Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions

<p>Abstract</p> <p>Background</p> <p>The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo).</p> <p>Results</p> <p>Here a scarless gene modification strategy based on the Red recombination system has been developed to modify <it>Pseudomonas </it>genome DNA via sequential deletion of multiple targets. This process was mediated by plasmid pRKaraRed encoding the Red proteins regulated by <it>P</it>_{<it>BAD </it>}promoter, which was functional in <it>P. aeruginosa </it>as well as in other bacteria. First the target gene was substituted for the <it>sacB</it>-<it>bla </it>cassette flanked by short homology regions (50 bp), and then this marker gene cassette could be replaced by the PCR fragment flanking itself, generating target-deleted genome without any remnants and no change happened to the surrounding region. Twenty genes involved in the synthesis and regulation pathways of the phenazine derivate, pyocyanin, were modified, including one single-point mutation and deletion of two large operons. The recombination efficiencies ranged from 88% to 98%. Multiple-gene modification was also achieved, generating a triple-gene deletion strain PCA (PAO1, <it>ΔphzHΔphzMΔphzS</it>), which could produce another phenazine derivate, phenazine-1-carboxylic acid (PCA), efficiently and exclusively.</p> <p>Conclusions</p> <p>This lambda Red-based technique can be used to generate scarless and sequential gene modification mutants of <it>P. aeruginosa </it>efficiently, using one-step PCR product flanked by short homology regions. Single-point mutation, scarless deletion of genes can be achieved easily in less than three days. This method may give a new way to construct genetically modified <it>P. aeruginosa </it>strains more efficiently and advance the regulatory network study of this organism.</p

Testing a linear ARMA Model against threshold-ARMA models : a Bayesian approach

We introduce a Bayesian approach to test linear autoregressive moving-average (ARMA) models against threshold autoregressive moving-average (TARMA) models. Firstly, the marginal posterior densities of all parameters, including the threshold and delay, of a TARMA model are obtained by using Gibbs sampler with Metropolis-Hastings algorithm. Secondly, reversible-jump Markov chain Monte Carlo (RJMCMC) method is adopted to calculate the posterior probabilities for ARMA and TARMA models: Posterior evidence in favor of TARMA models indicates threshold nonlinearity. Finally, based on RJMCMC scheme and Akaike information criterion (AIC) or Bayesian information criterion (BIC), the procedure for modeling TARMA models is exploited. Simulation experiments and a real data example show that our method works well for distinguishing a ARMA from a TARMA model and for building TARMA models

Automated Mapping of Ms 7.0 Jiuzhaigou Earthquake (China) Post-Disaster Landslides Based on High-Resolution UAV Imagery

The Ms 7.0 Jiuzhaigou earthquake that occurred on 8 August 2017 triggered hundreds of landslides in the Jiuzhaigou valley scenic and historic-interest area in Sichuan, China, causing heavy casualties and serious property losses. Quick and accurate mapping of post-disaster landslide distribution is of paramount importance for earthquake emergency rescue and the analysis of post-seismic landslides distribution characteristics. The automatic identification of landslides is mostly based on medium- and low-resolution satellite-borne optical remote-sensing imageries, and the high-accuracy interpretation of earthquake-triggered landslides still relies on time-consuming manual interpretation. This paper describes a methodology based on the use of 1 m high-resolution unmanned aerial vehicle (UAV) imagery acquired after the earthquake, and proposes a support vector machine (SVM) classification method combining the roads and villages mask from pre-seismic remote sensing imagery to accurately and automatically map the landslide inventory. Compared with the results of manual visual interpretation, the automatic recognition accuracy could reach 99.89%, and the Kappa coefficient was higher than 0.9, suggesting that the proposed method and 1 m high-resolution UAV imagery greatly improved the mapping accuracy of the landslide area. We also analyzed the spatial-distribution characteristics of earthquake-triggered landslides with the influenced factors of altitude, slope gradient, slope aspect, and the nearest faults, which provided important support for the further study of post-disaster landslide distribution characteristics, susceptibility prediction, and risk assessment.This work was funded by the National Key Research and Development Program of China (Project No. 2018YFC1505202), the National Natural Science Foundation of China (41941019), the State Key Laboratory of Geohazard Prevention and Geoenvironment Protection Independent Research Project (SKLGP2020Z012), the project on identification and monitoring of potential geological hazards with remote sensing in Sichuan Province (510201202076888) and the Everest Scientific Project at Chengdu University of Technology (2020ZF114103)

CrgA Protein Represses AlkB2 Monooxygenase and Regulates the Degradation of Medium-to-Long-Chain n-Alkanes in Pseudomonas aeruginosa SJTD-1

AlkB monooxygenases in bacteria are responsible for the hydroxylation of medium- and long-chain n-alkanes. In this study, one CrgA protein of Pseudomonas aeruginosa SJTD-1, a member of LysR family, was proved to regulate AlkB2 monooxygenase and the degradation of medium-to-long-chain n-alkanes (C14–C20) by directly binding to the upstream of alkB2 gene. Two specific sites for CrgA binding were found in the promoter region of alkB2 gene, and the imperfect mirror repeat (IIR) structure was proved critical for CrgA recognition and binding. Hexadecyl CoA and octadecyl CoA could effectively release the CrgA binding and start the transcription of alkB2 gene, implying a positive regulation of metabolic intermediate. In the presence of medium-to-long-chain n-alkanes (C14–C20), deletion of crgA gene could enhance the transcription and expression of AlkB2 monooxygenase significantly; and in n-octadecane culture, strain S1ΔalkB1&amp;crgA grew more vigorously than strain S1ΔalkB1&amp;crgA. Almost no regulation of CrgA protein was observed to alkB1 gene in vitro and in vivo. Therefore, CrgA acted as a negative regulator for the medium-to-long-chain n-alkane utilization in P. aeruginosa SJTD-1. The work will promote the regulation mechanism study of n-alkane degradation in bacteria and help the bioremediation method development for petroleum pollution

An Efficient Electro-Competent Cells Generation Method of Xanthomonas campestris pv. campestris: Its Application for Plasmid Transformation and Gene Replacement

Abstract A simple and rapid method to prepare efficient electro-competent cells of Xanthomonas campestris pv. campestris was generated, with up to 100-fold transformation efficiencies over the existing procedures. The overnight cultures were treated with sucrose solution and micro-centrifuged at room temperature; the entire electro-competent cells generation process can be completed in 15 minutes. It overcomes the complication and time-consuming shortcomings of the traditional conjugation or electro-transformation methods in this strain. Both the replicative plasmids and non-replicative plasmids could be transformed or integrated efficiently using this method. And the DNA concentration, cells growth stage, field strength and recovery time all had influences on the transformation efficiency. In the optimal conditions, the transformation efficiency for the replicative plasmids was 10 9 transformants per microgram DNA, and for non-replicative plasmids was 150 transformants per microgram DNA. Further with the homology sequences, two chromosomal target genes were deleted efficiently and the knockout strains were obtained easily

Construction of a Bacterial Assay for Estrogen Detection Based on an Estrogen-Sensitive Intein ▿ †

Escherichia coli strain DIER was constructed for estrogen detection by inserting an estrogen-sensitive intein (VMAER intein) into the specific site of the constitutively expressed chromosomal lacZ gene. This VMAER intein was generated by replacing the endonuclease region of the Saccharomyces cerevisiae VMA intein with the estrogen binding region of the human estrogen receptor α (hERα). When there were estrogens or analogs, the splicing of the VMAER intein was induced to produce the mature LacZ protein, which was detected through a β-galactosidase colorimetric assay. Eight typical chemicals (17-β-estradiol, bisphenol A, chrysene, 6-OH-chrysene, benz[a]anthracene, pyrene, progesterone, and testosterone) were detected using this DIER strain, and the whole detection procedure was accomplished in 2 h. Their 50% effective concentrations (EC50), relative estrogenic activities, and estradiol equivalency factors were calculated and were quite consistent with those detected with the yeast estrogen screening (YES) system. Furthermore, the estrogenic activities of the synthetic musk samples extracted from the wastewater and waste sludge of a sewage treatment plant of Shanghai (China) were detected, and their results were comparable to those obtained from the YES system and gas chromatography-mass spectrometry (GC-MS). In conclusion, the DIER bioassay could fill a niche for the efficient, rapid, high-throughput screening of estrogenic compounds and has potential for the remote, near-real-time monitoring of environmental estrogens

Bayesian analysis of two-regime threshold autoregressive moving average model with exogenous inputs

We consider Bayesian analysis of threshold autoregressive moving average model with exogenous inputs (TARMAX). In order to obtain the desired marginal posterior distributions of all parameters including the threshold value of the two-regime TARMAX model, we use two different Markov chain Monte Carlo (MCMC) methods to apply Gibbs sampler with Metropolis-Hastings algorithm. The first one is used to obtain iterative least squares estimates of the parameters. The second one includes two MCMC stages for estimate the desired marginal posterior distributions and the parameters. Simulation experiments and a real data example show support to our approaches

Experimental study on the control form of fin stabilizer at zero speed.

The zero-speed fin stabilizer is used to stabilize the roll motion of the ship at full speed. It has two working modes, that is the lift-based normal anti-rolling mode and the drag-based zero-speed anti-rolling mode. Different force generation mechanisms cause different control methods, especially in the form of control. The zero-speed fin stabilizer works in a sinusoidal fashion in normal mode, the same as the conventional fin stabilizer. However, its control form at zero speed is not particularly clear. This paper aims to investigate the control form of fin stabilizer at zero speed through a composite method of theoretical analysis and experimental research. Based on the established reaction force model, the forces generated on the fin flapped in sinusoidal and trapezoidal forms are compared and analyzed. It is found that the trapezoidal form flapping with a small half-cycle ratio generates larger force than the sinusoidal flapping form when the flapping amplitude is the same. The forced rolling tank tests with the fins flapping in sinusoidal and trapezoidal forms were conducted. The test results are consistent with the theoretical analysis results, and the trapezoidal flapping form with a proper small half-cycle ratio is recommended for fin stabilizers at zero speed. The control strategy of fin stabilizer at zero speed is obtained based on the further analysis of the force characteristics of the trapezoidal flapping form and the limitations of the actual fin stabilizer actuation system. The model and full scale roll reduction tests at zero speed were conducted, achieving more than 70% and 60% of the anti-rolling effect respectively. The test results further verify the effectiveness, applicability and practicability of the obtained control form and strategy for fin stabilizers in at-anchor conditions, and can be a reference for engineering practice and other similar studies

Characterization of the Tellurite-Resistance Properties and Identification of the Core Function Genes for Tellurite Resistance in <i>Pseudomonas citronellolis</i> SJTE-3

Tellurite is highly toxic to bacteria and commonly used in the clinical screening for pathogens; it is speculated that there is a potential relationship between tellurite resistance and bacterial pathogenicity. Until now, the core function genes of tellurite resistance and their characteristics are still obscure. Pseudomonas citronellolis SJTE-3 was found able to resist high concentrations of tellurite (250 μg/mL) and formed vacuole-like tellurium nanostructures. The terZABCDE gene cluster located in the large plasmid pRBL16 endowed strain SJTE-3 with the tellurite resistance of high levels. Although the terC and terD genes were identified as the core function genes for tellurite reduction and resistance, the inhibition of cell growth was observed when they were used solely. Interestingly, co-expression of the terA gene or terZ gene could relieve the burden caused by the expression of the terCD genes and recover normal cell growth. TerC and TerD proteins commonly shared the conserved sequences and are widely distributed in many pathogenic bacteria, highly associated with the pathogenicity factors

Study on Transmission Characteristics and Bandgap Types of Plasma Photonic Crystal

A plasma photonic crystal (PPC) was formed using an array of discharge plasma tubes. The transmission spectra and bandstructure of PPCs with different lattice types under different polarization modes were studied through simulation and measurement. To study the types of bandgap in PPCs, the bandstructure of the PPC is calculated using symplectic finite difference time domain (SFDTD), a modified plane wave expansion (PWE) method, and a finite element method (FEM) based on weak form equations. The bandstructure of the PPC is compared with the transmission curve results. The results show that the bandgap is stable in the PPC, and the experimental and numerical results of the transmission spectra agree well. There are different types of bandgap in the PPC; the bandgap under TE-like polarization is caused by localized surface plasmon (LSP) and Bragg scattering. The bandgap under TM-like polarization is caused by the cutoff effect of plasma on the electromagnetic wave and Bragg scattering. The lattice type also affects the position and number of the bandgap. The three methods have their advantages and disadvantages when calculating bandstructure. Therefore, it is necessary to combine the results of three methods and experimental results to accurately determine the bandgap type of the PPC