Journal: Journal of Bacteriology

Article Title: Enantioselective Regulation of Lactate Racemization by LarR in Lactobacillus plantarum

doi: 10.1128/JB.02192-14

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: TABLE 1 Strain or plasmid Characteristic(s) a Source or reference L. plantarum strains NCIMB8826 Wild type NCIMB b TF101 NCIMB8826 Δ ldhL 27 LR0002 NCIMB8826 Δ ldhL Δ larR nisRK This study L. lactis NZ3900 MG1363 derivative 19 E. coli TOP10 F − mcrA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 nupG recA1 araD 139 Δ( ara-leu ) 7697 galE15 galK16 rpsL (Str r ) endA1 λ − Invitrogen Plasmids pMEC10 Em r Amp r ; pMC1 derivative for integration of the nisRK genes at tRNA Ser 28 pUC18Ery Em r Amp r ; pUC18 derivative with a 1.1-kb insert containing the erm gene 17 pGIZ850 Em r Amp r Cm r ; pUC18Ery with a 4.215-kb insert containing the loxD gene disrupted by a cat gene under the control of the P32 promoter 26 pNZ5348 Em r ; pGID023 derivative containing cre under the control of the lp_1144 promoter 23 pGIR002 Em r Amp r ; pUC18Ery with a 4.32-kb insert containing the lox66 -P32- cat-lox71 cassette from pNZ5319 surrounded by the larR upstream and downstream regions This study pBADHisA Amp r P araB Invitrogen pGIR991 Amp r ; pBADHisA with a 0.82-kb insert containing the P araB -StrepII- larR translational fusion This study pNZ8048 Cm r P nisA 20 pGIR090 Cm r ; pNZ8048 derivative with a 0.78-kb insert containing the P nisA -larR translational fusion This study pGIR091 Cm r ; pNZ8048 derivative with a 0.82-kb insert containing the P nisA -StrepII- larR translational fusion This study pSIP409 Em r ; low-copy-no. gusA reporter plasmid 18 pGIR003 Em r ; pSIP409 derivative with the P larA -gusA translational fusion This study pGIR003B pGIR003 harboring one point mutation in the Lar box (mutation RRb) This study pGIR003C pGIR003 harboring one point mutation in the half-Lar box 1 (P larA side 1, mutation RRc) This study pGIR003D pGIR003 harboring two point mutations in half-Lar boxes 1 and 2 (P larA side 1 and 2, mutations RRc and RRd) This study Open in a separate window a Em r , Amp r , and Cm r indicate resistance to erythromycin, ampicillin, and chloramphenicol, respectively. b NCIMB, National Collections of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland.

Techniques: Plasmid Preparation, Control, Mutagenesis

Journal: Journal of Bacteriology

Article Title: Enantioselective Regulation of Lactate Racemization by LarR in Lactobacillus plantarum

doi: 10.1128/JB.02192-14

Figure Lengend Snippet: Mutagenesis of the larR-larA intergenic region. (A) Native (WT) and mutated DNA sequences (RRb, RRc, and RRc+RRd) of the region, including the Lar box and the −35 box of PlarA. The inverted repeat of the Lar box (black arrows) and the two half-Lar boxes on the PlarA side (gray arrows) are shown below the sequence. The mutations in the right repeats are highlighted in black. The arrow corresponding to a mutated right repeat was removed to indicate its alteration. (B) EMSA of 127-bp 32P-radiolabeled probes of the wild type (WT) and mutated larR-larA intergenic regions of L. plantarum in the presence of increasing amounts of purified rLarR. The lower-molecular-weight rLarR-DNA complex (C1), unbound probe (P), and high-molecular-weight complexes C2 and C3 (C2+3) are indicated. (C) Relative abundances of P, C1, and C2+3 in EMSA such as that shown in panel B. Data are from 4 replicates run in parallel on the same gel from one representative experiment. The error bars represent the standard deviations. (D) Ratios of C2+3 to C1 at 30 pmol of LarR in EMSA such as that shown in panel B. Data are from 4 replicates run in parallel on the same gel from one representative experiment. The error bars represent the standard deviations. Significance of the results was determined by Student's t test: **, P < 0.01. (E) Gus activities measured with the mutated larA promoters after induction by 200 mM dl-Lac or l-Lac. ND, not detected (<0.1 mU). The values shown are the mean results of 3 repetitions from 1 significant experiment out of 2 experiments showing similar results. The error bars give the confidence intervals at 95% (Student's t test).

Article Snippet: TABLE 1 Strain or plasmid Characteristic(s) a Source or reference L. plantarum strains NCIMB8826 Wild type NCIMB b TF101 NCIMB8826 Δ ldhL 27 LR0002 NCIMB8826 Δ ldhL Δ larR nisRK This study L. lactis NZ3900 MG1363 derivative 19 E. coli TOP10 F − mcrA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 nupG recA1 araD 139 Δ( ara-leu ) 7697 galE15 galK16 rpsL (Str r ) endA1 λ − Invitrogen Plasmids pMEC10 Em r Amp r ; pMC1 derivative for integration of the nisRK genes at tRNA Ser 28 pUC18Ery Em r Amp r ; pUC18 derivative with a 1.1-kb insert containing the erm gene 17 pGIZ850 Em r Amp r Cm r ; pUC18Ery with a 4.215-kb insert containing the loxD gene disrupted by a cat gene under the control of the P32 promoter 26 pNZ5348 Em r ; pGID023 derivative containing cre under the control of the lp_1144 promoter 23 pGIR002 Em r Amp r ; pUC18Ery with a 4.32-kb insert containing the lox66 -P32- cat-lox71 cassette from pNZ5319 surrounded by the larR upstream and downstream regions This study pBADHisA Amp r P araB Invitrogen pGIR991 Amp r ; pBADHisA with a 0.82-kb insert containing the P araB -StrepII- larR translational fusion This study pNZ8048 Cm r P nisA 20 pGIR090 Cm r ; pNZ8048 derivative with a 0.78-kb insert containing the P nisA -larR translational fusion This study pGIR091 Cm r ; pNZ8048 derivative with a 0.82-kb insert containing the P nisA -StrepII- larR translational fusion This study pSIP409 Em r ; low-copy-no. gusA reporter plasmid 18 pGIR003 Em r ; pSIP409 derivative with the P larA -gusA translational fusion This study pGIR003B pGIR003 harboring one point mutation in the Lar box (mutation RRb) This study pGIR003C pGIR003 harboring one point mutation in the half-Lar box 1 (P larA side 1, mutation RRc) This study pGIR003D pGIR003 harboring two point mutations in half-Lar boxes 1 and 2 (P larA side 1 and 2, mutations RRc and RRd) This study Open in a separate window a Em r , Amp r , and Cm r indicate resistance to erythromycin, ampicillin, and chloramphenicol, respectively. b NCIMB, National Collections of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland.

Techniques: Mutagenesis, Sequencing, Purification, Molecular Weight, High Molecular Weight

Journal: Clinical and Experimental Gastroenterology

Article Title: Quantification of Intestinal Lactobacillus Species in Children with Functional Constipation by Quantitative Real-Time PCR

doi: 10.2147/CEG.S250755

Figure Lengend Snippet: Specificity of the L. Casei Primer Pair Used in This Study

Article Snippet: Seven Lactobacillus reference strains ( L. casei ATCC 39392, L. paracasei ATCC 25598, L. rhamnosus ATCC 7469, L. plantarum ATCC 8014, L. reuteri ATCC 23272, L. fermentum ATCC 9338, L. acidophilus ATCC 4356) used to validate the assays in the study were ordered from the Iranian Research Organization for Science and Technology (IROST, Iran).

Techniques: Real-time Polymerase Chain Reaction

Journal: Clinical and Experimental Gastroenterology

Article Title: Quantification of Intestinal Lactobacillus Species in Children with Functional Constipation by Quantitative Real-Time PCR

doi: 10.2147/CEG.S250755

Figure Lengend Snippet: Prevalence of Lactobacillus Species, Detected by Quantitative PCR

Article Snippet: Seven Lactobacillus reference strains ( L. casei ATCC 39392, L. paracasei ATCC 25598, L. rhamnosus ATCC 7469, L. plantarum ATCC 8014, L. reuteri ATCC 23272, L. fermentum ATCC 9338, L. acidophilus ATCC 4356) used to validate the assays in the study were ordered from the Iranian Research Organization for Science and Technology (IROST, Iran).

Techniques:

Journal: Clinical and Experimental Gastroenterology

Article Title: Quantification of Intestinal Lactobacillus Species in Children with Functional Constipation by Quantitative Real-Time PCR

doi: 10.2147/CEG.S250755

Figure Lengend Snippet: Quantity of seven Lactobacillus species in the feces of constipated children and healthy controls. Statistical significance of observed differences in the amount of Lactobacillus species between both constipated and healthy groups was measured by the Mann–Whitney U -test. Bars represent standard errors. P < 0.05 was marked with one asterisk (∗), and P < 0.2 with two asterisks (∗∗). One-way ANOVA testing shows the significant differences in quantity (log 10 CFU/gram) between Lactobacillus species.

Article Snippet: Seven Lactobacillus reference strains ( L. casei ATCC 39392, L. paracasei ATCC 25598, L. rhamnosus ATCC 7469, L. plantarum ATCC 8014, L. reuteri ATCC 23272, L. fermentum ATCC 9338, L. acidophilus ATCC 4356) used to validate the assays in the study were ordered from the Iranian Research Organization for Science and Technology (IROST, Iran).

Techniques: MANN-WHITNEY

Journal: Frontiers in Microbiology

Article Title: Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1

doi: 10.3389/fmicb.2020.01822

Figure Lengend Snippet: Bacterial strains, plasmids, and primers used in this study.

Article Snippet: For peptide identification, the protein reference database of Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) for peptides and proteins identification downloaded from UniProt was used.

Techniques: Clone Assay, Acetylene Reduction Assay, Mutagenesis, Plasmid Preparation, Sequencing

Journal: Frontiers in Microbiology

Article Title: Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1

doi: 10.3389/fmicb.2020.01822

Figure Lengend Snippet: Secreted proteins extracted from L. plantarum WCFS1 (WT) and its lgt deletion derivative (Δ lgt ; NZ3565Cm). Proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. On the right side, the protein size marker (M), Precision Plus Protein Dual Color Standards (Bio-Rad).

Article Snippet: For peptide identification, the protein reference database of Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) for peptides and proteins identification downloaded from UniProt was used.

Techniques: SDS Page, Staining, Marker

Journal: Frontiers in Microbiology

Article Title: Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1

doi: 10.3389/fmicb.2020.01822

Figure Lengend Snippet: Relative abundance of secreted proteins in L. plantarum WCFS1 wild*type (WT) and the lgt deletion strain (Δ lgt ; NZ3565Cm). The relative abundance is defined as the ratios in the label free quantitation (LFQ, log 10 value) of detected proteins in wild-type and the deletion strain in tandem mass spectrometry (MS/MS) analysis. The abundance was compared with all proteins detected in both samples, or within specific protein groups. The ratios from specific proteins groups were compared against all proteins to test for significant differences using one-way ANOVA followed by Tukey’s multiple comparison correction and significant differences are indicated; *** P ≤ 0.001.

Article Snippet: For peptide identification, the protein reference database of Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) for peptides and proteins identification downloaded from UniProt was used.

Techniques: Quantitation Assay, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Frontiers in Microbiology

Article Title: Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1

doi: 10.3389/fmicb.2020.01822

Figure Lengend Snippet: TLR1_2 and TLR2_6 signaling capacities of NZ3400Cm, a L. plantarum WCFS1 derivative with a chromosomal integration of the cat cassette in a neutral chromosomal locus (NZ3400Cm, ) and the lgt deletion mutant, NZ3565Cm (Δ lgt ; NZ3565Cm). TLR1_2 (A) and TLR2_6 (B) activation were determined using TLR-expressing HEK cell lines, containing a NF-kB responsive luciferase reporter system. Measurements were performed in triplicate and are presented as log 10 luminescence units, and individually displayed ( n = 3), while the bar indicates the median. PBS serves as a negative control, while Pam3CysSK4 (Pam3) and Pam2CysSK4 (Pam2) are the positive stimulus of TLR1_2 (A) and TLR2_6 (B) activation, respectively. Data comparison of the wild-type and the deletion derivative was tested for significant differences using one-way ANOVA followed by Tukey’s multiple comparison correction and samples with significant different NF-kB activation are indicated with different letters.

Article Snippet: For peptide identification, the protein reference database of Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) for peptides and proteins identification downloaded from UniProt was used.

Techniques: Mutagenesis, Activation Assay, Expressing, Luciferase, Negative Control

Journal: Frontiers in Microbiology

Article Title: Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1

doi: 10.3389/fmicb.2020.01822

Figure Lengend Snippet: Immunomodulatory effect of NZ3400Cm, a L. plantarum WCFS1 derivative with a chromosomal integration of the cat cassette in a neutral chromosomal locus (NZ3400Cm, ), and the lgt deletion strain NZ3565Cm (Δ lgt ; NZ3565Cm). Cytokine production was determined in human PBMCs ( n = 3 donors) after 24 h co-incubation with the bacterial cells. The IL12 (A) , TNFα (B) , IL10 (C) , IL1β (D) , IL8 (E) , and IL6 (F) cytokine production levels are presented as Log values. The cytokine levels for individual donors stimulated with the strains were connected by a line to focus the read-outs on changes elicited by the deletion. Significant differences between cytokine levels induced by wild-type strains and their corresponding mutants (paired t -test) are indicated; * P ≤ 0.05; ** P ≤ 0.01; the P value of the difference of IL10 production by the NZ3400Cm and Δ lgt strains is indicated in the corresponding panel (C) .

Article Snippet: For peptide identification, the protein reference database of Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) for peptides and proteins identification downloaded from UniProt was used.

Techniques: Incubation