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.

Functional Characterization of a Global Virulence Regulator Hfq and Identification of Hfq-Dependent sRNAs in the Plant Pathogen Pantoea ananatis

“…Similarly, in P. carotovorum (Wang et al, 2018) and Serratia sp. ATCC 39006 (Wilf et al, 2013; Hampton et al, 2016), attenuation of flagellar motility was observed in hfq -deletion mutant strains, as an expression of the flhD C genes was dependent on Hfq. Given that sRNAs namely, ArcZ, OmrAB, and RyeB/SdsR, were also identified in our sRNA sequencing data (Supplementary Table S2), and that their wild-type transcript levels are dependent on the functional copy of hfq (Figure 9), we hypothesize that Hfq, in conjunction with the identified sRNAs, may regulate the flagellar motility of P. ananatis in a similar manner as the other enterobacterial species.…”

The FloR master regulator controls flotation, virulence and antibiotic production in Serratia sp. ATCC 39006

“…Proteomic analysis showed that, in addition to the gas vesicle proteins, RsmA abundance was negatively affected, whilst RbsR and SmaI/SmaR were not detected. RsmA also inhibits flagellar motility and prodigiosin production in S39006 via negative control of the flagella master regulator, FlhDC (Williamson et al, 2008;Wilf et al, 2013;Hampton et al, 2016). In a floR mutant, we observed increased RsmA levels and the high levels of motility related proteins.…”

“…RbsR stimulates gas vesicle biogenesis, and prodigiosin, carbapenem, and cellulase production, whilst inhibiting flagellar motility. The mRNA binding protein RsmA promotes expression of both gas vesicle operons, while repressing prodigiosin production and motility (Ramsay et al, 2011;Hampton et al, 2016). In addition, the quorum sensing system (SmaI/SmaR) controls gas vesicle assembly, motility and secondary metabolite elaboration (Thomson et al, 2000;Fineran et al, 2005b;Williamson et al, 2008;Ramsay et al, 2011;Tashiro et al, 2016).…”

Insights Into Non-coding RNAs as Novel Antimicrobial Drugs

“…To date, a few CRISPR guide RNAs have been designed to target virulence factors, antimicrobials determinants or essential chromosomal genes from specific pathogens, such as E. coli or Staphylococcus aureus (Bikard et al, 2014; Citorik et al, 2014; Gomaa et al, 2014). These systems were employed to efficiently target a particular DNA sequence resulting in the introduction of chromosome deletions in different pathogens, which consequently led to cell death or to the reduction in the population of unwanted bacteria (Vercoe et al, 2013; Bikard et al, 2014; Citorik et al, 2014; Gomaa et al, 2014; Hampton et al, 2016). Vercoe et al (2013) observed that a guide or CRISPR RNA (crRNA) programmed to target a large horizontally acquired island in Pectobacterium atrosepticum activated the endogenous CRISPR-Cas system and promoted the loss of both islands and the accessory genes encoded within.…”