Production of 3',3'-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme, Bd0367, regulates exit from prey by gliding motility.
Production of 3',3'-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme, Bd0367, regulates exit from prey by gliding motility.
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DOI：
10.1371/journal.pgen.1010164
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发表时间：
2022-05
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影响因子：
4.5
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细菌第二信使对于调节不同细菌的生活方式很重要。环状二-GMP (c-di-GMP) 由二鸟苷酸环化酶产生，称为 GGDEF 蛋白，广泛存在于细菌中。最近，在一些细菌中描述了混合混杂 (Hypr) GGDEF 蛋白，其产生 c-di-GMP 和最近鉴定的细菌第二信使 3',3'-环状 GMP-AMP (cGAMP)。其中一种蛋白质是在捕食性噬菌蛭弧菌 Bd0367 中发现的。研究发现，bd0367 GGDEF 基因缺失菌株可以进入猎物细胞，但一旦捕食者细胞分裂完成，就无法通过在固体表面上的滑动运动留下耗尽的猎物残余物。然而，尚不清楚哪个信号调节这一过程。我们表明，cGAMP 信号传导在食菌芽孢杆菌内活跃，并且除了产生 c-di-GMP 和一些 c-di-AMP 之外，Bd0367 也是体内 cGAMP 的主要生产者。将丝氨酸 214 定点诱变为天冬氨酸，使 Bd0367 主要转化为 c-di-GMP 合酶。 B. bacteriovorus 菌株 bd0367S214D 通过无法在固体表面上滑动来复制 bd0367 缺失菌株的表型，从而导致新的子代无法在复制后从猎物细胞中退出。因此，该过程受cGAMP 调节。还发现 bd0367 的删除与野生型鞭毛生物发生不相容，这是鞭毛蛋白伴侣基因同源物 fliS 发生获得性突变的结果，表明 c-di-GMP 参与游泳运动的调节。因此，单个 Bd0367 酶通过相同 GGDEF 结构域的作用产生两个第二信使，这是第一个报道的体内调节多个第二信使的合酶的例子。与这些信号分子在其他细菌中的作用不同，这些信号分子向两个独立的运动系统（滑动系统和鞭毛系统）发出信号，这对于完成细菌捕食周期和食菌芽孢杆菌猎物的退出至关重要。二级信使是细菌中重要的信号分子，最近发现了一种称为 cGAMP 的分子，最近显示它是由一些酶产生的，而这些酶以前已知会产生另一种二级信使 c-di-GMP。其中一种“混合混杂”酶 (Bd0367) 存在于噬菌蛭弧菌中，这种细菌捕食其他细菌，在它们内部挖洞并从内部消耗它们。先前的基因删除工作表明，Bd0367 对于蛭弧菌在捕食完成后通过滑行运动离开猎物细胞残余物的信号传导至关重要，并且人们认为这是由于 c-di-GMP 信号传导所致。然而，在这里，我们表明这种滑行运动实际上是由 cGAMP 信号传导调节的，并且 c-di-GMP 信号传导参与游泳运动。一种酶产生两种不同的分子，向两个离散的运动系统发出信号，这两种运动系统都是细菌在固体表面或液体中成功完成捕食生活方式所必需的。
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. Secondary messengers are important signalling molecules in bacteria and a recently discovered one, called cGAMP, has recently been shown to be made by some enzymes which had previously been known to produce another secondary messenger, c-di-GMP. One of these “hybrid promiscuous” enzymes (Bd0367) is found in Bdellovibrio bacteriovorus, a bacterium that preys upon other bacteria, burrowing inside them and consuming them from within. Previous gene deletion work had shown that Bd0367 was essential in signalling for Bdellovibrio to leave the remains of its prey cell by gliding motility after predation was complete and it was thought that this was due to c-di-GMP signalling. However, here, we show that this gliding motility is actually regulated by cGAMP signalling and that c-di-GMP signalling is involved in swimming motility. A single enzyme produces two different molecules, signalling to two discrete motility systems, both of which are required for successful completion of the bacterium’s predatory lifestyle in prey on solid surfaces or in liquids.