Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It grows when cheeses are ripened in a warm room (about 24°C). Cheeses with an acceptable eye formation level are transferred to a cold room (about 4°C), inducing a marked slowdown of propionic fermentation, but P. freudenreichii remains active in the cold. To investigate the P. freudenreichii strategies of adaptation and survival in the cold, we performed the first global gene expression profile for this species. The time-course transcriptomic response of P. freudenreichii CIRM-BIA1T strain was analyzed at five times of incubation, during growth at 30°C then for 9 days at 4°C, under conditions preventing nutrient starvation. Gene expression was also confirmed by RT-qPCR for 28 genes. In addition, proteomic experiments were carried out and the main metabolites were quantified. Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from 30°C to 4°C (P<0.05 and |fold change|>1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1T strain over-expressed genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis, and in glycogen synthesis. Interestingly, the expression of different genes involved in the formation of important cheese flavor compounds, remained unchanged at 4°C. This could explain the contribution of P. freudenreichii to cheese ripening even in the cold. In conclusion, P. freudenreichii remains metabolically active at 4°C and induces pathways to maintain its long-term survival.
References
[1]
Falentin H, Postollec F, Parayre S, Henaff N, Le Bivic P, et al. (2010) Specific metabolic activity of ripening bacteria quantified by real-time reverse transcription PCR throughout Emmental cheese manufacture. Int J Food Microbiol 144: 10–19.
[2]
Ganesan B, Stuart MR, Weimer BC (2007) Carbohydrate starvation causes a metabolically active but nonculturable state in Lactococcus lactis. Appl Environ Microbiol 73: 2498–2512.
[3]
Ta?bi A, Dabour N, Lamoureux M, Roy D, LaPointe G (2011) Comparative transcriptome analysis of Lactococcus lactis subsp. cremoris strains under conditions simulating Cheddar cheese manufacture. Int J Food Microbiol 146: 263–275.
[4]
Yvon M, Gitton C, Chambellon E, Bergot G, Monnet V (2011) The initial efficiency of the proteolytic system of Lactococcus lactis strains determines their responses to a cheese environment. Int Dairy J 21: 335–345.
[5]
Langsrud T, Reinbold GW (1973) Flavor development and microbiology of Swiss cheese- A review. III. Ripening and flavor production. J Milk Food Technol 36: 593–609.
[6]
Thierry A, Maillard MB, Richoux R, Kerjean JR, Lortal S (2005) Propionibacterium freudenreichii strains quantitatively affect production of volatile compounds in Swiss cheese. Lait 85: 57–74.
[7]
Cretenet M, Laroute V, Ulve V, Jeanson S, Nouaille S, et al. (2011) Dynamic analysis of the Lactococcus lactis transcriptome in cheeses made from milk concentrated by ultrafiltration reveals multiple strategies of adaptation to stresses. Appl Environ Microbiol 77: 247–257.
[8]
Fr?hlich-Wyder MT, Bachmann HP (2004) Cheeses with propionic acid fermentation. In: Fox PF, McSweeney PLH, Cogan TM, Guinee TP, editors. Cheese. Chemistry, Physics and Microbiology. London: Elsevier. pp. 141–156.
[9]
Thierry A, Salvat-Brunaud D, Madec MN, Michel F, Maubois JL (1998) Affinage de l'emmental: dynamique des populations bactériennes et évolution de la composition de la phase aqueuse. Lait 78: 521–542.
[10]
Malik AC, Reinbold GW, Vedamuthu ER (1968) An evaluation of the taxonomy of Propionibacterium. Can J Microbiol 14: 1185–1191.
[11]
Crow VL (1986) Metabolism of aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on lactate fermentation. Appl Environ Microbiol 52: 359–365.
[12]
Thierry A, Maillard MB, Yvon M (2002) Conversion of L-leucine to isovaleric acid by Propionibacterium freudenreichii TL 34 and ITGP23. Appl Environ Microbiol 68: 608–615.
[13]
Crow VL (1986) Utilization of lactate isomers by Propionibacterium freudenreichii subsp. shermanii: regulatory role for intracellular pyruvate. Appl Environ Microbiol 52: 352–358.
[14]
Liu H, Tarima S, Borders A, Getchell T, Getchell M, et al. (2005) Quadratic regression analysis for gene discovery and pattern recognition for non-cyclic short time-course microarray experiments. BMC Bioinformatics 6: 106.
[15]
Thieringer HA, Jones PG, Inouye M (1998) Cold shock and adaptation. Bioessays 20: 49–57.
[16]
Chan YC, Wiedmann M (2009) Physiology and genetics of Listeria monocytogenes survival and growth at cold temperatures. Crit Rev Food Sci Nutr 49:
[17]
Anastasiou R, Leverrier P, Krestas I, Rouault A, Kalantzopoulos G, et al. (2006) Changes in protein synthesis during thermal adaptation of Propionibacterium freudenreichii subsp. shermanii. Int J Food Microbiol 108: 301–314.
[18]
Leverrier P, Vissers JPC, Rouault A, Boyaval P, Jan G (2004) Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii. Arch Microbiol 181: 215–230.
[19]
Li SK, Xiao X, Sun P, Wang FP (2008) Screening of genes regulated by cold shock in Shewanella piezotolerans WP3 and time course expression of cold-regulated genes. Arch Microbiol 189: 549–556.
[20]
Piette F, D'Amico S, Mazzucchelli G, Danchin A, Leprince P, et al. (2011) Life in the cold: a proteomic study of cold-repressed proteins in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. Appl Environ Microbiol 77: 3881–3883.
[21]
Rodrigues DF, Tiedje JM (2008) Coping with our cold planet. Appl Environ Microbiol 74: 1677–1686.
[22]
Shivaji S, Prakash JSS (2010) How do bacteria sense and respond to low temperature? Arch Microbiol 192: 85–95.
[23]
Dherbécourt J, Maillard MB, Catheline D, Thierry A (2008) Production of branched-chain aroma compounds by Propionibacterium freudenreichii: links with the biosynthesis of membrane fatty acids. J Appl Microbiol 105: 977–985.
[24]
Hofherr LA, Hammond EG, Glatz BA, Ross PF (1983) Relation of growth temperature to fatty acid composition of Propionibacterium strains. J Dairy Sci 66: 1622–1629.
[25]
Pessione A, Lamberti C, Pessione E (2010) Proteomics as a tool for studying energy metabolism in lactic acid bacteria. Mol Biosyst 6: 1419–1430.
[26]
Clark JE, Beegen H, Wood HG (1987) Isolation of intact chains of polyphosphate from Propionibacterium shermanii grown on glucose or lactate. J Bacteriol 168: 1212–1219.
[27]
Gomez-Garcia MR, Losada M, Serrano A (2003) Concurrent transcriptional activation of ppa and ppx genes by phosphate deprivation in the cyanobacterium Synechocystis sp. strain PCC 6803. Biochem Bioph Res Co 302: 601–609.
[28]
Seufferheld MJ, Alvarez HM, Farias ME (2008) Role of polyphosphates in microbial adaptation to extreme environments. Appl Environ Microbiol 74: 5867–5874.
[29]
Deborde C, Rolin DB, Boyaval P (1999) In vivo 13C NMR study of the bidirectional reactions of the wood-werkman cycle and around the pyruvate node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici. Metab Eng 1: 309–319.
[30]
Ballicora MA, Iglesias AA, Preiss J (2003) ADP-Glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis. Microbiol Mol Biol R 67: 213–225.
[31]
Eydallin G, Montero M, Almagro G, Sesma MT, Viale AM, et al. (2010) Genome-wide screening of genes whose enhanced expression affects glycogen accumulation in Escherichia coli. DNA Res 17: 61–71.
[32]
Thierry A, Deutsch SM, Falentin H, Dalmasso M, Cousin FJ, et al. (2011) New insights into physiology and metabolism of Propionibacterium freudenreichii. Int J Food Microbiol 149: 19–27.
[33]
Dherbécourt J, Falentin H, Canaan S, Thierry A (2008) A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese. Microb Cell Fact 7:
[34]
Cousin FJ, Mater DDG, Foligné B, Jan G (2011) Dairy propionibacteria as human probiotics: a review of recent evidence. Dairy Sci Technol 91: 1–26.
[35]
Falentin H, Deutsch SM, Jan G, Loux V, Thierry A, et al. (2010) The complete genome of Propionibacterium freudenreichii CIRM-BIA1T, a hardy Actinobacterium with food and probiotic applications. PLoS ONE 5: e11748.
[36]
Deutsch SM, Le Bivic P, Herve C, Madec MN, LaPointe G, et al. (2010) Correlation of the capsular phenotype in Propionibacterium freudenreichii with the level of expression of gtf, a unique polysaccharide synthase-encoding gene. Appl Environ Microbiol 76: 2740–2746.
[37]
Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, et al. (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:
[38]
Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W, editors. Bioinformatics and computational biology solutions using R and Bioconductor. New York: Springer. pp. 397–420.
[39]
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate - A practical and powerful approach to multiple testing. J Roy Stat Soc B Met 57: 289–300.
[40]
Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, et al. (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35: W71–W74.
[41]
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.
[42]
Jan G, Leverrier P, Pichereau V, Boyaval P (2001) Changes in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichii. Appl Environ Microbiol 67: 2029–2036.
[43]
Gilad O, Jacobsen S, Stuer-Lauridsen B, Pedersen MB, Garrigues C, et al. (2010) Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a model of their utilization. Appl Environ Microbiol 76: 7285–7291.
[44]
Le Marechal C, Jardin J, Jan G, Even S, Pulido C, et al. (2011) Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis. Vet Res 42: 35.
[45]
Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, et al. (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cel Proteomics 4: 1265–1272.
[46]
Dupierris V, Masselon C, Court M, Kieffer-Jaquinod S, Bruley C (2009) A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25: 1980–1981.