Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.     

Cloning and in vitro and in vivo expression of plant glutathione S-transferase zeta class genes

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) (GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.     

Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides

N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the alpha1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnT-I activity was 40 degrees C, but the enzyme was active between 0-70 degrees C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent K(m) value of 0.483 mM and a V(max) of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.     

Host-dependent activation of IS1203v excision in Shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.     

Cloning of a thermostable xylanase from Actinomadura sp. S14 and its expression in Escherichia coli and Pichia pastoris

A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-β-xylanase, β-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-β-xylanase activity on xylan and xylooligosaccharides than on xylotriose.     

大肠杆菌生物膜关键组分缺失对细胞性能的影响

大肠杆菌胞外存在复杂且种类繁多的生物膜组分,这些生物膜组分不仅在合成和组装过程中耗费大量的能源和底物,且某些组分还存在巨大的致病风险。为减少这些不利影响,作者采用Crisp-Cas9基因编辑技术,从基因水平分别去除大肠杆菌MG1655中非必需生物膜组分,构建了一系列非必需生物膜组分缺失突变菌株。对突变株的细胞特性进行考察,筛选具有优异特性的突变菌株。结果显示,敲除鞭毛fliE-R、fliY-T、flhE-D 、4型荚膜、唾液酸和聚- β-1,6-葡萄糖胺基因簇能促进菌株在M9培养基中的生长;敲除鞭毛基因簇flgN-L和胞外多糖类组分有利于PHB合成;敲除胞外多糖类组分基因簇或鞭毛的4个基因簇,能增强菌株膜通透性;去除大肠杆菌核心多糖能重塑代谢调控,促进克拉酸的生产。     

In vitro Survival and Growth of Salmonella Typhimurium inoculated on Yolk Membrane after long Term refrigerated Storage of Shell Eggs

Salmonella enterica serovar Typhimurium has been isolated from commercial egg production facilities in the United States. Given its importance as a causative organism for food-borne salmonellosis, identifying approximate timelines for bacterial invasion of the egg is needed. The objective of this study was to examine net growth of S. Typhimurium in egg components over time. In trial 1 eggs were collected over a 24 hour period from a flock of single comb white leghorn hens while in trial 2 eggs were picked up from a commercial laying source once a week over the course of eight weeks and stored. Eggs were held at refrigeration temperature and each week, subsets of eggs were cracked, separated into yolk and albumen components, and inoculated with 10⁸ CFU/ml of novobiocin and nalidixic acid (NO/NA) resistant S. Typhimurium onto the vitelline membrane of the egg. Yolks were then covered with albumen. Eggs were incubated for twenty-four hours at 25°C. After incubation eggs were again separated into albumen, yolk, and vitelline membrane samples. In trial 1, S. Typhimurium net growth occurred in albumen by the second week and continued from 4 to 8 weeks while in trial 2 net growth only occurred at week 5 and 7. S. Typhimurium net growth on vitelline membranes occurred by 2 weeks and continued from 4 to 8 weeks in trial 1 while no net growth occurred in trial 2 over the 8 week period. Yolk samples showed no net increases in S. Typhimurium populations over the 8 week period. An erratum to this article is available at .     

A novel apparatus applying long term intermittent cyclic hydrostatic pressure to in vitro cell cultures

A novel apparatus has been designed that allows for application of long term intermittent cyclic hydrostatic pressure to in vitro cell cultures, while maintaining cell viability on par with a standard incubator. The system has been tested with monolayer cultures, but could also accommodate three dimensional constructs.     

Generation of a green fluorescent protein gene chromosomal insertion containing Escherichia coli strain for gene induction-based quantification of bioavailable lysine

The importance of lysine determination in feed materials is crucial for the feed industry because this amino acid can be limiting in many of the cereal materials used for animal feeds. The bacterial gene induction-based assay developed in this study aimed to measure lysine bioavailability in feeds as an alternative analytical method for animal assays. The advantages of a gene induction-based approach include rapid and quantitative estimation of many samples and results that relate a bacterial response to a biological response observed in animals. A whole-cell biosensor strain was constructed using a fluorescent E. coli strain that has an inducible fluorescent phenotype sensitive to extracellular lysine contents. A genetic fusion that links the promoter of cad operon with a green fluorescent protein encoding gene (gfp) was constructed, and a fluorescent assay was developed. A standard lysine curve (R² = 0.95) was generated and used for lysine bioavailability quantification of four feed ingredients (whole egg protein, blood-, soybean-, and meat and bone meal). Quantities as low as 50 μg/ml protein of digested samples were sufficient for analyses using the biosensor, except for meat and bone meal. Because of the low levels of free lysine in non-digested samples, fluorescence of these protein sources containing lower than 500 μg/ml protein was not detected (except for soybean meal). The results using enzymatically digested protein sources showed that the test strain emitted a fluorescent response that was proportional to the level of lysine present in the feed samples.     

Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extract from the signal-deleted lipL gene (lipL) in the presence of its N-terminal hydrophobic fragment-truncated modulator (rLimL) that was purified from the overexpressing E. coli cells. The specific activity of the lipase thus synthesized was 125 times higher than that of the purified one from Pseudomonas sp. 109. No lipase activity was detected in the absence of rLimL, even though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene (rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. The addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found during its folding process.     

Fate of Escherichia coli O157:H7 in field-inoculated lettuce

Impact of drip and overhead sprinkler irrigation on the persistence of attenuated Escherichia coli O157:H7 in the lettuce phyllosphere was investigated using a split-plot design in four field trials conducted in the Salinas Valley, California, between summer 2007 and fall 2009. Rifampicin-resistant attenuated E. coli O157:H7 ATCC 700728 (BLS1) was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old lettuce plant foliage with a spray bottle at a level of 7 log CFU ml⁻¹. When E. coli O157:H7 was inoculated onto 2-week-old plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation, respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7 declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks post-inoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on lettuce.     

High hydrostatic pressure treatment impairs AcrAB-TolC pump resulting in differential loss of deoxycholate tolerance in Escherichia coli

We reported previously that high hydrostatic pressure-injured stationary phase cells of Escherichia coli K-12 lost their intrinsic deoxycholate tolerance. The AcrAB-TolC multi-drug resistance pump driven by proton motive force has been argued to be responsible for the tolerance to deoxycholate. In this report, we tested the sensitivity of the AcrAB-TolC (three components) pump to high hydrostatic pressure treatment (HPT). E. coli K-12 treated with HPT became sensitive to AcrAB-TolC-specific drugs such as ethidium bromide, but not to tetracycline which is pumped out by a one-component transporter, Tet. Only E. coli K-12 overproducing both AcrAB and TolC exhibited restored tolerance to deoxycholate after HPT but not E. coli overproducing either TolC or AcrAB. These observations strongly suggest that three-component pumps such as AcrAB-TolC are more susceptible to HPT than one-component pumps such as Tet, resulting in the differential loss of deoxycholate tolerance in high hydrostatic pressure-injured E. coli cells.     

Nature of the inactivation of Escherichia coli suspended in an orange juice and milk beverage

The killing effect of pulsed electric fields (PEF) on Escherichia coli (ATCC 8739) suspended in an orange juice and milk beverage was studied. Bipolar square pulses with a pulse width of 2.5 μs were applied. Electric field strength and treatment times ranged from 15 to 40 kV/cm, and from 0 to 700 μs, respectively. A maximum of 3.83 log reductions was achieved at 15 kV/cm and 700 μs. The experimental data were fitted to Bigelow and Hülsheger models and Weibull distribution function. Results indicated that Weibull function best described the experimental data (lowest mean square error). As there were no significant differences in the values of the shape factor (n) at the electric field strength of 25–40 kV/cm, the number of parameters in the Weibull model were reduced, leading to a simplified model with a fit similar to that obtained with the full model.     

2018—2020年上海市致泻大肠埃希氏菌感染的流行病学特征分析

目的 了解上海市致泻大肠埃希氏菌感染的流行病学特征,为致泻大肠埃希氏菌感染的预防和控制提供科学依据。方法 应用描述流行病学的方法,对收集的2018—2020年来自上海市30家食源性疾病监测哨点医院感染性腹泻病例的流行病学信息和致泻大肠埃希氏菌的检测结果进行统计分析。结果 2018—2020年共收集到13 672例有自诉可疑食物史腹泻病例及其标本,致泻大肠埃希氏菌阳性检出率为5.71%(781/13 672),其毒力基因型以ETEC和EAEC为主。7岁以下年龄组和职业为农民的就诊病例致泻大肠埃希氏菌的阳性检出率最高,7～9月是致泻大肠埃希氏菌阳性检出最高的月份,有发热症状、无呕吐症状、腹泻次数在3～9次/d的就诊病例致泻大肠埃希氏菌阳性检出率明显高于其他病例。自诉可疑食物类别为水果及其制品的就诊病例致泻大肠埃希氏菌阳性检出率相对较高。结论 本市致泻大肠埃希氏菌感染毒力基因型以ETEC和EAEC为主,感染高峰出现在夏季。通过对特定临床症状的询问,对疾病临床诊断和治疗具有一定的指导作用。应加强对餐饮服务业和街头食品的监督管理,加强对公众食品安全意识和良好食品安全习惯的宣传与培训,降低致泻大肠...     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar against E. coli O157:H7

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (about 10(6) CFUg(-1)) of E. coli O157:H7 and Salmonella enterica serovars Typhimurium on shredded iceberg lettuce and water samples individually, they were stored at 4 degrees C for 14 days and 22 degrees C for 7 days to monitor the growth and survival of pathogens. The results showed that at the end of 4 degrees C storage, populations of two pathogens in lettuce and water decreased approximately 1 log CFUg(-1). However, microbial levels on shredded lettuce increased 3 logs within 3 days at 22 degrees C. Vinegar (acetic acid) had been used to reduce populations of foodborne pathogens in foods; hence, the antimicrobial effect of rice vinegar on the survival of E. coli O157:H7 in inoculated lettuce (10(4) and 10(7) CFUg(-1)) is examined in this study. Results were observed that the treatment of inoculated lettuce (10(7) CFUg(-1)) with commercial vinegar containing 5% acetic acid (pH 3.0) for 5 min would reduce 3 logs population at 25 degrees C. Less than a 1-log decrease in bacterial numbers was recovered during 5 min exposure to 0.5% (pH 3.26) acetic acid.     

In vitro antimicrobial susceptibility of Escherichia coli isolates from clinical bovine mastitis in Finland and Israel

Minimal inhibition concentration (MIC) values of 100 Finnish and 100 Israeli Escherichia coli isolated from clinical bovine mastitis were determined for ampicillin, cephalexin, ceftazidime, dihydrostreptomycin, gentamicin, tetracycline, trimethoprim-sulfadiazine, and ciprofloxacin by an agar dilution method. The in vitro antimicrobial susceptibility of the E. coli isolates was high; only 27% showed resistance to one or more tested antimicrobial agents. Fifteen percent of the Israeli isolates and 14% of the Finnish isolates were resistant to tetracycline, 3 and 16% to cephalexin, 10 and 7% to ampicillin, 13 and 9% to dihydrostreptomycin, and 4 and 2% to trimethoprim-sulfadiazine. No gentamicin-, ceftazidime-, or ciprofloxacin-resistant isolates were detected. Eleven percent of all the isolates were resistant to two or more antimicrobial agents. Tetracycline was most often associated with multiresistant patterns. Most of the multiresistant isolates had very high MIC values, whereas most of those that were resistant to only one tested antibiotic had MIC values close to the susceptibility breakpoint. Antimicrobial resistance appeared to pose no problem in E. coli isolated from mastitic milk of both countries. This is probably due to the controlled use of antimicrobial agents in the treatment of dairy herds. Some differences were present in the resistance patterns, which may reflect the different use of antimicrobial agents in these two countries.     

枸杞多糖体外调节人体肠道菌群的功能研究

为研究枸杞多糖(LBP)对人体肠道菌群结构和代谢产物的影响,对6名健康人的粪便提取物进行单独样本与混合样本的体外模拟厌氧发酵,利用16S rRNA基因高通量测序技术对发酵后肠道菌群进行结构和功能分析,并利用超高效液相色谱(UPLC)检测发酵液中的短链脂肪酸(SCFAs)浓度。结果表明,LBP能够明显改变人体肠道菌群结构与功能,提高肠道菌群中益生菌乳酸杆菌属与双歧杆菌属的丰度,并促进了SCFAs的产生。因此,LBP能够显著影响人体肠道菌群结构与功能。