Comparative study of the protein composition
of three strains of <i>Streptococcus 
thermophilus</i> grown either in M17 medium or in milk

Guimont, Christiane; Chopard, Marie-Amélie; Gaillard, Jean‐Luc; Chamba, Jean-François

doi:10.1051/lait:2002039

Cited by 13 publications

(11 citation statements)

References 34 publications

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“…Trisodium citrate (1 M) was added at a final concentration of 0.25 M to the cell suspensions, which were maintained at 4°C for 5 min under these conditions before centrifugation. This treatment, adapted from that described previously (16), limits the casein precipitation in the milk and was applied to the three media. The bacteria were harvested by centrifugation (10,000 ϫ g, 10 min, 4°C).…”

Section: Methodsmentioning

confidence: 99%

Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk

Gitton

Meyrand

Wang

et al. 2005

Appl Environ Microbiol

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We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in onecarbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.The bacterium Lactococcus lactis is the main source of mesophilic starters used for the manufacture of fermented dairy products, and strong research efforts have been dedicated in the past 20 years to the isolation and description of functions required for proper development in milk (7,22,24). Dairy lactococci present half a dozen amino acid auxotrophies, whereas milk is not an abundant source of free amino acids (34). Also, the hydrolysis of caseins by a cell-wall-attached protease (PrtP) is required to achieve a final biomass of approximately 2 ϫ 10 9 CFU/ml (3, 24). A limited number of the resulting peptides are internalized by the oligopeptide transport system (OppA) and degraded to amino acids by a pool of cytoplasmic peptidases (23). Logically, the activities of both PrtP and OppA have been demonstrated to be crucial for optimal growth of lactococci in milk (3, 39). Another essential property of dairy lactococci is their capacity to internalize lactose by use of a phosphotransferase system (LacEF) and to degrade lactose-6-phosphate by the tagatose pathway (7). The genes encoding the lactose phosphotransferase system (lacEF), the phospho-␤-galactosidase (lacG), and the enzymes of the tagatose phosphate pathway (lacABCD) are organized in an operon that is also located on the protease plasmid (3). Besides the capacity to use casein and lactose efficiently, a small number of enzymes have been re...

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Section: Methodsmentioning

confidence: 99%

Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk

Gitton

Meyrand

Wang

et al. 2005

Appl Environ Microbiol

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“…Before centrifugation, a 0.33 volume of 1 M trisodium citrate and a 0.13 volume of buffered saline solution (0.145 M sodium chloride, 0.016 M sodium ␤-glycerophosphate, 0.1% Tween 80, pH 7) were added to cultures. This procedure, described by Guimont et al (23), avoids the precipitation of caseins at the time of centrifugation. Cells were harvested by centrifugation (8,000 ϫ g for 10 min at 4°C), and the cell pellet was washed twice with ice-cold extraction buffer, pH 7.0 (5 mM sodium phosphate, 1 mM EDTA, 2 mM ␤-mercaptoethanol), and resuspended in 20 mM Na phosphate buffer, pH 6.4.…”

Section: Methodsmentioning

confidence: 99%

“…S. thermophilus total RNA was prepared from 200-ml cultures. Before harvesting the cells, a 0.33 volume of 1 M trisodium citrate and a 0.13 volume of buffered saline solution (0.145 M sodium chloride, 0.016 M sodium ␤-glycerophosphate, 0.1% Tween 80, pH 7) were added to the media (23). Cells were collected by centrifugation for 5 min at 4°C at 10,000 ϫ g, washed with 100 ml ice-cold extraction buffer, pH 7.0 (5 mM sodium phosphate, 1 mM EDTA, 2 mM ␤-mercaptoethanol), shock frozen, and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Proteome Analysis of Streptococcus thermophilus Grown in Milk Reveals Pyruvate Formate-Lyase as the Major Upregulated Protein

Derzelle

Bolotin

Mistou

et al. 2005

Appl Environ Microbiol

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We investigated the adaptation to milk of Streptococcus thermophilus LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that pfl transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce pfl expression, to suppress PFL synthesis, and to stimulate growth of S. thermophilus. These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes.The thermophilic bacteria Streptococcus thermophilus is one of the most widely used lactic acid bacteria (LAB) in the dairy fermentation industry for yoghurt and cheese production. In the industrial implementation of S. thermophilus, fast-growing capacity is crucial to enable rapid and intense acidification of milk. Identification of functions activated during growth in milk should help to understand the molecular adaptation of this bacterium to milk and provide the basis for targeted strain selection.In contrast with other LAB, the only environment from which S. thermophilus has been isolated is milk (38). In line with this restricted ecological niche, lactose, not glucose, is the preferred carbon source for S. thermophilus (31). The capacity to ferment lactose, the main sugar of milk, into lactic acid is essential for growth in milk and depends on a non-phosphotransferase system lactose permease (LacS) and a beta-galactosidase (LacZ) (40). Concerning the capacity of S. thermophilus to fulfill its amino acid needs during growth in milk, the species displays only a few amino acid auxotrophies compared to the model LAB, L. lactis (29,33). The growth of Lactococcus lactis in milk depends largely on the activity of a cell-wallbound proteinase (PrtP) that is responsible for casein hydrolysis, the main source of amino a...

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“…; pH 7) (Derzelle et al 2005;Guimont et al 2002). After centrifugation for 15 min at 3900 g and 4°C, pellets were frozen in liquid nitrogen at −80°C.…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Acquisition of PrtS in Streptococcus thermophilus is not enough in certain strains to achieve rapid milk acidification

Galia

Jameh

Perrin

et al. 2016

Dairy Sci. & Technol.

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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.

show abstract

Comparative study of the protein composition of three strains of Streptococcus thermophilus grown either in M17 medium or in milk

Cited by 13 publications

References 34 publications

Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk

Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk

Proteome Analysis of Streptococcus thermophilus Grown in Milk Reveals Pyruvate Formate-Lyase as the Major Upregulated Protein

Acquisition of PrtS in Streptococcus thermophilus is not enough in certain strains to achieve rapid milk acidification

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