"Early" protein synthesis of Lactobacillus delbrueckii ssp bulgaricus in milk revealed by [S-35]methionine labeling and two-dimensional gel electrophoresis

The proteomes of exponentially growing and stationary cells of Lactobacillu s delbrueckii ssp. bulgaricus grown in rich medium (MRS) were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified af ter Coomassie staining. Stationary cells grown in MRS were inoculated in re constituted skim milk, and "early" protein synthesis during the first 30 mi n of fermentation in milk was monitored by [S-35]methionine labeling and 2- DE. In contrast to exponentially growing or stationary cells, the predomina nt "early" proteins were small (< 15 kDa) and of low pI (< 5.3). Quantifica tion of the proteome of the "early" tag phase based on 47 "spots" revealed that only three "early" proteins accounted for more than 80% of the total l abel. They were identified as pI 4.7 and 4.9 isoforms of the heat-stable ph osphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectiv ely, and an unknown protein called EPr1 ("early" protein 1) with 26.6% of t otal label. Although an N-terminal sequence of 19 amino acids was obtained, no homologs to EPr1 could be found. De novo synthesis of the 10 and 60 kDa heat shock proteins (GroES and GroEL) was considerably lower (0.04 and 0.9 % of total label, respectively), indicating only low levels of stress. Synt hesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes r eached only 0.08% of total label. Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, im poses only little need for synthesis of stress or glycolytic enzymes, as su fficient proteins are present in the stationary, MRS-grown cells. The high level of expression of the pI 4.7 isoform of HPr suggests a regulatory func tion of the presumed Ser-46 phosphorylated form of HPr.