s aureus newman m smegmatis mc2 155 e faecium atcc 19434 e faecium 36235a e  (ATCC)

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: Differential expression of M. smegmatis lcp homologs under various stress factors. Fold change in lcp gene expression analysis by RT-qPCR in the WT, single- and double-knockouts (SKO and DKO respectively) under lysozyme stress at a concentration of 0.0625 mg mL -1 ( A ), SDS stress at a concentration of 0.25% ( B ), acid stress at pH 6.2 ( C ) and, oxidative stress at 2.5 mM H 2 O 2 ( D ). The fold change in WT under all stress is with respective to untreated gene expression levels, whereas the fold change in SKO and DKO is with respective to WT gene expression levels for the same concentration of the stressor used. The data highlights a fold increase of lcpA expression by the double knockout mutants under stress conditions. rpoD (SigA) was used as the reference gene to determine relative expression of M. smegmatis lcp homologs. The presented data is an average of three independent experiments each performed in triplicate, and the error bars represent ± SD between the experiments. Statistical significance for all the strains treated with various stress factors, was determined by two-way ANOVA with Tukey’s post-hoc analysis (95% confidence level)

Article Snippet: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

Techniques: Quantitative Proteomics, Gene Expression, Quantitative RT-PCR, Concentration Assay, Expressing, Double Knockout

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: Detection of cell wall defects and impact on cell surface hydrophobicity in the absence of both lcpB and lcpC in M. smegmatis genome. A Growth curve. All the strains under study were grown for 56 h under continuous shaking conditions at 37 °C, and the absorbance (OD 600 ) recorded every 8 h. The represented data is an average of four independent experiments with error bars indicating standard deviation. Ordinary one-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level) was used to determine statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcp C) at mid-log phase. B The doubling time was determined from the optical density curves of all the strains from T = 0 h to T = 24 h (mid-log phase). The middle line of the violin plots indicates the average of four independent biological replicates each performed in triplicate. The bottom and top line represent the standard deviation of the average. Statistical significance was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction, as compared with the WT strain. C Scanning electron microscopy (SEM). SEM images of WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) were captured at 10 kV and 15,000X magnification. D The SEM images were quantified for its length and width mean of 100 individual cells, using GraphPad prism. The error bars represent ± SD of the 100 individual cells from three independent bacterial cultures grown up to OD 600 = 1. Ordinary one-way ANOVA with Dunnett’s analysis (95% confidence level) was used to determine statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) strains. E Colonies on plate were attained by pipetting 5 µL of overnight cultures adjusted to OD 600 = 0.5 onto 7H10 plates supplemented with Congo Red (100 µg mL − 1 ). F Quantification of colonies on Congo Red plates. The diameter of each colony was measured from two perpendicular angles, and the average recorded. The violin plots represent comparison of the distribution of colony diameter profiles across all the strains under study from four independent experiments each performed in triplicate. The plots illustrate summary statistics (median, first and third quartiles, with whiskers representing the minimum and maximum values within 1.5× the interquartile (IQR) ranges from the first and third quartiles). The statistical significance was determined using unpaired two-tailed T-test (95% confidence level) with Welch’s correction in comparison with the WT strain. G Quantification of biofilms adhering to 96-well polystyrene plate with 1% crystal violet solution and measurement of the absorbance at OD 550 . The represented violin plots are indicative of three independent experiments each performed in triplicate. The plots illustrate median, first and third quartiles, with whiskers representing the minimum and maximum values with < 1.5 IQR ranges from the first and third quartiles. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test (95% confidence level). H Aggregation assay using the WT strain, mutants and the complemented strains at a time window of 15 min. The represented data is an average of three independent experiments each performed in triplicate, and statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) is determined around the midpoint (T = 8 min) and endpoint (T = 15 min) of the aggregation plot, using unpaired two-tailed T-test with Welch’s correction at 95% confidence level. I Nile red was used to determine the permeability of the strains by fluorescence levels of dye accumulation (Ex at 540 nm and Em at 630 nm) at midpoint (T = 24 h) and endpoint (T = 48 h). The represented data was normalized to T = 0 for each sample well. The data is an average of three independent experiments each performed in triplicate. The error bars represent standard deviation, and statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) strains was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction, as compared with WT. The statistical comparisons between the WT /∆lcpB∆lcpC ( $ ) and WT/c-( lcpB + lcpC ) ( $$ ) has been indicated with an arrow at midpoint and endpoint of the assay time window for both D and E

Article Snippet: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

Techniques: Cell Surface Hydrophobicity, Standard Deviation, Two Tailed Test, Electron Microscopy, Comparison, Permeability, Fluorescence

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: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

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: Hypersensitivity of ΔlcpBΔlcpC to stress. A Heat map displaying lower MIC values of major anti-TB drugs used in this study (PEN-penicillin; EMB-ethambutol; INH-isoniazid; VAN-vancomycin; BAC-bacitracin; NOV- novobiocin; RIF-rifampicin; PZA-pyrazinamide; ERY-erythromycin; STR-streptomycin). The MIC values are described in the supplementary information (Table S3). B Box plot showing impact on the survivability of the mutants as colonies after single- or double- lcp gene deletion, in comparison with the WT under no additional stress. The statistical significance was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction. The relative expression of the four M. smegmatis Lcp homologs under native conditions were determined by RT-qPCR. The data is represented as mean ± standard deviation and statistics performed by one-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level). C - F Separated box and whiskers with Tukey plots determining the impact of various stressors [C - Lysozyme at 0.125 mg mL − 1 conc.; D − 0.25% SDS conc.; E - Acid stress at pH 6.2 and, F - oxidative stress at 2.5 mM H 2 O 2 ] on the mutants and complemented strains in comparison with the untreated condition of the respective strain. All the box plot centres from B - F represent median, and box represents IQR. Whiskers extend to most extreme data point < 1.5 IQR. Statistical significance was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction

Article Snippet: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

Techniques: Comparison, Two Tailed Test, Expressing, Quantitative RT-PCR, Standard Deviation

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: Interplay of M. smegmatis Lcp proteins. A Evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method . The bootstrap consensus tree inferred from 10,000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches . The evolutionary distances were computed using the p-distance method (scale bar value: 0.05) and are in units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). This analysis involved 19 amino acid sequences. All ambiguous positions were removed for each sequence par (pairwise deletion). There were a total of 1038 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [ , ]. B Interaction of Lcp paralogs in M. smegmatis . Co-immunoprecipitation showing interplay of essential (i) and non-essential (ii) Lcp proteins. The data is a representative of three technical replicates. Full-length blots are presented in Supplementary file-2, Fig. 5B. C (i) Interaction between LcpB-LcpC as predicted by dimplot module of LigPlot + v 2.2.9. Hydrogen bonds are shown as a lime green dotted line, and hydrophobic bonds are represented in red. Common amino acid residues involved in individual LcpB and LcpC dimer complexes as well as conserved sites are encircled in red dashed lines. Red arrow represents one of the amino acid residues in LcpC commonly interacting with LcpA/B/D complex, for which a point mutant was generated; (ii) co-immunoprecipitation showing loss of LcpC-LcpA/B/D interaction with LcpC W327A mutation. The data is a representative of two technical replicates. Full-length blots are presented in Supplementary file-2, Fig. 5C

Article Snippet: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

Techniques: Sequencing, Immunoprecipitation, Mutagenesis, Generated

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: In vitro catalytic activity of M. smegmatis Lcp proteins. A Binding pockets (Pocket 1: LcpA Msmeg , LcpC Msmeg , LcpD Msmeg , LcpA Mtb , LcpD Mtb ; Pocket 2: LcpB Msmeg , LcpB Mtb , LcpC Mtb ) on the surface of Lcp homologs in M. smegmatis and M. tuberculosis showing the bound substrate, GGPP (geranylgeranyl pyrophosphate). The Lcp and LytR_C domains of the full-length Lcp proteins are represented by pink or yellow colored α-helix/β-sheet regions respectively, whereas the rest of the regions are represented in grey. 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 the binding regions buried inside the protein pockets. The binding energy of Lcp-GGPP is indicated in the sublet. The pocket dimensions and related information are provided in Table S5A and data source. B The in vitro phosphotransferase activity of Lcp homologs in both M. tuberculosis and M. smegmatis was determined by ADP-Glo™ kinase assay using purified Lcp proteins and GGPP as substrate. The net amount of ADP released by the Lcp enzymes upon phosphorylation was measured here. C The inorganic phosphate release (P i ) in the pyrophosphatase enzymatic activity was determined and compared for all the four Lcp homologs in M. tuberculosis and M. smegmatis (i) and, Mg + 2 dependence of the Lcp homologs in M. smegmatis was confirmed by using EDTA as the metal scavenger (ii). The P i upon hydrolysis of the pyrophosphate phosphoanhydride bond of GPP was determined by measuring the absorbance at 560 nm. Inorganic pyrophosphatase (PP i ) from yeast was used as the positive control (PC) and recombinant Nectin-4 was used as the negative control (NC). The data represents the detected P i at highest absorbance i.e., T = 2 h. The represented data is an average of four (B) and three (C) independent experiments respectively, each of them performed at least in duplicate. Error bars represent standard deviation, and statistical significance is determined by two-way ANOVA and Tukey post-hoc analysis (95% confidence level). D (i) Interaction between LcpA Msmeg - GGPP as predicted by ligplot module of LigPlot + v 2.2.9. Hydrogen bonds are shown as a lime green dotted line with corresponding distances in Å unit labeled over it. (ii) Stacked sequence logo showing enriched Lcp-GGPP binding residues of LcpA Msmeg generated (without gaps) after multiple pairwise structural alignment with closely related PDB structures of Z score > 24.5, in the Dali webserver . Star symbol represents mutated arginine residue in this study which is an Lcp-GGPP common binding site (Table S5B and Fig S3, C) as well as a charged residue that binds the pyrophosphate moiety of the bound substrate . Enzymatic activity of the mutated Lcp Msmeg R→A in comparison with the WT homologs showing net ADP released (a) and net inorganic phosphate released (b) per µM enzyme. The presented data is an average of four independent experiments each performed in duplicate, and the error bars represent ± SD between the experiments. Statistical significance for all the WT and mutated Lcp homologs was determined by one-way ANOVA with Holm-Šídák post-hoc analysis (95% confidence level)

Article Snippet: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

Techniques: In Vitro, Activity Assay, Binding Assay, Kinase Assay, Purification, Phospho-proteomics, Positive Control, Recombinant, Negative Control, Standard Deviation, Labeling, Sequencing, Generated, Residue, Comparison

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: The genome of M. smegmatis strain mc 2 155 (ATCC 607) harbors four Lcp proteins which we designated as LcpA (MSMEG_1824), LcpB (MSMEG_0107), LcpC (MSMEG_5775) and LcpD (MSMEG_6421) (Table ), with sequence identities of 69.8%, 41.3%, 56% and 68.1% respectively to the M. tuberculosis homologs.

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: 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: Differential expression of M. smegmatis lcp homologs under various stress factors. Fold change in lcp gene expression analysis by RT-qPCR in the WT, single- and double-knockouts (SKO and DKO respectively) under lysozyme stress at a concentration of 0.0625 mg mL -1 ( A ), SDS stress at a concentration of 0.25% ( B ), acid stress at pH 6.2 ( C ) and, oxidative stress at 2.5 mM H 2 O 2 ( D ). The fold change in WT under all stress is with respective to untreated gene expression levels, whereas the fold change in SKO and DKO is with respective to WT gene expression levels for the same concentration of the stressor used. The data highlights a fold increase of lcpA expression by the double knockout mutants under stress conditions. rpoD (SigA) was used as the reference gene to determine relative expression of M. smegmatis lcp homologs. The presented data is an average of three independent experiments each performed in triplicate, and the error bars represent ± SD between the experiments. Statistical significance for all the strains treated with various stress factors, was determined by two-way ANOVA with Tukey’s post-hoc analysis (95% confidence level)

Article Snippet: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

Techniques: Quantitative Proteomics, Gene Expression, Quantitative RT-PCR, Concentration Assay, Expressing, Double Knockout

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: Detection of cell wall defects and impact on cell surface hydrophobicity in the absence of both lcpB and lcpC in M. smegmatis genome. A Growth curve. All the strains under study were grown for 56 h under continuous shaking conditions at 37 °C, and the absorbance (OD 600 ) recorded every 8 h. The represented data is an average of four independent experiments with error bars indicating standard deviation. Ordinary one-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level) was used to determine statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcp C) at mid-log phase. B The doubling time was determined from the optical density curves of all the strains from T = 0 h to T = 24 h (mid-log phase). The middle line of the violin plots indicates the average of four independent biological replicates each performed in triplicate. The bottom and top line represent the standard deviation of the average. Statistical significance was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction, as compared with the WT strain. C Scanning electron microscopy (SEM). SEM images of WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) were captured at 10 kV and 15,000X magnification. D The SEM images were quantified for its length and width mean of 100 individual cells, using GraphPad prism. The error bars represent ± SD of the 100 individual cells from three independent bacterial cultures grown up to OD 600 = 1. Ordinary one-way ANOVA with Dunnett’s analysis (95% confidence level) was used to determine statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) strains. E Colonies on plate were attained by pipetting 5 µL of overnight cultures adjusted to OD 600 = 0.5 onto 7H10 plates supplemented with Congo Red (100 µg mL − 1 ). F Quantification of colonies on Congo Red plates. The diameter of each colony was measured from two perpendicular angles, and the average recorded. The violin plots represent comparison of the distribution of colony diameter profiles across all the strains under study from four independent experiments each performed in triplicate. The plots illustrate summary statistics (median, first and third quartiles, with whiskers representing the minimum and maximum values within 1.5× the interquartile (IQR) ranges from the first and third quartiles). The statistical significance was determined using unpaired two-tailed T-test (95% confidence level) with Welch’s correction in comparison with the WT strain. G Quantification of biofilms adhering to 96-well polystyrene plate with 1% crystal violet solution and measurement of the absorbance at OD 550 . The represented violin plots are indicative of three independent experiments each performed in triplicate. The plots illustrate median, first and third quartiles, with whiskers representing the minimum and maximum values with < 1.5 IQR ranges from the first and third quartiles. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test (95% confidence level). H Aggregation assay using the WT strain, mutants and the complemented strains at a time window of 15 min. The represented data is an average of three independent experiments each performed in triplicate, and statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) is determined around the midpoint (T = 8 min) and endpoint (T = 15 min) of the aggregation plot, using unpaired two-tailed T-test with Welch’s correction at 95% confidence level. I Nile red was used to determine the permeability of the strains by fluorescence levels of dye accumulation (Ex at 540 nm and Em at 630 nm) at midpoint (T = 24 h) and endpoint (T = 48 h). The represented data was normalized to T = 0 for each sample well. The data is an average of three independent experiments each performed in triplicate. The error bars represent standard deviation, and statistical significance between the WT, ∆lcpB∆lcpC and c-( lcpB + lcpC ) strains was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction, as compared with WT. The statistical comparisons between the WT /∆lcpB∆lcpC ( $ ) and WT/c-( lcpB + lcpC ) ( $$ ) has been indicated with an arrow at midpoint and endpoint of the assay time window for both D and E

Article Snippet: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

Techniques: Cell Surface Hydrophobicity, Standard Deviation, Two Tailed Test, Electron Microscopy, Comparison, Permeability, Fluorescence

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: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

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: Hypersensitivity of ΔlcpBΔlcpC to stress. A Heat map displaying lower MIC values of major anti-TB drugs used in this study (PEN-penicillin; EMB-ethambutol; INH-isoniazid; VAN-vancomycin; BAC-bacitracin; NOV- novobiocin; RIF-rifampicin; PZA-pyrazinamide; ERY-erythromycin; STR-streptomycin). The MIC values are described in the supplementary information (Table S3). B Box plot showing impact on the survivability of the mutants as colonies after single- or double- lcp gene deletion, in comparison with the WT under no additional stress. The statistical significance was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction. The relative expression of the four M. smegmatis Lcp homologs under native conditions were determined by RT-qPCR. The data is represented as mean ± standard deviation and statistics performed by one-way ANOVA with Dunnett’s post-hoc analysis (95% confidence level). C - F Separated box and whiskers with Tukey plots determining the impact of various stressors [C - Lysozyme at 0.125 mg mL − 1 conc.; D − 0.25% SDS conc.; E - Acid stress at pH 6.2 and, F - oxidative stress at 2.5 mM H 2 O 2 ] on the mutants and complemented strains in comparison with the untreated condition of the respective strain. All the box plot centres from B - F represent median, and box represents IQR. Whiskers extend to most extreme data point < 1.5 IQR. Statistical significance was determined by unpaired two-tailed T-test (95% confidence level) with Welch’s correction

Article Snippet: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

Techniques: Comparison, Two Tailed Test, Expressing, Quantitative RT-PCR, Standard Deviation

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: Interplay of M. smegmatis Lcp proteins. A Evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method . The bootstrap consensus tree inferred from 10,000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches . The evolutionary distances were computed using the p-distance method (scale bar value: 0.05) and are in units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). This analysis involved 19 amino acid sequences. All ambiguous positions were removed for each sequence par (pairwise deletion). There were a total of 1038 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [ , ]. B Interaction of Lcp paralogs in M. smegmatis . Co-immunoprecipitation showing interplay of essential (i) and non-essential (ii) Lcp proteins. The data is a representative of three technical replicates. Full-length blots are presented in Supplementary file-2, Fig. 5B. C (i) Interaction between LcpB-LcpC as predicted by dimplot module of LigPlot + v 2.2.9. Hydrogen bonds are shown as a lime green dotted line, and hydrophobic bonds are represented in red. Common amino acid residues involved in individual LcpB and LcpC dimer complexes as well as conserved sites are encircled in red dashed lines. Red arrow represents one of the amino acid residues in LcpC commonly interacting with LcpA/B/D complex, for which a point mutant was generated; (ii) co-immunoprecipitation showing loss of LcpC-LcpA/B/D interaction with LcpC W327A mutation. The data is a representative of two technical replicates. Full-length blots are presented in Supplementary file-2, Fig. 5C

Article Snippet: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

Techniques: Sequencing, Immunoprecipitation, Mutagenesis, Generated

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: In vitro catalytic activity of M. smegmatis Lcp proteins. A Binding pockets (Pocket 1: LcpA Msmeg , LcpC Msmeg , LcpD Msmeg , LcpA Mtb , LcpD Mtb ; Pocket 2: LcpB Msmeg , LcpB Mtb , LcpC Mtb ) on the surface of Lcp homologs in M. smegmatis and M. tuberculosis showing the bound substrate, GGPP (geranylgeranyl pyrophosphate). The Lcp and LytR_C domains of the full-length Lcp proteins are represented by pink or yellow colored α-helix/β-sheet regions respectively, whereas the rest of the regions are represented in grey. 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 the binding regions buried inside the protein pockets. The binding energy of Lcp-GGPP is indicated in the sublet. The pocket dimensions and related information are provided in Table S5A and data source. B The in vitro phosphotransferase activity of Lcp homologs in both M. tuberculosis and M. smegmatis was determined by ADP-Glo™ kinase assay using purified Lcp proteins and GGPP as substrate. The net amount of ADP released by the Lcp enzymes upon phosphorylation was measured here. C The inorganic phosphate release (P i ) in the pyrophosphatase enzymatic activity was determined and compared for all the four Lcp homologs in M. tuberculosis and M. smegmatis (i) and, Mg + 2 dependence of the Lcp homologs in M. smegmatis was confirmed by using EDTA as the metal scavenger (ii). The P i upon hydrolysis of the pyrophosphate phosphoanhydride bond of GPP was determined by measuring the absorbance at 560 nm. Inorganic pyrophosphatase (PP i ) from yeast was used as the positive control (PC) and recombinant Nectin-4 was used as the negative control (NC). The data represents the detected P i at highest absorbance i.e., T = 2 h. The represented data is an average of four (B) and three (C) independent experiments respectively, each of them performed at least in duplicate. Error bars represent standard deviation, and statistical significance is determined by two-way ANOVA and Tukey post-hoc analysis (95% confidence level). D (i) Interaction between LcpA Msmeg - GGPP as predicted by ligplot module of LigPlot + v 2.2.9. Hydrogen bonds are shown as a lime green dotted line with corresponding distances in Å unit labeled over it. (ii) Stacked sequence logo showing enriched Lcp-GGPP binding residues of LcpA Msmeg generated (without gaps) after multiple pairwise structural alignment with closely related PDB structures of Z score > 24.5, in the Dali webserver . Star symbol represents mutated arginine residue in this study which is an Lcp-GGPP common binding site (Table S5B and Fig S3, C) as well as a charged residue that binds the pyrophosphate moiety of the bound substrate . Enzymatic activity of the mutated Lcp Msmeg R→A in comparison with the WT homologs showing net ADP released (a) and net inorganic phosphate released (b) per µM enzyme. The presented data is an average of four independent experiments each performed in duplicate, and the error bars represent ± SD between the experiments. Statistical significance for all the WT and mutated Lcp homologs was determined by one-way ANOVA with Holm-Šídák post-hoc analysis (95% confidence level)

Article Snippet: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

Techniques: In Vitro, Activity Assay, Binding Assay, Kinase Assay, Purification, Phospho-proteomics, Positive Control, Recombinant, Negative Control, Standard Deviation, Labeling, Sequencing, Generated, Residue, Comparison

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: M. smegmatis str. mc 2 155 (ATCC 607) was purchased from China General Microbiological Culture Collection Center (CGMCC).

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