In Pseudomonas aeruginosa, the orphan two-component sensor SagS contributes both to transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug-tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment since glucose-6-phosphate likewise enhanced biofilm formation and also enhanced the expression of select biofilm marker genes. Moreover, exposure to glucose-6-phosphate coincided with decreased swarming motility but increased cellular cyclic-di-GMP (c-di-GMP) levels in biofilms. No such response was noted for compounds modulating attachment and biofilm formation in a manner independent of SagS. Modulation of c-di-GMP in response to glucose-6-phosphate was due to the diguanylate cyclase NicD, with NicD also being required for enhanced biofilm formation. The latter was independent of the sensory domain of NicD but dependent on NicD activity, SagS, and the interaction between NicD and SagS. Our findings indicate that glucose-6-phosphate likely mimics a signal or conditions sensed by SagS to activate its motile-sessile switch function. In addition, our findings provide new insight into the interfaces between the ligand-mediated two-component system signaling pathway and c-di-GMP levels.

中文翻译：

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6-磷酸葡萄糖作为 SagS 的细胞外信号调节铜绿假单胞菌 c-di-GMP 水平、附着和生物膜形成

在铜绿假单胞菌中，孤儿双组分传感器 SagS 有助于过渡到生物膜形成和生物膜细胞获得对抗菌素的更高耐受性。然而，对于 SagS 感应到的信号或条件的身份以诱导转变为固着的、耐药的生长模式，我们知之甚少。使用改良的 Biolog 表型分析来筛选以 SagS 依赖性方式调节附着的化合物，我们确定了 6-磷酸葡萄糖以依赖于 6-磷酸葡萄糖浓度和 SagS 的方式增强附着。刺激作用不仅限于附着，因为 6- 磷酸葡萄糖同样增强了生物膜的形成，并且还增强了选择的生物膜标记基因的表达。而且，暴露于 6-磷酸葡萄糖与降低蜂群运动性同时增加生物膜中的细胞环二 GMP (c-di-GMP) 水平。对于以独立于 SagS 的方式调节附着和生物膜形成的化合物，没有注意到这种反应。c-di-GMP 对葡萄糖-6-磷酸的调节是由于二鸟苷酸环化酶 NicD，NicD 也是增强生物膜形成所必需的。后者独立于 NicD 的感觉域，但依赖于 NicD 活动、SagS 以及 NicD 和 SagS 之间的相互作用。我们的研究结果表明，6-磷酸葡萄糖可能会模仿 SagS 感知到的信号或条件，以激活其运动-静止开关功能。此外，