Citrus yellow vein clearing virus infection triggers phloem remobilization of iron- and zinc-nicotianamine in citrus.

Citrus yellow vein clearing virus (CYVCV) is a worldwide and highly destructive disease of citrus, but the mechanisms involved in CYVCV-inhibited plant growth are not well understood. This study examined nutrient levels and their cellular distribution in different organs of healthy and CYVCV-affected citrus (Citrus reticulata 'Kanpei') plants. We found that CYVCV-infected plants exhibit characteristic symptoms, including a significant reduction in iron (Fe) and other elemental nutrients in the shoots. Our data suggest that CYVCV-induced chlorosis in citrus leaf veins is primarily due to iron deficiency, leading to reduced chlorophyll synthesis. Further analysis revealed a marked decrease in iron concentration within the pith and xylem of citrus petioles post-CYVCV infection, contrasting with increased Fe and zinc (Zn) concentrations in the phloem. Moreover, a substantial accumulation of starch granules was observed in the pith, xylem, and phloem vessels of infected plants, with vessel blockage due to starch accumulation reaching up to 81%, thus significantly obstructing Fe transport in the xylem. Additionally, our study detected an upregulation of genes associated with nicotinamide metabolism and Fe and Zn transport following CYVCV infection, leading to increased levels of nicotinamide metabolites. This suggests that CYVCV-infected citrus plants may induce nicotinamide synthesis in response to Fe deficiency stress, facilitating the transport of Fe and Zn in the phloem as nicotinamide-bound complexes. Overall, our findings provide insight into the mechanisms of long-distance Fe and Zn transport in citrus plants in response to CYVCV infection and highlight the role of nutritional management in mitigating the adverse effects of CYVCV, offering potential strategies for cultivating CYVCV-resistant citrus varieties.

Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable. To address this issue, the DNA replication and repair machinery of C. glutamicum was targeted in this study. DnaQ, the homolog of ϵ subunit of DNA polymerase III responsible for proofreading in Escherichia coli, was proven irrelevant to DNA replication fidelity in C. glutamicum. However, the histidinol phosphatase (PHP) domain of DnaE1, the α subunit of DNA polymerase III, was characterized as the key proofreading element and certain variants with PHP mutations allowed elevated spontaneous mutagenesis. Repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of newly screened NucS variants also impaired the DNA replication fidelity. Simultaneous interference with the DNA replication and repair machinery generated a binary genetic mutator capable of increasing the mutation rate by up to 2352-fold. The mutators facilitated rapid evolutionary engineering of C. glutamicum to acquire stress tolerance and protein overproduction phenotypes. This study provides efficient tools for evolutionary engineering of C. glutamicum and could inspire the development of mutagenesis strategy for other microbial hosts.

Serendipita indica Drives Sulfur-Related Microbiota in Enhancing Growth of Hyperaccumulator Sedum alfredii and Facilitating Soil Cadmium Remediation.

Endophytic fungus Serendipita indica can bolster plant growth and confer protection against various biotic and abiotic stresses. However, S. indica-reshaped rhizosphere microecology interactions and root-soil interface processes in situ at the submicrometer scale remain poorly understood. We combined amplicon sequencing and high-resolution nano X-ray fluorescence (nano-XRF) imaging of the root-soil interface to reveal cadmium (Cd) rhizosphere processes. S. indica can successfully colonize the roots of Sedum alfredii Hance, which induces a remarkable increase in shoot biomass by 211.32% and Cd accumulation by 235.72%. Nano-XRF images showed that S. indica colonization altered the Cd distribution in the rhizosphere and facilitated the proximity of more Cd and sulfur (S) to enter the roots and transport to the shoot. Furthermore, the rhizosphere-enriched microbiota demonstrated a more stable network structure after the S. indica inoculation. Keystone species were strongly associated with growth promotion and Cd absorption. For example, Comamonadaceae are closely related to the organic acid cycle and S bioavailability, which could facilitate Cd and S accumulation in plants. Meanwhile, Sphingomonadaceae could release auxin and boost plant biomass. In summary, we construct a mutualism system for beneficial fungi and hyperaccumulation plants, which facilitates high-efficient remediation of Cd-contaminated soils by restructuring the rhizosphere microbiota.

Cadmium inhibits powdery mildew colonization and reconstructs microbial community in leaves of the hyperaccumulator plant Sedum alfredii.

Understanding the influence of the heavy metal cadmium (Cd) on the phyllosphere microbiome of hyperaccumulator plants is crucial for enhancing phytoremediation. The characteristics of the phyllosphere of Sedum alfredii Hance, a hyperaccumulator plant, were investigated using 16S rRNA and internal transcribed spacer amplicon sequencing of powdery mildew-infected leaves treated or untreated with Cd. The results showed that the colonization of powdery mildew caused severe chlorosis and necrosis in S. alfredii leaves, and the relative abundance of Leotiomycetes in infected leaves increased dramatically and significantly decreased phyllosphere microbiome diversity. However, S. alfredii preferentially accumulated higher concentrations of Cd in the leaves of infected plants than in uninfected plants by powdery mildew, which in turn significantly inhibited powdery mildew colonization in leaves; the relative abundance of the fungal class Leotiomycetes in infected leaves decreased, and alpha and beta diversities of the phyllosphere microbiome significantly increased with Cd treatment in the infected plants. In addition, the inter-kingdom networks in the microbiota of the infected leaves treated with Cd presented many nodes and edges, and the highest inter-kingdom modularity compared to the untreated infected leaves, indicating a highly connected microbial community. These results suggest that Cd significantly inhibits powdery mildew colonization by altering the composition of the phyllosphere microbiome in S. alfredii leaves, paving the way for efficient heavy metal phytoremediation and providing a new perspective on defense strategies against heavy metals.

Crystal structure of 5-Aminolevulinate synthase HemA from Rhodopseudomonas palustris presents multiple conformations.

5-ALA is the precursor of all tetrapyrroles. 5-Aminolevulinate synthase (ALAS) catalyzes the production of 5-aminolevulinic acid (5-ALA) from glycine and succinyl-CoA. HemA from Rhodopseudomonas palustris (Rp-HemA) was reported to be a highly active ALAS. To understand the catalytic mechanism of Rp-HemA, the 2.05 Å resolution crystal structure of Rp-HemA was solved. Open, half close and close conformations were observed in the substrate-free structures. Structure comparison and sequence alignment suggest the newly observed half close conformation may also be conserved in ALAS family. The pre-existed close and half close conformations in Rp-HemA may play a key role for its high activity.

Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli.

5-Aminolevulinic acid (ALA) is a value-added compound with potential applications in the fields of agriculture and medicine. Although massive efforts have recently been devoted to building microbial producers of ALA through metabolic engineering, few studies focused on the cellular response and tolerance to ALA. In this study, we demonstrated that ALA caused severe cell damage and morphology change of Escherichia coli via generating reactive oxygen species (ROS), which were further determined to be mainly hydrogen peroxide and superoxide anion radical. ALA treatment activated the native antioxidant defense system by upregulating catalase (CAT) and superoxide dismutase (SOD) expression to combat ROS. Further overexpressing CAT (encoded by katG and katE) and SOD (encoded by sodA, sodB, and sodC) not only improved ALA tolerance but also its production level. Notably, coexpression of katE and sodB in an ALA synthase expressing strain enhanced the biomass and final ALA titer by 81% and 117% (11.5 g/L) in a 5 L bioreactor, respectively. This study demonstrates the importance of tolerance engineering in strain development. Reinforcing the antioxidant defense system holds promise to improve the bioproduction of chemicals that cause oxidative stress.

Enhancing thermostability and removing hemin inhibition of Rhodopseudomonas palustris 5-aminolevulinic acid synthase by computer-aided rational design.

OBJECTIVE: To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. RESULTS: Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 µM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. CONCLUSION: H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.

Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs.

Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other biochemicals. However, due to good genomic stability, C. glutamicum is more difficult to engineer than genetically tractable hosts. Herein, a synthetic small regulatory RNA (sRNA)-based gene knockdown strategy was developed for C. glutamicum. The RNA chaperone Hfq from Escherichia coli and a rationally designed sRNA consisting of the E. coli MicC scaffold and a target binding site were proven to be indispensable for repressing green fluorescent protein expression in C. glutamicum. The synthetic sRNA system was applied to improve glutamate production through knockdown of pyk, ldhA, and odhA, resulting almost a threefold increase in glutamate titer and yield. Gene transcription and enzyme activity were down-regulated by up to 80%. The synthetic sRNA system developed holds promise to accelerate C. glutamicum metabolic engineering for producing valuable chemicals and fuels.

Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869.

Corynebacterium glutamicum is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of C. glutamicum with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far. Herein, we reported that engineering of peptidoglycan synthesis could significantly increase the transformation efficiency of C. glutamicum Comparative analysis of C. glutamicum wild-type strain ATCC 13869 and a mutant with high electrotransformation efficiency revealed nine mutations in eight cell wall synthesis-related genes. Among them, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA dramatically increased the electrotransformation of strain ATCC 13869 by 19.25-fold in the absence of cell wall-weakening agents, with no inhibition on growth. The Y489C mutation had no effect on the membrane localization of PonA but affected the peptidoglycan structure. Deletion of the ponA gene led to more dramatic changes to the peptidoglycan structure but only increased the electrotransformation by 4.89-fold, suggesting that appropriate inhibition of cell wall synthesis benefited electrotransformation more. Finally, we demonstrated that the PonAY489C mutation did not cause constitutive or enhanced glutamate excretion, making its permanent existence in C. glutamicum ATCC 13869 acceptable. This study demonstrates that genetic engineering of genes involved in cell wall synthesis, especially peptidoglycan synthesis, is a promising strategy to improve the electrotransformation efficiency of C. glutamicumIMPORTANCE Metabolic engineering and synthetic biology are now the key enabling technologies for manipulating microorganisms to suit the practical outcomes desired by humankind. The introduction of exogenous DNA into cells is an indispensable step for this purpose. However, some microorganisms, including the important industrial workhorse Corynebacterium glutamicum, possess a complex cell wall structure to shield cells against exogenous DNA. Although genes responsible for cell wall synthesis in C. glutamicum are known, engineering of related genes to improve cell competency has not been explored yet. In this study, we demonstrate that mutations in cell wall synthesis genes can significantly improve the electrotransformation efficiency of C. glutamicum Notably, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA increased electrotransformation efficiency by 19.25-fold by affecting peptidoglycan synthesis.

[Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum].

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Cardiac-specific overexpression of CREM-IbΔC-X via CRISPR/Cas9 in mice presents a new model of atrial cardiomyopathy with spontaneous atrial fibrillation.

Atrial cardiomyopathy (ACM) forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. However, generating stable animal models that accurately replicate the entire progression of atrial lesions, particularly the onset of AF, presents significant challenges. In the present study, we found that the isoform of CRE-binding protein modulator (CREM-IbΔC-X), which is involved in the regulation of cardiac development and atrial rhythm, was highly expressed in atrial biopsies from patients with AF. Building upon this finding, we employed CRISPR/Cas9 technology to create a mouse model with cardiac-specific overexpression of CREM-IbΔC-X (referred to as CS-CREM mice). This animal model effectively illustrated the development of ACM through electrophysiological and structural remodelings over time. Proteomics and Chip-qPCR analysis of atrial samples revealed significant upregulation of cell-matrix adhesion and extracellular matrix structural components, alongside significant downregulation of genes related to atrial functions in the CS-CREM mice. Furthermore, the corresponding responses to anti-arrhythmia drugs, i.e., amiodarone and propafenone, suggested that CS-CREM mice could serve as an ideal in vivo model for drug testing. Our study introduced a novel ACM model with spontaneous AF by cardiac-specifically overexpressing CREM-IbΔC-X in mice, providing valuable insights into the mechanisms and therapeutic targets of ACM.

Follicle-stimulating hormone promotes atrial fibrosis in menopausal women with atrial fibrillation.

BACKGROUND: Postmenopausal women with atrial fibrillation (AF) exhibit a higher level of atrial fibrosis and a higher recurrence rate after ablation compared with men. However, the underlying mechanism remains unclear. OBJECTIVE: The purpost of this study was to investigate the mechanism through which menopause promotes atrial fibrosis. METHODS: In a prospective cohort of women with AF, regression analyses were conducted to assess the relationship between low-voltage area (LVA) and sex hormone levels. CREM-IbΔC-X mice, a spontaneous AF model, underwent bilateral ovariectomy (OVX). Electrocardiograms, echocardiograms, and Masson staining were performed. Follicle-stimulating hormone (FSH) stimulation was applied in male mice for 3 months. OVX was also applied in an angiotensin II (Ang II)-induced pressure overload mouse model, after programmed electrical stimulation and structural analyses. Bulk RNA sequencing (RNA-seq) was performed to elucidate potential mechanisms. RESULTS: Women demonstrated a significantly higher LVA burden than men (P < .001). A positive correlation was observed between LVA burden and FSH level (P = .002). Mice in the OVX group exhibited a significantly higher incidence of AF (P = .040) and atrial fibrosis (P = .021) compared with the Sham group, which could be attenuated by adeno-associated virus encoding small interfering RNA against Fshr. In male CREM-IbΔC-X mice, FSH stimulation promoted the occurrence of AF (P = .035) and atrial fibrosis (P = .002). In Ang II-induced female mice, OVX prompted atrial fibrosis, increased AF inducibility, and shortened atrial effective refractory period, which could be attenuated with knockdown of Fshr. RNA-seq indicated mitochondrial dysfunction. CONCLUSION: Postmenopausal women exhibited a higher LVA burden than men, which was positively correlated with FSH level. FSH promoted atrial fibrosis through oxidative stress.

Inoculation of multi-metal-resistant Bacillus sp. to a hyperaccumulator plant Sedum alfredii for facilitating phytoextraction of heavy metals from contaminated soil.

Co-contamination of soil by multiple heavy metals is a significant global challenge. An effective strategy to address this issue involves using hyperaccumulators such as Sedum alfredii (S. alfredii). The efficiency of phytoremediation can be improved by supplementing with plant growth-promoting bacteria (PGPB). However, bacteria resources of PGPB resistant to multi-heavy metal contamination are still lacking. This study focused nine different strains of Bacillus and screened for resistance to heavy metals including cadmium (Cd), zinc (Zn), copper (Cu), and lead (Pb). A superior strain, Bacillus subtilis PY79 (B. subtilis), showed tolerance for all tested metals. Inoculation with B. subtilis in the rhizosphere of S. alfredii increased the accumulation of Cd, Zn, Cu, and Pb by 88.02%, 58.99%, 90.22%, and 54.97% in the plant shoots after 30 days respectively. B. subtilis application lowered the pH of the rhizosphere soil, thereby increasing the bioavailability of nutrients and heavy metals. Furthermore, B. subtilis helped S. alfredii recruit PGPB and heavy metal-resistant bacteria such as Edaphobacter, Niastella, and Chitinophaga, enhancing the growth and phytoremediation efficiency. Moreover, inoculation with B. subtilis not only upregulated genes of the ABC, HMA, ZIP, and MTP families involved in the translocation and detoxification of heavy metals but also increased the secretion of antioxidants within the cells. These findings indicate that B. subtilis enhances the tolerance, uptake, and translocation of heavy metals in S. alfredii, offering valuable insights for the phytoremediation of multi-metal-contaminated soils.

Automated and integrated ultrahigh throughput screening for industrial strain enabled by acoustic-droplet-ejection mass spectrometry.

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Reconstruction the feedback regulation of amino acid metabolism to develop a non-auxotrophic L-threonine producing Corynebacterium glutamicum.

L-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation. The GRAS (generally regarded as safe) industrial workhorse Corynebacterium glutamicum is an attractive chassis for L-threonine production. However, the present L-threonine production in C. glutamicum cannot meet the requirement of industrialization due to the relatively low production level of L-threonine and the accumulation of large amounts of by-products (such as L-lysine, L-isoleucine, and glycine). Herein, to enhance the L-threonine biosynthesis in C. glutamicum, releasing the aspartate kinase (LysC) and homoserine dehydrogenase (Hom) from feedback inhibition by L-lysine and L-threonine, respectively, and overexpressing four flux-control genes were performed. Next, to reduce the formation of by-products L-lysine and L-isoleucine without the cause of an auxotrophic phenotype, the feedback regulation of dihydrodipicolinate synthase (DapA) and threonine dehydratase (IlvA) was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by L-lysine and L-isoleucine, respectively. The resulting strain maintained the capability of synthesizing enough amounts of L-lysine and L-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids. To further enhance L-threonine production and reduce the by-product glycine, L-threonine exporter and homoserine kinase were overexpressed. Finally, the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L (17.2% higher) L-threonine with a productivity of 1.20 g/L/h (108.0% higher) in fed-batch fermentation, along with significantly reduced by-product accumulation, representing the record for L-threonine production in C. glutamicum. In this study, we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic L-threonine producing C. glutamicum. The main end by-products including L-lysine, L-isoleucine, and glycine were almost eliminated in fed-batch fermentation of the engineered C. glutamicum strain. This strategy can also be used for engineering producing strains for other amino acids and derivatives.

Injectable and Conductive Nanomicelle Hydrogel with α-Tocopherol Encapsulation for Enhanced Myocardial Infarction Repair.

Substantial advancements have been achieved in the realm of cardiac tissue repair utilizing functional hydrogel materials. Additionally, drug-loaded hydrogels have emerged as a research hotspot for modulating adverse microenvironments and preventing left ventricular remodeling after myocardial infarction (MI), thereby fostering improved reparative outcomes. In this study, diacrylated Pluronic F127 micelles were used as macro-cross-linkers for the hydrogel, and the hydrophobic drug α-tocopherol (α-TOH) was loaded. Through the in situ synthesis of polydopamine (PDA) and the incorporation of conductive components, an injectable and highly compliant antioxidant/conductive composite FPDA hydrogel was constructed. The hydrogel exhibited exceptional stretchability, high toughness, good conductivity, cell affinity, and tissue adhesion. In a rabbit model, the material was surgically implanted onto the myocardial tissue, subsequent to the ligation of the left anterior descending coronary artery. Four weeks postimplantation, there was discernible functional recovery, manifesting as augmented fractional shortening and ejection fraction, alongside reduced infarcted areas. The findings of this investigation underscore the substantial utility of FPDA hydrogels given their proactive capacity to modulate the post-MI infarct microenvironment and thereby enhance the therapeutic outcomes of myocardial infarction.

Comprehensive screening of industrially relevant components at genome scale using a high-quality gene overexpression collection of Corynebacterium glutamicum.

Development of efficient microbial strains for biomanufacturing requires deep understanding of the biology and functional components responsible for the synthesis, transport, and tolerance of the target compounds. A high-quality controllable gene overexpression strain collection was constructed for the industrial workhorse Corynebacterium glutamicum covering 99.7% of its genes. The collection was then used for comprehensive screening of components relevant to biomanufacturing features. In total, 15 components endowing cells with improved hyperosmotic tolerance and l-lysine productivity were identified, including novel transcriptional factors and DNA repair proteins. Systematic interrogation of a subset of the collection revealed efficient and specific exporters functioning in both C. glutamicum and Escherichia coli. Application of the new exporters was showcased to construct a strain with the highest l-threonine production level reported for C. glutamicum (75.1 g/l and 1.5 g/l·h) thus far. The genome-scale gene overexpression collection will serve as a valuable resource for fundamental biological studies and for developing industrial microorganisms for producing amino acids and other biochemicals.

Cloning of two 5-aminolevulinic acid synthase isozymes HemA and HemO from Rhodopseudomonas palustris with favorable characteristics for 5-aminolevulinic acid production.

5-Aminolevulinic acid (ALA) synthase (ALAS) HemA from non-sulfur photosynthetic bacteria has been used for the ALA bioproduction, whereas the isoenzyme HemT/HemO is less studied and not used for ALA production. Two ALAS-encoding genes, hemA and hemO from Rhodopseudomonas palustris were cloned, purified and characterized. The ALASs had very high specific activity, 3.6 and 2.7 U/mg, respectively, and strong affinity for one of its substrates, succinyl-CoA, K m with values of 11 and 4.4 µM, respectively. HemO retained up to 60 % maximum activity within a broad range of concentrations of hemin, while HemA kept only 20 % at 10 µM hemin. Escherichia coli overexpressing HemA or HemO produced 5.7 and 6.3 g ALA/l, respectively, in a 5 l bioreactor.

Influence of lipid A acylation pattern on membrane permeability and innate immune stimulation.

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.

Construction of monophosphoryl lipid A producing Escherichia coli mutants and comparison of immuno-stimulatory activities of their lipopolysaccharides.

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine phosphorylated at the 1- and 4'-positions. It can be recognized by the TLR4/MD-2 complex of mammalian immune cells, leading to release of proinflammatory cytokines. The toxicity of lipid A depends on its structure. In this study, two E. coli mutants, HW001 and HW002, were constructed by deleting or integrating key genes related to lipid A biosynthesis in the chromosome of E. coli W3110. HW001 was constructed by deleting lacI and replacing lacZ with the Francisella novicida lpxE gene in the chromosome and only synthesizes monophosphoryl lipid A. HW002 was constructed by deleting lpxM in HW001 and synthesizes only the pentaacylated monophosphoryl lipid A. The structures of lipid A made in HW001 and HW002 were confirmed by thin layer chromatography and electrospray ionization mass spectrometry. HW001 and HW002 grew as well as the wild-type W3110. LPS purified from HW001 or HW002 was used to stimulate murine macrophage RAW264.7 cells, and less TNF-α were released. This study provides a feasible way to produce interesting lipid A species in E. coli.