Absence of KpsM (Slr0977) Impairs the Secretion of Extracellular Polymeric Substances (EPS) and Impacts Carbon Fluxes in Synechocystis sp. PCC 6803

Abstract

Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977. Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest. IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

“…Repression of the kpsM homologues did not significantly affect growth, compared to the Syn dCas9 (Figure 2A) and wild-type strains. However, similarly to the slr0977 knockout mutant, the 3-sgRNA kpsM mutant displayed a clumping phenotype at low cell densities [8]. Regarding total carbohydrates, the 3-sgRNA kpsM mutant produced approximately the same amount as the Syn dCas9 strain (Figure 2B).…”

“…Validation of the reference genes (rrn16S, petB, and rnpB) and data analysis were performed using the Bio-Rad CFX Maestro™ 1.1 software. The single sll0574 knockout mutant was generated via double homologous recombination, by partially replacing the gene with a kanamycin (Km) resistance cassette, as described by Santos et al [8]. The mutants were characterized in terms of growth (absorbance and chlorophyll a content) and carbohydrate production (total carbohydrates, CPS, and RPS) using the phenol-sulfuric acid method [24], as described previously by Santos et al [8].…”

“…The single sll0574 knockout mutant was generated via double homologous recombination, by partially replacing the gene with a kanamycin (Km) resistance cassette, as described by Santos et al [8]. The mutants were characterized in terms of growth (absorbance and chlorophyll a content) and carbohydrate production (total carbohydrates, CPS, and RPS) using the phenol-sulfuric acid method [24], as described previously by Santos et al [8]. Data were statistically analyzed with GraphPad Prism v5 (GraphPad Software) using analysis of variance (ANOVA), followed by Tukey's multiple-comparison test.…”

“…Regarding the ABC transporter-dependent pathway, Fisher et al reported knockout mutants of a putative transport permease (slr0977 (kpsM)) and its associated ATP-binding module (slr0982 (kpsT)), and a triple mutant (slr0977 (kpsM) and the putative pair sll0574 (kpsM)/sll0575 (kpsT)) produced EPS with different monosaccharidic composition compared to the EPS produced by the wild-type [14]. While Fisher et al [14] did not report on the amounts of EPS produced by these mutants, a recent extensive characterization of a slr0977 (kpsM) mutant showed that the absence of Slr0977 resulted in a significant reduction of RPS (50%) and a smaller decrease of CPS (20%) [8]. In addition, a mutant lacking Slr2107 (another KpsM homologue) did not show significant differences in the total carbohydrates, RPS, and CPS compared to the wild-type [13].…”

“…Such knowledge is therefore crucial to increase the production yields and to tailor polymer variants for a specific application. In addition, this knowledge can also aid efforts to redirect carbon fluxes from the high-energy-demanding EPS production towards the production of target/value-added compounds, when using cyanobacteria as green cell factories [7,8].…”