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. 2005 Dec;71(12):8597-605.
doi: 10.1128/AEM.71.12.8597-8605.2005.

Proteome analysis of Streptococcus thermophilus grown in milk reveals pyruvate formate-lyase as the major upregulated protein

Sylviane Derzelle  1 , Alexander Bolotin, Michel-Yves Mistou, Françoise Rul
Affiliations

Proteome analysis of Streptococcus thermophilus grown in milk reveals pyruvate formate-lyase as the major upregulated protein

Sylviane Derzelle et al. Appl Environ Microbiol. 2005 Dec.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
2-DE (pH range, 4 to 7) of intracellular extracts of S. thermophilus LMG18311 after growth in M17-Lac (A) or milk (B). Proteins (350 μg) were loaded on an IPG strip and stained with colloidal Coomassie blue. The proteins differentially expressed are indicated by circles (higher level in M17) or squares (higher level in milk).
FIG. 2.
FIG. 2.
Northern blot of RNA extracted after exponential growth in M17 (1) and after 15-min transfer in milk (2) and hybridized with a pfl probe. The size of the transcript (in kilobases) is shown on the left. On the right, the same membrane is shown stained with bromophenol blue.
FIG. 3.
FIG. 3.
mRNA and proteomic analyses of pfl of S. thermophilus LMG18311 after growth in milk. (A) Northern blot of RNA extracted during exponential growth (lanes 1 and 2) or stationary phase (lanes 3 to 5) in milk and hybridized with a pfl probe. The size of the transcript (in kilobases) is shown on the left. On the right, the same membrane is shown stained with bromophenol blue. (B) 2-DE of the intracellular proteins of S. thermophilus LMG18311 extracted during exponential growth (1 h, 30 min; lane 1) or stationary phase (4 h; lane 2) in milk. The picture represented a zoom of the Pfl region of a pH 4 to 7 gel.
FIG. 4.
FIG. 4.
mRNA and proteomic analyses of pfl of S. thermophilus LMG18311 after growth in milk supplemented or not with formate or adenine plus guanine. (A) Northern blot of RNA extracted from S. thermophilus LMG18311 after exponential growth in milk supplemented (lane 2) or not (lane 1) with 5 mM formate. The arrow indicates the 2.3-kb pfl transcript. On the right, the corresponding blots of extracted RNA after bromophenol blue coloration are presented. (B) 2-DE of the intracellular proteins of S. thermophilus LMG18311 extracted during exponential growth in milk in the presence (lane 2) or not (lane 1) of formate (5 mM) or adenine plus guanine (50 μM) (lane 3). The picture represented a zoom of the Pfl region of a pH 4 to 7 gel.
FIG. 5.
FIG. 5.
Growth and acidification curves of S. thermophilus LMG18311 in milk supplemented or not with formate or adenine plus guanine. The evolution of CFU (open symbols) and pH (filled symbols) in milk (squares) and milk supplemented with either 5 mM formate (triangles) or 50 μM adenine plus guanine (circles) is shown.
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