Methylthioadenosine Suppresses Salmonella Virulence.

In order to deploy virulence factors at appropriate times and locations, microbes must rapidly sense and respond to various metabolite signals. Previously, we showed a transient elevation of the methionine-derived metabolite methylthioadenosine (MTA) concentration in serum during systemic Salmonella enterica serovar Typhimurium infection. Here we explored the functional consequences of increased MTA concentrations on S Typhimurium virulence. We found that MTA, but not other related metabolites involved in polyamine synthesis and methionine salvage, reduced motility, host cell pyroptosis, and cellular invasion. Further, we developed a genetic model of increased bacterial endogenous MTA production by knocking out the master repressor of the methionine regulon, metJ Like MTA-treated S Typhimurium, the ΔmetJ mutant displayed reduced motility, host cell pyroptosis, and invasion. These phenotypic effects of MTA correlated with suppression of flagellar and Salmonella pathogenicity island 1 (SPI-1) networks. S Typhimurium ΔmetJ had reduced virulence in oral and intraperitoneal infection of C57BL/6J mice independently of the effects of MTA on SPI-1. Finally, ΔmetJ bacteria induced a less severe inflammatory cytokine response in a mouse sepsis model. Together, these data indicate that exposure of S Typhimurium to MTA or disruption of the bacterial methionine metabolism pathway suppresses S Typhimurium virulence.

A novel regulatory factor affecting the transcription of methionine biosynthesis genes in Escherichia coli experiencing sustained nitrogen starvation.

The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcriptional activator, NtrC. One of these NtrC-activated genes is yeaG, which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in ΔyeaG bacteria experiencing sustained N starvation. It appears the ability of MetJ, the master transcriptional repressor of methionine biosynthesis genes, to effectively repress transcription of genes under its control is compromised in ΔyeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. Although the aberrant biosynthesis does not appear to be a contributing factor for the compromised viability of ΔyeaG bacteria experiencing sustained N starvation, this study identifies YeaG as a novel regulatory factor in E. coli affecting the transcription of methionine biosynthesis genes under sustained N starvation.

CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production.

BACKGROUND: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet). RESULTS: Anthocyanin O-methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase (PhANS) and Arabidopsis thaliana anthocyanidin 3-O-glucosyltransferase (At3GT). Vitis vinifera AOMT (VvAOMT1) and fragrant cyclamen 'Kaori-no-mai' AOMT (CkmOMT2) were found to be the most effective AOMTs for production of the 3'-O-methylated product peonidin 3-O-glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of VvAOMT1 and CkmOMT2 expression conditions, production was further improved to 23 mg/L using VvAOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O-methylation of cyanidin 3-O-glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall. CONCLUSIONS: An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O-methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O-methyltransferases in pursuit of bespoke flower pigment compositions.

MetJ-Based Mutually Interfering SAM-ON/SAM-OFF Biosensors.

SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and is a controller of various physiological processes. Its availability is also believed to be a major bottleneck in the biological production of numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, an SAM-dependent transcriptional regulator, as a core component. SAM is a corepressor of MetJ, which suppresses the MetJ promoter with an increasing cellular concentration of SAM (SAM-OFF sensor). The application of transcriptional interference and evolutionary tuning effectively inverted its response, yielding a SAM-ON sensor (signal increases with increasing SAM concentration). By linking two genes encoding fluorescent protein reporters in such a way that their transcription events interfere with each other's and by placing one of them under the control of MetJ, we could increase the effective signal-to-noise ratio of the SAM sensor while decreasing the batch-to-batch deviation in signal output, likely by canceling out the growth-associated fluctuation in translational resources. By taking the ratio of SAM-ON/SAM-OFF signals and by resetting the default pool size of SAM, we could rapidly identify SAM synthetase (MetK) mutants with increased cellular activity from a random library. The strategy described herein should be widely applicable for identifying activity mutants, which would be otherwise overlooked because of the strong homeostasis of metabolic networks.