"Early" protein synthesis of Lactobacillus delbrueckii ssp. bulgaricus in milk revealed by [35S] methionine labeling and two-dimensional gel electrophoresis.
The proteomes of exponentially growing and stationary cells of Lactobacillus delbrueckii ssp. bulgaricus grown in rich medium (MRS) were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified after Coomassie staining. Stationary cells grown in MRS were inoculated in reconstituted skim milk, and "early" protein synthesis during the first 30 min of fermentation in milk was monitored by [35S]methionine labeling and 2-DE. In contrast to exponentially growing or stationary cells, the predominant "early" proteins were small (< 15 kDa) and of low pI (< 5.3). Quantification of the proteome of the "early" lag phase based on 47 "spots" revealed that only three "early" proteins accounted for more than 80% of the total label. They were identified as pI 4.7 and 4.9 isoforms of the heat-stable phosphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectively, and an unknown protein called EPr1 ("early" protein 1) with 26.6% of total 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. Synthesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes reached only 0.08% of total label. Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, imposes only little need for synthesis of stress or glycolytic enzymes, as sufficient 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 function of the presumed Ser-46 phosphorylated form of HPr.
Rechinger
K B
KB
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. kbr@kvl.dk
Siegumfeldt
H
H
Svendsen
I
I
Jakobsen
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D002417:Cattle; D018835:Chaperonin 10 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018834:Chaperonin 60 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D004396:Coloring Agents; D003470:Culture Media / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007091:Image Processing, Computer-Assisted; D007778:Lactobacillus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013058:Mass Spectrometry; D008892:Milk / Q000382:microbiology*; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D020033:Protein Isoforms / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D020543:Proteome; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012394:Rosaniline Dyes; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D016622:Silver Staining; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D014305:Triose-Phosphate Isomerase / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics*
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D004396:Coloring Agents; D003470:Culture Media; D020033:Protein Isoforms; D020543:Proteome; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012394:Rosaniline Dyes; C048139:Coomassie blue; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D014305:Triose-Phosphate Isomerase