Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays

Abstract

The transport and catabolic machinery involved in carbohydrate utilization by Lactobacillus acidophilus was characterized genetically by using whole-genome cDNA microarrays. Global transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose, and fructooligosaccharides. Hybridizations were carried out by using a round-robin design, and microarray data were analyzed with a two-stage mixed model ANOVA. Differentially expressed genes were visualized by hierarchical clustering, volcano plots, and contour plots. Overall, only 63 genes (3% of the genome) showed a >4-fold induction. Specifically, transporters of the phosphoenolpyruvate:sugar transferase system were identified for uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette transporters were identified for uptake of raffinose and fructooligosaccharides. A member of the LacS subfamily of galactoside-pentose hexuronide translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of monosaccharides, disaccharides, and polysaccharides into substrates of glycolysis were also found, including enzymatic machinery of the Leloir pathway. The transcriptome appeared to be regulated by carbon catabolite repression. Although substrate-specific carbohydrate transporters and hydrolases were regulated at the transcriptional level, genes encoding regulatory proteins CcpA, Hpr, HprK/P, and EI were consistently highly expressed. Genes central to glycolysis were among the most highly expressed in the genome. Collectively, microarray data revealed that coordinated and regulated transcription of genes involved in sugar uptake and metabolism is based on the specific carbohydrate provided. L. acidophilus's adaptability to environmental conditions likely contributes to its competitive ability for limited carbohydrate sources available in the human gastrointestinal tract.

Fig. 1.

Hierarchical clustering of gene expression patterns. Gene expression (vertically) during growth on eight carbohydrates (horizontally) is shown colorimetrically. (A) Global gene expression for 1,889 genes. (B) Select genes and operons. Least-squares means represent overall gene expression level: low, blue; high, red. FRU, fructose; GAL, galactose; GLC, glucose; LAC, lactose; RAF, raffinose; SUC, sucrose; TRE, trehalose.

Fig. 2.

Volcano plot comparison of gene expression between FOS and raffinose (RAFF). The x axis indicates the differential expression profiles, plotting the fold-induction ratios in a log-2 scale. The y axis indicates the statistical significance of the difference in expression (P value from a t test) in a log₁₀ scale. Genes within the raffinose msm locus are shown in green, genes within the FOS msm locus are shown in blue, and two genes within the trehalose tre locus are shown in red.

Fig. 3.

Contour plot comparison of gene expression among FOS, raffinose, and trehalose. Shown is a three-way plot of the least-squares means (Lsm) of all of the genes in the presence of FOS (x axis), raffinose (y axis), and trehalose (z axis). In the third dimension (z axis), the gene expression level is coded colorimetrically: blue, low gene expression; red, high gene expression. Also shown are differentially expressed genes: 1437–1442, raffinose msm₂ operon; 502–507, FOS msm operon; and 1012–1014, trehalose tre locus.

Fig. 4.

Carbohydrate utilization in L. acidophilus. (A) Pathway reconstruction as predicted by transcriptional profiles. PTS, red; GPH, yellow; ABC, green. (B) Genetic loci of interest: man, glucose–mannose; Fru, fructose; Suc, sucrose; raff, raffinose; Lac, lactose–galactose loci; tre, trehalose; CCR, carbon catabolite repression loci.