CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia
Hampton, Hannah G
McNeil, Matthew B
Paterson, Thomas J
Williamson, Neil R
Easingwood, Richard A
Fineran, Peter C
RsmA, RsmC and FlhDC regulate sdhEygfX in Serratia
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Hampton, H. G., McNeil, M. B., Paterson, T. J., Ney, B., Williamson, N. R., Easingwood, R. A., Bostina, M., et al. (2016). CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia. Microbiology, 162 1047-1058. https://doi.org/10.1099/mic.0.000283
SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase. In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterised family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators were identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilised endogenous CRISPR-Cas genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA, rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we demonstrate that SdhE and fumarate reductase are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.
prodigiosin, motility, secondary metabolism, antibiotic, CRISPR-Cas, SDH5/SdhAF2
This work was supported by the Marsden Fund, Royal Society of New Zealand (RSNZ) to PCF and a Strategic Grant from the Otago School of Medical Sciences (OSMS) to MB. HGH was supported by a University of Otago Doctoral Scholarship, MBM by a Division of Health Sciences Career Development Post-doctoral Fellowship, BN by a Dean's Prestigious Summer Scholarship from the OSMS and PCF was supported by a Rutherford Discovery Fellowship (RSNZ). NRW and GPCS were supported by Biotechnology and Biological Sciences Research Council (BBSRC), UK awards to the GPCS laboratory. We thank members of the Fineran and Cook laboratories for helpful discussions, Tim Blower for plasmid pTRB32 and for critically reading the manuscript.
External DOI: https://doi.org/10.1099/mic.0.000283
This record's URL: https://www.repository.cam.ac.uk/handle/1810/255027
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