Catabolite control of sugar metabolism in Streptococcus thermophilus

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

Sugar utilisation and conservation of the gal-lac gene cluster in Streptococcus thermophilus

The adaptation to utilise lactose as primary carbon and energy source is a characteristic for Streptococcus thermophilus. These organisms, however only utilise the glucose moiety of lactose while the galactose moiety is excreted into the growth medium. In this study we evaluated the diversity of sugar utilisation and the conservation of the gal-lac gene cluster in a collection of 18 S. thermophilus strains isolated from a variety of sources. For this purpose analysis was performed on DNA from these isolates and the results were compared with those obtained with a strain from which the complete genome sequence has been determined. The sequence, organisation and flanking regions of the S. thermophilus gal-lac gene cluster were found to be highly conserved among all strains. The vast majority of the S. thermophilus strains were able to utilize only glucose, lactose, and sucrose as carbon sources, some strains could also utilize fructose and two of these were able to grow on galactose. Molecular characterisation of these naturally occurring Gal+ strains revealed up-mutations in the galKTE promoter that were absent in all other strains. These data support the hypothesis that the loss of the ability to ferment galactose can be attributed to the low activity of the galKTE promoter, probably as a consequence of the adaptation to milk in which the lactose levels are in excess

Increased D-alanylation of lipoteichoic acid and a thickened septum are main determinants in the nisin resistance mechanism of Lactococcus lactis

Nisin is a post-translationally modified antimicrobial peptide produced by Lactococcus lactis which binds to lipid II in the membrane to form pores and inhibit cell-wall synthesis. A nisinresistant (NisR) strain of L. lactis, which is able to grow at a 75-fold higher nisin concentration than its parent strain, was investigated with respect to changes in the cell wall. Direct binding studies demonstrated that less nisin was able to bind to lipid II in the membranes of L. lactis NisR than in the parent strain. In contrast to vancomycin binding, which showed ring-like binding, nisin was observed to bind in patches close to cell-division sites in both the wild-type and the NisR strains. Comparison of modifications in lipoteichoic acid of the L. lactis strains revealed an increase in D-alanyl esters and galactose as substituents in L. lactis NisR, resulting in a less negatively charged cell wall. Moreover, the cell wall displays significantly increased thickness at the septum. These results indicate that shielding the membrane and thus the lipid II molecule, thereby decreasing abduction of lipid II and subsequent pore-formation, is a major defence mechanism of L. lactis against nisin