Functional Genomics Analyses of Carbohydrate Utilization by Lactobacillus acidopohilus

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Title: Functional Genomics Analyses of Carbohydrate Utilization by Lactobacillus acidopohilus
Author: Barrangou, Rodolphe
Advisors: Dahlia Nielsen, Committee Member
Greg Gibson, Committee Member
Robert M. Kelly, Committee Member
Todd Klaenhammer, Committee Chair
Abstract: Carbohydrates are a primary source of energy for microbes. Specifically, lactic acid bacteria have the ability to utilize a variety of nutrients available in their respective habitats. For probiotic microbes inhabiting the human gastrointestinal tract, the ability to utilize sugars non-digested by the host plays an important role in their survival. Lactobacillus acidophilus is a probiotic organism which can utilize a variety of mono-, di- and poly-saccharides, including prebiotic compounds such as fructooligosaccharides and raffinose. However, little information is available about the mechanisms and genes involved in carbohydrate utilization by lactobacilli. The transport and catabolic machinery involved in utilization of glucose, fructose, sucrose, FOS, raffinose, lactose, galactose and trehalose was characterized using global transcriptional profiling. Microarray hybridizations were carried out using a round-robin design and data analyzed using a two-stage mixed model ANOVA. Genes differentially expressed between treatments were visualized by hierarchical clustering, volcano plots, and 3-way contour plots. Globally, a small number of genes were highly induced, including a variety of carbohydrate transporters and sugar hydrolases. Members of the phosphoenolpyruvate sugar phosphotransferase system (PTS) family of transporters were identified for uptake of glucose, fructose, sucrose and trehalose. In contrast, transporters of the ATP binding cassette (ABC) family were identified for uptake of FOS and raffinose. A member of the LacS family of galactoside-pentose-hexuronide (GPH) translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of mono-, di- and poly- saccharides were also identified, including the enzymatic machinery of the Leloir pathway. Insertional inactivation of genes encoding sugar transporters and hydrolases confirmed microarray results. Quantitative RT-PCR was also used to confirm differential gene expression. Additional transcription experiments showed specific induction of genes encoding sugar transporters and hydrolases, and transcriptional repression by glucose. Collectively, microarray data revealed coordinated and regulated transcription of genes involved in sugar utilization based on carbohydrate availability, likely via carbon catabolite repression. The relationships between gene expression level, codon usage, chromosomal location and intrinsic gene parameters were investigated globally. Gene expression levels correlated most highly with GC content, codon adaptation index and gene size. In contrast, gene expression levels did not correlate with GC content at the third codon position. Perhaps the high correlation between GC content and gene expression is due to the low genomic GC composition of L. acidophilus. Analysis of variance was used to investigate the impact of chromosomal location on gene expression after data was segregated into four groups, by strand and orientation relative to the origin and terminus of replication. Results showed genes on the leading strand were more highly expressed. Also, genes pointing toward the terminus of replication showed higher expression levels. This preference allows for co-directional replication and transcription. Collectively, results showed a strong influence of chromosomal architecture, GC content and codon usage on gene transcription. Globally, analysis of gene expression in Lactobacillus acidophilus revealed orchestrated transcription, and adaptation to environmental conditions. Specifically, dynamic adaptation to carbohydrate sources available in the environment might contribute to competition with other commensal microbes for the limited nutrient sources available in the human gastrointestinal tract.
Date: 2006-11-17
Degree: PhD
Discipline: Functional Genomics
URI: http://www.lib.ncsu.edu/resolver/1840.16/4799


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