specific preimmune serum  (Agilent technologies)

Journal: Nature Communications

Article Title: Lsr2 acts as a cyclic di-GMP receptor that promotes keto-mycolic acid synthesis and biofilm formation in mycobacteria

doi: 10.1038/s41467-024-44774-6

Figure Lengend Snippet: A Spot colony morphology of the wide type and its ydeH -overexpressed strains, hadD Msm knock-out and its ydeH -overexpression strains. B Biofilm formation of the WT, ydeH , hadD Msm KO, and hadD Msm KO/ ydeH strains. C Quantitation of biofilm biomass by crystal violet staining of the WT, ydeH , hadD Msm KO, and hadD Msm KO/ ydeH strains ( n = 3, biological replicates). Two-tailed t-tests were performed for statistical analysis (*** p = 0.0004). D RT-PCR quantitation of hadD Msm transcription in the ydeH , ydeH (mut)-overexpressed M. smegmatis strains. Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). RT-PCR and gel analysis were performed under the same conditions and each experiment was performed three independent biological replicates. no RT was genomic DNA contamination control. E ChIP assays for the effect of c-di-GMP on the intracellular hadD Msm p-binding activity of Lsr2 Msm in M. smegmatis . The input (5%) indicated that the supernatant of disrupted cells was diluted to 5%, ChIP using preimmune (P) or immune (I) sera raised against HisLsr2 Msm . DNA of the input (5%), P, and I were used as temples for PCR (the right panel) ( n = 3, biological replicates) and RT-qPCR (the light panel). M: DNA Marker. Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). F β-galactosidase activity assays. The effect of c-di-GMP on Lsr2 Msm regulates the expression of hadD Msm was assayed by overexpressing pMV261- hadD Msm p- ydeH - lacZ and pMV261- hadD Msm p- ydeH (mut)- lacZ plasmids in the Msm/WT, lsr2 Msm KO strains ( n = 3, biological replicates). None promoter- lacZ and hsp60 p- lacZ plasmids overexpression were used as controls. Two-tailed Student’s t-tests were performed for statistical analysis (* p = 0.0414, **** p < 0.0001). Data were presented as mean ± SD of ( C ), ( D ), ( E ), ( F ). The source data were provided in the Source data file.

Article Snippet: The 900 µL supernatant was incubated with 1:2,000 dilution of mouse 6*His antibodies (#CSB-MA000011M0m, CUSABIO) or preimmune mouse serum (#NS03L, Sigma-Aldrich) for 3 h at 4 °C.

Techniques: Knock-Out, Over Expression, Quantitation Assay, Staining, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay, Quantitative RT-PCR, Marker, Expressing

Journal: Nature Communications

Article Title: Lsr2 acts as a cyclic di-GMP receptor that promotes keto-mycolic acid synthesis and biofilm formation in mycobacteria

doi: 10.1038/s41467-024-44774-6

Figure Lengend Snippet: A ITC assays for the interaction between Lsr2 Mtb and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots. B Spot colony morphology of the wide type, hadD Msm knock-out and hadD Mtb complementary strains. C Biofilm formation of the WT, hadD Msm KO, and hadD Mtb complementary strains. D Quantitation of biofilm biomass of the WT, hadD Msm KO, and hadD Mtb complementary strains by crystal violet staining ( n = 3, biological replicates). Two-tailed t-tests were performed for statistical analysis (**** p < 0.0001, *** p = 0.0004). E EMSA assays for the effect of c-di-GMP on hadD BCG promoter DNA-binding activity of Lsr2 BCG . hadD BCG p was co-incubated with increasing concentration of Lsr2 BCG (lanes 2–5). Three independent experiments were performed. F RT-PCR for transcriptional analysis of hadD BCG in the WT, lsr2 BCG KO M. bovis BCG strains ( n = 3, biological replicates). Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). RT-PCR and gel analysis were performed under the same conditions. G Spot colony morphology of the WT and lsr2 BCG knock-out BCG strains on 7H10 plates. H RT-PCR for transcriptional analysis of hadD BCG in the ydeH (mut), ydeH -overexpressed M. bovis BCG strains ( n = 3, biological replicates). Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). I ChIP assays for the effect of c-di-GMP on the intracellular DNA-binding activity of Lsr2 BCG in the M. bovis BCG strains. The input (5%) indicated that the supernatant of disrupted cells was diluted to 5%, ChIP using preimmune (P) or immune (I) sera raised against HisLsr2 BCG . DNA sample of the input (5%), P, and I were used as temples for PCR (the right panel) ( n = 3, biological replicates) and were quantified using RT-qPCR (the light panel). M: DNA marker. Two-tailed Student’s t-tests were performed to for statistical analysis (**** p < 0.0001). Data of figures ( D ), ( F ), ( H ), and ( I ) were presented as mean ± SD. The source data were provided in the Source data file.

Article Snippet: The 900 µL supernatant was incubated with 1:2,000 dilution of mouse 6*His antibodies (#CSB-MA000011M0m, CUSABIO) or preimmune mouse serum (#NS03L, Sigma-Aldrich) for 3 h at 4 °C.

Techniques: Titration, Knock-Out, Quantitation Assay, Staining, Two Tailed Test, Binding Assay, Activity Assay, Incubation, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Marker