Functional analysis of cyclic diguanylate-modulating proteins in Vibrio fischeri.

IF 5 2区 生物学 Q1 MICROBIOLOGY mSystems Pub Date : 2024-10-22 DOI:10.1128/msystems.00956-24
Ruth Y Isenberg, Chandler S Holschbach, Jing Gao, Mark J Mandel
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Abstract

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.

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费氏弧菌中环状二聚氰胺调节蛋白的功能分析
当细菌共生体从运动的自由生活状态过渡到无梗生物膜状态时,它们必须协调适合每种生活方式的行为变化。环状二胍酸盐(c-di-GMP)是一种细胞内信号分子,可以调节这种转变,它由二胍酸盐环化酶(DGC)合成,并由磷酸二酯酶(PDE)降解。一般来说,c-di-GMP 可抑制蠕动并促进生物膜的形成。虽然对病原体中的 c-di-GMP 及其代谢酶进行了深入研究,但对有益共生体中 c-di-GMP 调节的关注却少得多。费氏弧菌是夏威夷大尾乌贼(Euprymna scolopes)光器官的唯一有益共生菌,该菌需要同时具有运动性和形成生物膜才能有效定殖。鱼腥藻菌的基因组编码 50 种 DGC 和 PDE,虽然其中少数蛋白已经定性,但大多数蛋白尚未进行全面定性。在本研究中,我们利用蛋白质过表达系统地鉴定了所有 50 种 V. fischeri 蛋白的功能潜力。所有 28 个预测的 DGCs 和 14 个预测的 PDEs 中的 10 个都显示出至少一种与其预测功能一致的表型,其中大多数都显示出多种表型。最后,对可能同时具有 DGC 和 PDE 活性的蛋白质进行活性位点突变分析,发现了这些蛋白质的潜在活性。这项研究在系统水平上对可控动物共生体中的信号蛋白家族进行了功能分析,并将为今后鉴定单个蛋白在生活方式转变过程中的作用提供信息。这项研究提供了预测可调节 c-di-GMP 水平的 50 种蛋白质的系统级特征。通过结合多种检测方法,我们对哪些蛋白质有能力影响 c-di-GMP 水平和行为有了丰富的了解。我们的功能性方法让我们深入了解了具有合成和降解 c-di-GMP 功能域的蛋白质是如何影响细菌行为的。最后,我们整合了已发表的数据,为所分析的 50 种蛋白质中的每一种提供了更广泛的信息。这项研究将为今后的工作提供信息,以确定 c-di-GMP 调节共生行为和过渡的具体途径。
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来源期刊
mSystems
mSystems Biochemistry, Genetics and Molecular Biology-Biochemistry
CiteScore
10.50
自引率
3.10%
发文量
308
审稿时长
13 weeks
期刊介绍: mSystems™ will publish preeminent work that stems from applying technologies for high-throughput analyses to achieve insights into the metabolic and regulatory systems at the scale of both the single cell and microbial communities. The scope of mSystems™ encompasses all important biological and biochemical findings drawn from analyses of large data sets, as well as new computational approaches for deriving these insights. mSystems™ will welcome submissions from researchers who focus on the microbiome, genomics, metagenomics, transcriptomics, metabolomics, proteomics, glycomics, bioinformatics, and computational microbiology. mSystems™ will provide streamlined decisions, while carrying on ASM''s tradition of rigorous peer review.
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