Extremely Stable Perylene Bisimide-Bridged Regioisomeric Diradicals and Their Redox Properties.

Excellent stability is an essential premise for organic diradicals to be used in organic electronic and spintronic devices. We have attached two tris(2,4,6-trichlorophenyl)methyl (TTM) radical building blocks to the two sides of perylene bisimide (PBI) bridges and obtained two regioisomeric diradicals (1,6-TTM-PBI and 1,7-TTM-PBI). Both of the isomers show super stability rather than the monomeric TTM under ambient conditions, due to the increased conjugation and the electron-withdrawing effects of the PBI bridges. The diradicals show distinct and reversible multistep redox processes, and a spectro-electrochemistry investigation revealed the generation of organic mixed-valence (MV) species during reduction processes. The two diradicals have singlet ground states, very small singlet-triplet energy gaps (ΔES-T ) and a pure open-shell character (with diradical character y0 =0.966 for 1,6-TTM-PBI and 0.967 for 1,7-TTM-PBI). This work opens a window to developing very stable diradicals and offers the opportunity of their further application in optical, electronic and magnetic devices.

A pH/GSH/Glucose Responsive Nanozyme for Tumor Cascade Amplified Starvation and Chemodynamic Theranostics.

Nanozymes have brought enormous opportunities for disease theranostics. Here, a self-enhanced catalytic nanocrystal based on a bismuth-manganese core-shell nanoflower containing glucose oxide (GOx), termed BDS-GOx@MnOx, was designed for 4T1 tumor theranostics in vitro and in vivo. The BDS-GOx@MnOx nanozymes enable enhanced starvation treatment (ST) and chemotherapy (CDT) with high efficacy and exhibit sensitive tumor microenvironment (TME) responsive character for tumor therapy as well as for tumor-enhanced computer tomography (CT) and magnetic resonance (MR) diagnostic imaging. The characters and mechanism of the BDS-GOx@MnOx nanozymes have also been systematically studied and revealed.

Carboxymethyl chitosan/sodium alginate hydrogels with polydopamine coatings as promising dressings for eliminating biofilm and multidrug-resistant bacteria induced wound healing.

Microorganisms induced wound infection and the accompanying excessive inflammatory response is the daunting problems in wound treatment. Due to the lack of corresponding biological functions, traditional wound dressings cannot effectively protect the wound and are prone to induce local infection, excessive inflammation, and vascular damage, resulting in prolonged unhealing. Here, a mussel-inspired strategy was adopted to prepare a multifunctional hydrogel created by H2O2/CuSO4-induced rapid polydopamine (PDA) deposition on carboxymethyl chitosan (CMC)/sodium alginate (Alg) based hydrogel, termed as CAC/PDA/Cu(H2O2). The prepared CAC/PDA/Cu(H2O2) hydrogel features excellent biocompatibility, adequate mechanical properties, and good degradability. Moreover, the CAC/PDA/Cu(H2O2) hydrogel can not only realize antibacterial, and anti-inflammatory effects, but also promote angiogenesis to accelerate wound healing in vitro thanks to the composite PDA/Cu(H2O2) coatings. Significantly, CAC/PDA/Cu(H2O2) hydrogel illustrates excellent therapeutic effects in Methicillin-resistant Staphylococcus aureus (MRSA) induced-rat infection models, which can efficiently eliminate MRSA, dramatically reduce inflammatory expression, promote angiogenesis, and ultimately shorten the wound healing time. CAC/PDA/Cu(H2O2) hydrogel exhibited the best wound healing rate on days 7 (80.63 ± 2.44 %), 11 (92.45 ± 2.26 %), and 14 (97.86 ± 0.66 %). Thus, the multifunctional hydrogel provides a facile and efficient approach to wound management and represents promising potential in the therapy for wound healing.

Effect of atropine, orthokeratology and combined treatments for myopia control: a 2-year stratified randomised clinical trial.

PURPOSE: To investigate the 2-year efficacy of atropine, orthokeratology (ortho-k) and combined treatment on myopia. To explore the factors influencing the efficacy. METHODS: An age-stratified randomised controlled trial. Children (n=164) aged 8-12 years with spherical equivalent refraction of -1.00 to -6.00 D were stratified into two age subgroups and randomly assigned to receive placebo drops+spectacles (control), 0.01% atropine+spectacles (atropine), ortho-k+placebo (ortho-k) or combined treatment. Axial length was measured at baseline and visits at 6, 12, 18 and 24 months. The primary analysis was done following the criteria of intention to treat, which included all randomised subjects. RESULTS: All interventions can significantly reduce axial elongation at all visits (all p<0.05). Overall, the 2-year axial elongation was significantly reduced in combined treatment than in monotherapies (all p<0.05). After stratification by age, in the subgroup aged 8-10, the difference between combined treatment and ortho-k became insignificant (p=0.106), while in the subgroup aged 10-12, the difference between combined treatment and atropine became insignificant (p=0.121). A significant age-dependent effect existed in the ortho-k group versus the control group (p for interaction=0.013), and a significant age-dependent effect existed in the ortho-k group versus the atropine group (p for interaction=0.035), which indicated that ortho-k can achieve better efficacy in younger children. CONCLUSIONS: Atropine combined with ortho-k treatment can improve the efficacy of myopia control compared with monotherapy in children aged 8-12. Younger children might benefit more from ortho-k. TRIAL REGISTRATION NUMBER: ChiCTR1800015541.

Accommodation and vergence function in children using atropine combined with orthokeratology.

PURPOSE: This study aimed to evaluate binocular vision in terms of vergence and accommodative measurements in children treated with 0.01% atropine combined with orthokeratology (OK). METHODS: This was a prospective and randomized controlled clinical trial involving participants aged 8 to 12 years, with a spherical equivalent (SE) ranging from - 1.00 to - 6.00D. Participants were randomly divided into four groups: 1) a combination group using 0.01% atropine solution and OK lens; 2) an OK group using placebo solution and OK lens; 3) an atropine group using 0.01% atropine solution and wearing spectacles; and 4) a control group using placebo solution and wearing spectacles. Binocular vision was determined at baseline and at 3-month visits, with evaluations including horizontal phoria, fusional vergence, the accommodative convergence/accommodation (AC/A) ratio, accommodative lag, and accommodative amplitude (AA). The Wilcoxon signed-rank test was used to compare the changes in binocular vision in each group, and the Kruskal-Wallis test was used for comparisons of four groups. RESULTS: Sixty-two participants completed the study. There was no significant difference in baseline refraction, accommodation or vergence measurements among the groups (all P > 0.05). Three months later, the accommodative lag significantly decreased in the OK group (P = 0.002) but remained unchanged in the other three groups (all P > 0.05). In addition, binocular accommodative facilities and positive relative accommodations increased in the combination and OK groups (both P < 0.05) but remained unchanged in the atropine and control groups (both P > 0.05). Only the participants with esophoria in the OK group had a significant decrease in esophoria (P = 0.008). Moreover, the changes in fusional vergence and AC/A did not significantly differ between the four groups (all P > 0.05). CONCLUSION: Accommodative measurements changed similarly in the groups treated with OK. Changes in vergence measurements after treatment with 0.01% atropine were not significant.

Molecular characterization of a novel strain of Bacillus halotolerans protecting wheat from sheath blight disease caused by Rhizoctonia solani Kühn.

Rhizoctonia solani Kühn naturally infects and causes Sheath blight disease in cereal crops such as wheat, rice and maize, leading to severe reduction in grain yield and quality. In this work, a new bacterial strain Bacillus halotolerans LDFZ001 showing efficient antagonistic activity against the pathogenic strain Rhizoctonia solani Kühn sh-1 was isolated. Antagonistic, phylogenetic and whole genome sequencing analyses demonstrate that Bacillus halotolerans LDFZ001 strongly suppressed the growth of Rhizoctonia solani Kühn sh-1, showed a close evolutionary relationship with B. halotolerans F41-3, and possessed a 3,965,118 bp circular chromosome. Bioinformatic analysis demonstrated that the genome of Bacillus halotolerans LDFZ001 contained ten secondary metabolite biosynthetic gene clusters (BGCs) encoding five non-ribosomal peptide synthases, two polyketide synthase, two terpene synthases and one bacteriocin synthase, and a new kijanimicin biosynthetic gene cluster which might be responsible for the biosynthesis of novel compounds. Gene-editing experiments revealed that functional expression of phosphopantetheinyl transferase (SFP) and major facilitator superfamily (MFS) transporter genes in Bacillus halotolerans LDFZ001 was essential for its antifungal activity against R. solani Kühn sh-1. Moreover, the existence of two identical chitosanases may also make contribution to the antipathogen activity of Bacillus halotolerans LDFZ001. Our findings will provide fundamental information for the identification and isolation of new sheath blight resistant genes and bacterial strains which have a great potential to be used for the production of bacterial control agents. Importance: A new Bacillus halotolerans strain Bacillus halotolerans LDFZ001 resistant to sheath blight in wheat is isolated. Bacillus halotolerans LDFZ001 harbors a new kijanimicin biosynthetic gene cluster, and the functional expression of SFP and MFS contribute to its antipathogen ability.

The Structure and Function of Microbial Community in Rhizospheric Soil of American Ginseng (Panax quinquefolius L.) Changed with Planting Years.

The changes of microbial communities of rhizospheric soil in different ages are speculated to cause soil-borne diseases and replanting problem in American ginseng (Panax quinquefolius L.) cultivation. This study analyzed the physicochemical properties and microbial communities of rhizospheric soil during the planting of American ginseng in the Wendeng area of Weihai, China. The water content and organic matter content of American ginseng rhizospheric soil decreased year by year. A decline in the diversity of bacteria and fungi was observed in the rhizospheric soils planting American ginseng compared with the traditional crop wheat in the control group. During the later planting stage, the abundances of Proteobacteria, Actinobacteria, and Basidiomycota were lower, whereas that of Acidobacteria, Firmicutes, and Mucoromycota were higher. Through the correlation analysis between environmental factors and microbial community, it was found that the content of soil phosphorus was significantly positively correlated with the root rot pathogen Fusarium. The results of functional prediction showed that the decrease of secondary metabolite synthesis of rhizospheric soil bacteria and the increase of plant pathogenic fungi may be the important reasons for the increase of diseases in the later stage of American ginseng planting. This study revealed the evolution of rhizosphere microbial community and function in the process of American ginseng planting, which is valuable for planting management.

Customized Design 3D Printed PLGA/Calcium Sulfate Scaffold Enhances Mechanical and Biological Properties for Bone Regeneration.

Polylactic glycolic acid copolymer (PLGA) has been widely used in tissue engineering due to its good biocompatibility and degradation properties. However, the mismatched mechanical and unsatisfactory biological properties of PLGA limit further application in bone tissue engineering. Calcium sulfate (CaSO4) is one of the most promising bone repair materials due to its non-immunogenicity, well biocompatibility, and excellent bone conductivity. In this study, aiming at the shortcomings of activity-lack and low mechanical of PLGA in bone tissue engineering, customized-designed 3D porous PLGA/CaSO4 scaffolds were prepared by 3D printing. We first studied the physical properties of PLGA/CaSO4 scaffolds and the results showed that CaSO4 improved the mechanical properties of PLGA scaffolds. In vitro experiments showed that PLGA/CaSO4 scaffold exhibited good biocompatibility. Moreover, the addition of CaSO4 could significantly improve the migration and osteogenic differentiation of MC3T3-E1 cells in the PLGA/CaSO4 scaffolds, and the PLGA/CaSO4 scaffolds made with 20 wt.% CaSO4 exhibited the best osteogenesis properties. Therefore, calcium sulfate was added to PLGA could lead to customized 3D printed scaffolds for enhanced mechanical properties and biological properties. The customized 3D-printed PLGA/CaSO4 scaffold shows great potential for precisely repairing irregular load-bearing bone defects.

Biocompatible tellurium nanoneedles with long-term stable antibacterial activity for accelerated wound healing.

Tellurium (Te) nanomaterials (NMs) have emerged as a new antibacterial candidate to respond to the complex global health challenge of bacterial resistance. Herein, Te nanoneedles (NNs) that act both chemically and physically on bacteria are synthesized by a facile method using Na2TeO3, urea and glucose. It is found that the prepared Te NNs have a strong affinity to the cell membrane of bacteria and subsequently promote the generation of reactive oxygen species (ROS) in bacteria, resulting in an excellent antibacterial effect toward Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). What's more, this needle-like morphology also can physically damage the bacterial cell membranes. The Te NNs per se are inert in mammalian cells to produce ROS at a proper concentration, indicating considerable biocompatibility of this material. As a proof-of-concept, the antibacterial Te NNs were used as an anti-inflammatory reagent for promoting bacteria-infected wound healing in vivo, during which Te NNs caused no evident side effects to major organs in mice. Additionally, the antibacterial activity is maintained in the presence of surface oxidation of Te NNs after long-term dispersion in phosphate buffered saline solution. The needle-like Te NMs with long-term antibacterial stability and good biocompatibility have great potential for the treatment of associated infectious diseases.

EppR, a new LysR-family transcription regulator, positively influences phenazine biosynthesis in the plant growth-promoting rhizobacterium Pseudomonas chlororaphis G05.

Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and ß-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT-PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.

LysR-type transcriptional regulator FinR is required for phenazine and pyrrolnitrin biosynthesis in biocontrol Pseudomonas chlororaphis strain G05.

Phenazine-1-carboxylic acid and pyrrolnitrin, the two secondary metabolites produced by Pseudomonas chlororaphis G05, serve as biocontrol agents that mainly contribute to the growth repression of several fungal phytopathogens. Although some regulators of phenazine-1-carboxylic acid biosynthesis have been identified, the regulatory pathway involving phenazine-1-carboxylic acid synthesis is not fully understood. We isolated a white conjugant G05W03 on X-Gal-containing LB agar during our screening of novel regulator candidates using transposon mutagenesis with a fusion mutant G05Δphz::lacZ as a recipient. By cloning of DNA adjacent to the site of the transposon insertion, we revealed that a LysR-type transcriptional regulator (LTTR) gene, finR, was disrupted in the conjugant G05W03. To confirm the regulatory function of FinR, we constructed the finR-knockout mutant G05ΔfinR, G05Δphz::lacZΔfinR, and G05Δprn::lacZΔfinR, using the wild-type strain G05 and its fusion mutant derivatives as recipient strains, respectively. We found that the expressions of phz and prn operons were dramatically reduced in the finR-deleted mutant. With quantification of the production of antifungal metabolites biosynthesized by the finR-negative strain G05ΔfinR, it was shown that FinR deficiency also led to decreased yield of phenazine-1-carboxylic acid and pyrrolnitrin. In addition, the pathogen inhibition assay confirmed that the production of phenazine-1-carboxylic acid was severely reduced in the absence of FinR. Transcriptional fusions and qRT-PCR verified that FinR could positively govern the transcription of the phz and prn operons. Taken together, FinR is required for antifungal metabolite biosynthesis and crop protection against some fungal pathogens.Key points• A novel regulator FinR was identified by transposon mutagenesis.• FinR regulates antifungal metabolite production.• FinR regulates the phz and prn expression by binding to their promoter regions.

Rates of Myopia Development in Young Chinese Schoolchildren During the Outbreak of COVID-19.

Importance: During the outbreak of COVID-19, outdoor activities were limited and digital learning increased. Concerns have arisen regarding the impact of these environmental changes on the development of myopia. Objective: To investigate changes in the development of myopia in young Chinese schoolchildren during the outbreak of COVID-19. Design, Setting, and Participants: In this observational study, 2 groups of students from 12 primary schools in Guangzhou, China, were prospectively enrolled and monitored from grade 2 to grade 3. Comparisons between the exposure and nonexposure groups were made to evaluate any association between environmental changes during the COVID-19 outbreak period and development of myopia. The exposure group received complete eye examinations in November and December 2019 and November and December 2020. The nonexposure group received examinations in November and December 2018 and November and December 2019. Main Outcomes and Measures: Changes in cycloplegic spherical equivalent refraction (SER), axial length (AL) elongation, and myopia incidence from grade 2 to grade 3. Results: Among the 2679 eligible students in grade 2 (mean [SD] age, 7.76 [0.32] years; 1422 [53.1%] male), 2114 (1060 in the nonexposure group and 1054 in the exposure group) were reexamined in grade 3. Compared with the period from November and December 2018 to November and December 2019, the shift of SER, AL elongation, and myopia incidence from grade 2 to grade 3 from November and December 2019 to November and December 2020 was 0.36 D greater (95% CI, 0.32-0.41; P < .001), 0.08 mm faster (95% CI, 0.06-0.10; P < .001), and 7.9% higher (95% CI, 5.1%-10.6%; P < .001), respectively. In grade 3 students, the prevalence of myopia increased from 13.3% (141 of 1060 students) in November and December 2019 to 20.8% (219 of 1054 students) in November and December 2020 (difference [95% CI], 7.5% [4.3-10.7]; P < .001); the proportion of children without myopia and with SER greater than -0.50 D and less than or equal to +0.50 D increased from 31.1% (286 of 919 students) to 49.0% (409 of 835 students) (difference [95% CI], 17.9% [13.3-22.4]; P < .001). Conclusions and Relevance: In this study, development of myopia increased during the COVID-19 outbreak period in young schoolchildren in China. Consequently, myopia prevalence and the proportion of children without myopia who were at risk of developing myopia increased. Future studies are needed to investigate long-term changes in myopia development after the COVID-19 pandemic.

Association between the posterior ocular contour pattern and progression of myopia in children: A prospective study based on OCT imaging.

PURPOSE: This study aims to reveal the relationship between the posterior ocular contour and the subsequent progression of myopia in children. METHODS: Children aged 8-12 years with myopia received baseline measurements and were instructed to wear their glasses every day and return for a follow-up visit after one year. Axial length and other ocular parameters were measured using a noncontact biometer. The contour of the posterior eye was calculated and analysed based on images from spectral domain optical coherence tomography (SD-OCT). Univariate and multivariate linear regression models were created to analyse the relationship between the contour of the posterior eye and the progression of myopia. RESULTS: Baseline posterior ocular contour measurements correlated with baseline axial length and spherical equivalent refraction (SER) (all p < 0.05). Eyes that were more myopic tended to have a more prolate posterior ocular contour. Although the baseline contour of the retinal pigment epithelium (RPE) and chorioscleral interface (CSI) showed no significant relationship with the progression of myopia (all p > 0.05), interestingly, when the baseline contour of the RPE was more prolate than that of the CSI, the axial length increased during the following year (R2  = 0.62; p < 0.01). The multivariate model, when adjusted for other variables, further validated the independent role of this variable. CONCLUSIONS: The difference between the RPE and CSI contours correlated with the subsequent progression of myopia in children. This finding can help inform clinicians regarding the management of children at the onset of myopia and potentially provide an avenue for experimental research on the mechanism of myopia development.

Liquid-Exfoliated Mesostructured Collagen from the Bovine Achilles Tendon as Building Blocks of Collagen Membranes.

Mesoscaled assemblies are organized in native collagen tissues to achieve remarkable and diverse performance and functions. In this work, a facile, low-cost, and controllable liquid exfoliation method was applied to directly extract these collagen mesostructures from bovine Achilles tendons using a sodium hydroxide (NaOH)/urea aqueous system with freeze-thaw cycles and sonication. A series of collagen fibrils with diameters of 26-230 nm were harvested using this process, and in situ observations under polarizing microscopy (POM) and using molecular dynamics simulations revealed the influence of the NaOH/urea system on the tendon collagen. FTIR and XRD results confirmed that these collagen fibrils preserved typical structural characteristics of type I collagen. These isolated collagen fibrils were then utilized as building blocks to fabricate free-standing collagen membranes, which exhibited good stability in solvents and outstanding mechanical properties and transparency, with potential for utility in optical and electronic sensors. Moreover, in vitro and vivo evaluations demonstrated that these new resulting collagen membranes had good cytocompatibility, biocompatibility, and degradability for potential applications in biomedicine. This work provides a new approach for collagen processing by liquid exfoliation with utility for the formation of robust collagen materials that consist of native collagen mesostructures as building blocks.

Development and validation of a prediction model for axial length elongation in myopic children treated with overnight orthokeratology.

PURPOSE: To develop and validate a standardized prediction model aiming at 1-year axial length elongation and to guide the orthokeratology lens practice. METHODS: This retrospective study was based on medical records of myopic children treated with orthokeratology. Individuals aged 8-15 years (n = 1261) were included and divided into the primary cohort (n = 757) and validation cohort (n = 504). Feature selection was primarily performed to sort out influential predictors by high-throughput extraction. Then, the prediction model was developed using multivariable linear regression analysis completed by backward stepwise selection. Finally, the validation of the prediction model was performed by evaluation metrics (mean-square error, root-mean-square error, mean absolute error and R ad 2 ). RESULTS: No significant difference was found between primary and validation cohort (all p > 0.05). After the feature selection, the crude model was adjusted by demographic information in multivariable linear regression analysis, and five final predictors were identified (all p < 0.01). The interaction effect of age with 1-month change of zone-3 mm flat K was detected (p < 0.01); hence, two final prediction models were developed based on two age subgroups. The validation proved an acceptable performance. CONCLUSION: An effective multivariable prediction model aiming at 1-year axial length elongation was developed and validated. It can potentially help clinicians to predict orthokeratology efficacy and make valid adjustments. The influential variables revealed in this model can also provide designers directions to optimize the design of lens to improve the efficacy of myopia control.

Black Soldier Fly (Hermetia illucens) Larvae Significantly Change the Microbial Community in Chicken Manure.

Using black soldier fly (Hermetia illucens) larvae in treatment of livestock manure is a promising technology. In this study, high-throughput sequencing was used to analyze the microbial community in chicken manure before and after treatment with H. illucens larvae. In fresh chicken manure, the most abundant bacterial phylum was Firmicutes (55.58%) followed by Bacteroidetes (24.52%) and then Proteobacteria (12.29%). After treatment of the manure with H. illucens larvae for 15 days, the abundance of Firmicutes increased to 97.72% while that of Bacteroidetes and Proteobacteria decreased. Concomitantly, the most abundant genera of fungi in chicken manure changed from Kernia (46.19%) and Microascus (17.22%) to Penicillium (46.82%) and Aspergillus (45.22%). Correlation-network analysis showed the existence of strong and complex correlations between the dominant operational taxonomic units (OUT) of bacteria and fungi. While most of these correlations were positive, three specific genera, namely g_norank_f_Bacillaceae, Penicillium, and Aspergillus exhibited negative correlations with the remaining genera. These three genera were highly abundant in the intestines of H. illucens and in chicken manure treated with H. illucens larvae. Based on 16S rDNA microbiome-function predictions, the metabolic pathways associated with sugars, amino acids, and organic pollutants inside the intestinal tract of H. illucens were enriched versus those of the other three groups. In summary, the treatment of chicken manure with H. illucens larvae significantly reduced the microbial diversity, while strongly increasing organic metabolism in the intestinal bacteria. This technology shows the potential for applications in livestock manure treatment.

Pip serves as an intermediate in RpoS-modulated phz2 expression and pyocyanin production in Pseudomonas aeruginosa.

Pyocyanin, a main virulence factor that is produced by Pseudomonas aeruginosa, plays an important role in pathogen-host interaction during infection. Two copies of phenazine-biosynthetic operons on genome, phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2), contribute to phenazine biosynthesis. In our previous study, we found that RpoS positively regulates expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa PAO1. In this work, when a TetR-family regulator gene, pip, was knocked out, we found that pyocyanin production was dramatically reduced, indicating that Pip positively regulates pyocyanin biosynthesis. With further phenazines quantification and ß-galactosidase assay, we confirmed that Pip positively regulates phz2 expression, but does not regulate phz1 expression. In addition, while the rpoS gene was deleted, expression of pip was down-regulated. Expression of rpoS in the wild-type PAO1 strain, however, was similar to that in the Pip-deficient mutant PAΔpip, suggesting that expression of pip could positively be regulated by RpoS, whereas rpoS could not be regulated by Pip. Taken together, we drew a conclusion that Pip might serve as an intermediate in RpoS-modulated expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa.

Pathway construction and metabolic engineering for fermentative production of ß-alanine in Escherichia coli.

ß-Alanine is a naturally occurring ß-amino acid that has been widely applied in the life and health field. Although microbial fermentation is a promising method for industrial production of ß-alanine, an efficient microbial cell factory is still lacking. In this study, a new metabolically engineered Escherichia coli strain for ß-alanine production was developed through a series of introduction, deletion, and overexpression of genes involved in its biosynthesis pathway. First, the L-aspartate a-decarboxylase gene, BtADC, from Bacillus tequilensis, with higher catalytic activity to produce ß-alanine from aspartate, was constitutively expressed in E. coli, leading to an increased production of ß-alanine up to 2.76 g/L. Second, three native aspartate kinase genes, akI, akII, and akIII, were knocked out to promote the production of ß-alanine to a higher concentration of 4.43 g/L by preventing from bypass loss of aspartate. To increase the amount of aspartate, the native AspC gene was replaced with PaeAspDH, a L-aspartate dehydrogenase gene from Pseudomonas aeruginosa, accompanied with the overexpression of the native AspA gene, to further improve the production level of ß-alanine to 9.27 g/L. Last, increased biosynthesis of oxaloacetic acid (OAA) was achieved by a combination of overexpression of the native PPC, introduction of CgPC, a pyruvate decarboxylase from Corynebacterium glutamicum, and deletion of ldhA, pflB, pta, and adhE in E. coli, to further enhance the production of ß-alanine. Finally, the engineered E. coli strain produced 43.12 g/L ß-alanine in fed-batch fermentation. Our study will lay a solid foundation for the promising application of ß-alanine in the life and health field. KEY POINTS: • Overexpression of BtADC resulted in substantial accumulation of ß-alanine. • The native AspC was replaced with PaeAspDH to catalyze the transamination of OAA. • Deletion of gluDH prevented from losing carbon flux in TCA recycle. • A 43.12-g/L ß-alanine production in fed-batch fermentation was achieved. Graphical abstract.

LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.

Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA1 B1 C1 D1 E1 F1 G1 ) and phz2 (phzA2 B2 C2 D2 E2 F2 G2 ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria. In an other previous study, it was reported that RpoS deficiency caused overproduction of pyocyanin, a derivative of phenazine-1-carboxylic acid in P. aeruginosa PAO1. But it is not known how RpoS mediates the expression of each of two phz operons and modulates phenazine-1-carboxylic acid biosynthesis in detail. In this study, by deleting the rpoS gene in the mutant PNΔphz1 and the mutant PNΔphz2, we found that the phz1 operon contributes much more to phenazine-1-carboxylic acid biosynthesis than the phz2 operon in the absence of RpoS. With the construction of the translational and transcriptional fusion vectors with the truncated lacZ reporter gene, we demonstrated that RpoS negatively regulates the expression of phz1 and positively controls the expression of phz2, and the regulation of phenazine-1-carboxylic acid biosynthesis mediated by RopS occurs at the posttranscriptional level, not at the transcriptional level. Obviously, two copies of phz operons and their differential expression mediated by RpoS might help P. aeruginosa adapt to its diverse environments and establish infection in its hosts.