The tricarboxylic acid (TCA) cycle: a malleable metabolic network to counter cellular stress.

The tricarboxylic acid (TCA) cycle is a primordial metabolic pathway that is conserved from bacteria to humans. Although this network is often viewed primarily as an energy producing engine fueling ATP synthesis via oxidative phosphorylation, mounting evidence reveals that this metabolic hub orchestrates a wide variety of pivotal biological processes. It plays an important part in combatting cellular stress by modulating NADH/NADPH homeostasis, scavenging ROS (reactive oxygen species), producing ATP by substrate-level phosphorylation, signaling and supplying metabolites to quell a range of cellular disruptions. This review elaborates on how the reprogramming of this network prompted by such abiotic stress as metal toxicity, oxidative tension, nutrient challenge and antibiotic insult is critical for countering these conditions in mostly microbial systems. The cross-talk between the stressors and the participants of TCA cycle that results in changes in metabolite and nucleotide concentrations aimed at combatting the abiotic challenge is presented. The fine-tuning of metabolites mediated by disparate enzymes associated with this metabolic hub is discussed. The modulation of enzymatic activities aimed at generating metabolic moieties dedicated to respond to the cellular perturbation is explained. This ancient metabolic network has to be recognized for its ability to execute a plethora of physiological functions beyond its well-established traditional roles.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

A Metabolic Network Mediating the Cycling of Succinate, a Product of ROS Detoxification into α-Ketoglutarate, an Antioxidant.

Sulfur is an essential element for life. However, the soil microbe Pseudomonas (P.) fluorescens can survive in a low sulfur environment. When cultured in a sulfur-deficient medium, the bacterium reprograms its metabolic pathways to produce α-ketoglutarate (KG) and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KGDC) work in partnership to synthesize KG. This process is further aided by the increased activity of the enzymes glutamate decarboxylase (GDC) and γ-amino-butyrate transaminase (GABAT). The pool of succinate semialdehyde (SSA) generated is further channeled towards the formation of the antioxidant. Spectrophotometric analyses, HPLC experiments and electrophoretic studies with intact cells and cell-free extracts (CFE) pointed to the metabolites (succinate, SSA, GABA) and enzymes (SSADH, GDC, KGDC) contributing to this KG-forming metabolic machinery. Real-time polymerase chain reaction (RT-qPCR) revealed significant increase in transcripts of such enzymes as SSADH, GDC and KGDC. The findings of this study highlight a novel pathway involving keto-acids in ROS scavenging. The cycling of succinate into KG provides an efficient means of combatting an oxidative environment. Considering the central role of KG in biological processes, this metabolic network may be operative in other living systems.

Inter-dependent Centrosomal Co-localization of the cen and ik2 cis-Natural Antisense mRNAs in Drosophila.

Overlapping genes are prevalent in most genomes, but the extent to which this organization influences regulatory events operating at the post-transcriptional level remains unclear. Studying the cen and ik2 genes of Drosophila melanogaster, which are convergently transcribed as cis-natural antisense transcripts (cis-NATs) with overlapping 3' UTRs, we found that their encoded mRNAs strikingly co-localize to centrosomes. These transcripts physically interact in a 3' UTR-dependent manner, and the targeting of ik2 requires its 3' UTR sequence and the presence of cen mRNA, which serves as the main driver of centrosomal co-localization. The cen transcript undergoes localized translation in proximity to centrosomes, and its localization is perturbed by polysome-disrupting drugs. By interrogating global fractionation-sequencing datasets generated from Drosophila and human cellular models, we find that RNAs expressed as cis-NATs tend to co-localize to specific subcellular fractions. This work suggests that post-transcriptional interactions between RNAs with complementary sequences can dictate their localization fate in the cytoplasm.

Metabolic adaptation and NADPH homeostasis evoked by a sulfur-deficient environment in Pseudomonas fluorescens.

Sulfur is essential for all living organisms due to its ability to mediate a variety of enzymatic reactions, signalling networks, and redox processes. The interplay between sulfhydryl group (SH) and disulfide bond (S-S) is central to the maintenance of intracellular oxidative balance. Although most aerobic organisms succumb to sulfur starvation, the nutritionally versatile soil microbe Pseudomonas fluorescens elaborates an intricate metabolic reprogramming in order to adapt to this challenge. When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. While oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle, metabolic modules known to generate reactive oxygen species are impeded, the activities NADPH-producing enzymes such as malic enzyme, and glutamate dehydrogenase (GDH) NADP-dependent are increased. The α-ketoglutarate (KG) generated from glutamine rapidly enters the TCA cycle via α-ketoglutarate dehydrogenase (KGDH), an enzyme that was prominent in the control cultures. In the S-deficient media, the severely impeded KGDH coupled with the increased activity of the reversible isocitrate dehydrogenase (ICDH) that fixes KG into isocitrate in the presence of NADH and HCO3- ensures a constant supply of this critical tricarboxylic acid. The up-regulation of ICDH-NADP dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis, a reaction aided by the concomitant increase in NAD kinase activity. The latter converts NAD into NADP in the presence of ATP. Taken together, the data point to a metabolic network involving isocitrate, α-KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment.

Isocitrate Lyase and Succinate Semialdehyde Dehydrogenase Mediate the Synthesis of α-Ketoglutarate in Pseudomonas fluorescens.

Glycerol is an important by-product of the biodiesel industry and its transformation into value-added products like keto acids is being actively pursued in order to improve the efficacy of this renewable energy sector. Here, we report that the enhanced production of α-ketoglutarate (KG) effected by Pseudomonas fluorescens in a mineral medium supplemented with manganese (Mn) is propelled by the increased activities of succinate semialdehyde dehydrogenase (SSADH), γ-aminobutyric acid aminotransaminase (GABAT), and isocitrate lyase (ICL). The latter generates glyoxylate and succinate two key metabolites involved in this process. Fumarate reductase (FRD) also aids in augmenting the pool of succinate, a precursor of succinate semialdehyde (SSA). The latter is then carboxylated to KG with the assistance of α-ketoglutarate decarboxylase (KDC). These enzymes work in tandem to ensure copious secretion of the keto acid. When incubated with glycerol in the presence of bicarbonate ( H C O 3 - ), cell-free extracts readily produce KG with a metabolite fingerprint attributed to glutamate, γ-aminobutyric acid (GABA), succinate and succinate semialdehyde. Further targeted metabolomic and functional proteomic studies with high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and gel electrophoresis techniques provided molecular insights into this KG-generating machinery. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed the transcripts responsible for ICL and SSADH were elevated in the Mn-supplemented cultures. This hitherto unreported metabolic network where ICL and SSADH orchestrate the enhanced production of KG from glycerol, provides an elegant means of converting an industrial waste into a keto acid with wide-ranging application in the medical, cosmetic, and chemical sectors.

Whole mount RNA fluorescent in situ hybridization of Drosophila embryos.

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.