The prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, while treatment options are inadequate. Hypertension and obesity-related metabolic dysfunction contribute to HFpEF. Nitro-oleic acid (NO2-OA) impacts metabolic syndromes by improving glucose tolerance and adipocyte function. Here we show that treatment with NO2-OA ameliorates diastolic dysfunction and heart failure symptoms in a HFpEF mouse model induced by high-fat diet and inhibition of the endothelial nitric oxide synthase. Proteomic analysis of left ventricular tissue reveals that one-third of identified proteins, predominantly mitochondrial, are upregulated in hearts of NO2-OA-treated HFpEF mice compared to naïve and vehicle-treated HFpEF mice. Increased mitochondrial mass and numbers, and enhanced mitochondrial respiration are linked with this response, as assessed by transmission electron microscopy and high-resolution respirometry. Activation of the 5'-adenosine-monophosphate-activated-protein-kinase (AMPK) signaling pathway mediates the enhancement of mitochondrial dynamics in hearts of NO2-OA-treated HFpEF mice. These findings suggest that targeting mitochondrial function with NO2-OA may represent a promising therapeutic strategy for HFpEF.

Prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, while treatment options are inadequate. Hypertension and obesity-related metabolic dysfunctions contribute to HFpEF progression. Nitro-oleic acid (NO2-OA) impacts metabolic processes by improving glucose tolerance and adipocyte function. In this study, 4 week treatment with NO2-OA ameliorated diastolic dysfunction in a HFpEF mouse model induced by high-fat diet and inhibition of the endothelial nitric oxide synthase. A proteomic analysis of left ventricular tissue revealed, that one third of the identified proteins, mostly mitochondrial proteins, were upregulated in hearts of NO2-OA-treated HFpEF mice compared to controls and vehicle-treated HFpEF mice, which was confirmed by immunoblot. Activation of the 5’-adenosine-monophosphate-activated-protein-kinase (AMPK) signaling pathway mediated an enhancement of mitochondrial biogenesis in hearts of NO2-OA-treated HFpEF mice. In cardiomyocytes under metabolic stress, NO2-OA increased mitochondrial protein level accompanied by enhanced oxidative phosphorylation. In conclusion, targeting mitochondrial integrity in HFpEF leads to improved diastolic function.### Competing Interest StatementBAF acknowledges an interest in Creegh Pharmaceuticals, Inc. and FJS acknowledges an interest in Creegh Pharmaceuticals, Inc. and Furnaica, Inc. The other authors declare no conflicts.* 4APIX : apical four chamber view AMPK : 5’ adenosine monophosphate-activated protein kinase AngII : angiotensin 2 ANT : adenosine nucleotide translocase AT1R : angiotensin 2 type 1 receptor ATP : adenosine triphosphate AUC : area under the curve BDH1 : 3-hydroxybutyrate dehydrogenase 1 COX4 : cytochrome c oxidase subunit 4 CPT1B : carnitine O-palmitoyl-transferase 1 DAG : diacylglycerol eNOS : endothelial nitric oxide synthase ET-1 : endothelin-1 FA : fatty acid FAO : fatty acid oxidation FAT/CD36 : long chain fatty acid transporter gDNA : genomic DNA GLUT4 : glucose transporter, solute carrier family 2, member 4 GO : glucose oxidation HC : hydrocortisone HFD : high fat diet HFpEF : heart failure with preserved ejection fraction KEGG : Kyoto Encyclopedia of Genes and Genomes LC-MS : liquid chromatography followed by mass spectrometry L-NAME : N ω-nitro-L-arginine methyl ester hydrochloride LV : left ventricle LVEF : left ventricular ejection fraction LVPW : left ventricular posterior wall thickness mtDNA : mitochondrial DNA NAD : nicotinamide adenine dinucleotide NF-κB : nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells Nmnat1 : gene of nicotinamide nucleotide adenyl transferase 1 NO2-CLA : conjugated nitro-linoleic acid NO2-LA : nitro-linoleic acid NO2-OA : nitro-oleic acid Nos2 : gene of nitric oxide synthase, inducible Nrf2 : nuclear factor erythroid 2-related factor 2 OA : oleic acid OCR : oxygen consumption rate OXPHOS : oxidative phosphorylation PDK4 : pyruvate dehydrogenase kinase 4 PDH : pyruvate dehydrogenase PGC-1α : peroxisome proliferator-activated receptor gamma coactivator 1-alpha PPAR peroxisome-proliferator activated receptor PSAX : parasternal short-axis PSLAX : parasternal long-axis RPL32 : ribosomal protein L32 SD : standard deviation SEM : standard error of the mean SERCA : sarcoplasmic reticular calcium ATPase SGLT2 : sodium glucose cotransporter 2 SIRT : sirtuin Sirt 1 : gene of sirtuin 1 SUIT : substrate-uncoupler-inhibitor titration protocol TnT : troponin T TOM70 : translocase of outer membrane 70 Tomm70 : gene of translocase of outer membrane 70 UCP : uncoupling protein Xbp1s : gene of spliced form of X-box binding protein 1

BACKGROUND:Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production.RESULTS:In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production.CONCLUSIONS:This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production.

Coenzyme Q 10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para -hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli . CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Flavin-dependent halogenases selectively introduce halogen substituents into (hetero-)aromatic substrates and require only molecular oxygen and halide salts for this regioselective oxidative CH-functionalization. Genomic analysis of Xanthomonas campestris pv. campestris B100 identified three novel putative members of this enzyme class. They were shown to introduce halogen substituents into, e. g., substituted indoles, while preferring bromide over chloride.

Flavin-dependent halogenases can be used as biocatalysts because they regioselectively halogenate their substrates under mild reaction conditions. New halogenases with novel substrate specificities will add to the toolbox of enzymes available to organic chemists. HalX, the product of the xcc-b100_4193 gene, is a putative flavin-dependent halogenase from Xanthomonas campestris. The enzyme was recombinantly expressed and crystallized in order to aid in identifying its hitherto unknown substrate. Native data collected to a resolution of 2.5 Å showed indications of merohedral twinning in a hexagonal lattice. Attempts to solve the phase problem by molecular replacement failed. Here, a detailed analysis of the suspected twinning is presented. It is most likely that the crystals are trigonal (point group 3) and exhibit perfect hemihedral twinning so that they appear to be hexagonal (point group 6). As there are several molecules in the asymmetric unit, noncrystallographic symmetry may complicate twinning analysis and structure determination.

Background: Sigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown.Results: In this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and metabolites were investigated. Overexpression of sigD decreased the growth rate of C. glutamicum cultures, and induced several physiological effects such as reduced culture foaming, turbid supernatant and cell aggregation. Upon overexpression of sigD, the level of Cmt1 (corynomycolyl transferase) in the supernatant was notably enhanced, and carbohydrate-containing compounds were excreted to the supernatant. The real-time PCR analysis revealed that sigD overexpression increased the expression of genes related to corynomycolic acid synthesis (fadD2, pks), genes encoding corynomycolyl transferases (cop1, cmt1, cmt2, cmt3), L, D-transpeptidase (lppS), a subunit of the major cell wall channel (porH), and the envelope lipid regulation factor (elrF). Furthermore, overexpression of sigD resulted in trehalose dicorynomycolate accumulation in the cell envelope.Conclusions: This study demonstrated that SigD regulates the synthesis of corynomycolate and related compounds, and expanded the knowledge of regulatory functions of sigma factors in C. glutamicum.

Plant cryptochromes are photoreceptors that regulate flowering, circadian rhythm and photomorphogenesis in response to blue and UV‐A light. It has been demonstrated that the oxidized flavin cofactor is photoreduced to the neutral radical state via separate electron and proton transfer. Conformational changes have been found in the C‐terminal extension, but few studies have addressed the changes in secondary structure in the sensory photolyase homology region (PHR). Here, we investigated the PHR of the plant cryptochrome from the green alga Chlamydomonas reinhardtii by light‐induced infrared difference spectroscopy in combination with global 13C and 15N isotope labeling. Assignment of the signals is achieved by establishing a labeling strategy for cryptochromes that preserves the flavin at natural abundance. We demonstrate by UV/vis spectroscopy that the integrity of the sample is maintained and by mass spectrometry that the global labeling was highly efficient. As a result, difference bands are resolved at full intensity that at natural abundance are compensated by the overlap of flavin and protein signals. These bands are assigned to prominent conformational changes in the PHR by blue light illumination. We postulate that not only the partial unfolding of the C‐terminal extension but also changes in the PHR may mediate signaling events.

Bile salts such as cholate are surface-active steroid compounds with functions for digestion and signaling in vertebrates. Upon excretion into soil and water bile salts are an electron- and carbon-rich growth substrate for environmental bacteria. Degradation of bile salts proceeds via intermediates with a 3-keto-Δ1,4 -diene structure of the steroid skeleton as shown for e.g. Pseudomonas spp. Recently, we isolated bacteria degrading cholate via intermediates with a 3-keto-7-deoxy-Δ4,6 -structure of the steroid skeleton suggesting the existence of a second pathway for cholate degradation. This potential new pathway was investigated with Novosphingobium sp. strain Chol11. A 7α-hydroxysteroid dehydratase encoded by hsh2 was identified, which was required for the formation of 3-keto-7-deoxy-Δ4,6 -metabolites. A hsh2 deletion mutant could still grow with cholate but showed impaired growth. Cholate degradation of this mutant proceeded via 3-keto-Δ1,4 -diene metabolites. Heterologous expression of Hsh2 in the bile salt-degrading Pseudomonas sp. strain Chol1 led to the formation of a dead-end steroid with a 3-keto-7-deoxy-Δ4,6 -diene structure. Hsh2 is the first steroid dehydratase with an important function in a metabolic pathway of bacteria that use bile salts as growth substrates. This pathway contributes to a broad metabolic repertoire of Novosphingobium strain Chol11 that may be advantageous in competition with other bile salt-degrading bacteria.

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.

Proteins from the supernatant of Bradyrhizobium japonicum were separated by two-dimensional gel electrophoresis and stained with Coomassie. This revealed more than 100 protein spots. Sixty-eight proteins were identified by mass spectrometry. Thirty-five are predicted to contain an N-terminal signal peptide characteristic for proteins transported by the general secretory pathway. Most of these appear to be substrate-binding proteins of the ABC transporter family. Ten proteins were categorized as unclassified conserved or hypothetical. None of the proteins has similarity to proteins transported by a type I secretion system or to autotransporters. Three of the proteins might be located in the outer membrane. The addition of genistein led to changes in the spot pattern of three flagellar proteins and resulted in the identification of the nodulation outer protein Pgl. Moreover, the application of shot-gun mass spectrometry resulted in the first-time identification of NopB, NopH and NopT, which were present only after genistein induction. Replacing genistein with daidzein or coumestrol reduced the amount of the type III-secreted protein GunA2.

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium—genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix¯ nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photo-synthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.

Three of the most abundant proteins (OmpW, MopB and SodM) of the extracellular proteome of Xanthomonas campestris pv. campestris were analysed in a luminol-based oxidative burst assay to identify novel pathogen-associated molecular patterns (PAMP). Tobacco cell suspension cultures were used as a model system to monitor elicitor induced plant defence reaction. The candidate proteins were isolated from two-dimensional gels prior to application to the oxidative burst assay. The superoxide dismutase (SodM) was the only isolated protein that could elicit a notable hydrogen peroxide (H2O2) production in tobacco cell cultures indicating the initiation of plant defence. An alignment of the SodM sequences from X. campestris pv. campestris and Escherichia coli revealed 55.7% identity and 29% of the sequence were substitutions for amino acids with similar physico-chemical properties. By using a commercially available purified E. coli derived SodM preparation, it was possible to show that the amino acid sequence of this protein is responsible for the elicitation of an oxidative burst reaction in the tobacco cell culture model. This suggests that the bacterial superoxide dismutase is a novel pathogen-associated molecular pattern. The minimal elicitor active sequence, however, is still elusive.

A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula . A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition. S. meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step. Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS. The different compounds were identified by comparison with the NIST 98 database and available standards. From about 200 peaks in each chromatogram 65 compounds have been identified so far. A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools. A principal component analysis (PCA) was able to distinguish S. meliloti cells grown on different carbon sources based on their metabolite profile. A comparison of the metabolite composition of a S. meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant. Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine. This finding further emphasises the importance of integrating metabolic data into post-genomic research.