L. plantarum grown in a variety of different fermentation conditions
ABSTRACT: Lactobacillus plantarum WCFS1 was differentially fermented in 29 different fermentations according to a factorial, combinatorial scheme that included variations in growth temperature (28 or 37C), NaCl concentration (0 or 0.3M), pH (5.2, 5.8, 6.4), as well as oxygen (N2 or air) and amino acid availability (1.1 or 2x concentration). Samples were hybridized in a star design using five samples in the center all connected to each other. The remaining samples were linked (evenly distributed) to the samples in the inner circle in a loop design with additional links (shortcuts) to the center sample. All samples were hybridized in duplicate (at minimum) using each of the two dyes (Cy3/Cy5) at least once per sample.
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
Project description:Sortases are transpeptidase enzymes that couple surface proteins to the peptidoglycan of Gram-positive bacteria. Several sortase-dependent proteins (SDPs) have been identified that are crucial for bacterial pathogenesis and, although less frequently, for the physiology of non-pathogenic bacteria. We found that an isogenic sortase A (srtA) deletion derivative (NZ7104) of Lactobacillus plantarum WCFS1 did not express any residual SrtA activity, i.e. failed to cleave the LPQTDE SrtA recognition motif. Trypsination of intact bacterial cells, followed by mass spectrometry based peptide identification, revealed a significant decrease, but not complete loss of SDPs on the NZ7104 cell surface as compared to the wildtype. Using LiCl, we further found that several SDPs could be extracted from the cell surface of NZ7104 but not from the wildtype, demonstrating that SrtA is involved in the covalent coupling of these SDPs. Neither the gastrointestinal persistence of L. plantarum in mice, nor the cytokine secretion patterns induced in monocyte derived immature dendritic cells (iDCs) was significantly affected by the srtA deletion. However, contrary to the wild-type cells, LiCl washed NZ7104 cells induced drastically increased proinflammatory cytokine production in iDCs, indicating a role of the SDPs in attenuation of immune system stimulation. array were constructed in two subdesigns: one comparing all samples from logarithmic phase (WT vs mutant) and one comparing the stationary phase samples (WT vs mutant)
Project description:A mutant of L. plantarumWCFS1 (deletion of lp_2991) was compared with the wildtype grown in standard MRS broth. Cells were sampled at OD1 for mRNA extraction. Knockout vs wildtype. Technical replicates (same mRNA isolation) used for a dye swap.
Project description:The effect of nitrate reduction (anaerobic cultivation in the presence of heme, vitamin K2 and nitrate) was compared with anaerobic cultivation supplemented with citrate (Lactobacillus plantarum). The medium was chemically defined medium with mannitol as main carbon source Two-condition experiment, nitrate vs citrate reducing cells. Biological replicates: 4 nitrate reducing cultures, 4 citrate reducing cultures, independently grown and harvested. Two slides were used, each slide contained 8 Arrays. Citrate reducing cultures are called reactor 1-4, Nitrate reducing cultures are called reactor A-D
Project description:The effect of respiration (aerobic cultivation in the presence of heme and vitamin K2) was compared with unsupplemented aerobic cultivation with Lactobacillus plantarum. Two-condition experiment, aerobic vs respiring cells. Biological replicates: 3 aerobic cultures, 3 respiring cultures, independently grown and harvested. One replicate per array. Respiring cultures are called reactor 1-3; Aerobic cultures are called reactor 4-6 In the subsequent analysis data from reactor 4 were not used. There was likely a mistake made during quenching. This was concluded as new labeling/hybridisation gave same (bad) results (128a); slide 128b was dye-swap.
Project description:Specific Lactobacillus strains are marketed as probiotics i.e. health promoting organisms. As previously shown in vivo, L. plantarum WCFS1 modulates immune responses via nuclear factor (NF)kB in a growth phase dependent manner, likely caused by changed microbial cell surface characteristics. Here, we identified the differential cell-surface proteome composition of L. plantarum WCFS1 cells derived from the mid-logarithmic and late stationary growth phase by surface trypsination of intact bacterial cells, followed by a combined 1D- and 2D-LC-MS shotgun proteomics strategy, and growth phase dependent transcriptome analysis. Overall, these analyses led to the identification of 31 surface proteins differentially present in the logarithmic and stationary phase, whereas additional 17 and 33 proteins appeared solely present in stationary and logarithmic phase of growth, respectively. From 75% of predicted surface related genes displaying relatively high expression levels, the corresponding proteins were detected, demonstrating a high correlation between transcriptome and proteome profiles. The impact of cell-surface peptide fractions on cellular host responses was evaluated using NFkB promoter activation assays in intestinal epithelial cells, revealing that only late stationary phase derived peptides are capable of attenuating flagellin and cell envelope induced NFkB activation. Overall our data mechanistically expand earlier observations that revealed growth-phase dependent immunomodulation by L. plantarum WCFS1, leading to a typical adjuvant- or tolerance-like response after exposure to stationary phase harvested bacterial cells. Sub-fractionation of the late stationary phase peptide fraction revealed a sub-set of strong attenuator peptides able to manipulate NFkB related pathways to hereby attenuate pro-inflammatory signaling. Samples were hybridizedin a loop design linking each timepoint to the next as well as the previous timepoint. Additional hybridizations were introduced linking each sample three timepoints forward and three timepoints back. All samples were hybridized unsing at least each of the two dyes once.
Project description:Lactobacillus plantarum WCFS1 was grown under anaerobic carbon-limited conditions in a chemostat with complete biomass retention (retentostat). In this cultivation system, the biomass concentration progressively increases while the dilution rate is kept constant, resulting in decreased specific susbtrate availibility, and hence, a progressive decrease in the specific growth rate. During the progressive transition from growth to virtually no growth, the global changes occurring at the level of metabolism and gene expression were studied using a genome-scale metabolic model and DNA microarrays. Four different time-points are compared, corresponding to 4 different specific growth rates, and hence, 4 different ratios of energy used for maintenance and growth. The samples taken at the start of retentostat cultivation serves as a a reference sample, to which the three other samples (taken after 3 days, 17 days, and 31 days under retentostat conditions) are compared. No biological replicates: all samples were taken from the same retentostat fermentation.
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. loop design of the samples including two shortcuts
Project description:L. plantarum is known to possess an L-lactate inducible lactate racemase activity (Goffin et al. 2005. J. Bacteriol. 187:6750). In the present study, microarrays were used in order to identify all genes that are up-regulated by L-lactate, but not by a racemic mixture of D- and L-lactate. A mutant of L. plantarum NCIMB8826 deficient for NAD-dependent L-lactate activity (TF101; Ferain et al. 1994. 176:596), and thus producing no L-lactate, was grown in MRS medium at 28°C until mid-exponential phase (OD600nm 0.75). The culture was then divided into 3 sub-cultures. Optically pure sodium L-lactate (200 mM) was added to the first sub-culture (TF101 + L-lac 200 mM). An equimolar mixture of sodium D- and L-lactate (100 mM each) was added to the second sub-culture (TF101 + L/D-lac 200 mM). The third sub-culture was not treated (TF101; reference sample). The three sub-cultures were further incubated at 28°C for 1h30 (a time known to be sufficient for induction of lactate racemase activity by L-lactate). Cells were harvested by centrifugation. Microarray data were used ot identify genes that are specifically induced by L-lactate (comparison of TF101 with TF101 + L-lac 200 mM), but not by DL-lactate (comparison of TF101 with T101 + L/D-lac 200 mM). There are no biological replicates.
Project description:The bacterial cell envelope is a complex, multilayered structure that is not only essential to maintain cellular integrity, but also facilitates vital bacterial processes such as adaption, colonization and adhesion. Cell envelopes comprise a wide range of molecules, such as proteins and capsular polysaccharide (CPS), which collectively decorate the bacterial cell in a species- and strain specific manner. Here we characterized the 4 CPS gene cluster of L. plantarum and assessed the impact on CPS (combinatorial) gene deletions on surface polysaccharide composition. individual KOs of the cps clusters and the combination of all four together were compared with the WT expression in a loop design. The KO of 1-3 was directly compared to the WT (dye swapped)