Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk (original) (raw)
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The relevance of carbon dioxide metabolism in Streptococcus thermophilus
Microbiology (Reading, England), 2009
Streptococcus thermophilus is a major component of dairy starter cultures used for the manufacture of yoghurt and cheese. In this study, the CO 2 metabolism of S. thermophilus DSM 20617 T , grown in either a N 2 atmosphere or an enriched CO 2 atmosphere, was analysed using both genetic and proteomic approaches. Growth experiments performed in a chemically defined medium revealed that CO 2 depletion resulted in bacterial arginine, aspartate and uracil auxotrophy. Moreover, CO 2 depletion governed a significant change in cell morphology, and a high reduction in biomass production. A comparative proteomic analysis revealed that cells of S. thermophilus showed a different degree of energy status depending on the CO 2 availability. In agreement with proteomic data, cells grown under N 2 showed a significantly higher milk acidification rate compared with those grown in an enriched CO 2 atmosphere. Experiments carried out on S. thermophilus wild-type and its derivative mutant, which was inactivated in the phosphoenolpyruvate carboxylase and carbamoyl-phosphate synthase activities responsible for fixing CO 2 to organic molecules, suggested that the anaplerotic reactions governed by these enzymes have a central role in bacterial metabolism. Our results reveal the capnophilic nature of this micro-organism, underlining the essential role of CO 2 in S. thermophilus physiology, and suggesting potential applications in dairy fermentation processes.
PROTEOMICS, 2008
Streptococcus thermophilus is a thermophilic lactic acid bacterium widely used as starter in the manufacture of dairy products in particular in yoghurt manufacture in combination with Lactobacillus delbrueckii ssp. bulgaricus. However, in spite of its massive use, the physiological state of S. thermophilus in milk has hardly been investigated. We established the first map of the cytosolic proteome of S. thermophilus LMG18311 grown in milk. It comprises 203 identified proteins corresponding to 32% of theoretical proteome. In addition, using proteomic and transcriptomic approaches, we analyzed the physiology of LMG18311 during its late stage of growth in milk (between 2h30 and 5h30). It revealed the up-regulation of (i) peptides and AA transporters and of specific AA biosynthetic pathways notably for sulfur AA and (ii) genes and proteins involved in the metabolism of various sugars. These two effects were also observed in LMG18311 grown in milk in coculture with L. bulgaricus although the effect on sugar metabolism was less pronounced. It suggests that the stimulatory effect of Lactobacillus on the Streptococcus growth is more complex than AA or peptides supply.
Applied and Environmental Microbiology
We investigated the carbon dioxide metabolism of Streptococcus thermophilus, evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, ⌬ppc. The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with L-aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus. As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of L-aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus. It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a p ureI -gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions.
Applied and Environmental Microbiology, 2007
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Dairy Science & Technology
The acquisition of prtS by Streptococcus thermophilus strains allowed hydrolysis of caseins into peptides and then to increase their growth in milk. This leads to faster milk acidification, which is important in dairy industry. However, some strains harboring the same allele of prtS present different acidification rates, which could be explained by a difference in the regulation of prtS expression. We chose two strains with the same allele of prtS (including the same promoter region): one, PB302, is with high acidification rate while the other, PB18O, is without. They exhibited similar growth in M17, but not in milk, where PB302 showed better growth. The expression of prtS and activity of PrtS were lower in PB18O, in the two media tested. We demonstrated that other genes known to be involved in carbon and nitrogen metabolism were overexpressed in PB302. Interestingly, these genes were overexpressed in milk compared to M17. Nearly all these genes possessed a putative CodY-box in their promoter region. Taken together, difference of gene expression detected in PB302 between milk (low-peptide medium) and M17 (rich-peptide medium) and presence of a putative CodY-box is a feature of the transcriptional pattern of CodY-regulated genes. Altogether, our results propose that acquisition of prtS is not enough in certain strains to achieve rapid milk acidification. High transcriptional level of dtpT, amiF, ilvC, ilvB, bcaT, livJ, ackA, codY, and prtS in fast acidifying strain suggests that this transcriptional pattern could be required for fast milk acidification in Streptococcus thermophilus.
Microcalorimetric study of the growth of Streptococcus thermophilus in renneted milk
Frontiers in microbiology, 2015
The growth of Streptococcus thermophilus ST12 (ST12) in liquid milk, reconstituted from low-heat skim milk powder reconstituted skim milk (RSM) and in RSM with rennet addition (r-RSM) at 40°C was monitored by microcalorimetry. It was shown that the growth rate of bacteria decreased in renneted samples in comparison with liquid RSM starting from certain sizes of the colonies ("deviation moments"), which depended on the inoculation rates. The hydrolysis of lactose was delayed for about 1 h in the r-RSM in comparison with RSM but otherwise the metabolism of carbohydrates in the renneted and non-renneted milks was similar. The total free amino acids (TFAA) content by the end of fermentations was higher in r-RSM than in RSM presumably due to the enzymatic hydrolytic activity of rennet. The quantitatively dominating amino acids were remarkably different in the r-RSM and RSM indicating that the hydrolysis cascade of caseins and/or metabolism of amino acids by the bacteria functio...
Journal of Applied Microbiology, 2002
Aims: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods and Results: Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. Conclusions: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. Significance and Impact of the Study: The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.
Streptococcus thermophilus strain Yt3 was isolated from yoghurt starter BY145-18, strongly influenced by urea in milk. Its urease-deficient spontaneous mutant Yt3uD3-1 showed lack of urease activity. S. thermophilus Yt3 and its urease negative mutant Yt3uD3-1 were compared for their growth at different fermentation conditions of varying pH and temperature during the process of pH-controlled fermentation. The optimal pH and temperature resulting in maximal cell numbers of S. thermophilus Yt3 and its urease-deficient derivative Yt3uD3-1 were determined. A concentrated culture prepared from the urease-deficient mutant Yt3uD3-1 showed faster acidification rate in milk medium than the mother strain Yt3. The basic fermentation characteristics for the two bacterial strains determined in this study permit the large-scale production of concentrated cultures with a high number of viable cells in the bacterial concentrate.
diversity of Streptococcus thermophilus strains isolated from traditional Egyptian fermented dairy products and to characterize phenotypic traits of those strains related to their potential use in bioprocessing applications. Methods and Results: A novel, simplified approach was developed for genotyping Strep. thermophilus involving the analysis of nucleotide sequence variations within a housekeeping gene encoding the phosphoserine phosphatase (SerB). Using this method, it was possible to identify ten genotypes involving diverse serB alleles among 54 Strep. thermophilus isolates cultured from Egyptian dairy products. These isolates harboured five de novo serB alleles that have not been detected in other Strep. thermophilus strains, deposited in a multilocus sequence typing (MLST) database. To assess distinct genotypes of the organism with phenotypic traits relevant to their potential use in industry, Strep. thermophilus strains were all subjected to a series of phenotypic characterizations. The strains were found to exhibit phenotypic diversity in terms of their ability to ferment lactose and galactose, express urease activity, produce exopolysaccharides and develop acidity. Conclusions: The analysis of nucleotide sequence variations within the serB gene could serve as a suitable tool for probing diverse genotypes of Strep. thermophilus. Streptococcus thermophilus isolates associated with traditional Egyptian dairy products show high degree of genetic and phenotypic diversity. Significance and Impact of the Study: This study presents a novel, simplified procedure based on serB nucleotide sequencing for genotyping Strep. thermophilus. It also provides a pool of phenotypically diverse Strep. thermophilus cultures, from which certain strains could be selected for use in bioprocessing applications including the preparation of fermented dairy products.