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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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Schematic diagram for genome regions surrounding spxB, ulaE and xfp locus in diverse lactobacilli. (A), spxB. (B), ulaE. (C), xfp. Gene syntenies were explored using the web service SyntTax [27]. TDF-derived protein sequences were used to query the selected genomes. Genes corresponding to query proteins are drawn in bold. A consistent color coding allows identification of orthologs and paralogs. Some gene names are indicated. Normalized BLAST scores are visualized. Reference organisms: L. rhamnosus GG, L. casei ATCC 334, L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. bulgaricus ATCC 11842 and L. brevis ATCC 367. For ulaE, a conserved gene order was observed only in L. rhamnosus GG and L. casei ATCC 334.
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Figure 3: Schematic diagram for genome regions surrounding spxB, ulaE and xfp locus in diverse lactobacilli. (A), spxB. (B), ulaE. (C), xfp. Gene syntenies were explored using the web service SyntTax [27]. TDF-derived protein sequences were used to query the selected genomes. Genes corresponding to query proteins are drawn in bold. A consistent color coding allows identification of orthologs and paralogs. Some gene names are indicated. Normalized BLAST scores are visualized. Reference organisms: L. rhamnosus GG, L. casei ATCC 334, L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. bulgaricus ATCC 11842 and L. brevis ATCC 367. For ulaE, a conserved gene order was observed only in L. rhamnosus GG and L. casei ATCC 334.

Mentions: Notably, 3 genes encoding putative pyruvate oxidases are harbored in the completely sequenced genomes of L. rhamnosus GG and L. casei ATCC 334, whereas 4 and 5 pox genes were retrieved in the genome sequences of L. buchneri CD034 and L. plantarum WCFS1, respectively. Goffin et al. [36] reported that among the predicted pox genes encoded in the L. plantarum lp80 genome, only poxB and poxF appeared to be involved in the generation of acetate from lactate during the stationary phase of aerobic growth. Interestingly, poxB and poxF genes shared 63 and 61% amino acid similarity with TDF 93, respectively. To date, only one gene potentially encoding for pyruvate oxidase has been located in the complete genome sequences of the SLAB L. helveticus R0052 and L. delbrueckii subsp. bulgaricus ATCC 11842. The pyruvate oxidase gene of L. rhamnosus GG with the highest homology to TDF 93 is flanked by genes whose order and transcriptional orientation are partially shared with L. casei ATCC 334 but not with L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. bulgaricus ATCC 11842 and L. brevis ATCC 367 (Figure 3A). In particular, spxB locus in L. rhamnosus and L. casei genomes is preceded by three genes encoding putative hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and acetyl-CoA acetyltransferase. These enzymes are known to be involved in the mevalonate pathway, routing acetyl-CoA towards isoprenoid biosynthesis. However, whether these proteins are actually expressed in L. rhamnosus and play a role in deviating the flow of acetyl-CoA from the acetate production via PTA and ACK during cheese ripening still remain to be determined. According to PePPER, spxB gene from L. rhamnosus GG was predicted to be monocistronically transcribed. Phylogenetic tree showed a clear segregation of putative pyruvate oxidases from L. casei group (Figure 4A). As expected, a subgroup was represented by POX proteins from the SLAB L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. L. plantarum and L. pentosus homologues clustered together and close to L. buchneri. Multiple sequence alignment of TDF 93 and pyruvate oxidase protein sequences from several NSLAB and SLAB is shown in Additional file 1: Figure S1A.


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)

Schematic diagram for genome regions surrounding spxB, ulaE and xfp locus in diverse lactobacilli. (A), spxB. (B), ulaE. (C), xfp. Gene syntenies were explored using the web service SyntTax [27]. TDF-derived protein sequences were used to query the selected genomes. Genes corresponding to query proteins are drawn in bold. A consistent color coding allows identification of orthologs and paralogs. Some gene names are indicated. Normalized BLAST scores are visualized. Reference organisms: L. rhamnosus GG, L. casei ATCC 334, L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. bulgaricus ATCC 11842 and L. brevis ATCC 367. For ulaE, a conserved gene order was observed only in L. rhamnosus GG and L. casei ATCC 334.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Schematic diagram for genome regions surrounding spxB, ulaE and xfp locus in diverse lactobacilli. (A), spxB. (B), ulaE. (C), xfp. Gene syntenies were explored using the web service SyntTax [27]. TDF-derived protein sequences were used to query the selected genomes. Genes corresponding to query proteins are drawn in bold. A consistent color coding allows identification of orthologs and paralogs. Some gene names are indicated. Normalized BLAST scores are visualized. Reference organisms: L. rhamnosus GG, L. casei ATCC 334, L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. bulgaricus ATCC 11842 and L. brevis ATCC 367. For ulaE, a conserved gene order was observed only in L. rhamnosus GG and L. casei ATCC 334.
Mentions: Notably, 3 genes encoding putative pyruvate oxidases are harbored in the completely sequenced genomes of L. rhamnosus GG and L. casei ATCC 334, whereas 4 and 5 pox genes were retrieved in the genome sequences of L. buchneri CD034 and L. plantarum WCFS1, respectively. Goffin et al. [36] reported that among the predicted pox genes encoded in the L. plantarum lp80 genome, only poxB and poxF appeared to be involved in the generation of acetate from lactate during the stationary phase of aerobic growth. Interestingly, poxB and poxF genes shared 63 and 61% amino acid similarity with TDF 93, respectively. To date, only one gene potentially encoding for pyruvate oxidase has been located in the complete genome sequences of the SLAB L. helveticus R0052 and L. delbrueckii subsp. bulgaricus ATCC 11842. The pyruvate oxidase gene of L. rhamnosus GG with the highest homology to TDF 93 is flanked by genes whose order and transcriptional orientation are partially shared with L. casei ATCC 334 but not with L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. bulgaricus ATCC 11842 and L. brevis ATCC 367 (Figure 3A). In particular, spxB locus in L. rhamnosus and L. casei genomes is preceded by three genes encoding putative hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and acetyl-CoA acetyltransferase. These enzymes are known to be involved in the mevalonate pathway, routing acetyl-CoA towards isoprenoid biosynthesis. However, whether these proteins are actually expressed in L. rhamnosus and play a role in deviating the flow of acetyl-CoA from the acetate production via PTA and ACK during cheese ripening still remain to be determined. According to PePPER, spxB gene from L. rhamnosus GG was predicted to be monocistronically transcribed. Phylogenetic tree showed a clear segregation of putative pyruvate oxidases from L. casei group (Figure 4A). As expected, a subgroup was represented by POX proteins from the SLAB L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. L. plantarum and L. pentosus homologues clustered together and close to L. buchneri. Multiple sequence alignment of TDF 93 and pyruvate oxidase protein sequences from several NSLAB and SLAB is shown in Additional file 1: Figure S1A.

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