Journal: Molecular Microbiology

Article Title: Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

doi: 10.1111/j.1365-2958.2010.07472.x

Figure Lengend Snippet: In vitro activity of B. subtilis LtaS-type enzymes. A. Chemical structures of fluorescently labelled NBD-PG and NBD-DAG lipids with known S. aureus LtaS and B. cereus PLC cleavage site indicated by an arrow. B. Coomassie stained gel of purified B. subtilis LtaS-like proteins. Extracellular enzymatic domains of B. subtilis LtaS BS , YfnI, YqgS and YvgJ were purified as N-terminal His-tag fusion proteins and 10 µg purified protein separated on a 10% SDS-PAGE gel and visualized by staining with Coomassie brilliant blue. C. TLC analysis of B. subtilis LtaS BS , YfnI, YvgJ and YqgS in vitro reaction products. The NBD-PG lipid substrate was incubated with eLtaS BS , eYfnI, eYvgJ or eYqgS enzyme. Subsequently, lipids were extracted and separated by TLC and fluorescent lipid bands visualized by scanning plates with a fluorescence imager. As negative and positive controls, reactions were set up without enzyme or with the B. cereus PLC enzyme respectively. Note that only 10% of the PLC reaction was run on the TLC plate. Positions of NBD-PG and presumed NBD-DAG reaction product are indicated on the left and proteins added to each reaction are shown on the top of the panel.

Article Snippet: Briefly, 1.8 ml of 10 mM sodium succinate buffer, pH 6.0, ionic strength (µ) = 50 mM (adjusted with NaCl) was added to 25 µg of TLC-purified NBD-PG lipid (Avanti; order number 810163).

Techniques: In Vitro, Activity Assay, Staining, Purification, SDS Page, Incubation, Fluorescence

Journal: Molecular Microbiology

Article Title: Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

doi: 10.1111/j.1365-2958.2010.07472.x

Figure Lengend Snippet: Kinetic measurements for recombinant LtaS BS , YfnI, YqgS and YvgJ enzymes. A. Time-course experiment. Enzyme reactions were set up as described under Experimental procedures , aliquots removed at the indicated time points and reactions stopped by the addition of chloroform and methanol. Lipids were separated on TLC plates and the NBD-DAG reaction product quantified. For each time point and enzyme the average value and standard deviation of three values is plotted. Three independent experiments were performed and a representative graph is shown. B. Maximal enzyme activity of B. subtilis LtaS BS , YfnI, YqgS and YvgJ. The slope of the linear fit through the first three data points of the curve shown in (A) was used to calculate the maximal enzyme activity for each B. subtilis LtaS orthologue. Three independent time-course experiments were used to determine an average value and standard deviation for the maximal enzyme activity and these values are plotted.

Article Snippet: Briefly, 1.8 ml of 10 mM sodium succinate buffer, pH 6.0, ionic strength (µ) = 50 mM (adjusted with NaCl) was added to 25 µg of TLC-purified NBD-PG lipid (Avanti; order number 810163).

Techniques: Recombinant, Standard Deviation, Activity Assay

Journal: Molecular Microbiology

Article Title: Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

doi: 10.1111/j.1365-2958.2010.07472.x

Figure Lengend Snippet: Metal and substrate specificity of recombinant B. subtilis LtaS-type enzymes. A. B. subtilis LtaS-type enzymes require Mn 2+ for activity. In vitro enzyme assays were set up with NBD-PG lipid as the substrate in the presence of 10 mM MgCl 2 , MnCl 2 , CaCl 2 or ZnCl 2 and reactions were initiated by the addition of eLtaS BS . As controls, reactions were set up without enzyme or without metal ion added. Samples were incubated for 3 h at 37°C, lipids extracted and separated by TLC. Plates were scanned and signals of the reaction product quantified. Reactions were set up in triplicate and the average value and standard deviation plotted. The average fluorescence reading for the reactions set up with MnCl 2 was set to 1 and other values were adjusted accordingly. Similar results were obtained for B. subtilis YfnI, YqgS and YvgJ (see ). B. NBD-PG is the sole lipid substrate for B. subtilis LtaS BS , YfnI, YqgS and YvgJ. Standard enzyme reactions were set up using NBD-PG, NBD-PS, NBD-PE or NBD-PC as substrate (indicated on the left of the panel) and reactions were initiated by the addition of the different B. subtilis enzymes. As a negative control, lipid substrates were incubated without enzyme (no enz.) and as a positive control, a PLC reaction using NBD-PG as substrate was run alongside on each TLC plate in order to determine the mobility of the reaction product. Three independent experiments were performed and a representative result is shown. Note that only the upper part of the TLC plates is shown with the area of the reaction product.

Article Snippet: Briefly, 1.8 ml of 10 mM sodium succinate buffer, pH 6.0, ionic strength (µ) = 50 mM (adjusted with NaCl) was added to 25 µg of TLC-purified NBD-PG lipid (Avanti; order number 810163).

Techniques: Recombinant, Activity Assay, In Vitro, Incubation, Standard Deviation, Fluorescence, Negative Control, Positive Control

Journal: EMBO Molecular Medicine

Article Title: Integrated multi-omics mapping of mitochondrial dysfunction and substrate preference in Barth syndrome cardiac tissue

doi: 10.1038/s44321-025-00320-5

Figure Lengend Snippet: ( A ) Overview of cardiolipin biosynthesis and remodelling via the Tafazzin enzyme, which is dysfunctional in BTHS. ( B ) Relative intensity of CL(72:8), MLCL(50:2) and the ratio between MLCL(50:2) and CL(72:8) in both BTHS ( n = 5) and non-failing donors ( n = 24). Boxplots represent the interquartile range (IQR), with the box spanning the 25th to 75th percentiles and the median indicated by a horizontal line. Whiskers extend to the most extreme data points within 1.5×IQR. Statistical comparisons were performed using moderated t-tests from the limma package in R, with Benjamini–Hochberg correction. Annotated p -values are defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( C ) Volcano plot of CL species, depicting depleted CL species (blue) and accumulated CL species (red) in cardiac tissue of BTHS individuals ( n = 5) compared to non-failing donors ( n = 24). ( D ) Relative intensity of the total MLCL and DLCL classes in BTHS individuals ( n = 5) and non-failing donors ( n = 24). Boxplots are defined as in ( B ). Statistical comparisons were performed using moderated t-tests from the limma package in R, with Benjamini–Hochberg correction. Annotated p -values are defined as in ( B ). Exact p -values corresponding to all statistical comparisons are provided in: Dataset (STATISTICS LIPIDOMICS), Dataset (STATISTICS METABOLOMICS), and Dataset (STATISTICS PROTEOMICS).

Article Snippet: Cardiolipin CL(14:0)4 , Avanti Polar Lipids, Alabaster, AL, USA , 750332.

Techniques: