ethylmalonyl-CoA Pathway Research Articles

Fecal microbiota related to postoperative endoscopic recurrence in patients with Crohn's disease.

Postoperative recurrence (POR) remains a major challenge for patients with Crohn's disease (CD). Gut microbial dysbiosis has been reported to be involved in the pathogenesis of POR. This study aims to investigate the relationship between fecal microbiome and endoscopic recurrence in patients with CD after ileocolonic resection. This is a cross-sectional study. Fecal samples were collected from 52 patients with CD after surgical intervention from 6 to 12 months before endoscopic examination. Endoscopic recurrence was defined as Rutgeerts score ≥ i2. The microbiome was analyzed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. A total of 52 patients were included and classified into POR (n = 27) and non-POR (n = 25) groups. Compared with the non-POR group, the POR group had a significantly lower community richness (Chao1 index: 106.5 vs 124, P = 0.013) and separated microbial community (P = 0.007 for Adonis, P = 0.032 for Anosim), combined with different distribution of 16 gut microbiotas and decrease of 11 predicted metabolic pathways (P < 0.05). Lactobacillus and Streptococcus were identified to closely correlate to non-POR (P < 0.05) after controlling for confounding factors. Kaplan-Meier analysis indicated that the patients with higher abundance of Streptococcus experienced longer remission periods (P < 0.01), but this was not for Lactobacillus. The predicted ethylmalonyl-coA pathway related to increased amount of succinate was positively correlated with Streptococcus (r > 0.5, P < 0.05). The characteristic alterations of fecal microbiota are associated with postoperative endoscopic recurrence in patients with CD; particularly, high abundance of Streptococcus may be closely related to endoscopic remission.

The interactive effects of ocean acidification and warming on bioeroding sponge Spheciospongia vesparium microbiome indicated by metatranscriptomics

Global climate change will cause coral reefs decline and is expected to increase the reef erosion potential of bioeroding sponges. Microbial symbionts are essential for the overall fitness and survival of sponge holobionts in changing ocean environments. However, we rarely know about the impacts of ocean warming and acidification on bioeroding sponge microbiome. Here, the structural and functional changes of the bioeroding sponge Spheciospongia vesparium microbiome, as well as its recovery potential, were investigated at the RNA level in a laboratory system simulating 32 °C and pH 7.7. Based on metatranscriptome analysis, acidification showed no significant impact, while warming or simultaneous warming and acidification disrupted the sponge microbiome. Warming caused microbial dysbiosis and recruited potentially opportunistic and pathogenic members of Nesiotobacter, Oceanospirillaceae, Deltaproteobacteria, Epsilonproteobacteria, Bacteroidetes and Firmicutes. Moreover, warming disrupted nutrient exchange and molecular interactions in the sponge holobiont, accompanied by stimulation of virulence activity and anaerobic metabolism including denitrification and dissimilatory reduction of nitrate and sulfate to promote sponge necrosis. Particularly, the interaction between acidification and warming alleviated the negative effects of warming and enhanced the Rhodobacteraceae-driven ethylmalonyl-CoA pathway and sulfur-oxidizing multienzyme system. The microbiome could not recover during the experiment period after warming or combined stress was removed. This study suggests that warming or combined warming and acidification will irreversibly destabilize the S. vesparium microbial community structure and function, and provides insight into the molecular mechanisms of the interactive effects of acidification and warming on the sponge microbiome.

Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids

Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M.extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M.extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches.Key points• Substitutions at position F35 of YciAHI changed the productivity of YciA-based release of carboxylic acid products in M. extorquens AM1 and E. coli.• YciAHI F35N and F35L are improved variants for dicarboxylic production of 2-methylsuccinic acid and mesaconic acid with M. extorquens AM1.• In vitro enzyme assays did not reveal superior properties of the optimized protein variants.

Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

Comparative Genomic Analysis of a Methylorubrum rhodesianum MB200 Isolated from Biogas Digesters Provided New Insights into the Carbon Metabolism of Methylotrophic Bacteria.

Methylotrophic bacteria are widely distributed in nature and can be applied in bioconversion because of their ability to use one-carbon source. The aim of this study was to investigate the mechanism underlying utilization of high methanol content and other carbon sources by Methylorubrum rhodesianum strain MB200 via comparative genomics and analysis of carbon metabolism pathway. The genomic analysis revealed that the strain MB200 had a genome size of 5.7 Mb and two plasmids. Its genome was presented and compared with that of the 25 fully sequenced strains of Methylobacterium genus. Comparative genomics revealed that the Methylorubrum strains had closer collinearity, more shared orthogroups, and more conservative MDH cluster. The transcriptome analysis of the strain MB200 in the presence of various carbon sources revealed that a battery of genes was involved in the methanol metabolism. These genes are involved in the following functions: carbon fixation, electron transfer chain, ATP energy release, and resistance to oxidation. Particularly, the central carbon metabolism pathway of the strain MB200 was reconstructed to reflect the possible reality of the carbon metabolism, including ethanol metabolism. Partial propionate metabolism involved in ethyl malonyl-CoA (EMC) pathway might help to relieve the restriction of the serine cycle. In addition, the glycine cleavage system (GCS) was observed to participate in the central carbon metabolism pathway. The study revealed the coordination of several metabolic pathways, where various carbon sources could induce associated metabolic pathways. To the best of our knowledge, this is the first study providing a more comprehensive understanding of the central carbon metabolism in Methylorubrum. This study provided a reference for potential synthetic and industrial applications of this genus and its use as chassis cells.

Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain.

Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake

The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids.Key points• 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations.• Deletion of dctA2 leads to reduction of dicarboxylic acid uptake.• Transporter-deficient strains show improved production of citramalic acid.

Investigations into Multiple Acyl‐CoA Assimilation Pathways in a Single Organism

By 2050 there could be around 12 billion tons of plastic in landfills and the natural environment, which could be avoided by using a more environmentally friendly alternative: biologically produced plastic, or bioplastic. Polyhydroxybutyrate (PHB) is a bioplastic whose precursor is an intermediate in the ethylmalonyl‐CoA pathway (EMCP), a pathway for acetyl‐CoA assimilation in bacteria. The genes that encode the enzymes for two different acetyl‐CoA assimilation pathways, the glyoxylate bypass (GB) and the EMCP, have been observed in two PHB‐producing bacteria: Rhodobacter capsulatus and Paracoccus denitrificans. It was previously observed that P. denitrificans asynchronously uses both pathways when grown with acetate. We produced R. capsulatus fusion strains by fusing the gene for a fluorescent protein, mCherry, to the gene of a key enzyme of the EMCP or GB and observed the fluorescence during growth with acetate. Tracking fluorescence allowed us to track the accumulation of each key enzyme, which indirectly indicates the activity of the pathway. We observed that unlike P. denitrificans, the GB in R. capsulatus is not essential to growth with acetate. Understanding the use/occurrence of the EMCP in bacteria is a step to being able to manipulate the EMCP to produce commercially relevant levels of bioplastic and related products.

Phylogeny and Metabolic Potential of the Methanotrophic Lineage MO3 in Beijerinckiaceae from the Paddy Soil through Metagenome-Assembled Genome Reconstruction.

Although the study of aerobic methane-oxidizing bacteria (MOB, methanotrophs) has been carried out for more than a hundred years, there are many uncultivated methanotrophic lineages whose metabolism is largely unknown. Here, we reconstructed a nearly complete genome of a Beijerinckiaceae methanotroph from the enrichment of paddy soil by using nitrogen-free M2 medium. The methanotroph labeled as MO3_YZ.1 had a size of 3.83 Mb, GC content of 65.6%, and 3442 gene-coding regions. Based on phylogeny of pmoA gene and genome and the genomic average nucleotide identity, we confirmed its affiliation to the MO3 lineage and a close relationship to Methylocapsa. MO3_YZ.1 contained mxaF- and xoxF-type methanol dehydrogenase. MO3_YZ.1 used the serine cycle to assimilate carbon and regenerated glyoxylate through the glyoxylate shunt as it contained isocitrate lyase and complete tricarboxylic acid cycle-coding genes. The ethylmalonyl-CoA pathway and Calvin–Benson–Bassham cycle were incomplete in MO3_YZ.1. Three acetate utilization enzyme-coding genes were identified, suggesting its potential ability to utilize acetate. The presence of genes for N2 fixation, sulfur transformation, and poly-β-hydroxybutyrate synthesis enable its survival in heterogeneous habitats with fluctuating supplies of carbon, nitrogen, and sulfur.

Ethylmalonyl-CoA pathway involved in polyhydroxyvalerate synthesis in Candidatus Contendobacter

Here a stable glycogen accumulating organisms (GAOs) system was operated by anaerobic–aerobic mode in the sequencing batch reactor. We focused on the metabolic mechanisms of PHAs storage from GAOs. Our system showed the classic characteristic of glycogen accumulating metabolism (GAM). Glycogen consumption was followed by acetic acid uptake to synthesize poly-β-hydroxyalkanoates (PHAs) during the anaerobic period, and glycogen was synthesized by PHAs degradation in the aerobic stage. Microbial community structure indicated that Candidatus Contendobacter was the most prevalent GAOs. We found that the ethylmalonyl-CoA (EMC) pathway was the crucial pathway supplying the core substance propionyl-CoA for poly-β-hydroxyvalerate (PHV) synthesis in Candidatus Contendobacter. All genes in EMC pathway were mainly located in Candidatus Contendobacter by gene source analysis. The key genes expression of EMC pathway increased with Candidatus Contendobacter enrichment, further validating that propionyl-CoA was synthesized by Candidatus Contendobacter predominantly via EMC pathway. Our work revealed the novel mechanisms underlying PHV synthesis through EMC pathway and further improved the intercellular storage metabolism of GAOs.

Adaption and Degradation Strategies of Methylotrophic 1,4-Dioxane Degrading Strain Xanthobacter sp. YN2 Revealed by Transcriptome-Scale Analysis.

Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl–CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.

The ethylmalonyl-CoA pathway for methane-based biorefineries: a case study of using Methylosinus trichosporium OB3b, an alpha-proteobacterial methanotroph, for producing 2-hydroxyisobutyric acid and 1,3-butanediol from methane

Through genome-scale metabolic model simulations and transcriptomic analysis the ethylmalonyl-CoA pathway of an alpha-proteobacteria, Methylosinus trichosporium OB3b, was systematically assessed and exploited to produce 2-hydroxybutiric acid and 1,3-butanediol from methane.

Anti-Inflammatory Properties and Gut Microbiota Modulation of Porphyra tenera Extracts in Dextran Sodium Sulfate-Induced Colitis in Mice.

Porphyra tenera (PT) is a functional seaweed food that has been reported for health benefits such as antioxidant, immunostimulant, anti-inflammation, and hepatoprotective effects. In this study, we investigated the effect of PT extracts on gut microbiota modulation in colitis-induced mice. The mice experiment was designed as three groups including normal mice (CTL), dextran sodium sulfate (DSS)-fed mice, and DSS plus PT extracts-fed mice (PTE). DSS was administrated through drinking water containing DSS for 1 week, and the PT extract was ingested into the gastrointestinal tract in mice. PT extract ameliorated the decreased body weight and colon length and improved disease activity index and pro-inflammatory cytokine expression. In addition, PT extract significantly shifted the gut microbiota of mice. DSS treatment significantly increased the portion of harmful bacteria (i.e., Helicobacter, Mucipirillum, and Parasutterella) and decreased the butyrate producing bacteria (i.e., Acetatifactor, Alistipes, Oscillibacter, and Clostridium_XIVb). PT extract increased the abundance of genera Clostridium_XIVb and also enriched some of predicted metabolic activities such as glyoxylate cycle, ethylmalonyl-CoA pathway, nitrate reduction, creatinine degradation, and glycine betaine metabolism. These results suggest that PT extract may ameliorate the DSS-induced colitis inflammation through regulating the compositions and functions of gut microbiota in mice.

A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes

BackgroundThioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5–10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells.ResultsHere, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the l-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum.ConclusionsThe high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

New perspectives on butyrate assimilation in Rhodospirillum rubrum S1H under photoheterotrophic conditions

BackgroundThe great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied.ResultsIn this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate.ConclusionPhotoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.

Global Proteomic Analysis Reveals High Light Intensity Adaptation Strategies and Polyhydroxyalkanoate Production in Rhodospirillum rubrum Cultivated With Acetate as Carbon Source.

Purple non-sulfur bacteria (PNSBs) are well known for their metabolic versatility. Among them, Rhodospirillum rubrum can assimilate a broad range of carbon sources, including volatile fatty acids (VFAs), such as acetate, propionate or butyrate. These carbon sources are gaining increasing interest in bioindustrial processes since they allow reduction of the production costs. Recently, our lab discovered that, after long term cultivation with acetate as unique carbon source, Rs. rubrum got acclimated to this carbon source which resulted in a drastic reduction of the lag phase. This acclimation was characterized by the amplification of the genomic region containing, among others, genes belonging to the ethylmalonyl-CoA (EMC) pathway, which has been demonstrated to be required for acetate assimilation in Rs. rubrum. In this paper, we combined bacterial growth analysis with proteomic (SWATH -Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra-processing) investigation to better understand the bacterial response to a sudden increase of the light intensity. We compared the impact of suddenly increasing light intensity on the WT strain to that on the newly described acetate-competent strain in the presence of acetate. Contrary to what was observed with the WT strain, we observed that the acetate-competent strain was tolerant to the light stress. Proteomic analysis revealed that increasing light intensity had a significant impact on the photosynthetic apparatus, especially in the wild-type strain cultivated in the presence of acetate and low concentration of HCO3–. This phenomenon was accompanied by a relatively higher abundance of certain stress related proteins. Our results suggested that the production of PHA, but also potentially of branched chain amino acids synthesis, could be part of the mechanism used by Rs. rubrum to adapt to the light stress and the redox imbalance it triggered.

Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans.

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism's central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism.IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable "operating system" of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

Transcriptome analysis of Rhodobacter capsulatus grown on different nitrogen sources.

This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation.

Barriers to 3-Hydroxypropionate-Dependent Growth of Rhodobacter sphaeroides by Distinct Disruptions of the Ethylmalonyl Coenzyme A Pathway.

Rhodobacter sphaeroides is able to use 3-hydroxypropionate as the sole carbon source through the reductive conversion of 3-hydroxypropionate to propionyl coenzyme A (propionyl-CoA). The ethylmalonyl-CoA pathway is not required in this process because a crotonyl-CoA carboxylase/reductase (Ccr)-negative mutant still grew with 3-hydroxypropionate. Much to our surprise, a mutant defective for another specific enzyme of the ethylmalonyl-CoA pathway, mesaconyl-CoA hydratase (Mch), lost its ability for 3-hydroxypropionate-dependent growth. Interestingly, the Mch-deficient mutant was rescued either by introducing an additional ccr in-frame deletion that resulted in the blockage of an earlier step in the pathway or by heterologously expressing a gene encoding a thioesterase (YciA) that can act on several CoA intermediates of the ethylmalonyl-CoA pathway. The mch mutant expressing yciA metabolized only less than half of the 3-hydroxypropionate supplied, and over 50% of that carbon was recovered in the spent medium as free acids of the key intermediates mesaconyl-CoA and methylsuccinyl-CoA. A gradual increase in growth inhibition due to the blockage of consecutive steps of the ethylmalonyl-CoA pathway by gene deletions suggests that the growth defects were due to the titration of free CoA and depletion of the CoA pool in the cell rather than to detrimental effects arising from the accumulation of a specific metabolite. Recovery of carbon in mesaconate for the wild-type strain expressing yciA demonstrated that carbon flux through the ethylmalonyl-CoA pathway occurs during 3-hydroxypropionate-dependent growth. A possible role of the ethylmalonyl-CoA pathway is proposed that functions outside its known role in providing tricarboxylic acid intermediates during acetyl-CoA assimilation.IMPORTANCE Mutant analysis is an important tool utilized in metabolic studies to understand which role a particular pathway might have under certain growth conditions for a given organism. The importance of the enzyme and of the pathway in which it participates is discretely linked to the resulting phenotype observed after mutation of the corresponding gene. This work highlights the possibility of incorrectly interpreting mutant growth results that are based on studying a single unit (gene and encoded enzyme) of a metabolic pathway rather than the pathway in its entirety. This work also hints at the possibility of using an enzyme as a drug target although the enzyme may participate in a nonessential pathway and still be detrimental to the cell when inhibited.

The complete genome sequence of Rhodobaca barguzinensis alga05 (DSM 19920) documents its adaptation for life in soda lakes.

Soda lakes, with their high salinity and high pH, pose a very challenging environment for life. Microorganisms living in these harsh conditions have had to adapt their physiology and gene inventory. Therefore, we analyzed the complete genome of the haloalkaliphilic photoheterotrophic bacterium Rhodobaca barguzinensis strain alga05. It consists of a 3,899,419bp circular chromosome with 3624 predicted coding sequences. In contrast to most of Rhodobacterales, this strain lacks any extrachromosomal elements. To identify the genes responsible for adaptation to high pH, we compared the gene inventory in the alga05 genome with genomes of 17 reference strains belonging to order Rhodobacterales. We found that all haloalkaliphilic strains contain the mrpB gene coding for the B subunit of the MRP Na+/H+ antiporter, while this gene is absent in all non-alkaliphilic strains, which indicates its importance for adaptation to high pH. Further analysis showed that alga05 requires organic carbon sources for growth, but it also contains genes encoding the ethylmalonyl-CoA pathway for CO2 fixation. Remarkable is the genetic potential to utilize organophosphorus compounds as a source of phosphorus. In summary, its genetic inventory indicates a large flexibility of the alga05 metabolism, which is advantageous in rapidly changing environmental conditions in soda lakes.