Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.

Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis.

Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.

Regulatory effects of miR-138 and RUNX3 on Th1/Th2 balance in peripheral blood of children with cough variant asthma. / 咳嗽变异性哮喘患儿外周血miR-138及RUNX3对Th1/Th2平衡的调节作用.

OBJECTIVES: To study the expression levels of microRNA-138 (miR-138) and Runt-related transcription factor 3 (RUNX3) in peripheral blood of children with cough variant asthma (CVA) and their regulatory effects on Th1/Th2 balance. METHODS: Sixty-five children with CVA (CVA group) and 30 healthy children (control group) were enrolled. Peripheral venous blood samples were collected for both groups, and CD4+ T cells were isolated and cultured. Enzyme-linked immunosorbent assay was used to measure the levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-5, and IL-13 that were secreted by CD4+ T cells. Flow cytometry was used to determine the percentages of Th1 and Th2 cells. Quantitative real-time PCR was used to measure the level of RUNX3 mRNA in CD4+ T cells and the level of miR-138 in peripheral blood. Western blot was used to determine the protein expression of RUNX3 in CD4+ T cells. The dual-luciferase reporter assay was used to determine the targeting effects of miR-138 and RUNX3. The RUNX3-mimic plasmid was transfected into CD4+ T cells, and the effects on the levels of IFN-γ, IL-2, IL-4, IL-5, and IL-13 and the percentages of Th1 and Th2 cells were measured. RESULTS: Compared with the control group, the CVA group showed significantly decreased levels of IFN-γ and IL-2 from CD4+ T cells, significantly increased levels of IL-4, IL-5, and IL-13 from CD4+ T cells, significantly decreased Th1 cell percentage and Th1/Th2 ratio, and a significantly increased Th2 cell percentage (P<0.05). The CVA group showed significantly lower relative expression levels of RUNX3 mRNA and protein in CD4+ T cells in peripheral blood than the control group (P<0.001). The relative expression level of miR-138 was significantly higher in the CVA group than in the control group (P<0.001). MiR-138 could target the expression of RUNX3. Upregulating the expression of RUNX3 in CD4+ T cells induced significantly increased levels of IFN-γ and IL-2, significantly decreased levels of IL-4, IL-5, and IL-13, significantly increased Th1 cell percentage and Th1/Th2 ratio, and a significantly decreased Th2 cell percentage (P<0.05). CONCLUSIONS: MiR-138 regulates Th1/Th2 balance by targeting RUNX3 in children with CVA, providing a new direction for the treatment of CVA.

The escape of CRISPR-mediated gene editing in Zymomonas mobilis.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knockout by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene-editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene may be attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence, several strategies are proposed to enhance gene knockout and plasmid curing.

Annual hypoxia causing long-term seawater acidification: Evidence from low-molecular-weight organic acids in the Changjiang Estuary and its adjacent sea area.

In this study, components, concentrations, distribution characteristics, sources of low-molecular-weight organic acids (LMWOAs) and relationships among the annual hypoxia, LMWOAs and seawater acidification were investigated in the Changjiang Estuary and its adjacent sea area in July 2015. Lactic, acetic and formic acids were detected in the seawater samples in the study area, and their total concentrations (ΣLMWOAs) varied from 0 to 262.6 µmol·L-1, with an average value of 39.2 µmol·L-1. In the surface seawater, high concentration areas of ΣLMWOAs occurred in the sea area near the Changjiang Estuary and the Hangzhou Bay, and north of study area. In the sampling stations along transect A6, high concentration areas of ΣLMWOAs appeared in the bottom seawater of nearshore stations and middle seawater of offshore stations. The terrigenous inputs, especially the Changjiang runoff, were the dominant sources for LMWOAs in the sampling period. The consistency of hypoxia areas, high concentration areas of ΣLMWOAs and low pH value areas in winter and summer suggested that annual hypoxia could cause the long-term seawater acidification by producing LMWOAs in the Changjiang Estuary and its adjacent sea area.

Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition.

Zymomonas mobilis, a promising candidate for industrial biofuel production, is capable of nitrogen fixation naturally without hindering ethanol production. However, little is known about the regulation of nitrogen fixation in Z. mobilis. We herein conducted a high throughput analysis of proteome and protein acetylation in Z. mobilis under N2-fixing conditions and established its first acetylome. The upregulated proteins mainly belong to processes of nitrogen fixation, motility, chemotaxis, flagellar assembly, energy production, transportation, and oxidation-reduction. Whereas, downregulated proteins are mainly related to energy-consuming and biosynthetic processes. Our acetylome analyses revealed 197 uniquely acetylated proteins, belonging to major pathways such as nitrogen fixation, central carbon metabolism, ammonia assimilation pathway, protein biosynthesis, and amino acid metabolism. Further, we observed acetylation in glycolytic enzymes of central carbon metabolism, the nitrogenase complex, the master regulator NifA, and the enzyme in GS/GOGAT cycle. These findings suggest that protein acetylation may play an important role in regulating various aspects of N2-metabolism in Z. mobilis. This study provides new knowledge of specific proteins and their associated cellular processes and pathways that may be regulated by protein acetylation in Z. mobilis.

Responses of the marine carbonate system to a green tide: A case study of an Ulva prolifera bloom in Qingdao coastal waters.

As an environmental nuisance, Ulva prolifera green tides have occurred annually in the southern Yellow Sea since 2007. While it is expected that high levels of biological activity during these blooms can alter seawater carbonate chemistry, there has been little research on the responses of marine carbonate system to green tides. Here, the effects of the bloom on the carbonate system were examined on three cruises in June, July, and September, corresponding to the early-, late-, and after-bloom periods of the U. prolifera bloom in Qingdao coastal waters in 2018. Among these three stages, the pH (National Bureau of Standards scale), dissolved inorganic carbon (DIC), total alkalinity (TA), and partial pressure of CO2 (pCO2) were all affected by bloom, with the highest pH and lowest DIC and TA concentrations of the surface seawater occurring at the late-bloom stage. While pCO2 continuously increased from the beginning to the end of the bloom. TA increased by ∼40 µmol kg-1 between the early- and after-bloom periods likely due to the shifts in the carbonate system equilibrium caused by increased CO32- concentrations and the organic matter released by U. prolifera during decomposition. Compared to nearby areas with no U. prolifera bloom, the green tide, along with increasing temperature, reduced the pH and DIC but increased the TA and pCO2. This large-scale bloom also turned the coastal waters from being an atmospheric CO2 sink to a strong source, with the estimation of air-sea CO2 fluxes about 1.69 ± 1.70, 2.28 ± 1.16, and 7.44 ± 5.84 mmol m-2 d-1 during the early-, late-, and after-bloom periods, respectively. This bloom event also promoted the formation of CaCO3 and was an important source of low molecular weight organic acids. These new findings provide nuances for the current conversations on the role of biological processes in modulating marine carbonate system and the contribution of organic matter to alkalinity.

[Clinical study on the antioxidant effect of methylene blue in vivo].

OBJECTIVE: To investigate the relationship of oxygen free radical (OFR) with per-oxidative injury of erythrocyte induced by intravenous procaine in vivo and the effect of methylene blue (MB) in removal of nitric oxide (NO) and peroxynitrite (ONOO(-)). METHODS: Forty patients undergoing elective surgery were divided randomly into intravenous procaine anesthesia (IPA) group and fentanyl group. Blood sample was taken before anesthesia (T0), 120 minutes (T1) and 180 minutes (T2) after IPA and 30 minutes after treatment with MB (1-2 mg/kg, T3) to determine the changes in the levels of NO, OFR, lipid peroxide (LPO), superoxide dismutase (SOD), catalase (CAT), NADH-Cyt b5-reductase (Cyt b5-R) and methemoglobin (MHb). RESULTS: Compared with T0, the levels of NO, OFR, LPO, MHb in IPA group were significantly increased at T1,T2. At same time SOD, CAT and Cyt b5-R were significantly decreased. NO, OFR, MHb, SOD, CAT and Cyt b5-R were all reduced to the normal levels at T3. No changes in any determined parameters in fentanyl group during anesthesia. CONCLUSION: It is indicated that the metabolites of procaine consist of a large quantity of NO:ONOO(-), producing per-oxidative injury to erythrocyte. MB is effective in eliminating OFR in vivo, protecting tissue cells. It may act as an antioxidant drug in the treatment of critical illness.