Limits...
Growth phase-associated changes in the proteome and transcriptome of Lactobacillus rhamnosus GG in industrial-type whey medium.

Laakso K, Koskenniemi K, Koponen J, Kankainen M, Surakka A, Salusjärvi T, Auvinen P, Savijoki K, Nyman TA, Kalkkinen N, Tynkkynen S, Varmanen P - Microb Biotechnol (2011)

Bottom Line: Of the significantly differentially produced proteins, 61 were associated with alterations at the transcript level.The most remarkable growth phase-dependent changes occurred during the transition from the exponential to the stationary growth phase and were associated with the shift from glucose fermentation to galactose utilization and the transition from homolactic to mixed acid fermentation.Furthermore, several genes encoding proteins proposed to promote the survival and persistence of L. rhamnosus GG in the host and proteins that directly contribute to human health showed temporal changes in expression.

View Article: PubMed Central - PubMed

Affiliation: Research and Development, Valio Ltd, Helsinki, Finland.

Show MeSH

Related in: MedlinePlus

Gene expression patterns of selected genes and operons during growth in whey coding for (A) carbohydrate and pyruvate metabolic proteins, (B) proteinases, peptidases and amino acid transporters and (C) probiotic‐associated factors. Gene expression changes (averages of three biological replicates) between paired time points are represented colorimetrically, with dark red indicating an expression ratio of 5.0 and dark blue indicating an expression ratio of −5.0 on a log2 scale. The first four lanes show results of comparison of time points 12, 16, 20 and 31 h to the time point 4 h corresponding to the mid‐exponential phase. Statistically significant changes (P‐value ≤ 0.01) are marked with asterisks.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815411&req=5

f4: Gene expression patterns of selected genes and operons during growth in whey coding for (A) carbohydrate and pyruvate metabolic proteins, (B) proteinases, peptidases and amino acid transporters and (C) probiotic‐associated factors. Gene expression changes (averages of three biological replicates) between paired time points are represented colorimetrically, with dark red indicating an expression ratio of 5.0 and dark blue indicating an expression ratio of −5.0 on a log2 scale. The first four lanes show results of comparison of time points 12, 16, 20 and 31 h to the time point 4 h corresponding to the mid‐exponential phase. Statistically significant changes (P‐value ≤ 0.01) are marked with asterisks.

Mentions: Carbohydrate transport and metabolism. The whey medium used in this study contains glucose and galactose derived from hydrolysed lactose. In L. rhamnosus GG, there are two different pathways for the catabolism of the galactose moiety of lactose. The Leloir pathway (proteins encoded by the galKETRM operon and pgm) converts galactose to glucose‐6‐phosphate, and the tagatose‐6‐phosphate pathway (proteins encoded by the lacCDBAR operon) metabolizes galactose to glyceraldehyde‐3‐phosphate and dihydroxyacetone phosphate (Kankainen et al., 2009). Genes encoding enzymes from both the Leloir pathway and the tagatose‐6‐phosphate pathway showed a significant increase in expression at the mRNA level when the culture shifted from the exponential to the stationary phase of growth (Fig. 4A, Table S1). The same expression pattern was also evident at the protein level, as galactose utilization enzymes were more abundant in the early stationary growth phase (20 h) than in the exponential growth phase (4 or 12 h) (Tables 1 and 2). Furthermore, chemical analyses of the cultures showed that glucose was no longer detectable (detection limit 0.05%) in the early stationary phase of growth while galactose was still found in high amounts (1.0%). Therefore, increased expression of genes involved in galactose utilization at the stationary‐phase transition point was expected because L. rhamnosus GG first metabolizes the readily fermented glucose moiety; only after that does it metabolize the less easily exploited galactose portion of the hydrolysed lactose in the whey medium. Applying a proteomic approach, Cohen and colleagues (2006) also detected upregulation of the Leloir pathway in L. plantarum WCFS1 during stationary growth in the laboratory MRS medium, which possibly contained trace amounts of galactose. Our transcript results also indicated that genes encoding components of the phosphotransferase system (PTS), currently annotated as galactitol‐specific (LGG_00343, LGG_00345–00346), were significantly upregulated upon entry into the stationary growth phase and clustered together with lacCDBAR genes (cluster 2 in Fig. S1). Increased expression of the IIA component (LGG_00345) of that PTS system was also detected at the protein level (Tables 1 and 2). These results suggest that this particular PTS is involved in the transportation of galactose, and revision of its present annotation could be considered. It is noteworthy that current annotations of PTS transporters do not necessarily indicate their true role in the metabolism of particular sugars because the specificities of many lactobacilli PTS transporters are incorrectly annotated (Francl et al., 2010), and current computational methods are unreliable for the prediction of substrate specificity. Therefore, further experiments are needed to characterize the PTS transporter specificities of L. rhamnosus GG; for example, this could be done by studying transcript expression profiles in response to different carbohydrates.


Growth phase-associated changes in the proteome and transcriptome of Lactobacillus rhamnosus GG in industrial-type whey medium.

Laakso K, Koskenniemi K, Koponen J, Kankainen M, Surakka A, Salusjärvi T, Auvinen P, Savijoki K, Nyman TA, Kalkkinen N, Tynkkynen S, Varmanen P - Microb Biotechnol (2011)

Gene expression patterns of selected genes and operons during growth in whey coding for (A) carbohydrate and pyruvate metabolic proteins, (B) proteinases, peptidases and amino acid transporters and (C) probiotic‐associated factors. Gene expression changes (averages of three biological replicates) between paired time points are represented colorimetrically, with dark red indicating an expression ratio of 5.0 and dark blue indicating an expression ratio of −5.0 on a log2 scale. The first four lanes show results of comparison of time points 12, 16, 20 and 31 h to the time point 4 h corresponding to the mid‐exponential phase. Statistically significant changes (P‐value ≤ 0.01) are marked with asterisks.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815411&req=5

f4: Gene expression patterns of selected genes and operons during growth in whey coding for (A) carbohydrate and pyruvate metabolic proteins, (B) proteinases, peptidases and amino acid transporters and (C) probiotic‐associated factors. Gene expression changes (averages of three biological replicates) between paired time points are represented colorimetrically, with dark red indicating an expression ratio of 5.0 and dark blue indicating an expression ratio of −5.0 on a log2 scale. The first four lanes show results of comparison of time points 12, 16, 20 and 31 h to the time point 4 h corresponding to the mid‐exponential phase. Statistically significant changes (P‐value ≤ 0.01) are marked with asterisks.
Mentions: Carbohydrate transport and metabolism. The whey medium used in this study contains glucose and galactose derived from hydrolysed lactose. In L. rhamnosus GG, there are two different pathways for the catabolism of the galactose moiety of lactose. The Leloir pathway (proteins encoded by the galKETRM operon and pgm) converts galactose to glucose‐6‐phosphate, and the tagatose‐6‐phosphate pathway (proteins encoded by the lacCDBAR operon) metabolizes galactose to glyceraldehyde‐3‐phosphate and dihydroxyacetone phosphate (Kankainen et al., 2009). Genes encoding enzymes from both the Leloir pathway and the tagatose‐6‐phosphate pathway showed a significant increase in expression at the mRNA level when the culture shifted from the exponential to the stationary phase of growth (Fig. 4A, Table S1). The same expression pattern was also evident at the protein level, as galactose utilization enzymes were more abundant in the early stationary growth phase (20 h) than in the exponential growth phase (4 or 12 h) (Tables 1 and 2). Furthermore, chemical analyses of the cultures showed that glucose was no longer detectable (detection limit 0.05%) in the early stationary phase of growth while galactose was still found in high amounts (1.0%). Therefore, increased expression of genes involved in galactose utilization at the stationary‐phase transition point was expected because L. rhamnosus GG first metabolizes the readily fermented glucose moiety; only after that does it metabolize the less easily exploited galactose portion of the hydrolysed lactose in the whey medium. Applying a proteomic approach, Cohen and colleagues (2006) also detected upregulation of the Leloir pathway in L. plantarum WCFS1 during stationary growth in the laboratory MRS medium, which possibly contained trace amounts of galactose. Our transcript results also indicated that genes encoding components of the phosphotransferase system (PTS), currently annotated as galactitol‐specific (LGG_00343, LGG_00345–00346), were significantly upregulated upon entry into the stationary growth phase and clustered together with lacCDBAR genes (cluster 2 in Fig. S1). Increased expression of the IIA component (LGG_00345) of that PTS system was also detected at the protein level (Tables 1 and 2). These results suggest that this particular PTS is involved in the transportation of galactose, and revision of its present annotation could be considered. It is noteworthy that current annotations of PTS transporters do not necessarily indicate their true role in the metabolism of particular sugars because the specificities of many lactobacilli PTS transporters are incorrectly annotated (Francl et al., 2010), and current computational methods are unreliable for the prediction of substrate specificity. Therefore, further experiments are needed to characterize the PTS transporter specificities of L. rhamnosus GG; for example, this could be done by studying transcript expression profiles in response to different carbohydrates.

Bottom Line: Of the significantly differentially produced proteins, 61 were associated with alterations at the transcript level.The most remarkable growth phase-dependent changes occurred during the transition from the exponential to the stationary growth phase and were associated with the shift from glucose fermentation to galactose utilization and the transition from homolactic to mixed acid fermentation.Furthermore, several genes encoding proteins proposed to promote the survival and persistence of L. rhamnosus GG in the host and proteins that directly contribute to human health showed temporal changes in expression.

View Article: PubMed Central - PubMed

Affiliation: Research and Development, Valio Ltd, Helsinki, Finland.

Show MeSH
Related in: MedlinePlus