i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
Microbiologyopen. 2019 Jun;8(6):e00753. doi: 10.1002/mbo3.753. Epub 2019 Jan 23.
Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.
许多蓝藻产生细胞外聚合物质(EPS),主要由具有独特特性的杂多糖组成,使其适合生物技术应用。然而,由于对 EPS 生物合成/特性的产生途径和细菌物种之间的差异了解不足,对其进行操纵/优化受到了阻碍。在这项工作中,针对单细胞集胞藻 PCC 6803 中的蓝藻 EPS 聚合、组装和输出的不同途径的假定相关基因进行了缺失或截断操作。在蓝藻基因组中多次出现的基因(即 ∆wzy(∆sll0737)、∆wzx(∆sll5049)、∆kpsM(∆slr2107)和 ∆kpsM∆wzy(∆slr2107∆sll0737))的一些突变体没有观察到明显的表型变化,强烈表明存在功能冗余。相比之下,Δwzc(Δsll0923)和Δwzb(Δslr0328)影响 EPS 的数量和组成,表明 Wzc 参与了荚膜(CPS)和释放(RPS)多糖的产生,而 Wzb 影响 RPS 的产生。Wzb 的结构得到了解决(2.28Å),揭示了与其他参与 EPS 生产的磷酸酶的结构差异,并表明了不同的底物识别机制。此外,Wzc 表现出细菌酪氨酸激酶的 ATP 酶和自激酶活性。最重要的是,Wzb 能够在体外使 Wzc 去磷酸化,这表明酪氨酸磷酸化/去磷酸化在蓝藻 EPS 生产中起作用。