Lipases and carboxylesterases are involved in interspecific pheromone differences between two moth species

Moth sex pheromones are a classical model for studying sexual selection. Females produce a species-specific pheromone blend that attracts males. Revealing the enzymes involved in the interspecific variation in blend composition is key for understanding the evolution of these sexual communication systems. The nature of the enzymes involved in the variation of acetate esters, which are prominent compounds in moth pheromone blends, remains unclear. We identified enzymes involved in acetate metabolism in two closely related species: Heliothis (Chloridea) subflexa and H. (C.) virescens, which differ in production of acetate esters. Through comparative transcriptomic analyses and CRISPR/Cas9 knockouts, we showed that two lipases and two esterases induce lower levels of acetate esters in female pheromones. To place our findings in an evolutionary context, we explored the molecular evolution of related lipases and esterases in Lepidoptera. Together, our results show that lipases and carboxylesterases are unexpectedly involved in tuning Lepidoptera pheromones composition.

Nutritional Ketosis as a Potential Treatment for Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic, relapsing brain disorder, characterized by compulsive alcohol seeking and disrupted brain function. In individuals with AUD, abstinence from alcohol often precipitates withdrawal symptoms than can be life threatening. Here, we review evidence for nutritional ketosis as a potential means to reduce withdrawal and alcohol craving. We also review the underlying mechanisms of action of ketosis. Several findings suggest that during alcohol intoxication there is a shift from glucose to acetate metabolism that is enhanced in individuals with AUD. During withdrawal, there is a decline in acetate levels that can result in an energy deficit and could contribute to neurotoxicity. A ketogenic diet or ingestion of a ketone ester elevates ketone bodies (acetoacetate, β-hydroxybutyrate and acetone) in plasma and brain, resulting in nutritional ketosis. These effects have been shown to reduce alcohol withdrawal symptoms, alcohol craving, and alcohol consumption in both preclinical and clinical studies. Thus, nutritional ketosis may represent a unique treatment option for AUD: namely, a nutritional intervention that could be used alone or to augment the effects of medications.

Proteomic Analysis of a Syntrophic Coculture of Syntrophobacter fumaroxidans MPOBT and Geobacter sulfurreducens PCAT

We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems.

PSVII-18 Association of post-absorptive glucose and acetate metabolism with feed intake, growth, and efficiency in finishing beef heifers

Glucose and acetate are important nutrients for muscle and fat accretion in beef cattle. The objective of this experiment was to determine if the demand for acetate and glucose, as well as insulin response to glucose, are associated with dry matter intake (DMI), average daily gain (ADG), residual feed intake (RFI), and gain:feed (G:F). Charolais heifers (n = 16; initial BW = 412 ± 10 kg) were trained to close human contact and fed a finishing diet ad libitum in an Insentec feeding system. Following a 12-hour fast, a jugular catheter was inserted, and an acetate clearance test was performed by infusing acetate (2.18 mmol/kg BW0.75) and collecting blood samples over a 30-minute period. One hour after the conclusion of the acetate test, a glucose clearance test was performed by infusing glucose (7.57 mmol/kg BW0.75) and collecting samples over a two-hour period. Four days after the metabolic tests, heifers began an 84-d DMI and ADG test period. The area under the acetate, glucose, and insulin curves were calculated as were the clearance rate, peaks, nadir, and insulin time to peak. Pearson correlations were calculated for the metabolic parameters and production traits using SAS 9.4. Heifers gained 1.69 ± 0.03 kg/d and consumed 10.4 ± 0.19 kg/d. Acetate and glucose clearance rates were not associated with any production trait (P > 0.40). Insulin time to peak concentration after the glucose challenge was associated (r = 0.69; P = 0.003) with G:F, but peak concentration was not (P = 0.45). Additionally, there was a trend (r = 0.40; P = 0.13) for area under the insulin curve to be associated with G:F. Given the small sample size in this experiment, it is possible that decreased insulin sensitivity early in the finishing period is related to improved feed efficiency in finishing heifers.

Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

Involvement of the MxtR/ErdR (CrbS/CrbR) Two-Component System in Acetate Metabolism in Pseudomonas putida KT2440

MxtR/ErdR (also called CrbS/CrbR) is a two-component system previously identified as important for the utilization of acetate in Vibrio cholerae and some Pseudomonas species. In addition, evidence has been found in Pseudomonas aeruginosa for a role in regulating the synthesis and expression, respectively, of virulence factors such as siderophores and RND transporters. In this context, we investigated the physiological role of the MxtR/ErdR system in the soil bacterium Pseudomonas putida KT2440. To that end, mxtR and erdR were individually deleted and the ability of the resulting mutants to metabolize different carbon sources was analyzed in comparison to wild type. We also assessed the impact of the deletions on siderophore production, expression of mexEF-oprN (RND transporter), and the biocontrol properties of the strain. Furthermore, the MxtR/ErdR-dependent expression of putative target genes and binding of ErdR to respective promoter regions were analyzed. Our results indicated that the MxtR/ErdR system is active and essential for acetate utilization in P. putida KT2440. Expression of scpC, pp_0354, and acsA-I was stimulated by acetate, while direct interactions of ErdR with the promoter regions of the genes scpC, pp_0354, and actP-I were demonstrated by an electromobility shift assay. Finally, our results suggested that MxtR/ErdR is neither involved in regulating siderophore production nor the expression of mexEF-oprN in P. putida KT2440 under the conditions tested.

Mutant strains of Escherichia coli lacking global regulators, arcA and fis, demonstrate better growth fitness by pathway reprogramming under acetate metabolism

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A systems-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the substrates available in a range of nutrient niches such as the mammalian gut and high-fat diet. Therefore, investigating the study on acetate metabolism is highly significant. It is well known that the global regulators arcA and fis regulate acetate uptake genes in E. coli under glucose condition. In this study, we deciphered the growth and flux distribution of E.coli transcription regulatory knockout mutants ΔarcA, Δfis and double deletion mutant, ΔarcAfis under acetate using 13C-Metabolic Flux Analysis which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~1.2-1.6 fold) with a proportionate increase in acetate uptake rates compared to the wild-type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes, which conferred an advantage of faster growth with better carbon usage in all the mutants. Under acetate metabolism, the mutants exhibited higher fluxes in the TCA cycle (~18-90%) and lower gluconeogenesis flux (~15-35%) with the proportional increase in growth rate. This study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate to acetyl-CoA and therefore are potential candidates that can serve as a precursor for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products.

O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors.

Correlation of Key Physiological Properties of Methanosarcina Isolates with Environment of Origin

It is known that the physiology of Methanosarcina species can differ significantly, but the ecological impact of these differences is unclear. We recovered two strains of Methanosarcina from two different ecosystems with a similar enrichment and isolation method. Both strains had the same ability to metabolize organic substrates and participate in direct interspecies electron transfer, but also had major physiological differences. Strain DH-1, which was isolated from an anaerobic digester, could use H2 as an electron donor. Genome analysis indicated that it lacks an Rnf complex and conserves energy from acetate metabolism via intracellular H2 cycling. In contrast, strain DH-2, a subsurface isolate, lacks hydrogenases required for H2 uptake and cycling, and has an Rnf complex for energy conservation when growing on acetate. Further analysis of the genomes of previously described isolates, as well as phylogenetic and metagenomic data on uncultured Methanosarcina in anaerobic digesters and diverse soils and sediments, further revealed a physiological dichotomy that corresponded with environment of origin. The physiology of Type I Methanosarcina revolves around H2 production and consumption. In contrast, Type II Methanosarcina eschew H2, and have genes for an Rnf complex and the multi-heme, membrane-bound c-type cytochrome MmcA, shown to be essential for extracellular electron transfer. The distribution of Methanosarcina species in diverse environments suggests that the Type I H2-based physiology is well suited for high energy environments, like anaerobic digesters, whereas Type II Rnf/cytochrome-based physiology is an adaption to the slower, steady-state carbon and electron fluxes common in organic-poor anaerobic soils and sediments. Importance Biogenic methane is a significant greenhouse gas and the conversion of organic wastes to methane is an important bioenergy process. Methanosarcina species play an important role in methane production in many methanogenic soils and sediments as well as anaerobic waste digesters. The studies reported here emphasize that the genus Methanosarcina is composed of two physiologically distinct groups. This is important to recognize when interpreting the role of Methanosarcina in methanogenic environments, especially regarding H2 metabolism. Furthermore, the finding that Type I Methanosarcina predominate in environments with high rates of carbon and electron flux and that Type II Methanosarcina predominate in lower energy environments suggests that evaluating the relative abundance of Type I and Type II Methanosarcina may provide further insights into rates of carbon and electron flux in methanogenic environments.