Shining the Light on Cyclic di-GMP Dark Matter

Waters, Christopher M.

doi:10.1128/jb.00030-18

Cited by 3 publications

(2 citation statements)

References 19 publications

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“…In addition to the possibility of posttranscriptional differences in expression, our results suggest that local signaling effectse.g., localization, protein-protein interactions-may play a major role in how these factors mediate host interactions. There is growing evidence for local signaling and regulated activity of distinct DGCs and PDEs (74)(75)(76), and unique patterns in our data provide hints as to where such regulation could occur in V. fischeri.…”

Section: Discussionmentioning

confidence: 55%

Functional analysis of cyclic diguanylate-modulating proteins inVibrio fischeri

Isenberg

Holschbach

Mandel

2023

Preprint

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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 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.

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Section: Discussionmentioning

confidence: 55%

Functional analysis of cyclic diguanylate-modulating proteins inVibrio fischeri

Isenberg

Holschbach

Mandel

2023

Preprint

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“…This defect in colonization can be rescued by an epsA-O mutant, indicating that it is most likely acting upstream of EPS biosynthesis in the regulatory cascade [46]. The more well-known bacterial second messenger, cyclic-di-GMP, was first identified as a regulator of cellulose biosynthesis is Acetobacter xylinum [47] and has since been identified to regulate many cellular processes including biofilm formation in a diverse range of bacteria [reviewed in 48,49]. Biofilm formation in B. subtilis does not appear to be as strongly regulated by c-di-GMP as in other bacteria, but a role as been observed [50].…”

Section: Chapter 1: Introductionmentioning

confidence: 99%

Novel signaling mechanisms regulating biofilm formation in Bacillus subtilis

Greenwich¹

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Associate Professor of Biology would not be where I am today without all of you. To the LONG squad (past and present) -thank you for letting a scientist be a history nerd and play lawn games all these years. It means the world to me. To the many friends I made along the way-thank you for the love and support you have given me over the years. Dan-thanks for starting so many conversations we'll never finish, giving me an excuse to leave the lab, and of course, introducing me to my favorite brothers that I am not related to. I never would have gotten through grad school without them. To John and Hank Green, for never forgetting to be awesome and giving me an escape from grad school. To Hank, thank you for helping me identify what my passions are and being an inspirational science communicator.Finally, and most importantly, thank you to my family. Your unconditional love and support has brought me to where I am and I could not have done it without you. This degree belongs as much to you as it does to me, if not more so. Mom-you never let me give up, and I don't tell you often enough how much I appreciate that. Aunt Marge-thank you for everything you've done to support me all of these years. Joe-thanks for keeping me humble and reminding me to be normal. Sorry I'll never remember. Abstract of DissertationBacteria are capable of forming multicellular communities, known as biofilms. Bacillus subtilis is a Gram-positive, rod shaped, soil-dwelling bacterium often used as a model organism to study biofilm formation. Both extracellular and intracellular signals can trigger biofilm formation through a signaling cascade, which turns on genes involved in production of the biofilm matrix. In B. subtilis, the matrix consists of an exopolysaccharide (EPS) component and two protein components, which are regulated on a genetic level by two master regulators, SinR and AbrB.Biosynthesis of the EPS is also regulated at the post-translational level, by a bacterial tyrosine kinase (BY-kinase), EpsB, which is encoded within the same operon as the enzymes involved in the biosynthesis of EPS. EpsB is activated by EpsA, which senses EPS in a quorum-sensing-like manner. We show genetic evidence for EpsB activation by TkmA, a modulator of a different BYkinase can activate EpsB. This cross-talk allows B. subtilis to increase the signals to which it can respond.Intracellularly, serine starvation triggers biofilm formation. We elucidate the mechanism by which this occurs and investigate the regulation of serine biosynthesis. Expression of serA, which encodes SerA, the enzyme responsible for the first and rate-limiting step of serine biosynthesis decreases upon entry into stationary phase and is regulated by catabolite control. This regulation allows cells to trigger biofilm formation based on available nutrients as well as external stimuli.Accompanying this decrease in serine is a decrease in seryl-tRNA isoacceptors, responsible for adding serine to growing peptide chains in the ribosome. It was previously observed that ribosomes pause on specif...

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Shigella flexneri Diguanylate Cyclases Regulate Virulence

et al. 2021

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Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli , and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-guanosine monophosphate (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. C-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri . In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae , in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation, and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome, and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation, reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes Importance The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella species is on the rise. Here, we explore how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We find that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella ’s ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

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Shining the Light on Cyclic di-GMP Dark Matter

Cited by 3 publications

References 19 publications

Functional analysis of cyclic diguanylate-modulating proteins inVibrio fischeri

Functional analysis of cyclic diguanylate-modulating proteins inVibrio fischeri

Novel signaling mechanisms regulating biofilm formation in Bacillus subtilis

Shigella flexneri Diguanylate Cyclases Regulate Virulence

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