title: 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.
abstract: SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized 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 have been 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 utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) 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 or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD 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.
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Hannah G
authors.initials: HG
authors.lastname: Hampton
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: McNeil
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Paterson
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Blair
authors.initials: B
authors.lastname: Ney
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Williamson
authors.affiliation: Otago Centre for Electron Microscopy, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Easingwood
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.|Otago Centre for Electron Microscopy, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Mihnea
authors.initials: M
authors.lastname: Bostina
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
authors.forename: George P C
authors.initials: GPC
authors.lastname: Salmond
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fineran
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D005407:Flagella / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics*; D011353:Prodigiosin / Q000096:biosynthesis; D016601:RNA-Binding Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D012705:Serratia / Q000235:genetics* / Q000472:pathogenicity; D013385:Succinate Dehydrogenase / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D013385:Succinate Dehydrogenase; D008780:Methyltransferases; D011353:Prodigiosin