Journal: Journal of Oral Microbiology

Article Title: Streptococcus tigurinus is frequent among gtfR -negative Streptococcus oralis isolates and in the human oral cavity, but highly virulent strains are uncommon

doi: 10.1080/20002297.2017.1307079

Figure Lengend Snippet: Primers and polymerase chain reaction conditions used in this study.

Article Snippet: DNA was extracted from S. oralis SN 16495, SN 17127, SN 28194, SN 31376, SN 37569, SN 37737, SN 39325, SN 40525, SN 45448, SN 48861, SN 50746, SN 51446, SN 54788, SN 58364, SN 58746, SN 59433, SN 60579, SN 63707, S. infantis SN 54787, SN 57625, SN 17128, SN 19640, ‘S. tigurinus’ SN 62386 (all proven and epidemiologically unrelated endocarditis isolates collected by the National Reference Center for Streptococci, Aachen), and from four S. tigurinus reference strains, including type strain AZ_3a, as well as small colony variants (2425, 2426) of parental strain 1366 [ ] kindly provided by A. Zbinden (Zürich).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Applied Microbiology and Biotechnology

Article Title: Comparative genomics of Streptococcus parauberis : new target for molecular identification of serotype III

doi: 10.1007/s00253-020-10683-z

Figure Lengend Snippet: Multiplex PCR products of S. parauberis strains. Lanes: MM, Generuler DNA ladder 1 kb (Fermentas); 1, (subserotype Ia); 2, subserotype Ib; 3, subserotype Ic; 4, serotype II; 5, serotype III; 6 and 7, non-typeable strains; 8, NCDO2020 (serotype IV)

Article Snippet: DNA isolated from pure cultures of the reference strain S. parauberis NCIMB 703043, which belongs to serotype III, was used as a positive control in qPCR reactions.

Techniques: Multiplex Assay

Journal: Applied Microbiology and Biotechnology

Article Title: Comparative genomics of Streptococcus parauberis : new target for molecular identification of serotype III

doi: 10.1007/s00253-020-10683-z

Figure Lengend Snippet: Determination of C q values obtained from the amplification of tenfold dilutions of a purified amplicon of the cps3K gene of the strain NCIMB 703043 obtained by qPCR

Article Snippet: DNA isolated from pure cultures of the reference strain S. parauberis NCIMB 703043, which belongs to serotype III, was used as a positive control in qPCR reactions.

Techniques: Amplification, Purification, Concentration Assay, Intra Assay, Inter Assay

Journal: Applied Microbiology and Biotechnology

Article Title: Comparative genomics of Streptococcus parauberis : new target for molecular identification of serotype III

doi: 10.1007/s00253-020-10683-z

Figure Lengend Snippet: Standard curves obtained by qPCR from the amplification of 10-fold dilutions of purified amplicon obtained using SP3-130F and SP3-130R primers and DNA from the reference strain S. parauberis NCIMB 703043 (black circles) and a clinical isolate of S. parauberis (SK451/04) (gray squares) ( a ) and the corresponding electrophoresis gel showing the band intensities correlating with the number of copies of the cps3K gene ( b ). Lanes: MM, Generuler DNA ladder 1-kb (Fermentas); 1, 2.67 × 10 8 copies; 2, 2.67 × 10 7 copies; 3, 2.67 × 10 6 copies; 4, 2.67 × 10 5 copies; 5, 2.67 × 10 4 copies; 6, 2.67 × 10 3 copies; 7, 2.67 × 10 2 copies

Article Snippet: DNA isolated from pure cultures of the reference strain S. parauberis NCIMB 703043, which belongs to serotype III, was used as a positive control in qPCR reactions.

Techniques: Amplification, Purification, Electrophoresis

Journal: Applied Microbiology and Biotechnology

Article Title: Comparative genomics of Streptococcus parauberis : new target for molecular identification of serotype III

doi: 10.1007/s00253-020-10683-z

Figure Lengend Snippet: Samples tested by qPCR for the detection of S. parauberis serotype III

Article Snippet: DNA isolated from pure cultures of the reference strain S. parauberis NCIMB 703043, which belongs to serotype III, was used as a positive control in qPCR reactions.

Techniques: Concentration Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates

doi: 10.3389/fcimb.2023.1177857

Figure Lengend Snippet: S. pneumoniae strains and oligonucleotide primers used in this study.

Article Snippet: The wildtype S. pneumoniae strains used in this study ( ) were provided to us by the German National Reference Center for Streptococci (Aachen) ( ).

Techniques: Sequencing, Mutagenesis

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates

doi: 10.3389/fcimb.2023.1177857

Figure Lengend Snippet: spnIII allele frequencies of 60B and 60CSF recovered post intranasal murine infection. Mice were intranasally challenged with 10 8 CFU S. pneumoniae serotype 15C ST8711 blood isolate (60B) or CSF isolate (60CSF). At 24 h, mice from each group were humanely euthanized and pneumococci in the nasal tissue were harvested. spnIII allele quantification was performed on DNA extracted from the original inoculum and colonies grown from nasopharynx samples. Allele percentages for original inoculum and bacteria recovered from noses are represented. Statistical analysis was performed using two-way analysis of variance and a Tukey post-comparison test; *P < 0.05.

Article Snippet: The wildtype S. pneumoniae strains used in this study ( ) were provided to us by the German National Reference Center for Streptococci (Aachen) ( ).

Techniques: Infection, Bacteria, Comparison

Journal: eLife

Article Title: An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis

doi: 10.7554/eLife.57322

Figure Lengend Snippet: ( A ) Schematic diagram of the isd operon of S. lugdunensis N920143. Coding sequences, direction of transcription and Fur-binding sites are indicated. ABC membrane-transporters are shown in green. lhaS - SLUG_00900; lhaT - SLUG_00910; lhaA - SLUG_00920 ( B ) Iron-regulated expression of Lha: S. lugdunensis was grown overnight in TSB, TSB + 200 µM EDDHA or TSB + 200 µM EDDHA + 200 µM FeSO 4 . Gene expression was quantified by qPCR. Expression was normalized to 5srRNA and to the TSB standard condition using the ΔΔCt method. Fold differences in gene expression are shown. Data represent mean and SD of four independent experiments. Statistical evaluation was performed using students unpaired t-test (lhaS: t = 7,045, df = 6; lhaA: t = 2,979, df = 6) C/D Growth curves of S. lugdunensis N920143 and isogenic mutants. The wild type (WT) S. lugdunensis N920143 strain and the indicated isogenic null mutant strains were grown in the presence of 20 µM FeSO 4 ( C ) or 150 nM heme ( D ) as a sole source of iron. 500 µl of bacterial cultures were inoculated to an OD 600 = 0,05 in 48 well plates and OD 600 was monitored every 15 min using an Epoch1 plate reader. For reasons of clarity values taken every 5 hr are displayed. Mean and SD of three experiments are shown. Statistical analysis was performed using one-way ANOVA followed by Dunett’s test for multiple comparisons. * - p<0,05, ****p<0,00001.

Article Snippet: Strain, strain background ( S. lugdunensis ) , N920143 , National Reference Center for Staphylococci, Lyon, France ( ) , , .

Techniques: Binding Assay, Membrane, Expressing, Gene Expression, Mutagenesis

Journal: eLife

Article Title: An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis

doi: 10.7554/eLife.57322

Figure Lengend Snippet: ( A/B ) Proliferation of S. lugdunensis N920143 ∆ isd pRB473 and ∆ isd pRB473: lhaSTA strains. The indicated strains were grown in the absence of nutritional iron ( A ) or in the presence of 20 µM FeSO 4 ( B ). 500 µl of cultures were inoculated to an OD 600 = 0,05 in 48 well plates and OD 600 was monitored every 15 min using a Epoch1 plate reader. For reasons of clarity values taken every 5 hr are displayed. Mean and SD of three experiments are shown. Statistical analysis was performed using students unpaired t-test.

Article Snippet: Strain, strain background ( S. lugdunensis ) , N920143 , National Reference Center for Staphylococci, Lyon, France ( ) , , .

Techniques:

Journal: eLife

Article Title: An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis

doi: 10.7554/eLife.57322

Figure Lengend Snippet: ( A ) LhaSTA-dependent proliferation. S. lugdunensis N920143 deletion mutant strains lacking the entire isd operon and expressing LhaSTA (∆ isd pRB473: lhaSTA ) or not (∆ isd pRB473) from the plasmid pRB473 were grown in the presence of 200 nM heme as a sole source of iron. 500 µl of cultures were inoculated to an OD 600 = 0,05 in 48 well plates and OD 600 was monitored every 15 min using an Epoch1 plate reader. For reasons of clarity values taken every 5 hr are displayed. Mean and SD of three experiments are shown. Statistical analysis was performed using students unpaired t-test. ***p<0,0001 ( B ) Intracellular accumulation of iron. Strains were grown in iron limited medium to OD 600 = 0,6 and 5 µM heme were added for 3 hr. Cell fractionation of 1 ml OD 600 = 50 was performed and the iron content of the cytosolic fraction was determined using the ferrozine assay. Data represent the mean and SD of three independent experiments. Statistical analysis was performed using students unpaired t-test (t = 5,12729, df = 4).

Article Snippet: Strain, strain background ( S. lugdunensis ) , N920143 , National Reference Center for Staphylococci, Lyon, France ( ) , , .

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Cell Fractionation, Ferrozine Assay

Journal: eLife

Article Title: An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis

doi: 10.7554/eLife.57322

Figure Lengend Snippet: ( A ) Schematic diagram of known heme acquisition systems in the S. lugdunensis mutant strains lacking either the genes encoding LhaSTA ( ∆lhaSTA , left) or the entire isd operon and expressing LhaSTA from the plasmid pRB473 ( ∆isd pRB473: lhaSTA ). ABC membrane transporters are shown in green. Cell wall-anchored proteins of the Isd-system are shown in yellow. Heme/hemoglobin-binding NEAT motifs within each protein are indicated as black boxes. Black arrows indicate the transfer of heme. he: heme; hb: hemoglobin; PG: peptidoglycan. ( B ) Growth of S. lugdunensis N920143 wild type (WT) and ∆ lhaSTA . Strains were grown in the presence of 20 µM FeSO 4 or 2.5 µg/ml human hemoglobin (hHb) or 10 µg/ml equine myoglobin (eqMb) or 117 nM hemoglobin-haptoglobin complex (Hb-Hap) as a sole source of iron. 500 µl of cultures were inoculated to an OD 600 = 0,05 in 48 well plates and OD 600 was measured after 30 hr using an Epoch1 plate reader. Mean and SD of three experiments are shown. Statistical analysis was performed using students unpaired t-test. hHb - t = 6,0007, df = 4; eqMb – t = 20,52, df = 4; Hb-Hap – t = 1,978, df = 4. ( C ) Growth of S. lugdunensis N920143 ∆ isd pRB473 and ∆ isd pRB473: lhaSTA . Strains were grown in the presence of 20 µM FeSO 4 or 2.5 µg/ml hHb or 2.5 µg/ml murine hemoglobin (mHb) or 10 µg/ml human myoglobin (hMb) or 10 µg/ml eqMb or 200 nM human hemopexin (Hpx) or 117 nM Hb-Hap as a sole source of iron. 500 µl of cultures were inoculated to an OD 600 = 0,05 in 48 well plates and OD 600 was measured after 30 hr using an Epoch1 plate reader. Mean and SD of three experiments are shown. Statistical analysis was performed using students unpaired t-test hHb – t = 18,5, df = 4; mHb – t = 29,03, df = 4; hMb – t = 25,98, df = 4; eqMb – t = 3,442, df = 4; Hpx – t = 77,12 df = 4; Hb-Hap t = 2758 df = 4. ( D ) TMBZ-H 2 O 2 stain of TGX gels for heme-associated peroxidase activity. Membrane vesicles were saturated with excess of hemoprotein (5.6 µM heme, 476 µg/ml hHb, 437 µg/ml eqMb, 5.6 µM Hpx, 476 µg/ml Hb-Hap) or no hemoprotein (-) for 10 min at RT. LhaS was purified, 15 µg protein was loaded on a TGX gel and stained for peroxidase activity with TMBZ-H 2 O 2 (upper panel). Gels were destained and subsequently stained with BlueSafe (lower panel) to confirm the presence of the protein in all conditions.

Article Snippet: Strain, strain background ( S. lugdunensis ) , N920143 , National Reference Center for Staphylococci, Lyon, France ( ) , , .

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Membrane, Binding Assay, Staining, Activity Assay, Purification

Journal: eLife

Article Title: An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis

doi: 10.7554/eLife.57322

Figure Lengend Snippet:

Article Snippet: Strain, strain background ( S. lugdunensis ) , N920143 , National Reference Center for Staphylococci, Lyon, France ( ) , , .

Techniques: Mutagenesis, Recombinant, Expressing, Plasmid Preparation, Protein Purification