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Supplementary data

Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis, by M. Kumar and D. Chatterji

Microbiology vol. 154, part 10, pp. 2942-2955

Table S1. List of the primers used in the present study.

Fig. S1. MALDI-TOF MS analysis of DGC assay reaction mixtures for detection and identification of c-di-GMP and pGpG. (a) Control (no protein); (b) with PleD protein; (c, d) with MSDGC-1. The following major ions were detected: for GTP 524, [M+H]⁺, 546 [M+Na]⁺, 568 [M+2Na]⁺; for c-di-GMP, 691 [M+H]⁺, 713 [M+Na]⁺; for pGpG, 709 [M+H]⁺, 731 [M+Na]⁺, 753 [M+2Na]⁺.

Fig. S2. Genotype confirmation for the deletion of MSDGC-1. (a) Colony PCR of wild-type (wt) and DMSDGC knockout (DDGC) strains. The amplicons differ in their size because of the insertion of a kanamycin cassette. (b) Colony PCR using primers from the kanamycin cassette and downstream sequences which are not part of the construct for deletion. No amplification is observed from the wild-type in the absence of the kanamycin cassette sequence. This confirms the replacement of the gene at the desired place. (c) Southern hybridization. Genomic DNA from M. smegmatis mc²155 (wt) and the ΔMSDGC-1 knockout was digested with PvuII and run on a 0.8 % agarose gel. Digestion with PvuII gives two fragments of 3.9 and 0.5 kb in the wild-type, and 4.7 and 0.5 kb in the mutant strain. The probe was a 1.8 kb fragment of the MSMEG_2196 gene.

Fig. S3. The ability to form biofilms remains unchanged in the ΔMSDGC-1 knockout strain. We tested biofilm formation by two methods. (a) The pellicle form of the biofilm at the water&endash;air interface was grown in Petri dishes containing Sauton's medium and photographed on day 7. (b) To quantify biofilm formation and its ability to bind to a surface and grow, cultures were grown in 96-well tissue culture plates. After incubation in static conditions, biofilms were stained with crystal violet and analysed as described in Methods. Grey bars, wild-type; white bars, ΔMSDGC-1.

Fig. S4. Declumping of an M. smegmatis culture grown in MB7H9 with 0.02 % glucose and 0.05 % Tween 80. Cells were observed under a microscope before (a) and after declumping (b) as described in Methods. This is a representative photograph of the wild-type strain grown for 20 days; a similar observation was made with ΔMSDGC-1.

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