fructose; DHAP: dyhidroxyacetone phosphate; GAP: glyceraldehyde 3-phosphate; PEP: phosphoenolpyruvate; NagA, N-acetylglucosamine-6P deacetylase; NagB: glucosamine-6P deaminase; Pfk: 6-phosphofructo-1-kinase; Gk, glucokinase; Pgi, phosphoglucose isomerase; LacAB: galactose-6P isomerase; LacC: tagatose-6P kinase; LacD: tagatose-1,6 P aldolase; Fba: fructose-1,6 P aldolase; Tpi: triose phosphate isomerase.

Figure 2

Growth curves of Lactobacillus casei wild type strain BL23 on sugar-free MRS without carbon source (black), with N-acetyllactosamine (LacNAc) (red), lactose (blue) or glucose (green). Data presented are mean values based on at least three replicates. Error bars indicate standard deviations.

Figure 3

Growth curves of Lactobacillus casei mutant strain BL385 (gnbG) (a) on sugar-free MRS without carbon source (black) or with N-acetyllactosamine (LacNAc) (red). Data presented are mean values based on at least three replicates. Error bars indicate standard deviations. Growth curves of L. casei mutant strain BL153 (lacE) (b), L. casei mutant strain BL155 (lacF) (c) and L. casei mutant strain BL126 (pstI) (d) on sugar-free MRS without carbon source (black), with N-acetyllactosamine (LacNAc) (red), glucose (green) or lactose (blue). Data presented are mean values based on at least three replicates. Error bars indicate standard deviations.

Figure 4

Growth curves of Lactobacillus casei mutant strain BL400 (lacG) on sugar-free MRS without carbon source (black), with N-acetyllactosamine (LacNAc) (red), glucose (green) or lactose (blue). Data presented are mean values based on at least three replicates. Error bars indicate standard deviations.

Figure 5

Growth curves of Lactobacillus casei wild type strain BL23 (blue) and mutant strain BL393 (lacAB) (cyan) on sugar-free MRS with lactose. BL23 (red) and mutants BL393 (lacAB) (pink) and BL388 (nagA) (green) on sugar-free MRS supplemented with N-acetyllactosamine (LacNAc). Data presented are mean values based on at least three replicates. Error bars indicate standard deviations.

Figure 6

Transcriptional analysis by RT-qPCR of the expression of lacT (blue bars), lacE (green bars), lacG (black bars) and lacF (red bars) in Lactobacillus casei wild type strain BL23 grown in sugar-free MRS containing galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), lactose (Lac), a mix of glucose and lactose (Glc + Lac) or a mix of glucose and N-acetyllactosamine (Glc + LacNAc). Cells grown in sugar-free MRS supplemented with glucose were used as reference condition. Data presented are mean values based on three replicates of at least two biological independent samples. Bars indicate standard errors. For each lac gene, significantly different values (P < 0.05) among culture conditions are marked by different lower-case letters.

Figure 7

Growth curves of Lactobacillus casei mutant strain BL195 (lacT) on sugar-free MRS without carbon source (black), with N-acetyllactosamine (LacNAc) (red), glucose (green) or lactose (blue). Data presented are mean values based on at least three replicates. Error bars indicate standard deviations.