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Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese.

Lazzi C, Turroni S, Mancini A, Sgarbi E, Neviani E, Brigidi P, Gatti M - BMC Microbiol. (2014)

Bottom Line: The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways.The cDNA-AFLP results were validated on selected regulated genes by qPCR.By a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Food Science, Parma University, Parco Area delle Scienze 48/A, 43124 Parma, Italy. camilla.lazzi@unipr.it.

ABSTRACT

Background: Lactobacillus rhamnosus is a non-starter lactic acid bacterium that plays a significant role during cheese ripening, leading to the formation of flavor. In long-ripened cheeses it persists throughout the whole time of ripening due to its capacity to adapt to changing environmental conditions. The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways. However, the molecular mechanisms allowing the growth of L. rhamnosus in the cheese dairy environment are still poorly understood. The aim of the present study was to identify genes potentially contributing to the growth ability of L. rhamnosus PR1019 in cheese-like medium (CB) using a transcriptomic approach, based on cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time reverse transcription-PCR (qPCR).

Results: Using three primer combinations, a total of 89 and 98 transcript-derived fragments were obtained for L. rhamnosus PR1019 grown in commercial MRS medium and CB, respectively. The cDNA-AFLP results were validated on selected regulated genes by qPCR. In order to investigate the main adaptations to growth in a cheese-mimicking system, we focused on 20 transcripts over-expressed in CB with respect to MRS. It is worth noting the presence of transcripts involved in the degradation of pyruvate and ribose. Pyruvate is a intracellular metabolite that can be produced through different metabolic routes starting from the carbon sources present in cheese, and can be released in the cheese matrix with the starter lysis. Similarly the ribonucleosides released with starter lysis could deliver ribose that represents a fermentable carbohydrate in environments, such as cheese, where free carbohydrates are lacking.Both pyruvate degradation and ribose catabolism induce a metabolite flux toward acetate, coupled with ATP production via acetate kinase. Taking into account these considerations, we suggest that the energy produced through these pathways may concur to explain the great ability of L. rhamnosus PR1019 to grow on CB.

Conclusions: By a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.

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Degradation of ribose. Enzymes showing differences in protein (*) or transcript abundance for L. rhamnosus PR1019 grown in CB compared to MRS are highlighted. Dark green, expression ratio CB versus MRS 5 to 10. Transcript data are from the present study. Protein data are from Bove et al. [16].
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Figure 5: Degradation of ribose. Enzymes showing differences in protein (*) or transcript abundance for L. rhamnosus PR1019 grown in CB compared to MRS are highlighted. Dark green, expression ratio CB versus MRS 5 to 10. Transcript data are from the present study. Protein data are from Bove et al. [16].

Mentions: Facultatively heterofermentative LAB, like L. rhamnosus, degrade hexoses via the Embden-Meyerhoff-Parnas pathway and pentoses via the phosphoketolase pathway (PKP). Xylulose 5-phosphate phosphoketolase is the central enzyme of PKP. In the presence of inorganic phosphate this enzyme converts xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetylphosphate (FigureĀ 5) [47]. Recently, McLeod et al. [48] studied the transcriptome response of L. sakei during growth on ribose, demonstrating that the ribose uptake and catabolic machinery are highly regulated and closely linked with the catabolism of nucleotides. It is known that ribonucleosides are source of ribose as a fermentable carbohydrate in environments where free carbohydrates are lacking. For example, in the meat, a rich environment but carbohydrate-poor substrate for microorganisms, the ability of L. sakei to use nucleosides offers a competitive advantage [49]. Nucleosides represent a potential energy source also in the cheese environment, where microbial autolysis occurs, releasing ribose- and desoxyribose-containing nucleic acids [14]. Notably, it has been observed that ribose released after lysis of SLAB decreased steadily in parallel with the growth of facultatively heterofermentative lactobacilli, strongly suggesting that these bacteria used ribose as a growth substrate [14].


Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese.

Lazzi C, Turroni S, Mancini A, Sgarbi E, Neviani E, Brigidi P, Gatti M - BMC Microbiol. (2014)

Degradation of ribose. Enzymes showing differences in protein (*) or transcript abundance for L. rhamnosus PR1019 grown in CB compared to MRS are highlighted. Dark green, expression ratio CB versus MRS 5 to 10. Transcript data are from the present study. Protein data are from Bove et al. [16].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3928093&req=5

Figure 5: Degradation of ribose. Enzymes showing differences in protein (*) or transcript abundance for L. rhamnosus PR1019 grown in CB compared to MRS are highlighted. Dark green, expression ratio CB versus MRS 5 to 10. Transcript data are from the present study. Protein data are from Bove et al. [16].
Mentions: Facultatively heterofermentative LAB, like L. rhamnosus, degrade hexoses via the Embden-Meyerhoff-Parnas pathway and pentoses via the phosphoketolase pathway (PKP). Xylulose 5-phosphate phosphoketolase is the central enzyme of PKP. In the presence of inorganic phosphate this enzyme converts xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetylphosphate (FigureĀ 5) [47]. Recently, McLeod et al. [48] studied the transcriptome response of L. sakei during growth on ribose, demonstrating that the ribose uptake and catabolic machinery are highly regulated and closely linked with the catabolism of nucleotides. It is known that ribonucleosides are source of ribose as a fermentable carbohydrate in environments where free carbohydrates are lacking. For example, in the meat, a rich environment but carbohydrate-poor substrate for microorganisms, the ability of L. sakei to use nucleosides offers a competitive advantage [49]. Nucleosides represent a potential energy source also in the cheese environment, where microbial autolysis occurs, releasing ribose- and desoxyribose-containing nucleic acids [14]. Notably, it has been observed that ribose released after lysis of SLAB decreased steadily in parallel with the growth of facultatively heterofermentative lactobacilli, strongly suggesting that these bacteria used ribose as a growth substrate [14].

Bottom Line: The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways.The cDNA-AFLP results were validated on selected regulated genes by qPCR.By a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Food Science, Parma University, Parco Area delle Scienze 48/A, 43124 Parma, Italy. camilla.lazzi@unipr.it.

ABSTRACT

Background: Lactobacillus rhamnosus is a non-starter lactic acid bacterium that plays a significant role during cheese ripening, leading to the formation of flavor. In long-ripened cheeses it persists throughout the whole time of ripening due to its capacity to adapt to changing environmental conditions. The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways. However, the molecular mechanisms allowing the growth of L. rhamnosus in the cheese dairy environment are still poorly understood. The aim of the present study was to identify genes potentially contributing to the growth ability of L. rhamnosus PR1019 in cheese-like medium (CB) using a transcriptomic approach, based on cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time reverse transcription-PCR (qPCR).

Results: Using three primer combinations, a total of 89 and 98 transcript-derived fragments were obtained for L. rhamnosus PR1019 grown in commercial MRS medium and CB, respectively. The cDNA-AFLP results were validated on selected regulated genes by qPCR. In order to investigate the main adaptations to growth in a cheese-mimicking system, we focused on 20 transcripts over-expressed in CB with respect to MRS. It is worth noting the presence of transcripts involved in the degradation of pyruvate and ribose. Pyruvate is a intracellular metabolite that can be produced through different metabolic routes starting from the carbon sources present in cheese, and can be released in the cheese matrix with the starter lysis. Similarly the ribonucleosides released with starter lysis could deliver ribose that represents a fermentable carbohydrate in environments, such as cheese, where free carbohydrates are lacking.Both pyruvate degradation and ribose catabolism induce a metabolite flux toward acetate, coupled with ATP production via acetate kinase. Taking into account these considerations, we suggest that the energy produced through these pathways may concur to explain the great ability of L. rhamnosus PR1019 to grow on CB.

Conclusions: By a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.

Show MeSH
Related in: MedlinePlus