ABSTRACT
Among the group of lactic acid bacteria, Streptococcus thermophilus has found a wide application in industrial processes used for the manufacture of dairy products. Taking advantage of different proteome extraction and subfractionation protocols, bacterial cytosolic and membrane proteins were isolated and resolved by independent gel-free and gel-based separation procedures. Whole cytosolic fraction and its acid, basic and low molecular mass protein components were separated by different resolutive 2-DE and tricine 1-DE gels and identified by MALDI-TOF PMF and/or microLC-ESI-IT-MS/MS. Membrane proteins were resolved by 2-DE and SDS-PAGE gels and similarly identified by PMF and TMS analysis. In parallel, whole extract was trypsinized and resulting peptides were identified by shotgun 2-D LC-ESI-IT-MS/MS analysis. Using this combined approach, expression products corresponding to 458 different genes were identified, which cover almost a third of the predicted vegetative proteome. Relative protein concentration and hydrophobicity affected protein detection. Broad recognition was obtained for enzymes involved in carbohydrate, fatty acid, amino acid and nucleotide metabolism, replication, transcription, translation, cell wall synthesis, as well as for proteins affecting bacterial functions important for industrial applications, i.e. milk sugar import and exopolysaccharide biosynthesis. By providing detailed reference electrophoretic/chromatographic maps to be used in future comparative proteomic investigations on bacteria grown under various experimental conditions or on different bacterial strains, our results will favour dedicated studies on S. thermophilus metabolism and its regulation or on detection of biomarkers for selection of optimal strains for industrial applications.
Subject(s)
Bacteriolysis , Proteome/chemistry , Streptococcus thermophilus/chemistry , Amino Acid Sequence , Cytosol/chemistry , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Membrane Proteins/chemistry , Metabolic Networks and Pathways , Molecular Sequence Data , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.
