High concentration of 2,4-DAPG in the cells impaired the growth of the rsmA rsmE double mutant
Previous study showed that the growth of the rsmA rsmE double mutant was severely impaired compared with the wild-type strain 2P24. Since high levels of 2,4-DAPG was toxic to the producing bacterium [20], to assess if the growth of the rsmA rsmE double mutant was impaired by the overdose of 2,4-DAPG in the cells, we overexpressed the phlG gene which encoding the 2,4-DAPG hydrolase and then measured the growth of 2P24 and its derivatives. As expected, introduction of the phlG gene cloned in the pRK415 plasmid (p415-phlG), into the rsmA rsmE double mutant, resulted in repression of 2,4-DAPG production (Fig. 1A). The growth rate of the rsmA rsmE double mutant with p415-phlG could be partially restored to that of the wild-type strain 2P24 (Fig. 1B). Furthermore, by deleting the phlD gene in the rsmA rsmE double mutant, the growth curve of the rsmA rsmE phlD triple mutant was significantly improved compared to that of the rsmA rsmE double mutant although was slightly lower compared to the wild-type strain 2P24 or the phlD mutant (Fig. 1C). Thus, these data suggested that overproduction of 2,4-DAPG contributes to the reduced growth of the rsmA rsmE double mutant.
RNA sequencing reveals the P. fluorescens RsmA and RsmE regulon.
To insight into the role of RsmA and RsmE in P. fluorescens, RNA sequencing (RNA-seq) was performed to define the RsmA and RsmE regulon of P. fluorescens. The genes that are significantly downregulated or upregulated are summarized in Table S1 & S2. We defined the genes that showed a > 2-fold change of expression as differentially expressed genes (DEGs). In the rsmA rsmE double mutant, 621 genes were upregulated and 304 genes were downregulated compared to the wild-type strain (Table S1 & S2).
Based on the RNA-seq results, we observed that the expression of genes in phl operon (phlACBD) was increased by 145 to 587-fold, which was consistent with that of the phlA′-′lacZ translational fusion assay [15]. Many regulatory elements, including Gcd [23], Hfq [24], PsrA [25], RpoS [26], and PhlG which directly or indirectly influence 2,4-DAPG biosynthesis, were regulated by the RsmA and RsmE. As expected, transcriptional fusion assays showed that both of the expression of phlG and phlF were significantly increased in the rsmA rsmE double mutant compared with that in the wild-type strain (Fig. 2). These results indicated a sophisticated role for the RsmA family proteins RsmA and RsmE in the production of 2,4-DAPG in P. fluorescens.
Among the genes upregulated in the rsmA rsmE double mutant, 21 encoded proteins that are associate with type six secretion system (T6SS), which is known as an important mechanism in interactions and pathogenesis against bacterial and eukaryotic cells. In addition, the RNA-seq data revealed that a significant number of genes influenced by RsmA and RsmE were involved in fatty acid metabolism (fadA, fadB, fabG, fadH, psrA), energy and carbon metabolism (glpD, zwf, fahA, gcd, gltK), and cell motility (flaG, fliT, fliS, motA, motC, flgE). Collectively, our data suggested that RsmA and RsmE are the pleiotropic regulators of secondary metabolism, cell motility, and other physiological processes.
RsmA and RsmE negatively regulated the Type six secretion system (T6SS)
Bacterial T6SS plays an important role in both virulence and inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial habitats [27]. Since many genes related to T6SS were up-regulated in the rsmA rsmE double mutant, we assayed the effect of the RsmA family protein on the production of T6SS structure protein Hcp1. Consistent with the RNA-seq data in the rsmA rsmE double mutant, Western blot analysis showed that this mutant produced higher amount of the Hcp1 protein than wild-type strain P. fluorescens 2P24 (Fig. 3A). Previously studies showed that the T6SSs could inject the T6SS toxins into bacterial preys [28]. We then performed the antibacterial assays using E. coli carrying the plasmid pHSG299 as prey and P. fluorescens 2P24 or its derivatives as predators. Similar to the retS mutant which triggered the T6SS [29], The rsmA rsmE double mutant caused a significant increase in survival of E. coli (Fig. 3B). Taken together, our results indicate that RsmA and RsmE repress the T6SS activity and play an important role for the inter-bacterial competition.
The effect of RsmA and RsmE on cell motility and biofilm formation
Analysis of the rsmA rsmE double mutant RNA-seq data showed that expression of 20 genes involved in flagella biosynthesis and assembly was significantly changed, indicated a decrease in cell motility. To confirm this result, the motility of strain 2P24 and its derivatives was measured. Compared to the wild-type strain, the swimming motility was reduced by 72% in the rsmA rsmE double mutant (Fig. 4A). Motility is crucial in cell-to-cell adherence and attachment in early biofilm development. Whereas our data revealed a positive influence of the RsmA family proteins on biofilm formation (Fig. 4B). All observed phenotypes in the rsmA rsmE double mutant could be partly complemented by introducing the plasmid pBBR-rsmE (Fig. 4). Taken together, these results demonstrated that RsmA and RsmE are crucial for cell motility and biofilm formation in strain 2P24.