In Vitro BioTransformation (ivBT): Definitions, Opportunities, and Challenges

Regulation of translation by methylation multiplicity of 18S rRNA

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation

AbstractAutophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFImin splicing and SAM metabolism.

Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction

ABSTRACTThe signaling ligand (p)ppGpp binds diverse targets across bacteria, yet the mechanistic and evolutionary basis underlying these ligand-protein interactions remains poorly understood. Here we identify a novel (p)ppGpp binding motif in the enzyme HPRT, where (p)ppGpp shares identical binding residues for PRPP and nucleobase substrates to regulate purine homeostasis. Intriguingly, HPRTs across species share the conserved binding site yet strongly differ in ligand binding, from strong inhibition by basal (p)ppGpp levels to weak regulation at induced concentrations. Surprisingly, strong ligand binding requires an HPRT dimer-dimer interaction that allosterically opens the (p)ppGpp pocket. This dimer-dimer interaction is absent in the common ancestor but evolved to favor (p)ppGpp binding in the vast majority of bacteria. We propose that the evolutionary plasticity of oligomeric interfaces enables allosteric adjustment of ligand regulation, bypassing constraints of the ligand binding site. Since most ligands bind near protein-protein interfaces, this principle likely extends to other protein-ligand interactions.

Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations

Many bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities. Bacillus subtilis is a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for single B. subtilis cells. Our analysis shows that c-di-GMP levels are conspicuously different among B. subtilis cellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.

The U6 snRNA m 6 A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

ABSTRACT The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis , (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel Ef ) and the small alarmone synthetase (SAS) RelQ Ef . Although Rel is the main enzyme responsible for SR activation in Firmicutes , there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ Ef synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQ Ef also efficiently utilized GMP to form GMP 3′-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ Ef was activated only by ppGpp. Furthermore, enzymatic activity of RelQ Ef is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of “long” RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQ Ef ), we found that, in addition to (p)ppGpp, RelQ Ef is an efficient producer of pGpp (GMP 3′-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQ Ef and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Diversity in (p)ppGpp metabolism and effectors

Molecular Mechanism and Evolution of Guanylate Kinase Regulation by (p)ppGpp

Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli

SummaryDuring amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re‐programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three Escherichia coli strains, each expressing a different RelA‐fluorescent protein (RelA‐FP) construct: RelA‐YFP, RelA‐mEos2 and RelA‐Dendra2. Single‐molecule localization and tracking studies were carried out under normal growth conditions and during amino acid starvation. Study of three labeling schemes enabled us to assess potential problems with FP labeling of RelA. The diffusive trajectories and axial spatial distributions indicate that amino acid starvation induces net binding of all three RelA‐FP constructs to 70S ribosomes. The data are most consistent with a model in which RelA synthesizes (p)ppGpp while bound to the 70S ribosome. We suggest a ‘short hopping time’ model of RelA activity during starvation. Our results contradict an earlier study of RelA‐Dendra2 diffusion that inferred off‐ribosome synthesis of (p)ppGpp. The reasons for the discrepancy remain unclear.

Basal Levels of (p)ppGpp in Enterococcus faecalis: the Magic beyond the Stringent Response

ABSTRACT The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis , production of (p)ppGpp is controlled by the bifunctional protein RSH (for “Rel SpoT homologue”; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh , relQ , or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the ∆ rsh ∆ relQ [(p)ppGpp 0 ] strain, suggesting that a lack of basal (p)ppGpp places the cell in a “transcriptionally relaxed” state. Alterations in the fermentation profile and increased production of H 2 O 2 in the (p)ppGpp 0 strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis , (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions. IMPORTANCE Drug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogen Enterococcus faecalis as a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.

Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance

Escherichia coli O157:H7 of Genotype Lineage-Specific Polymorphism Assay 211111 and Clade 8 Are Common Clinical Isolates Within Pennsylvania