Vfm a new quorum sensing system controls the virulence of Dickeya dadantii
Article first published online: 11 DEC 2012
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Plant–Microbe Interactions
Volume 15, Issue 3, pages 865–880, March 2013
How to Cite
Nasser, W., Dorel, C., Wawrzyniak, J., Van Gijsegem, F., Groleau, M.-C., Déziel, E. and Reverchon, S. (2013), Vfm a new quorum sensing system controls the virulence of Dickeya dadantii. Environmental Microbiology, 15: 865–880. doi: 10.1111/1462-2920.12049
- Issue published online: 4 MAR 2013
- Article first published online: 11 DEC 2012
- Accepted manuscript online: 21 NOV 2012 01:35AM EST
- Manuscript Accepted: 10 NOV 2012
- Manuscript Revised: 30 OCT 2012
- Manuscript Received: 3 OCT 2012
- Centre National de la Recherche Scientifique (CNRS)
- French ‘ANR blanc Régupath 2007 Program. Grant Number: N°ANR-07-BLAN-0212
Table S1. Characteristics of the predicted gene products of the vfm cluster.
Table S2. Bacterial strains, plasmids and phage.
Table S3. Oligonucleotides.
Fig. S1. Identification of a new locus modulating extracellular enzymes production in D. dadantii. A mutagenesis of the D. dadantii wild-type 3937 strain was performed by using transposon Tn5-B21. Screening for extracellular enzymes production was made on agar plates allowing detection of pectinases, cellulases and proteases respectively.
A. Comparison of enzymes production by the wild-type strain 3937 and the two mutants A3470 and A3473 is shown for each type of enzyme.
B. Restoration of the protease− phenotype of mutants A3470 and A3473 by complementation using cell-free supernatant of the D. dadantii signal producing strain A3997 which is protease deficient due to a prtE mutation. Cell-free supernatant was incorporated into the agar medium at a final concentration of 10% v/v.
Fig. S2. The vfm genes are required for high transcription of CWDE genes. Impact of the four vfm mutations (vfmA, vfmE, vfmH and vfmK) representative of the four transcriptional units of the vfm cluster on pelE, pelB, celZ and prtC transcript levels was monitored by qRT-PCR. The pelE and pelB genes encode pectate lyases (a family of pectinase); celZ gene encodes a cellulase and prtC gene encodes a protease. Expression of the pelE, pelB, celZ and prtC genes in the vfmA, vfmE, vfmH and vfmK mutants was measured as transcript levels relative to wild-type strain (WT) at early stationary growth phase from cultures in M63 minimal medium supplemented with sucrose and PGA. Gene expression was normalized using lpxC and hemF mRNAs as controls. Values represent average gene expression ± SD from three independent experiments. Results obtained with the vfmA, vfmE, vfmH and vfmK mutants are different from the wild-type results, with P-values < 0.05 in a one sample t-test. Fold changes (FC) are expressed as the ratio of the specific gene expression in the studied mutant, compared with that in the wild-type strain. Negative FC values represent genes downregulated in the studied vfm mutant compared with the wild-type strain.
Fig. S3. Transcriptional organization of the vfm locus. Transcription units were analysed by RT-PCR using the Access RT-PCR System kit from Promega. For each pair of primers, three reactions were performed. The first reaction (1) corresponds to the RT-PCR assay. The second reaction (2) corresponds to a negative control without reverse transcriptase. The third reaction (3) corresponds to a positive control using genomic DNA instead of cDNA. For the vfmKLMNOPQRSTUVW transcriptional unit, the amplification of two overlapping fragments A and B confirmed the presence of a long polycistronic RNA. No RT-PCR amplification product (fragment C) could be detected using the vfmW-ID16066 pair of primers, whereas a RT-PCR amplification product (fragment D) was observed using the pair of primers spanning ID16066. These results indicate that ID16066 is a monocistronic transcriptional unit. The lane M corresponds to DNA ladder.
Fig. S4. Kinetic of Vfm signal production in D. dadantii 3937 wild-type strain and Vfm signal dose-dependent response of vfmE gene expression.
A. Accumulation of the Vfm signal in culture supernatant from D. dadantii. Kinetic of the Vfm signal production was analysed by measuring the expression of the strongly signal-sensitive vfmE::uidA reporter fusion from strain A5243. Cell-free supernatants from the D. dadantii wild-type strain and vfmA mutant grown in M63 minimal medium supplemented with sucrose 0.2% were collected throughout the growth curve. Bacterial growth was estimated by OD600 nm. The D. dadantii vfmE::uidA strain A5243 was grown overnight in M63 sucrose 0.2% medium in the presence of 10% (v/v) cell-free culture supernatants from each time point of the wild-type strain and vfmA mutant growth curve. Following A5243 strain growth, β-glucuronidase assays were performed in triplicate. Values represent average of the vfmE gene expression ± SD.
B. Expression of vfmE gene is dependent of the Vfm signal concentration. Dickeya dadantii A5243 vfmE::uidA mutant was grown overnight in M63 sucrose 0.2% medium in the presence of various amounts of cell-free supernatant prepared from early stationary-phase culture of the 3937 wild-type strain. Following growth, β-glucuronidase assays were performed in triplicate. Values represent average of the vfmE gene expression ± SD.
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