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Molecular and physiological adaptations of Lactococcus lactis at near-zero growth rates

ABSTRACT: This paper describes the molecular and physiological adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. Metabolic and transcriptomic analyses revealed that metabolic patterns shifted between homolactic and mixed-acid fermentation during the retentostat cultivation, which appeared to be controlled at the transcription level of the corresponding pyruvate-dissipation enzyme pathway encoding genes. Furthermore, during extended retentostat cultivation, cells continued to consume several amino acids, but also produced specific amino acids subsets, which may derive from the conversion of glycolytic intermediates. Under conditions of extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly up-regulated, which was confirmed by enhanced initial acidification rates on various sugar substrates in cells obtained from near-zero growth cultures. Moreover, the expression of genes involved in multiple stress response mechanisms was gradually induced during extended retentostat cultivation, supporting the strong molecular focus on maintenance of cellular function and viability. The present integrated transcriptome and metabolome study provides molecular understanding of the adaptation of Lactococcus lactis KF147 to near-zero growth rate conditions, and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates. loop design of the samples including two shortcuts

ORGANISM(S): Lactococcus lactis

SUBMITTER: Michiel Wels

PROVIDER: E-GEOD-51494 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Molecular and physiological adaptations of Lactococcus lactis at near-zero growth rates

Project description:This paper describes the molecular and physiological adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. Metabolic and transcriptomic analyses revealed that metabolic patterns shifted between homolactic and mixed-acid fermentation during the retentostat cultivation, which appeared to be controlled at the transcription level of the corresponding pyruvate-dissipation enzyme pathway encoding genes. Furthermore, during extended retentostat cultivation, cells continued to consume several amino acids, but also produced specific amino acids subsets, which may derive from the conversion of glycolytic intermediates. Under conditions of extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly up-regulated, which was confirmed by enhanced initial acidification rates on various sugar substrates in cells obtained from near-zero growth cultures. Moreover, the expression of genes involved in multiple stress response mechanisms was gradually induced during extended retentostat cultivation, supporting the strong molecular focus on maintenance of cellular function and viability. The present integrated transcriptome and metabolome study provides molecular understanding of the adaptation of Lactococcus lactis KF147 to near-zero growth rate conditions, and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates.

2014-01-01 | GSE51494 | GEO

pBL1 impairs cellobiose metabolism in Lactococcus lactis

Project description:pBL1 is a Lactococcus lactis theta-replicating 10.9-kbp plasmid that encodes the synthetic machinery of the bacteriocin Lcn972. In this work, the transcriptomes of exponentially growing L. lactis with and without pBL1 were compared. A discrete response was observed with a total of ten genes showing significantly changed expression. Up-regulation of the lactococcal oligopeptide uptake system (opp) was observed, likely linked to a higher nitrogen demand required for Lcn972 biosynthesis. Striking, celB coding for the membrane porter IIC of the cellobiose-PTS and the upstream gene llmg0186 were down-regulated. Growth profiles for L. lactis strains MG1363, MG1363/pBL1 and MG1363ΔcelB grown in CDM-cellobiose confirmed slower growth of pBL1 and ΔcelB while no differences were scored on glucose. The presence of pBL1 shifted the fermentation products towards a mixed acid profile and promoted substantial changes in intracellular pool sizes for glycolytic intermediates in cellobiose-growing cells as determined by HPLC and NMR. Overall, these data support the genetic evidence of a constriction in cellobiose uptake. Notably, several cell wall precursors accumulated, while other UDP-activated sugars pools were lower, which could reflect rerouting of precursors towards the production of structural or storage polysaccharides. Moreover, slow cellobiose-growing cells and those lacking celB were more tolerant to Lcn972 than cellobiose adapted cells. Thus, down-regulation of celB could help to build-up a response against the antimicrobial activity of Lcn972 enhancing self-immunity of the producer cells. The transcriptomes of Lactococcus lactis MG1614 with and without the bacteriocinogenic plasmid pBL1, grown under laboratory conditions, were compared using three biological replicates.

2011-09-14 | E-GEOD-30625 | biostudies-arrayexpress

Transcriptional changes associated to resistance to the cell wall active bacteriocin Lcn972

Project description:Lcn972 is a non-modified bacteriocin that targets exclusively Lactococcus sp. Addition of Lcn972 inhibits cell wall biosynthesis at the septum by binding to the cell wall precursor lipid II. Resistance to Lcn972 develops upon selection with subinhibitory concentrations. The transcriptome of the highly resistant L. lactis D1 derived from the susceptible strain L. lactis MG1614 was compared. Fourteen genes were significantly up-regulated and 29 were down-regulated (expression change > 2-fold, p<0.001). Down-regulation was mostly found in sugar catabolic genes. Up-regulated genes included members of the cell envelope stress (CesR) regulon, the penicillin-binding protein pbpX and llmg2447, which may encode a putative extracytoplasmic function (ECF) anti-sigma factor located downstream of a non-functional ECF-sigX. Up-regulation of llmg2447 was linked to integration of IS981 that exchanged the -35 promoter region of llmg2447. Over-expression of llmg2447 resulted in highly Lcn972-resistant L. lactis transformants. The transcriptomes of Lactococcus lactis MG1614 susceptible to Lcn972 and L. lactis D1 resistant to Lcn972, grown under laboratory conditions, were compared using four biological replicates.

2012-12-06 | E-GEOD-38349 | biostudies-arrayexpress

Modulation of Lactobacillus plantarum gastrointestinal robustness by fermentation conditions enables identification of bacterial robustness markers

Project description:Transcriptome profiles of control Lactobacillus plantarum WCFS1 cells were compared with a lp_1669 deletion mutant in MRS and 2*CDM media. A continuous culture was performed on MRS and 2*CDM media and whole genome transcriptome analysis was perfomred on the WCFS1 and NZ3417CM (harboring a chlooramphenicol resistance marker (cat) replacement mutation in lp_1669) strains.

2012-01-01 | E-GEOD-31254 | biostudies-arrayexpress

The CtsR and HrcA regulon of Lactobacillus plantarum WCFS1 at normal and elevated temperature Nov 01, 2011 approved

Project description:Transcriptome profiles of control Lactobacillus plantarum WCFS1 cells were compared with ctsR, hrcA, and ctsR-hrcA deletion mutants grown in MRS media at 28 and 40 degrees Celcius. Continuous cultures were performed in duplo on MRS media and whole genome transcriptome analysis was performed on the WCFS1 (WT), NZ3410CM (harboring a chlooramphenicol resistance marker (cat) replacement mutation in ctsR, dc), NZ3425CM (harboring a cat replacement mutation in hrcA, dh), and NZ3423 (harboring deletion mutation in ctsR and a cat replacement mutation inhrcA, dcdh) strains.

2012-01-01 | E-GEOD-31253 | biostudies-arrayexpress

The Transcriptional and Gene Regulatory Network of Lactococcus lactis MG1363 during Growth in Milk

Project description:In the present study we examine the changes in the expression of genes of Lactococcus lactis subspecies cremoris MG1363 during growth in milk. To reveal which specific classes of genes (pathways, operons, regulons, COGs) are important, we performed a transcriptome time series experiment. Global analysis of gene expression over time showed that L. lactis adapted quickly to the environmental changes. Using upstream sequences of genes with correlated gene expression profiles, we uncovered a substantial number of putative DNA binding motifs that may be relevant for L. lactis fermentative growth in milk. All available novel and literature-derived data were integrated into network reconstruction building blocks, which were used to reconstruct and visualize the L. lactis gene regulatory network. This network enables easy mining in the chrono-transcriptomics data. A freely available website at http://milkts.molgenrug.nl gives full access to all transcriptome data, to the reconstructed network and to the individual network building blocks. 12 time points

2012-09-12 | E-GEOD-40780 | biostudies-arrayexpress

Cellular responses of Saccharomyces cerevisiae at near-zero growth rates: transcriptome analysis of anaerobic retentostat cultures

Project description:Extremely low specific growth rates (below 0.01 h-1) represent a largely unexplored area of microbial physiology. Retentostats enable controlled, energy-limited cultivation at near-zero specific growth rates while avoiding starvation. In this study, anaerobic, glucose-limited retentostats were used to analyze physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. Cultures at near-zero specific growth rates exhibited several characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in storage metabolism, autophagy and exit from the replicative cell cycle into G0. Analysis of transcriptome data from glucose-limited retentostat and chemostat cultures showed, as specific growth rate was decreased, quiescence-related transcriptional responses already set in at specific growth rates above 0.025 h-1. Many genes involved in mitochondrial processes were specifically upregulated at near-zero specific growth rates, possibly reflecting an increased turn-over of organelles under these conditions. Prolonged (> 2 weeks) cultivation in retentostat cultures led to induction of several genes that were previously implicated in chronological ageing. These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal. Independent duplicate retentostat cultures were subjected to microarray analysis at four time points after switching the effluent line to the filter unit (2, 9, 16 and 22 d). Microarray analysis of independent, triplicate anaerobic glucose-limited chemostat cultures grown at a specific growth rate of 0.025 h-1 (t = 0) were also performed as part of this study, resulting in a dataset of 11 arrays.

2011-08-22 | E-GEOD-22574 | biostudies-arrayexpress

Cellular responses of Saccharomyces cerevisiae at near-zero growth rates: transcriptome analysis of anaerobic retentostat cultures

2011-08-23 | GSE22574 | GEO

Comparative analysis of bacterial transcriptome using DNA microarray and next generation sequencing technologies

Project description:Validation of Lactobacillus plantarum WCFS1 transcriptome profile using RNA sequencing (direct cDNA sequencing and 3'-UTR sequencing) in comparison with DNA microarray as a reference platform. Transcriptome analysis was performed on total RNA and enriched mRNA of L. plantarum WCFS1 grown in CDM or MRS media. Samples were hybridized in a loop design, hybridizing each sample twice, once for each dye This Series represents the DNA microarray experiments only.

2012-07-09 | E-GEOD-35754 | biostudies-arrayexpress

tagF1-F2 and dlt mutant study

Project description:Several strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of specific cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in probiotic-host interactions remains obscure. Through genome mining and mutagenesis we constructed derivatives of L. plantarum WCFS1 that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. Transcriptome analysis revealed the genetic determinants involved in backbone switching. Human dendritic cells secreted drastically decreased levels of pro-inflammatory cytokines after stimulation with the WTA mutants, and indicated LTA contributes to TLR-2/6 signalling, whereas WTA attenuates TLR-2 and TLR-1/2 signalling in a backbone-alditol dependent manner. Overall, the engineering of WTA and its consequences for immune system interaction advances our molecular understanding of host-microbe communication, and underpins the strain-specificity of probiotics. All samples were hybridized twice (each dye once) in a triangular design, hybridizing all samples

2011-05-01 | E-GEOD-27683 | biostudies-arrayexpress

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