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1. Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

   https://doi.org/10.7554/eLife.43959  

   Hallberg, Zachary F; Chan, Chi Ho; Wright, Todd A; Kranzusch, Philip J; Doxzen, Kevin W; Park, James J; Bond, Daniel R; Hammond, Ming C (April 2019, eLife)

   Microscopic organisms known as bacteria are found in virtually every environment on the planet. One reason bacteria are so successful is that they are able to form communities known as biofilms on surfaces in animals and other living things, as well as on rocks and other features in the environment. These biofilms protect the bacteria from fluctuations in the environment and toxins. For over 30 years, a class of enzymes called the GGDEF enzymes were thought to make a single signal known as cyclic di-GMP that regulates the formation of biofilms. However, in 2016, a team of researchers reported that some GGDEF enzymes, including one from a bacterium called Geobacter sulfurreducens, were also able to produce two other signals known as cGAMP and cyclic di-AMP. The experiments involved making the enzymes and testing their activity outside the cell. Therefore, it remained unclear whether these enzymes (dubbed ‘Hypr’ GGDEF enzymes) actually produce all three signals inside cells and play a role in forming bacterial biofilms. G. sulfurreducens is unusual because it is able to grow on metallic minerals or electrodes to generate electrical energy. As part of a community of microorganisms, they help break down pollutants in contaminated areas and can generate electricity from wastewater. Now, Hallberg, Chan et al. – including many of the researchers involved in the 2016 work – combined several experimental and mathematical approaches to study the Hypr GGDEF enzymes in G. sulfurreducens. The experiments show that the Hypr GGDEF enzymes produced cGAMP, but not the other two signals, inside the cells. This cGAMP regulated the ability of G. sulfurreducens to grow by extracting electrical energy from the metallic minerals, which appears to be a new, biofilm-less lifestyle. Further experiments revealed how Hypr GGDEF enzymes have evolved to preferentially make cGAMP over the other two signals. Together, these findings demonstrate that enzymes with the ability to make several different signals, are capable of generating specific responses in bacterial cells. By understanding how bacteria make decisions, it may be possible to change their behaviors. The findings of Hallberg, Chan et al. help to identify the signaling pathways involved in this decision-making and provide new tools to study them in the future. 

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2. A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling

   https://doi.org/10.1038/s41467-021-26240-9  

   Banerjee, Daipayan; Langberg, Kurt; Abbas, Salar; Odermatt, Eric; Yerramothu, Praveen; Volaric, Martin; Reidenbach, Matthew A.; Krentz, Kathy J.; Rubinstein, C. Dustin; Brautigan, David L.; et al (December 2021, Nature Communications)

   null (Ed.)

   Abstract Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD + ; meanwhile, restoring NAD + levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN ( Crassostrea virginica and Nematostella vectensis ), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity. 

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3. Second messengers and divergent HD‐GYP phosphodiesterases regulate 3′,3′‐cGAMP signaling

   https://doi.org/10.1111/mmi.14412  

   Wright, Todd A.; Jiang, Lucy; Park, James J.; Anderson, Wyatt A.; Chen, Ge; Hallberg, Zachary F.; Nan, Beiyan; Hammond, Ming C. (November 2019, Molecular Microbiology)

   Summary 3′,3′‐cyclic GMP‐AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB fromMyxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di‐GMP, which provides evidence for cross‐regulation between different CDN pathways. Finally, we reveal that the HD‐GYP enzyme PmxA is a cGAMP‐specific phosphodiesterase (GAP) that promotes resistance to osmotic stress inM. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non‐canonical HD‐GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling. 

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4. Engineered citrate synthase improves citramalic acid generation in Escherichia coli

   https://doi.org/10.1002/bit.27450  

   Wu, Xianghao; Tovilla‐Coutiño, D_Brisbane; Eiteman, Mark_A (June 2020, Biotechnology and Bioengineering)

   Abstract The microbial product citramalic acid (citramalate) serves as a five‐carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized inEscherichia colidirectly by the condensation of pyruvate and acetyl‐CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl‐CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in thegltAgene coding citrate synthase prevents acetyl‐CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study theE. colicitrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl‐CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed‐batch process using MEC626/pZE12‐cimAharboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals. 

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5. Bacterial Cyclic Dinucleotides and the cGAS–cGAMP–STING Pathway: A Role in Periodontitis?

   https://doi.org/10.3390/pathogens10060675  

   Elmanfi, Samira; Yilmaz, Mustafa; Ong, Wilson W.; Yeboah, Kofi S.; Sintim, Herman O.; Gürsoy, Mervi; Könönen, Eija; Gürsoy, Ulvi K. (June 2021, Pathogens)

   null (Ed.)

   Host cells can recognize cytosolic double-stranded DNAs and endogenous second messengers as cyclic dinucleotides—including c-di-GMP, c-di-AMP, and cGAMP—of invading microbes via the critical and essential innate immune signaling adaptor molecule known as STING. This recognition activates the innate immune system and leads to the production of Type I interferons and proinflammatory cytokines. In this review, we (1) focus on the possible role of bacterial cyclic dinucleotides and the STING/TBK1/IRF3 pathway in the pathogenesis of periodontal disease and the regulation of periodontal immune response, and (2) review and discuss activators and inhibitors of the STING pathway as immune response regulators and their potential utility in the treatment of periodontitis. PubMed/Medline, Scopus, and Web of Science were searched with the terms “STING”, “TBK 1”, “IRF3”, and “cGAS”—alone, or together with “periodontitis”. Current studies produced evidence for using STING-pathway-targeting molecules as part of anticancer therapy, and as vaccine adjuvants against microbial infections; however, the role of the STING/TBK1/IRF3 pathway in periodontal disease pathogenesis is still undiscovered. Understanding the stimulation of the innate immune response by cyclic dinucleotides opens a new approach to host modulation therapies in periodontology. 

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