fluorescent d amino acid hada  (MedChemExpress)

Journal: BMC Microbiology

Article Title: Impairment of two non-essential LytR-cpsA-psr (Lcp) cell wall ligases decelerate cell wall assembly in Mycolicibacterium smegmatis

doi: 10.1186/s12866-026-04722-4

Figure Lengend Snippet: Depletion of AG-PG cross-linkers in ΔlcpBΔlcpC . A Immuno dot blot analysis of the anti-glucan antibody binding to the whole bacterial cell and the ⍺-glucans in capsular polysaccharide extracts. This is a representative of two independent experiments. Full-length blots of the replicates are presented in Supplementary file 2 (Fig. 2A, Experiment-2). B (i) Schematic representation of the EB-A2 Mab binding to the exposed Galf-β-(1→5) units of the AG chain in ΔlcpBΔlcpC mutant. Unlike the presence of numerous branched AG chains linking the PG layer in native bacterium, only one branched chain of AG has been shown here for clarity. Abbreviations: MA – Mycolic acids, AG – arabinogalactan, PG – peptidoglycan, PM – plasma membrane. (ii) Data represents six independent experiments (each in triplicate) of direct ELISA showing the significant binding of EB-A2 Mab to the whole bacterial cell of ΔlcpBΔlcpC . Error bars indicate standard deviation, and statistical significance was determined by student’s T-test, in comparison with the WT strain. C Phase contrast and fluorescent images of HADA-stained WT, ΔlcpBΔlcpC and c-( lcpB + lcpC ) strains harvested at mid-log (OD 600 = 0.8) (i) and late-log phase (OD 600 = 1.5) (ii), showing major fluorescent binding of the dye at septum and poles of the mycobacterial strains. All images were minimally processed in ImageJ by simply subtracting the background at a rolling ball radius of 50 pixels with a dark background. The inbuilt CellSens dimension software of the Olympus BX53 was used to measure the length of the individual cells ( n = 300 for mid-log and n = 60 for late-log, across three independent experiments). Error bars represent standard deviation, and the statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level). D Determination of major sugar components in AG-PG complex of WT, ΔlcpBΔlcpC and c-( lcpB + lcpC ) M. smegmatis strains by TLC. PBS was used as blank. This is a representative of three independent experiments. E - F Quantification of AG-PG markers and major sugar components in the AG-PG complex of WT, ΔlcpBΔlcpC and c-( lcpB + lcpC ) strains by HILIC-MS. The intensity of the peaks for each component were measured in comparison to the peak of the standards (refer peak intensity histograms in Fig S2B). The data are presented as mean ± standard deviation and statistical significance determined by two-way ANOVA with Tukey post-hoc analysis (95% confidence level)

Article Snippet: In order to label the nascent PG in actively growing M. smegmatis strains, a final concentration of 250 μM fluorescent D-amino acid HADA (3-[7-hydroxycoumarin]-carboxamide-D-Alanine) (MedChemExpress, China) were incubated with the bacterial cells harvested at mid-log phase (OD 600 = 0.8) and at late-log phase (OD 600 = 1.5), on a shaking platform for 30 min at 37 °C [ ].

Techniques: Dot Blot, Binding Assay, Mutagenesis, Clinical Proteomics, Membrane, Direct ELISA, Standard Deviation, Comparison, Staining, Software, Hydrophilic Interaction Liquid Chromatography

Journal: BMC Microbiology

Article Title: Impairment of two non-essential LytR-cpsA-psr (Lcp) cell wall ligases decelerate cell wall assembly in Mycolicibacterium smegmatis

doi: 10.1186/s12866-026-04722-4

Figure Lengend Snippet: Effective bactericidal activity by combined drug strategy. A LcpA Mtb inhibition by kinase inhibitors. IC 50 determination of Lcp inhibitors, Entrectinib (i) and Sorafenib (ii) by in vitro ADP-Glo™ kinase assay using 2 µM purified LcpA Mtb protein, 135 µM GGPP and 10 µM ATP. The data is an average of three independent experiments each in duplicate and presented as mean ± standard deviation. B (i) MIC determination of Lcp inhibitors, cell-wall acting TB inhibitors and a combination of both on M. smegmatis plate culture. Abbreviation: VAN – vancomycin, INH – isoniazid, ENT – entrectinib and SORA – sorafenib. Final concentration of the inhibitor has been indicated, where combined inhibitors are in a ratio of 1:3 (VAN/INH: ENT/SORA); (ii) superimposed scatter plot showing M. smegmatis inhibition by combined drug strategy. Combined inhibitors are in a ratio of 1:3 (VAN/INH: ENT/SORA). Individual experimental values are plotted and represented as median with interquartile range, and statistical significance ($ − 50 µM, $$ − 100 µM and $$$ − 200 µM) determined by two-way ANOVA with Tukey post-hoc analysis (95% confidence level); (iii) phase contrast and fluorescent images of HADA-stained untreated and treated WT mc 2 155 strains harvested at mid-log phase (OD 600 = 0.8), showing major fluorescent binding of the dye at septum and poles. All images were minimally processed in ImageJ by simply subtracting the background at a rolling ball radius of 50 pixels with a dark background. The inbuilt CellSens dimension software of the Olympus BX53 was used to measure the length (iv) and width (v) of the individual cells ( n = 50 across three independent experiments). Error bars represent standard deviation, and the statistical significance was determined by two-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level). C HILIC-MS quantification of AG and PG markers (i-ii), and a representative intensity plot showing reduced PG components, GlcNAc and MurNAc in M. smegmatis sample treated with a combined dose of VAN and ENT in the ratio of 1:3 (iii); quantification of major sugar components in the AG-PG purified complex of untreated and treated WT mc 2 155 strains (iv-v). The intensity of the peaks for each component were measured in comparison to the peak of the standards (refer peak intensity histograms in Fig S5). The data is an average of three independent experiments and presented as mean ± standard deviation and statistical significance determined by two-way ANOVA with Tukey post-hoc analysis (95% confidence level). D Western blot images showing effective inhibition of M. smegmatis Lcp paralogs by combined drug strategy. 20 µg of the total protein lysate was used, and final concentration of the inhibitor has been indicated. For combined drug strategy, a constant concentration of 0.3 µM VAN/INH was used. Anti-flag Mab was used as the primary antibody. Full-length blots are presented in Supplementary file-2, Fig. 7D. E A representation of the M. smegmatis Lcp proteins interacting with two kinase inhibitors, Entrectinib (blue stick model) and Sorafenib (yellow stick model) (vii). The interacting amino acid residues are indicated for each interaction. Each protein chain is colored by individual colors as indicated. F Binding pockets of Lcp Msmeg in the catalytic Lcp domain showing LcpA-LcpD and LcpB-LcpC interacting residues that are common inhibitory targets of ENT/SORA. The Lcp domain of each Lcp Msmeg protein is represented as grey α-helix/β-sheet. The pocket surface is colored by hydrophobicity values in the Kyte-Doolittle scale (dodger blue for the most hydrophilic; white at 0.0; orange red for the most hydrophobic) in CASTpFold webserver . Sublets show magnified images of ENT/SORA binding sites as ball and stick model (ENT target site: Yellow; SORA target site: Green; Both ENT and SORA target site: Red)

Article Snippet: In order to label the nascent PG in actively growing M. smegmatis strains, a final concentration of 250 μM fluorescent D-amino acid HADA (3-[7-hydroxycoumarin]-carboxamide-D-Alanine) (MedChemExpress, China) were incubated with the bacterial cells harvested at mid-log phase (OD 600 = 0.8) and at late-log phase (OD 600 = 1.5), on a shaking platform for 30 min at 37 °C [ ].

Techniques: Activity Assay, Inhibition, In Vitro, Kinase Assay, Purification, Standard Deviation, Concentration Assay, Staining, Binding Assay, Software, Hydrophilic Interaction Liquid Chromatography, Comparison, Western Blot

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A) Organization of the operon containing sepF . The location targeted by CRISPRi sgRNAs are indicated. Both sepF and divIVA sgRNAs target the non-template strand. (B) A C. difficile strain was generated harboring a plasmid-encoded xylose-inducible CRISPRi sepF -KD cassette in addition to a CRISPRi-resistant sepF im complementation cassette integrated in the genome under control of an aTc-inducible P tet promoter. Culturing in 2.5% xylose results in induction of the sepF -KD cassette, and addition of 2.5 ng/mL aTc induces expression of the sepF im complementation construct. Cells were labeled with HADA to visualize sites of peptidoglycan synthesis and/or remodeling, then fixed for microscopy. Yellow arrows indicate division septa. Scale bars = 5 μm. (C) A C. difficile strain harboring a plasmid-encoded xylose-inducible CRISPRi divIVA -KD cassette was cultured in the presence of 2.5% xylose, then labeled with HADA and fixed for microscopy. Yellow arrows indicate division septa. Scale bars = 5 μm. (D) C. difficile harboring a plasmid-encoded xylose-inducible sepF -KD or divIVA -KD CRISPRi cassette were cultured in the absence (-) or presence (+) of 2.5% xylose for approximately 6 hr (∼8 doublings) and western blotting was performed for DivIVA with GDH as a loading control. Data are representative of at least two independent experiments.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Generated, Plasmid Preparation, Control, Expressing, Construct, Labeling, Microscopy, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A-B) C. difficile strains were made harboring expression cassettes to produce mScarlet-I3 (mScI3) fusion proteins under control of an aTc-inducible P tet promoter from an ectopic site in the genome. To induce production of the fusion protein, logarithmically growing cells were cultured in the presence of the following concentrations of aTc for 1 hr: 0.5 ng/mL aTc for FtsZ-mScI3 and mScI3-ZapA, 1 ng/mL aTc for SepF-mScI3, and 2.5 ng/mL aTc for mScI3-PBP1. Cells were labeled with HADA for 10 min to visualize sites of peptidoglycan synthesis and/or remodeling and then fixed for fluorescence microscopy. (A) Merged images containing phase and mScI3 signal (top) or phase and HADA signal (bottom) are shown, and yellow arrows point to examples of divisome protein foci. Scale bars = 5 μm. (B) Demographs were generated using MicrobeJ to visualize the medial axis fluorescence profile of ≥500 cells. Cells are ordered by length, and the mScI3 or HADA signal along the distance of the cell relative to mid-cell is shown in magenta and cyan, respectively. Data in A-B are representative of at least three independent experiments.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Expressing, Control, Cell Culture, Labeling, Fluorescence, Microscopy, Generated

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A-E) Plasmid-encoded xylose-inducible CRISPRi-KD cassettes that either (A) are non-targeting (negative control), or target (B) ftsZ , (C) zapA , (D) sepF , or (E) pbp1 , were introduced into C. difficile strains carrying chromosomally-encoded complementation constructs that are “immune” to CRISPRi targeting. These CRISPRi-immune constructs were expressed under control of an aTc-inducible P tet promoter. The indicated strains of C. difficile were cultured for ∼6 hr in the presence of 2.5% xylose, to induce the CRISPRi-KD cassette, and aTc ( ftsZ /negative control = 1 ng/mL; zapA = 50 ng/mL; sepF = 2.5 ng/mL; pbp1 = 2 ng/mL), to induce expression of the CRISPRi-immune complementation construct. The cultures were then labeled with HADA to visualize sites of peptidoglycan synthesis and/or remodeling and fixed for microscopy. Merged images containing phase/HADA (blue) or phase/mScI3 (magenta) are shown. The HADA signal is normalized across all images, but the mScI3 signal was scaled such that the localization for each individual fusion protein was more easily detected. The yellow arrow in C points to an example of a HADA “spiral”; these structures were only observed in zapA -KD/ mScI3 - zapA im cells. More examples of these structures can be found in Fig S3. All data are representative of at least two independent experiments. Scale bars = 5 μm.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Plasmid Preparation, Negative Control, Construct, Control, Cell Culture, Expressing, Labeling, Microscopy

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A-B) Constructs encoding tagged divisome proteins were each expressed from an ectopic locus under the control of an aTc-inducible P tet promoter; xylose-inducible CRISPRi-KD constructs are encoded on a plasmid. Cells were cultured with 2.5% xylose for 6 hr and pulsed with aTc (FtsZ-mScI3 = 0.5 ng/mL; mScI3-ZapA = 0.5 ng/mL; SepF-mScI3 = 1 ng/mL; mScI3-PBP1 = 2.5 ng/mL). for the last hour of the treatment before labeling with HADA and fixing cells for microscopy. (A) Control C. difficile strains producing the indicated divisome protein-mScarlet-I3 (mScI3) fusion and harboring a negative control CRISPRi cassette with non-targeting sgRNA were visualized by microscopy to ensure that the CRISPRi cassette itself does not impact the protein localization. Yellow arrows point to divisome protein foci. (B) C. difficile cells producing divisome protein-mScI3 fusions upon the indicated CRISPRi ftsZ -KD, zapA -KD, sepF -KD, and pbp1 -KD cassettes were visualized by microscopy. Yellow arrows point to divisome protein foci. The HADA signal is scaled equally across all images, but the mScI3 signal was scaled independently for optimal protein localization The data in A-B are representative of at least two independent experiments. Scale bars = 5 μm. (C) The order of assembly of the essential divisome proteins is depicted. The dotted line indicates that while SepF can form foci without ZapA, the foci appear irregular and diffuse, suggesting that ZapA is partially required for proper SepF localization.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Construct, Control, Plasmid Preparation, Cell Culture, Labeling, Microscopy, Negative Control

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A-B) Constructs encoding mScarlet-I3 (mScI3) fusion proteins under the control of an aTc-inducible P tet promoter were expressed from an ectopic site in the genome. To induce production of the fusion protein, logarithmically growing cells were cultured in the presence of aTc for 1 hr (mScI3-MldA = 1 ng/mL; mScI3-MldB = 0.5 ng/mL; mScI3-MldC = 0.5 ng/mL; DivIVA-mScI3 = 0.5 ng/mL; mScI3-FtsK = 5 ng/mL; mScI3-PBP3 = 5 ng/mL). Cells were labeled with HADA for 10 min to visualize sites of peptidoglycan synthesis and/or remodeling and then fixed for fluorescence microscopy. (A) Merged images containing phase and mScI3 signal (top) or phase and HADA signal (bottom) are shown, and yellow arrows point to examples of divisome protein foci. Scale bars = 5 μm. (B) Demographs were generated using MicrobeJ to visualize the medial axis fluorescence profile of ≥500 cells. Cells are ordered by length, and the mScI3 or HADA signal along the distance of the cell relative to mid-cell is shown in magenta and cyan, respectively. Data in A-B are representative of at least three independent experiments.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Construct, Control, Cell Culture, Labeling, Fluorescence, Microscopy, Generated

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A) Constructs encoding fluorescent protein fusions to divisome proteins were expressed from an ectopic locus under the control of an aTc-inducible P tet promoter during xylose-inducible CRISPRi-KD of the indicated genes. The CRISPRi-KD constructs were expressed from a plasmid. Cells were cultured with 2.5% xylose for 6 hr and pulsed with aTc for the last hour of the treatment (mScI3-MldA = 1 ng/mL; mScI3-MldB = 0.5 ng/mL; mScI3-MldC = 0.5 ng/mL; DivIVA-mScI3 = 0.5 ng/mL; mScI3-FtsK = 5 ng/mL; mScI3-PBP3 = 5 ng/mL) before labeling with HADA and fixing cells for microscopy. Divisome protein foci are indicated by arrows. Cyan arrows represent divisome protein foci that are co-localized with septa in the pbp1 -KD strain, which is likely due to incomplete depletion of PBP1 protein leading to a low-level of septum synthesis. White arrows represent divisome protein foci within cells that did not label efficiently with HADA, so the presence or absence of septa at the site of protein localization is unknown. Yellow arrows indicate divisome protein foci that localize in the absence of septa, specifically in cells that label well with HADA. Data are representative of at least two independent experiments. Scale bars = 5 μm. (B) The order of divisome assembly is depicted. The dotted line indicates that ZapA is only partially required for proper SepF localization. The proteins labeled with an * indicate that the protein only formed foci when septa were detectable; these septa likely form because PBP1 is only partially depleted.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Construct, Control, Plasmid Preparation, Cell Culture, Labeling, Microscopy

Journal: bioRxiv

Article Title: Defining the order of assembly of the Clostridioides difficile divisome complex

doi: 10.64898/2026.01.19.700290

Figure Lengend Snippet: (A-B) The indicated clean deletion strains of C. difficile were cultured to logarithmic phase and labeled with HADA. (A) yellow arrows point to division septa. (B) The Δ mldA or Δ divIVA mutants were complemented with either an empty vector integrated into an ectopic locus “-“ or the indicated expression construct. Scale bars = 5 μm.

Article Snippet: The fluorescent D-amino acid HADA (Tocris) was used to label sites of peptidoglycan synthesis and/or remodeling.

Techniques: Cell Culture, Labeling, Plasmid Preparation, Expressing, Construct