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Streptococcus thermophilus is used in many industrial dairy fermentations that require processing of milk at elevated temperatures. Its primary function is the rapid conversion of lactose to lactate while it also contributes to important sensory qualities. S. thermophilus strain CNRZ302 is unable to ferment galactose, neither the free sugar, nor when it is generated intracellularly by lactose hydrolysis. Nevertheless, sequence analysis demonstrated that strain CNRZ302 contained structurally intact genes for the Leloir pathway enzymes. These genes appeared to be organized in an operon with the order galKTE , which was preceded by a divergently transcribed regulator gene galR , and followed by a galM gene and the lactose operon lacSZ . This S. thermophilus gal-lac gene cluster is very conserved in sequence, organization and flanking regions among strains isolated from various fermented products. The structural gal genes were found to be transcribed weakly by strain CNRZ302, and only in medium containing lactose, reflecting the Gal -phenotype. Indeed, the upregulation of the galKTE promoter seems to suffice for a galactose-fermenting phenotype of S. thermophilus .A catabolite responsive element ( cre ) was identified in the promoter of the lacSZ operon indicating a possible role for CcpA in regulation of transcription of this operon. CcpA has been studied in many low G+C Gram-positive bacteria where it mediates catabolite repression. S. thermophilus , unlike many other Gram-positive bacteria, prefers lactose to glucose as the primary carbon and energy source. To assess the role of CcpA mediated global regulation in S. thermophilus the ccpA gene was cloned and sequenced. Transcription analysis of the lacSZ operon showed relief of repression in the absence of a functional CcpA when cells were grown on lactose. In strains carrying a disrupted ccpA gene lactose transport was increased significantly while lactate production was reduced relative to wild-type cells. Global control of carbon metabolism in bacteria is primarily modulated by intracellular concentrations of glycolytic intermediates. The efficiency of glycolytic steps were determined by glycolytic intermediate dynamics analysis in resting cells isolated from various stages of growth. This showed a change in flux through glycolysis coinciding with this transition in growth by the lactose availability level indicated that the efficiency of several glycolytic steps were growth phase dependently regulated. This regulation was lost in ccpA mutant cells. Moreover, Northern analysis showed that CcpA acts as an activator of the pfk-pyk operon as well as of the ldh gene in S. thermophilus . CcpA not only fine-tunes lactose uptake and conversion when cells are grown in excess lactose, but is also involved in derepression of transport activity and reduction of glycolytic capacity to allow cellular adaptation to conditions where lactose becomes limiting. By over-expressing wild-type S. thermophilusptsH and specifically mutated ptsH alleles in a ptsH deletion strain of L. lactis the functional role of S. thermophilus HPr was confirmed and specific amino acid residues were identified that are essential for its function in utilization of PTS and non-PTS carbohydrates. However, we hypothesize that the role of HPr in S. thermophilus may have been adapted to the preferential lactose metabolism similar to what was found for CcpA in this organism.The work described in this thesis can provide the basis for metabolic engineering of fermentation properties of this lactic acid bacterium, which is used in many dairy fermentations and is of great commercial importance. |
