Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3.

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. Here, we uncover a histone modification induced by S. pneumoniae infection maintained for at least 9 days upon clearance of bacteria with antibiotics. Di-methylation of histone H3 on lysine 4 (H3K4me2) is induced in an active manner by bacterial attachment to host cells. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3 or me1, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses to subsequent infections and promoting infection.

Myeloid-specific Hdac10 deletion protects against LPS-induced acute lung injury via P62 acetylation at lysine 165.

BACKGROUND: Aberrant activation of macrophages is associated with pathogenesis of acute lung injury (ALI). However, the potential pathogenesis has not been explored. OBJECTIVES: We aimed to identify whether histone deacetylase (HDAC) 10 is involved in lipopolysaccharide (LPS)-exposed ALI and reveal the underlying pathogenesis by which it promotes lung inflammation in LPS-exposed ALI via modifying P62 with deacetylation. METHODS: We constructed an ALI mice model stimulated with LPS to determine the positive effect of Hdac10 deficiency. Moreover, we cultured murine alveolar macrophage cell line (MH-S cells) and primary bone marrow-derived macrophages (BMDMs) to explore the pro-inflammatory activity and mechanism of HDAC10 after LPS challenge. RESULTS: HDAC10 expression was increased both in mice lung tissues and macrophage cell lines and promoted inflammatory cytokines production exposed to LPS. Hdac10 deficiency inhibited autophagy and inflammatory response after LPS stimulation. In vivo, Hdac10fl/fl-LysMCre mice considerably attenuated lung inflammation and inflammatory cytokines release exposed to LPS. Mechanistically, HDAC10 interacts with P62 and mediates P62 deacetylation at lysine 165 (K165), by which it promotes P62 expression and increases inflammatory cytokines production. Importantly, we identified that Salvianolic acid B (SAB), an HDAC10 inhibitor, reduces lung inflammatory response in LPS-stimulated ALI. CONCLUSION: These results uncover a previously unknown role for HDAC10 in regulating P62 deacetylation and aggravating lung inflammation in LPS-induced ALI, implicating that targeting HDAC10 is an effective therapy for LPS-exposed ALI.

[Lysine Succinylation:A New Perspective in the Treatment of Cardiovascular Diseases].

With the continuous development of identification technologies such as mass spectrometry,omics,and antibody technology,post-translational modification (PTM) has demonstrated increasing potential in medical research.PTM as a novel chemical modification method provides new perspectives for the research on diseases.Succinylation as a novel modification has aroused the interest of more and more researchers.The available studies about succinylation mainly focus on a desuccinylase named sirtuin 5.This enzyme plays a key role in modification and has been preliminarily explored in cardiovascular studies.This paper summarizes the influencing factors and regulatory roles of succinylation and the links between succinylation and other PTMs and reviews the research progress of PTMs in the cardiovascular field,aiming to deepen the understanding about the role of this modification and give new insights to the research in this field.

Release profile of amino acids encapsulated in solid lipid particles during in vitro oro-gastrointestinal digestion.

Some amino acids are known to mediate immune responses through gut microbiota metabolism in both humans and monogastric animals. However, through the diet, most free amino acids are absorbed in the small intestine and only a small quantity reaches the microbiota-rich colon. To enhance microbial metabolism of amino acids and their potential health benefits, encapsulation strategies are developed for their protection and delivery to the colon. So far, the main encapsulation systems for amino acids are based on solid lipid particles, but their fate within the digestive tract has never been fully clarified. In this study, we investigated the release of various amino acids (branched-chain amino acid mixture, or lysine, or tryptophan) loaded in solid lipid particles during in vitro oro-gastrointestinal digestion mimicking the piglet. The loaded solid lipid particles were fully characterized for their composition, thermal behavior, molecular structure, crystalline state, surface morphology, and particle size distribution. Moreover, we investigated the effect of particle size by sieving solid lipid particles into two non-overlapping size fractions. We found that amino acid release was high during the gastric phase of digestion, mainly controlled by physical parameters, namely particle size and crystalline state including surface morphology. Large particle size and/or smooth ordered particle indeed led to slower and lower release. Although lipid hydrolysis was significant during the intestinal phase of digestion, the impact of the crystalline state and surface morphology was also observed in the absence of enzymes, pointing to a dominant water/solute diffusion mechanism through these porous solid lipid particles.

Prostate Cancer-Specific Lysine 53 Acetylation of Cytochrome c Drives Metabolic Reprogramming and Protects from Apoptosis in Intact Cells.

Cytochrome c (Cytc) is important for both mitochondrial respiration and apoptosis, both of which are altered in cancer cells that switch to Warburg metabolism and manage to evade apoptosis. We earlier reported that lysine 53 (K53) of Cytc is acetylated in prostate cancer. K53 is conserved in mammals that is known to be essential for binding to cytochrome c oxidase and apoptosis protease activating factor-1 (Apaf-1). Here we report the effects of this acetylation on the main functions of cytochrome c by expressing acetylmimetic K53Q in cytochrome c double knockout cells. Other cytochrome c variants analyzed were wild-type, K53R as a control that maintains the positive charge, and K53I, which is present in some non-mammalian species. Intact cells expressing K53Q cytochrome c showed 49% decreased mitochondrial respiration and a concomitant increase in glycolytic activity (Warburg effect). Furthermore, mitochondrial membrane potential was decreased, correlating with notably reduced basal mitochondrial superoxide levels and decreased cell death upon challenge with H2O2 or staurosporine. To test for markers of cancer aggressiveness and invasiveness, cells were grown in 3D spheroid culture. K53Q cytochrome c-expressing cells showed profoundly increased protrusions compared to WT, suggesting increased invasiveness. We propose that K53 acetylation of cytochrome c is an adaptive response that mediates prostate cancer metabolic reprogramming and evasion of apoptosis, which are two hallmarks of cancer, to better promote tumor survival and metastasis.

The Rhododendron Chrysanthum Pall.s' Acetylation Modification of Rubisco Enzymes Controls Carbon Cycling to Withstand UV-B Stress.

Lysine acetylation of proteins plays a critical regulatory function in plants. A few advances have been made in the study of plant acetylproteome. However, until now, there have been few data on Rhododendron chrysanthum Pall. (R. chrysanthum). We analyzed the molecular mechanisms of photosynthesis and stress resistance in R. chrysanthum under UV-B stress. We measured chlorophyll fluorescence parameters of R. chrysanthum under UV-B stress and performed a multi-omics analysis. Based on the determination of chlorophyll fluorescence parameters, R. chrysanthum Y(NO) (Quantum yield of non-photochemical quenching) increased under UV-B stress, indicating that the plant was damaged and photosynthesis decreased. In the analysis of acetylated proteomics data, acetylated proteins were found to be involved in a variety of biological processes. Notably, acetylated proteins were significantly enriched in the pathways of photosynthesis and carbon fixation, suggesting that lysine acetylation modifications have an important role in these activities. Our findings suggest that R. chrysanthum has decreased photosynthesis and impaired photosystems under UV-B stress, but NPQ shows that plants are resistant to UV-B. Acetylation proteomics revealed that up- or down-regulation of acetylation modification levels alters protein expression. Acetylation modification of key enzymes of the Calvin cycle (Rubisco, GAPDH) regulates protein expression, making Rubisco and GAPDH proteins expressed as significantly different proteins, which in turn affects the carbon fixation capacity of R. chrysanthum. Thus, Rubisco and GAPDH are significantly differentially expressed after acetylation modification, which affects the carbon fixation capacity and thus makes the plant resistant to UV-B stress. Lysine acetylation modification affects biological processes by regulating the expression of key enzymes in photosynthesis and carbon fixation, making plants resistant to UV-B stress.

Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation.

Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8 null , H4 K20R/A , and l(3)mbt mutant Drosophila melanogaster We found that the gene expression profiles of H4 K20A and H4 K20R larvae are markedly different than Set8 null larvae despite similar reductions in H4K20me1. Set8 null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4 K20 , indicating that the developmental defects of Set8 null animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8 null and H4 K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.

Vibrio alginolyticus PEPCK Mediates Florfenicol Resistance through Lysine Succinylation Modification.

Protein succinylation modification is a common post-translational modification (PTM) that plays an important role in bacterial metabolic regulation. In this study, quantitative analysis was conducted on the succinylated proteome of wild-type and florfenicol-resistant Vibrio alginolyticus to investigate the mechanism of succinylation regulating antibiotic resistance. Bioinformatic analysis showed that the differentially succinylated proteins were mainly enriched in energy metabolism, and it was found that the succinylation level of phosphoenolpyruvate carboxyl kinase (PEPCK) was highly expressed in the florfenicol-resistant strain. Site-directed mutagenesis was used to mutate the lysine (K) at the succinylation site of PEPCK to glutamic acid (E) and arginine (R), respectively, to investigate the function of lysine succinylation of PEPCK in the florfenicol resistance of V. alginolyticus. The detection of site-directed mutagenesis strain viability under florfenicol revealed that the survival rate of the E mutant was significantly higher than that of the R mutant and wild type, indicating that succinylation modification of PEPCK protein may affect the resistance of V. alginolyticus to florfenicol. This study indicates the important role of PEPCK during V. alginolyticus antibiotic-resistance evolution and provides a theoretical basis for the prevention and control of vibriosis and the development of new antibiotics.

Skeletal muscle metabolic dysfunction with circulating carboxymethyl-lysine in dietary food additive-induced leaky gut.

Impaired intestinal permeability induces systemic inflammation and metabolic disturbance. The effect of a leaky gut on metabolism in skeletal muscle, a major nutrient consumer, remains unclear. In this study, we aimed to investigate the glucose metabolic function of the whole body and skeletal muscles in a mouse model of diet-induced intestinal barrier dysfunction. At Week 2, we observed higher intestinal permeability in mice fed a titanium dioxide (TiO2)-containing diet than that of mice fed a normal control diet. Subsequently, systemic glucose and insulin tolerance were found to be impaired. In the skeletal muscle, glucose uptake and phosphorylation levels in insulin signaling were lower in the TiO2 group than those in the control group. Additionally, the levels of pro-inflammatory factors were higher in TiO2-fed mice than those in the control group. We observed higher carboxymethyl-lysin (CML) levels in the plasma and intestines of TiO2-fed mice and lower insulin-dependent glucose uptake in CML-treated cultured myotubes than those in the controls. Finally, soluble dietary fiber supplementation improved glucose and insulin intolerance, suppressed plasma CML, and improved intestinal barrier function. These results suggest that an impaired intestinal barrier leads to systemic glucose intolerance, which is associated with glucose metabolism dysfunction in the skeletal muscles due to circulating CML derived from the intestine. This study highlights that the intestinal condition regulates muscle and systemic metabolic health.

Abrogating K458 acetylation enhances hepatocyte nuclear factor 4α (HNF4α)-induced differentiation therapy for hepatocellular carcinoma.

OBJECTIVES: In this study we aimed to assess the impact of acetylation of hepatocyte nuclear factor 4α (HNF4α) on lysine 458 on the differentiation therapy of hepatocellular carcinoma (HCC). METHODS: Periodic acid-Schiff (PAS) staining, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and senescence-associated ß-galactosidase (SA-ß-gal) activity analysis were performed to assess the differentiation of HCC cells. HNF4α protein was detected by western blot and immunohistochemistry (IHC). The effects of HNF4α-K458 acetylation on HCC malignancy were evaluated in HCC cell lines, a Huh-7 xenograft mouse model, and an orthotopic model. The differential expression genes in Huh-7 xenograft tumors were screened by RNA-sequencing analysis. RESULTS: K458R significantly enhanced the inhibitory effect of HNF4α on the malignancy of HCC cells, whereas K458Q reduced the inhibitory effects of HNF4α. Moreover, K458R promoted, while K458Q decreased, HNF4α-induced HCC cell differentiation. K458R stabilized HNF4α, while K458Q accelerated the degradation of HNF4α via the ubiquitin proteasome system. K458R also enhanced the ability of HNF4α to inhibit cell growth of HCC in the Huh-7 xenograft mouse model and the orthotopic model. RNA-sequencing analysis revealed that inhibiting K458 acetylation enhanced the transcriptional activity of HNF4α without altering the transcriptome induced by HNF4α in HCC. CONCLUSION: Our data revealed that inhibiting K458 acetylation of HNF4α might provide a more promising candidate for differential therapy of HCC.

Combined assessment of lysine and N-acetyl cadaverine levels assist as a potential biomarker of the smoker periodontitis.

Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers.

Substrate and Functional Diversity of Protein Lysine Post-translational Modifications.

Lysine post-translational modifications (PTMs) are widespread and versatile protein PTMs that are involved in diverse biological processes by regulating the fundamental functions of histone and non-histone proteins. Dysregulation of lysine PTMs is implicated in many diseases, and targeting lysine PTM regulatory factors, including writers, erasers, and readers, has become an effective strategy for disease therapy. The continuing development of mass spectrometry (MS) technologies coupled with antibody-based affinity enrichment technologies greatly promotes the discovery and decoding of PTMs. The global characterization of lysine PTMs is crucial for deciphering the regulatory networks, molecular functions, and mechanisms of action of lysine PTMs. In this review, we focus on lysine PTMs, and provide a summary of the regulatory enzymes of diverse lysine PTMs and the proteomics advances in lysine PTMs by MS technologies. We also discuss the types and biological functions of lysine PTM crosstalks on histone and non-histone proteins and current druggable targets of lysine PTM regulatory factors for disease therapy.

Effect of supplementing lysins and methionine to low-protein diets on growth performance, hepatic antioxidant capacity, immune status, and glycolytic activity of tibetan sheep.

Reducing the levels of dietary protein is an effective nutritional approach in lowering feed cost and nitrogen emissions in ruminants. The purpose of this study was to evaluate the effects of dietary Lys/Met ratio in a low protein diet (10%, dry matter basis) on the growth performance and hepatic function (antioxidant capacity, immune status, and glycolytic activity) in Tibetan lambs. Ninety two-month-old rams with an average weight of 15.37 ± 0.92 kg were randomly assigned to LP-L (dietary Lys/Met = 1:1), LP-M (dietary Lys/Met = 2:1) and LP-H (dietary Lys/Met = 3:1) treatments. The trial was conducted over 100 d, including 10 d of adaption to the diets. Hepatic phenotypes, antioxidant capacity, immune status, glycolytic activity and gene expression profiling was detected after the conclusion of the feeding trials. The results showed that the body weight was higher in the LP-L group when compared to those on the LP-M group (P < 0.05). In addition, the activities of the catalase (CAT) and glutathione peroxidase (GSH-Px) in the LP-L group were significantly increased compared with the LP-M group (P < 0.05), while the malondialdehyde (MDA) levels in LP-H group were significantly decreased (P < 0.05). Compared with LP-H group, both hepatic glycogen (P < 0.01) and lactate dehydrogenase (LDH) (P < 0.05) were significantly elevated in LP-L group. For the LP-L group, the hepatocytes were arranged radially with the central vein in the center, and hepatic plates exhibited tight arrangement. Transcriptome analysis identified 29, 179, and 129 differentially expressed genes (DEGs) between the LP-M vs. LP-L, LP-H vs. LP-M, and LP-H vs. LP-L groups, respectively (Q-values < 0.05 and |log2Fold Change| > 1). Gene Ontology (GO) and correlation analyses showed that in the LP-L group, core genes (C1QA and JUNB) enriched in oxidoreductase activity were positively correlated with antioxidant indicators, while the MYO9A core gene enriched in the immune response was positively associated with immune indicators, and core genes enriched in molecular function (PDK3 and PDP2) were positively correlated with glycolysis indicators. In summary, low-protein diet with a low Lys/Met ratio (1:1) could reduce the hepatic oxidative stress and improve the glycolytic activity by regulating the expression of related genes of Tibetan sheep.

Delivery of mRNA with Histidine-Lysine Peptides.

Transfection with mRNA has been considered superior to that with plasmids since the mRNA can be translated to a protein in the cytosol without entering the nucleus. One disadvantage of using mRNA is its susceptibility to enzymatic biodegradability, and consequently, significant research has occurred to determine nonviral carriers that will sufficiently stabilize this nucleic acid for cellular transport. Histidine-lysine peptides (HK) are one such class of mRNA carriers, which we think serves as a model for other peptides and polymeric carrier systems. When the HK peptide and mRNA are mixed and interact through ionic and nonionic bonds, mRNA polyplexes are formed, which can transfect cells. In contrast to linear HK peptides, branched HK peptides protected and efficiently transfected mRNA into cells. After describing the preparation and biophysical characterization of these polyplexes, we will provide protocols for in vitro and in vivo transfection for these mRNA polyplexes.

Genetically Encoded Epitope Tag for Probing Lysine Acylation-Mediated Protein-Protein Interactions.

Histone lysine acetylation (Kac) and crotonylation (Kcr) marks mediate the recruitment of YEATS domains to chromatin. In this way, YEATS domain-containing proteins such as AF9 participate in the regulation of DNA-templated processes. Our previous study showed that the replacement of Kac/Kcr by a 2-furancarbonyllysine (Kfu) residue led to greatly enhanced affinity toward the AF9 YEATS domain, rendering Kfu-containing peptides useful chemical tools to probe the AF9 YEATS-Kac/Kcr interactions. Here, we report the genetic incorporation of Kfu in Escherichia coli and mammalian cells through the amber codon suppression technology. We develop a Kfu-containing epitope tag, termed RAY-tag, which can robustly and selectively engage with the AF9 YEATS domain in vitro and in cellulo. We further demonstrate that the fusion of RAY-tag to different protein modules, including fluorescent proteins and DNA binding proteins, can facilitate the interrogation of the histone lysine acylation-mediated recruitment of the AF9 YEATS domain in different biological contexts.

Nuclear position and local acetyl-CoA production regulate chromatin state.

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

SLC7A2-Mediated Lysine Catabolism Inhibits Immunosuppression in Triple Negative Breast Cancer.

Breast cancer is one of the most common malignant tumors worldwide. SLC7A2 is abnormally expressed in multiple cancers. However, its potential in triple negative breast cancer (TNBC) is still unclear. The purpose of this study was to investigate the roles of SLC7A2 and its underlying molecular mechanisms in TNBC. mRNA expression was detected by RT-qPCR. Protein expression was detected by western blot. Co-localization of ACOX1 and TCF1 was determined using FISH assay. Histone crotonylation was performed using in vitro histone crotonylation assay. Functional analysis was performed using CCK-8 and flow cytometry assays. Xenograft assay was conducted to further verify the role of SLC7A2 in TNBC. CD8A expression was detected using immunohistochemistry. We found that SLC7A2 is downregulated in TNBC tumors. Low levels are associated with advanced stages and lymph node metastasis. SLC7A2 expression is positively correlated with CD8A. SLC7A2-mediated lysine catabolism drives the activation of CD8+ T cells. Moreover, SLC7A2 promotes histone crotonylation via upregulating ACOX1. It also promotes interaction between ACOX1 and TCF1, thus promoting antitumor T cell immunity. Additionally, overexpression of SLC7A2 activates CD8+ T cells and enhances the chemosensitivity of anti-PD-1 therapies in vivo. In conclusion, SLC7A2 may function as an antitumor gene in TNBC by activating antitumor immunity, suggesting SLC7A2/ACOX1/TCF1 signaling as a promising therapeutic strategy.

Acetylation of histones and non-histone proteins is not a mere consequence of ongoing transcription.

In all eukaryotes, acetylation of histone lysine residues correlates with transcription activation. Whether histone acetylation is a cause or consequence of transcription is debated. One model suggests that transcription promotes the recruitment and/or activation of acetyltransferases, and histone acetylation occurs as a consequence of ongoing transcription. However, the extent to which transcription shapes the global protein acetylation landscapes is not known. Here, we show that global protein acetylation remains virtually unaltered after acute transcription inhibition. Transcription inhibition ablates the co-transcriptionally occurring ubiquitylation of H2BK120 but does not reduce histone acetylation. The combined inhibition of transcription and CBP/p300 further demonstrates that acetyltransferases remain active and continue to acetylate histones independently of transcription. Together, these results show that histone acetylation is not a mere consequence of transcription; acetyltransferase recruitment and activation are uncoupled from the act of transcription, and histone and non-histone protein acetylation are sustained in the absence of ongoing transcription.

A new lysine biosynthetic enzyme from a bacterial endosymbiont shaped by genetic drift and genome reduction.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.

H3K9 methylation regulates heterochromatin silencing through incoherent feedforward loops.

Histone H3 lysine-9 methylation (H3K9me) is a hallmark of the condensed and transcriptionally silent heterochromatin. It remains unclear how H3K9me controls transcription silencing and how cells delimit H3K9me domains to avoid silencing essential genes. Here, using Arabidopsis genetic systems that induce H3K9me2 in genes and transposons de novo, we show that H3K9me2 accumulation paradoxically also causes the deposition of the euchromatic mark H3K36me3 by a SET domain methyltransferase, ASHH3. ASHH3-induced H3K36me3 confers anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2. These results demonstrate that H3K9me2 not only facilitates but orchestrates silencing by actuating antagonistic silencing and anti-silencing pathways, providing insights into the molecular basis underlying proper partitioning of chromatin domains and the creation of metastable epigenetic variation.