Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis.

MAIN CONCLUSION: Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as ß-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or ß-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with ß-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of ß-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

High-level production of Rhodiola rosea characteristic component rosavin from D-glucose and L-arabinose in engineered Escherichia coli.

Rosavin is the characteristic component of Rhodiola rosea L., an important medicinal plant used widely in the world that has been reported to possess multiple biological activities. However, the endangered status of wild Rhodiola has limited the supply of rosavin. In this work, we successfully engineered an Escherichia coli strain to efficiently produce rosavin as an alternative production method. Firstly, cinnamate: CoA ligase from Hypericum calycinum, cinnamoyl-CoA reductase from Lolium perenne, and uridine diphosphate (UDP)-glycosyltransferase (UGT) from Bacillus subtilis (Bs-YjiC) were selected to improve the titer of rosin in E. coli. Subsequently, four UGTs from the UGT91R subfamily were identified to catalyze the formation of rosavin from rosin, with SlUGT91R1 from Solanum lycopersicum showing the highest activity level. Secondly, production of rosavin was achieved for the first time in E. coli by incorporating the SlUGT91R1 and UDP-arabinose pathway, including UDP-glucose dehydrogenase, UDP-xylose synthase, and UDP-xylose 4-epimerase, into the rosin-producing stain, and the titer reached 430.5 ± 91.4 mg/L. Thirdly, a two-step pathway derived from L-arabinose, composed of L-arabinokinase and UDP-sugar pyrophosphorylase, was developed in E. coli to further optimize the supply of the precursor UDP-arabinose. Furthermore, 1203.7 ± 32.1 mg/L of rosavin was produced from D-glucose and L-arabinose using shake-flask fermentation. Finally, the production of rosavin reached 7539.1 ± 228.7 mg/L by fed-batch fermentation in a 5-L bioreactor. Thus, the microbe-based production of rosavin shows great potential for commercialization. This work provides an effective strategy for the biosynthesis of other valuable natural products with arabinose-containing units from D-glucose and L-arabinose.

Expression of chicken epidermal growth factor (cEGF) in Escherichia coli regulates the microflora structure of the duodenum to improve growth performance and intestinal morphogenesis in broilers.

1. A study used gene synthesis to obtain the functional domains of chicken epidermal growth factor (cEGF) and examined their impact on broiler growth performance, small intestinal morphology, digestive enzyme activities in the intestinal contents and the structure of duodenal microflora.2. The pET-32a-cEGF recombinant expression vector was constructed. The specific band at 26 KDa was shown by SDS-PAGE analysis and WB results. The purified protein content was shown to be 1687 µg/ml by assay.3. A total of 180 healthy, one-day-old Arbor Acres male, white-feathered broilers were randomly divided into three dietary treatment groups (six replicate pens, 10 birds per replicate): A control diet (ND); cEGF diet (cEGF), control supplemented with 250 mg/kg cEGF and the control diet (CD) supplemented with 250 mg/kg chlortetracycline.4. The results showed that feeding the cEGF and CD diet reduced FCR of broilers aged 1-21 d, average daily feed intake (ADFI) at 22-42 d, and the FCR in the whole period (1-42 d; p < 0.05). Compared with the ND group, the cEGF diet increased duodenal α-amylase and alkaline phosphatase activities in the 1-21 d, duodenal lipase, alkaline phosphatase, and ileal alkaline phosphatase activities in the post-period and increased villus height in the duodenum and ileum (p < 0.05). In addition, the ACE and Chao1 index for the birds fed cEGF were higher than the ND group (p < 0.05). At the phyla level, Firmicutes and Proteobacteria were dominant in all groups. At the genus level, the dominant genus was Lactobacillus. The LEfSe analysis showed that the cEGF group was enriched by 11 species including Brevibacillus, Eisenbergiella, Cloacibacterium, Butyricoccus spp.5. The addition of 250 mg/kg cEGF to the diet can increase growth performance by improving intestinal development and digestive enzyme activity, which may be related to the duodenal intestinal microflora. Therefore, cEGF is an effective alternative to antibiotics in broiler farming.

Exploring of pyrazinamidase recombinant activity from PZA-sensitive and resistant Mycobacterium tuberculosis expressed in Escherichia coli BL21 (DE3).

The mutations of pncA gene encoding pyrazinamidase/PZase in Mycobacterium tuberculosis are often associated with pyrazinamide/PZA resistance. The H and R1 isolates showed significant phenotypic differences to PZA. The H isolate was PZA sensitive, but R1 was PZA resistant up to 100 ug/ml. The paper reports the pncA profile for both isolates and the activity of their protein expressed in Escherichia coli BL21(DE3). The 0.6 kb of each pncA genes have been subcloned successfully into the 5.4 kb pET30a vector and formed the pET30a-pncA recombinant with a size of 6.0 kb. The pncAR1 profile exhibited base mutations, but not for pncAH against to pncA from the PZA-sensitive M. tuberculosis H37RV published in Genbank ID: 888260. Three mutations were found in pncAR1, ie T41C, G419A, and A535G that subsequently changed amino acids of Cys14Arg, Arg140His and Ser179Gly in its protein level. The mutant PZase R1 that expressed as a 21 kDa protein in E. coli Bl21(DE3) lost 32% of its performance in activating PZA drug to pyrazinoic acid/POA compared to the wild-type PZase H. The mutation in the pncAR1 gene that followed by the decreasing of its PZase activity underlies the emergence of pyrazinamide resistance in the clinical isolate. Structural studies for the R1 mutant PZase protein should be further developed to reveal more precise drug resistance mechanisms and design more effective TB drugs.

CFTR is required for zinc-mediated antibacterial defense in human macrophages.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.

rBet v 1a-BanLecwt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors.

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.

Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L-cysteine.

L-Cysteine production through fermentation stands as a promising technology. However, excessive accumulation of L-cysteine poses a challenge due to the potential to inflict damage on cellular DNA. In this study, we employed a synergistic approach encompassing atmospheric and room temperature plasma mutagenesis (ARTP) and adaptive laboratory evolution (ALE) to improve L-cysteine tolerance in Escherichia coli. ARTP-treated populations obtained substantial enhancement in L-cysteine tolerance by ALE. Whole-genome sequencing, transcription analysis, and reverse engineering, revealed the pivotal role of an effective export mechanism mediated by gene eamB in augmenting L-cysteine resistance. The isolated tolerant strain, 60AP03/pTrc-cysEf , achieved a 2.2-fold increase in L-cysteine titer by overexpressing the critical gene cysEf during batch fermentation, underscoring its enormous potential for L-cysteine production. The production evaluations, supplemented with L-serine, further demonstrated the stability and superiority of tolerant strains in L-cysteine production. Overall, our work highlighted the substantial impact of the combined ARTP and ALE strategy in increasing the tolerance of E. coli to L-cysteine, providing valuable insights into improving L-cysteine overproduction, and further emphasized the potential of biotechnology in industrial production.

A sustainable strategy for biosynthesis of Rebaudioside D using a novel glycosyltransferase of Solanum tuberosum.

Bioconversion of Rebaudioside D faces high-cost obstacles. Herein, a novel glycosyltransferase StUGT converting Rebaudioside A to Rebaudioside D was screened and characterized, which exhibits stronger affinity and substrate specificity for Rebaudioside A than previously reported enzymes. A whole-cell catalytic system was thus developed using the StUGT strain. The production of Rebaudioside D was enhanced significantly by enhancing cell permeability, and the maximum production of 6.12 g/L and the highest yield of 98.08% by cell catalyst was obtained by statistical-based optimization. A new cascade process utilizing this recombinant strain and E. coli expressing sucrose synthase was further established to reduce cost through replacing expensive UDPG with sucrose. A StUGT-GsSUS1 system exhibited high catalytic capability, and 5.27 g L-1 Rebaudioside D was achieved finally without UDPG addition by systematic optimization. This is the best performance reported in cell-cascaded biosynthesis, which paves a new cost-effective strategy for sustainable synthesis of scarce premium sweeteners from biomass.

Metabolome analysis of metabolic burden in Escherichia coli caused by overexpression of green fluorescent protein and delta-rhodopsin.

Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E. coli strains have distinct metabolic capacities. In this study, two proteins were overexpressed in four E. coli strains (MG1655(DE3), W3110(DE3), BL21star(DE3), and Rosetta(DE3)), and their effects on metabolic burden were investigated. Metabolomic analysis showed that E. coli strains overexpressing green fluorescent protein had decreased levels of several metabolites, with a positive correlation between the number of reduced metabolites and green fluorescent protein expression levels. Moreover, nucleic acid-related metabolites decreased, indicating a metabolic burden in the E. coli strains, and the growth rate and protein expression levels were improved by supplementation with the five nucleosides. In contrast, two strains overexpressing delta rhodopsin, a microbial membrane rhodopsin from Haloterrigena turkmenica, led to a metabolic burden and decrease in the amino acids Ala, Val, Leu, Ile, Thr, Phe, Asp, and Trp, which are the most frequent amino acids in the delta rhodopsin protein sequence. The metabolic burden caused by protein overexpression was influenced by the metabolic capacity of the host strains and the sequences of the overexpressed proteins. Detailed characterization of the effects of protein expression on the metabolic state of engineered cells using metabolomics will provide insights into improving the production of target compounds.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

The metabolic slowdown caused by the deletion of pspA accelerates protein aggregation during stationary phase facilitating antibiotic persistence.

Entering a dormant state is a prevailing mechanism used by bacterial cells to transiently evade antibiotic attacks and become persisters. The dynamic progression of bacterial dormancy depths driven by protein aggregation has been found to be critical for antibiotic persistence in recent years. However, our current understanding of the endogenous genes that affects dormancy depth remains limited. Here, we discovered a novel role of phage shock protein A (pspA) gene in modulating bacterial dormancy depth. Deletion of pspA of Escherichia coli resulted in increased bacterial dormancy depths and prolonged lag times for resuscitation during the stationary phase. ∆pspA exhibited a higher persister ratio compared to the wild type when challenged with various antibiotics. Microscopic images revealed that ∆pspA showed accelerated formation of protein aggresomes, which were collections of endogenous protein aggregates. Time-lapse imaging established the positive correlation between protein aggregation and antibiotic persistence of ∆pspA at the single-cell level. To investigate the molecular mechanism underlying accelerated protein aggregation, we performed transcriptome profiling and found the increased abundance of chaperons and a general metabolic slowdown in the absence of pspA. Consistent with the transcriptomic results, the ∆pspA strain showed a decreased cellular ATP level, which could be rescued by glucose supplementation. Then, we verified that replenishment of cellular ATP levels by adding glucose could inhibit protein aggregation and reduce persister formation in ∆pspA. This study highlights the novel role of pspA in maintaining proteostasis, regulating dormancy depth, and affecting antibiotic persistence during stationary phase.

Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2.

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC). Notably, the purified Pq3-O-UGT2 showed substantial activity toward Rh2 and F2, catalyzing the formation of Rg3 and Rd, respectively. This activity was discernible within a pH range of 4.0-9.0 and temperature range of 30-55 °C, with optimal conditions observed at pH 7.0-8.0 and 37 °C. The catalytic efficiency of Pq3-O-UGT2 toward Rh2 and F2 was 31.43 s-1 mΜ-1 and 169.31 s-1 mΜ-1, respectively. We further crystalized Pq3-O-UGT2 in both its apo form and co-crystalized forms with UDPG, Rh2 and F2, respectively. High-quality crystals were obtained and X-ray diffraction data was collected for all co-crystalized samples. Analysis of the diffraction data revealed that the crystal of Pq3-O-UGT2 co-crystalized with UDP-Glc belonged to space group P1, while the other two crystals belonged to space group P212121. Together, this study has laid a robust foundation for subsequent structural analysis of Pq3-O-UGT2.

New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Cellular toxicity and DNA damage induced by Newbouldia laevis used for male infertility treatment in prokaryotic and eukaryotic models.

Leaves of Newbouldia laevis have been extensively used in solving problems associated with infertility and childbirth in many African countries. Yet, information is very limited on the DNA damaging potential of this plant. This study evaluated the cytogenotoxic effect of the aqueous extract of N. laevis leaf using prokaryotic models (Ames Salmonella fluctuation test using TA100 and TA98 strains of Salmonella typhimurium and SOS Chromotest with Escherichia coli PQ37) and eukaryotic model (Allium cepa root cells). Identification of the volatile organic compounds (VOCs) and phytochemical screening of the plant extract were also performed. Onion bulbs were grown on each concentration (1 to 50%; v/v, extract/tap water) of the extract for chromosomal aberrations and root growth analyses. Results of the Ames test indicated that the extract is mutagenic while the SOS Chromotest results showed good complementation to the Ames test results, although the E. coli PQ37 system showed slightly higher sensitivity in the detection of mutagenicity and genotoxicity of the extract. The plant extract was cytotoxic when compared to the control, inducing a significant (p < 0.05) concentration-dependent inhibition of root growth from 5 to 50% concentrations. At 50% concentration, the extract completely inhibited cell division in the A. cepa. Also, chromosomal aberration increased significantly (p < 0.05) in exposed onions from 5 to 20% concentrations. The mutagenicity and cytogenotoxicity recorded in this report were believed to be caused by the presence of VOCs such as 1,2,3-benzene-triol, 1,2-benzenediol, and 5-hydroxymethylfurfural, and alkaloids in the extract an indication of the cytogenotoxicity of the aqueous extract of N. laevis leaf even at low concentration.

The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.

Identification of Pla a 7 as a novel pollen allergen group in Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is recognized as a source of allergenic pollen worldwide. Currently, five Platanus acerifolia pollen allergens belonging to different protein families have been identified, in which profilin and enolase were characterized by our group recently. Besides, we also screened and identified a novel allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens. However, the role of this novel allergen group in Platanus acerifolia pollen allergy was unclear. Therefore, we further investigated the allergenicity and clarify its clinical relevance in this study. METHODS: The natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test. RESULTS: A novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients' sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test. CONCLUSIONS: Triosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy.

Identification and functional characterization of two trans-isopentenyl diphosphate synthases and one squalene synthase involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.

Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics.

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon-controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.