1887

Abstract

Cyclic di-GMP (c-di-GMP) plays an important role in bacterial adaptation to enable survival in changing environments. It orchestrates various pathways involved in biofilm formation, changes in the cell surface, host colonization and virulence. In this article, we report the presence of c-di-GMP in , and its role in the long-term survival of the organism. has a single bifunctional protein with both GGDEF and EAL domains, which show diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activity, respectively, . We named this protein MSDGC-1. Deletion of the gene encoding MSDGC-1 did not affect growth and biofilm formation in , but long-term survival under conditions of nutritional starvation was affected. Most of the proteins that contain GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. To gain further insight into the regulation of the protein, we cloned the individual domains, and tested their respective activities. MSDGC-1, the full-length protein, is required for activity, as its GGDEF and EAL domains are inactive when separated.

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2008-10-01
2019-11-22
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List of the primers used in the present study.

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List of the primers used in the present study.

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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] .

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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 mc 155 (wt) and the ΔMSDGC-1 knockout was digested with II and run on a 0.8 % agarose gel. Digestion with II 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 gene.

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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.

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