School of Life Sciences, University of Nottingham, Medical School, Nottingham, United Kingdom.
University of California, Berkeley, Department of Chemistry, Berkeley, California, United States of America.
PLoS Genet. 2022 May 27;18(5):e1010164. doi: 10.1371/journal.pgen.1010164. eCollection 2022 May.
Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3',3'-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.
细菌的第二信使对于调节各种细菌的生活方式非常重要。环二鸟苷酸（c-di-GMP）是由二鸟苷酸环化酶酶产生的，这些酶被命名为 GGDEF 蛋白，它们在细菌中广泛存在。最近，一些细菌中描述了混合混杂（Hypr）的 GGDEF 蛋白，这些蛋白既能产生 c-di-GMP，也能产生最近发现的另一种细菌第二信使，3'，3'-环鸟苷酸-AMP（cGAMP）。这些蛋白之一存在于捕食性细菌蛭弧菌 Bd0367 中。发现 bd0367 GGDEF 基因缺失菌株能够进入猎物细胞，但一旦捕食细胞分裂完成，它就无法通过在固体表面滑行运动来离开耗尽的猎物残留物。然而，尚不清楚哪种信号调节了这一过程。我们表明 cGAMP 信号在 B. bacteriovorus 中是活跃的，除了产生 c-di-GMP 和一些 c-di-AMP 外，Bd0367 还是体内 cGAMP 的主要生产者。将丝氨酸 214 突变为天冬氨酸使 Bd0367 主要成为 c-di-GMP 合酶。B. bacteriovorus 菌株 bd0367S214D 通过无法在固体表面滑行而类似于 bd0367 缺失菌株，导致新后代在复制后无法从猎物细胞中退出。因此，这个过程受 cGAMP 调节。还发现 bd0367 的缺失与野生型鞭毛生物发生不兼容，这是由于鞭毛伴侣蛋白基因同源物 fliS 获得了突变，这表明 c-di-GMP 参与了游泳运动的调节。因此，单个 Bd0367 酶通过相同 GGDEF 结构域的作用产生两种第二信使，这是体内调节多种第二信使的第一个报道的例子。与这些信号分子在其他细菌中的作用不同，它们作用于两个独立的运动系统，滑行和鞭毛，这对于 B. bacteriovorus 完成细菌捕食周期和猎物退出至关重要。
