Figures
Citation: Rangarajan AA, Waters CM (2022) Double take: A dual-functional Hypr GGDEF synthesizes both cyclic di-GMP and cyclic GMP—AMP to control predation in Bdellovibrio bacteriovorus. PLoS Genet 18(7): e1010263. https://doi.org/10.1371/journal.pgen.1010263
Editor: Sean Crosson, Michigan State University, UNITED STATES
Published: July 21, 2022
Copyright: © 2022 Rangarajan, Waters. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (NIH) grants GM139537 and AI58433 to C.M.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Bacteria possess well-orchestrated signal transduction mechanisms, such as nucleotide second messengers, to tune gene regulation for adaption and survival in variety of environmental conditions [1,2]. Over the last 2 decades, our knowledge of cyclic di-nucleotide (cdNs) second messengers has exploded, revealing that these molecules are key signals that carry out this function in bacteria and eukaryotes [3]. A recent PLOS Genetics paper by Lowry and colleagues adds a new twist to cdN signaling, demonstrating for the first time that a cdN synthase can synthesize in vivo 2 cdNs that each have a distinct physiological role [4].
There are 3 families of cdN synthases that regulate a multitude of biological functions (Fig 1). Cyclic di-GMP (c-di-GMP), the earliest discovered cdN, is synthesized by diguanylate cyclase (DGC) enzymes containing a GGDEF motif [5,6]. C-di-GMP regulates a variety of cellular processes including virulence, motility, biofilm formation, and even cell shape [6–8]. DGCs are generally highly specific for making c-di-GMP, but some DGCs, known as Hybrid promiscuous (Hypr) GGDEFs, which consist of less than 0.2% of all GGDEF domains, synthesize 3′3′ cyclic GMP–AMP (cGAMP) instead of c-di-GMP [9]. Cyclic di-AMP (c-di-AMP) is synthesized by diadenylate cyclase (DAC) domains, and no DACs have been discovered that synthesize other cdNs [10,11]. Cyclic di-AMP plays a crucial role in DNA repair, cell wall homeostasis, osmotic homeostasis, sporulation, and biofilm formation in predominantly gram-positive bacteria [12,13]. Another broadly classified group of enzymes, the cGAS/DncV-like nucelotidyl transferase (CD-NTase), which are also widely present in several bacterial phyla, produces a variety of purine, pyrimidine, and purine-pyrimidine hybrid cdNs and even cyclic tri-nucleotides [14]. This family of enzymes includes DncV in Vibrio cholerae and cGAS in metazoans, and it is the most promiscuous of the cdN synthase family [14,15]. cGAMP synthesized by CD-NTases mediates bacterial phage defense through an abortive infection mechanism, while cGAMP synthesized by Hypr GGDEFs controls exoelectrogenesis in Geobacter sulfurreducens [15–21].
CD-NTase, cGAS/DncV-like nucelotidyl transferase; cdN, cyclic di-nucleotide; DAC, diadenylate cyclase; DGC, diguanylate cyclase.
Lowry and colleagues have expanded our understanding of these 3 cdN synthase enzyme families by discovering a novel Hypr GGDEF protein in Bdellovibrio bacteriovorus, Bd0367, which synthesizes both cGAMP and c-di-GMP to regulate gliding and swimming motility, respectively (Fig 2) [4]. B. bacteriovorus is gram-negative bacterium that preys upon other bacteria by invading their periplasm and stealing the host cell’s nutrients for growth before ultimately escaping the exhausted prey using gliding motility [22,23]. While a Δbd0367 mutant of B. bacteriovorus could enter prey cells, it was unable to glide and escape from the prey to complete the predatory life cycle, and this defect was previously attributed to a loss of c-di-GMP synthesis. [24]. However, Bd0367 is actually a Hypr GGDEF because it encodes a key amino-terminal serine (S124) in its GGDEF domain that allows utilization of GTP and ATP while standard GGDEFs encode an aspartate at this position [18]. Whereas the WT Bd0367 enzyme primarily synthesizes 3′3′-cGAMP in vitro, generation of the Bd0367S124D mutant enzyme rendered it solely able to synthesize c-di-GMP. Moreover, complementation of the Δbd0367 mutant with the bd0367S124D allele abolished in vivo cGAMP synthesis and did not restore functional gliding motility or escape from prey cells [18]. This is an exciting finding as it expands the realm of bacterial cGAMP signaling into Bdellovibrio. For reasons that are not yet understood, Δbd0367 mutants only grow outside of the host if they acquire a null suppressor mutation in the flagellar chaperone fliS. However, the bd0367S124D mutant does not mimic the Δbd0367 mutant as it can grow outside of the host without the need for additional suppressor mutations in fliS, suggesting c-di-GMP synthesis by Bd0367 has a functional role in the cell that abrogates the need for fliS suppressor mutations during host independent growth. This is the first in vivo example showing a single Hypr GGDEF containing enzyme produces in vivo 2 different cdNs, 3′3′-cGAMP and c-di-GMP, in which each regulate a distinct physiological process.
Bd0367 can produce cGAMP and c-di-GMP, which control gliding motility and swimming motility, respectively. The signals that regulate synthesis of c-di-GMP or cGAMP by Bd0367 are unknown.
The predatory bacterium B. bacteriovorus undergoes a complex life cycle involving several sequential events: hunt and attachment to prey, prey entry, intracellular growth forming a bdelloplast, replication inside the bdelloplast, rupture, and exit of the prey cell [22,23]. The physiological conditions and the growth phase that triggers Bd0367 enzymes to produce cGAMP or c-di-GMP is unknown, but it could depend on several factors including the specific host, ATP/GTP concentrations in that host, dimerization of the protein, or unidentified signals affecting the amino-terminal receiver domain. B. bacteriovorus has only 4 GGDEF containing proteins, whereas other bacteria like Vibrios, Escherichia coli, or G. sulfurreducens have dozens of DGCs [23–26]. Due to the small number of GGDEF proteins present in B. bacteriovorus, Bd0367 may have evolved to produce more than 1 cdN to increase signaling flexibility, allowing it to respond to specific signals to successfully complete its predatory lifecycle.
The well-characterized Hypr GGDEF protein GacA in G. sulfurreducens synthesizes c-di-GMP, cGAMP, and c-di-AMP in vitro but only an in vivo role for cGAMP has been determined [18]. Likewise, CD-NTases from many bacteria produce more than 1 dinucleotide signal in vitro, but their in vivo activity is less characterized [14]. Hypr GGDEF containing proteins are present across different genera [9]. It is possible that these Hypr GGDEF and other CD-NTases produce more than 1 functional cdN in vivo under specific physiological conditions. This might be especially true for predatory bacteria like Bdellovibrio or species that encode fewer GGDEF or Hypr GGDEF containing proteins, allowing an expansion of the cdN repertoire with fewer synthases. The research of Lowry and colleagues raises the possibility that cdN synthases could produce multiple signals, each with a distinct regulatory role, and it will be intriguing to discover which synthases have this capability [4].
References
- 1. Hengge R, Gründling A, Jenal U, Ryan R, Yildiz F. Bacterial signal transduction by cyclic di-GMP and other nucleotide second messengers. J Bacteriol. 2016;198:15–26. pmid:26055111
- 2. Krasteva PV, Sondermann H. Versatile modes of cellular regulation via cyclic dinucleotides. Nat Chem Biol. 2017;13:350–9. pmid:28328921
- 3. Yoon S, hun, Waters CM. The ever-expanding world of bacterial cyclic oligonucleotide second messengers. Curr Opin Microbiol. 2021;60:96–103. pmid:33640793
- 4. Lowry RC, Hallberg ZF, Till R, Simons TJ, Nottingham R, Want F, et al. Production of 3’,3’-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme, Bd0367, regulates exit from prey by gliding motility. PLoS Genet. 2022:1–24. pmid:35622882
- 5. Römling U, Galperin MY, Gomelsky M. Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger. Microbiol Mol Biol Rev. 2013;77:1–52. pmid:23471616
- 6. Jenal U, Reinders A, Lori C. Cyclic di-GMP: Second messenger extraordinaire. Nat Rev Microbiol. 2017;15:271–84. pmid:28163311
- 7. Conner JG, Zamorano-Sánchez D, Park JH, Sondermann H, Yildiz FH. The ins and outs of cyclic di-GMP signaling in Vibrio cholerae. Curr Opin Microbiol. 2017;36:20–9. pmid:28171809
- 8. Fernandez NL, Hsueh BY, Nhu NTQ, Franklin JL, Dufour YS, Waters CM. Vibrio cholerae adapts to sessile and motile lifestyles by cyclic di-GMP regulation of cell shape. Proc Natl Acad Sci U S A. 2020;117:29046–54. pmid:33139575
- 9. Hallberg ZF, Wang XC, Wright TA, Nan B, Ad O, Yeo J, et al. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3’, 3’-cGAMP). Proc Natl Acad Sci U S A. 2016;113:1790–5. pmid:26839412
- 10. Commichau FM, Heidemann JL, Ficner R, Stülke J. Making and breaking of an essential poison: The cyclases and phosphodiesterases that produce and degrade the essential second messenger cyclic di-AMP in bacteria. J Bacteriol. 2019;201. pmid:30224435
- 11. Mudgal S, Manikandan K, Mukherjee A, Krishnan A, Sinha KM. Cyclic di-AMP: Small molecule with big roles in bacteria. Microb Pathog. 2021;161:105264. pmid:34715302
- 12. Corrigan RM, Gründling A. Cyclic di-AMP: Another second messenger enters the fray. Nat Rev Microbiol. 2013;11:513–24. pmid:23812326
- 13. Stülke J, Krüger L. Cyclic di-AMP Signaling in Bacteria. Annu Rev Microbiol. 2020;74:159–79. pmid:32603625
- 14. Whiteley AT, Eaglesham JB, de Oliveira Mann CC, Morehouse BR, Lowey B, Nieminen EA, et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature. 2019;567:194–9. pmid:30787435
- 15. Davies BW, Bogard RW, Young TS, Mekalanos JJ. Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence. Cell. 2012;149:358–70. pmid:22500802
- 16. Gao D, Wu J, Wu Y, Du F, Aroh C, Yan N, et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science. 2013;1375:903–7. pmid:23929945
- 17. Li F, Cimdins A, Rohde M, Jänsch L, Kaever V, Nimtz M, et al. DncV synthesizes cyclic GMP-AMP and regulates biofilm formation and motility in Escherichia coli ECOR31. mBio. 2019;10. pmid:30837338
- 18. Hallberg ZF, Chan CH, Wright TA, Kranzusch PJ, Doxzen KW, Park JJ, et al. Structure and mechanism of a hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration. Elife. 2019;8:1–36. pmid:30964001
- 19. Severin GB, Ramliden MS, Hawver LA, Wang K, Pell ME, Kieninger AK, et al. Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae. Proc Natl Acad Sci U S A. 2018;115:E6048–55. pmid:29891656
- 20. Nelson JW, Sudarsan N, Phillips GE, Stav S, Lünse CE, McCown PJ, et al. Control of bacterial exoelectrogenesis by c-AMP-GMP. Proc Natl Acad Sci U S A. 2015;112:5389–94. pmid:25848023
- 21. Cohen D, Melamed S, Millman A, Shulman G, Oppenheimer-Shaanan Y, Kacen A, et al. Cyclic GMP–AMP signalling protects bacteria against viral infection. Nature. 2019;574:691–5. pmid:31533127
- 22. Rendulic S, Jagtap P, Rosinus A, Eppinger M, Baar C, Lanz C, et al. A Predator Unmasked: Life Cycle of Bdellovibrio bacteriovorus from a Genomic Perspective. Science. 2004;303:689–92. pmid:14752164
- 23. Rotema O, Pasternak Z, Shimoni E, Belausov E, Porat Z, Pietrokovski S, et al. Cell-cycle progress in obligate predatory bacteria is dependent upon sequential sensing of prey recognition and prey quality cues. Proc Natl Acad Sci U S A. 2015;112:E6028–37. pmid:26487679
- 24. Hobley L, Fung RKY, Lambert C, Harris MATS, Dabhi JM, King SS, et al. Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus. PLoS Pathog. 2012;8. pmid:22319440
- 25. Massie JP, Reynolds EL, Koestler BJ, Cong JP, Agostoni M, Waters CM. Quantification of high-specificity cyclic diguanylate signaling. Proc Natl Acad Sci U S A. 2012;109:12746–51. pmid:22802636
- 26. Galperin MY. Bacterial signal transduction network in a genomic perspective. Environ Microbiol. 2004;6:552–67. pmid:15142243