A proteomic view of probiotic Lactobacillus rhamnosus GG

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CONTENTS Abstract List of abbreviations List of original publications 1. Review of the literature ..........................................................................................1 1.1. Probiotic bacteria ..................................................................................................... 1 1.1.1. Common properties of probiotic bacteria .............................................. 1 1.1.1.1. Probiotic mechanisms .................................................................. 1 1.1.1.2. Main properties of probiotic bacteria ......................................... 2 1.1.1.3. Health-benefi cial eff ects of probiotics ........................................ 2 1.1.2. Lactobacilli .................................................................................................. 3 1.1.2.1. Lactobacillus rhamnosus GG ........................................................ 3 1.1.3. Bifi dobacteria and propionibacteria ........................................................ 5 1.2. Proteomics ................................................................................................................. 5 1.2.1. Proteomic methods ........................................................................................ 5 1.3. Proteomics of potential probiotic bacteria ............................................................ 6 1.3.1. Basic proteome research .......................................................................... 10 1.3.1.1. Proteome maps ............................................................................ 10 1.3.1.2. Comparison of growth phases ................................................... 11 1.3.1.3. Comparison of diff erent strains and growth media ............... 11 1.3.1.4. Development of proteomic methods ........................................ 12 1.3.2. Proteomics of stress responses of potentially probiotic bacteria ........ 13 1.3.2.1. Bile stress ...................................................................................... 13 1.3.2.2. Acid stress .................................................................................... 22 1.3.2.3. Other stresses ............................................................................... 23 1.3.3. Studies of the cell envelope proteomes and secretomes of potentially probiotic bacteria .................................................................. 24 1.3.3.1. Cell envelope proteome .............................................................. 24 1.3.3.2. Secretome ..................................................................................... 26 1.3.3.3. Surfome and secretome predictions ......................................... 27 1.3.4. Other proteomic studies of potentially probiotic bacteria ..................... 28 2. Aims of the study ..................................................................................................30
3. Materials and methods .........................................................................................31 3.1. Bacterial strain and growth conditions ............................................................... 31 3.2. Proteomic methods ................................................................................................ 31 3.2.1. Protein extraction ..................................................................................... 31 3.2.2. Labeling of protein samples .................................................................... 31 3.2.3. 2-D GE and image analysis ..................................................................... 32 3.3. Protein identifi cation by MS ................................................................................. 32 3.4. Transcriptomic methods ....................................................................................... 32 3.4.1. Experimental design, RNA methods, cDNA synthesis, and labeling 32 3.4.2. Scanning, image analysis, and data analyses ......................................... 33 3.5. Analysis of the phosphoproteome ........................................................................ 33 3.5.1. Protein extraction ..................................................................................... 33 3.5.2. 2-D GE and image analysis ..................................................................... 33 3.5.3. Phosphopeptide identifi cation ................................................................ 33 4. Results and discussion ..........................................................................................34 4.1. Eff ect of the growth medium on the proteome of L. rhamnosus GG .............. 34 4.2. Growth phase-associated changes in L. rhamnosus GG .................................... 35 4.3. Bile stress response of L. rhamnosus GG ............................................................. 37 4.4. Eff ect of growth pH on L. rhamnosus GG ........................................................... 38 4.5. Protein phosphorylation in L. rhamnosus GG .................................................... 39 4.6. Proteome map of L. rhamnosus GG ..................................................................... 39 5. Concluding remarks and future prospects ..........................................................43 6. Acknowledgements ...............................................................................................45 7. References .........................................................................................................46 Original publications
Page 1 and 2: Department of Veterinary Bioscience
Page 6 and 7: ABSTRACT Lactobacillus rhamnosus GG
Page 8 and 9: LIST OF ABBREVIATIONS 2-D DIGE two-
Page 11 and 12: 1. REVIEW OF THE LITERATURE 1.1. Pr
Page 13 and 14: ingestion of Lactobacillus gasseri
Page 15 and 16: 1.1.3. Bifi dobacteria and propioni
Page 17 and 18: Table 1. Proteomic studies of probi
Page 19 and 20: Topic Separation and detection meth
Page 21 and 22: 1.3.1.2. Comparison of growth phase
Page 23 and 24: ecause of the complex nutrient requ
Page 25 and 26: Bile stress Acid stress Heat stress
Page 31 and 32: on the metabolic processes of probi
Page 33 and 34: esistant mutant strain was also abl
Page 35 and 36: Glyceraldehyde-3-phosphate dehydrog
Page 37 and 38: nine proteins that are potentially
Page 39 and 40: down-regulated the chemotaxis syste
Page 41 and 42: 3. MATERIALS AND METHODS All materi
Page 43 and 44: μg of RNA was reverse transcribed
Page 45 and 46: L. rhamnosus GG in MRS medium, and
Page 47 and 48: (EPS) biosynthesis and D-alanylatio
Page 49 and 50: that exports protons from the cell
Page 51 and 52: Results and Discussion 41
Page 53 and 54:
5. CONCLUDING REMARKS AND FUTURE PR
Page 55 and 56:
6. ACKNOWLEDGEMENTS Acknowledgement
Page 57 and 58:
Castaldo C., Vastano V., Siciliano
Page 59 and 60:
Hickson M., D’Souza A. L., Muthu
Page 61 and 62:
Lee K. & Pi K. (2010) Effect of tra
Page 63 and 64:
Adaptation and response of Bifidoba
Page 65:
Wu R., Wang W., Yu D., Zhang W., Li
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A proteomic view of probiotic Lactobacillus rhamnosus GG

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