Local metabolic response of Escherichia coli to the module genetic perturbations in l-methionine biosynthetic pathway

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Evidence for internalization of Escherichia coli into the aerial parts of maize via the root system

Escherichia coli introduced into the hydroponic growing medium of maize plants was detected 48 h later in the shoot. Decapitation of root tips or severing of the plant root system at the root-shoot junction enhanced bacterial internalization. The density of the bacteria in shoots of plants with damaged roots or removed root systems was 27.8 and 23.9 times higher than that in plants with intact roots, respectively. The concentration of viable cells in the hydroponic solution decreased over time from 9.3 x 10(6) CFU/ml at the time of inoculation to 8.5 x 10(1) CFU/ml 4 days thereafter. The number of E. coli cells associated with the roots also decreased with time, but a significant decline appeared only at 4 days postinoculation. At the time of sampling for E. coli presence in the shoot, 10(2) CFU/ml was present in the nutrient solution and 8 x 10(3) CFU/g was associated with the roots. The present study is the first to demonstrate internalization of E. coli via the root in a monocotyledonous plant.     

大肠杆菌代谢工程生产芳香族化合物研究进展

芳香族化合物广泛应用于化工、食品及医药等领域,多来自于化工合成或天然产物提取。天然微生物也具有合成芳香族化合物的能力,以维持自身生命活动代谢需求,但其积累能力较低。近几年利用代谢工程的方法对微生物特别是大肠杆菌进行途径优化、设计、改造等方法在提高其芳香族化合物的发酵产量方面取得了显著成效,并且创新地生产出多种有价值的芳香族衍生物。这些研究成果对于未来以合成生物学和细胞工厂为基础利用可再生资源进行工业生物制造,解决化石能源危机和天然产物提取等问题具有重要意义。     

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.     

精氨酸—还原糖体系中1,2-二羰基化合物的形成和抑制研究

建立精氨酸—还原糖体系,考察影响该体系中1,2-二羰基化合物形成的因素,并探究形成过程中反应物与产物变化的动力学以及抑制剂染料木黄酮抑制1,2-二羰基化合物的动力学。对建立的精氨酸—还原糖模拟体系,采用气相色谱法评价温度、还原糖种类、pH和抑制剂染料木黄酮等各种因素对1,2-二羰基化合物形成的影响,并分析其变化规律,进而分析了反应体系中染料木黄酮介入前后反应物精氨酸、葡萄糖及产物1,2-二羰基化合物的含量变化规律。结果显示:影响1,2-二羰基化合物形成的最主要因素是时间;该体系中精氨酸和葡萄糖的浓度随着反应时间的延长而降低,且随着温度升高快速下降;1,2-二羰基化合物含量随着温度的升高和反应时间的延长而不断升高。时间和温度对丙酮醛(MGO)和乙二醛(GO)的产生有一定的影响,染料木黄酮通过降低反应体系中1,2-二羰基化合物的反应速率常数而抑制其产生。当染料木黄酮的添加量为50mmol/L时,对1,2-二羰基化合物的抑制率最高,均50%。LC-MS证实染料木黄酮在氨基酸体系中抑制1,2-二羰基化合物的机制是通过捕获MGO形成染料木黄酮-MGO加合产物,从而达到消除效果。     

Biosorption of nonpolar hydrophobic organic compounds to Escherichia coli facilitated by metal and proton surface binding

We observed that the presence of transition metal ion, Ag+, Cu2+, or Fe3+, at a concentration of 3 mg L(-1) increases sorption of two nonpolar hydrophobic organic compounds (HOCs), phenanthrene (PHEN), and 1,2,4,5-tetrachlorobenzene (TeCB) by 1.5-4 times to Gram-negative bacteria Escherichia coli. Complexation of transition metals with the deprotonated functional groups (mainly carboxyl) of bacterial cell walls neutralizes the negative charge, making the bacterial surface less hydrophilic and enhancing hydrophobic partition of HOCs. This is evidenced by the fact that the zeta potential (zeta) value of bacteria becomes less negative when a transition metal is present. Furthermore, the observed higher sorption of PHEN than TeCB at low pH (3.8) cannot be fully explained by the pH-dependent hydrophobic effects. The results led us to propose two specific sorption mechanisms for pi-donor compounds: cation-pi interactions with protonated amines and pi H-bonding with protonated carboxyls. The biosorption of PHEN was best described as pi-donor compared to the biosorption of TeCB considered non-pi-donor. Results of the present study highlight that the presence of coexisting transition metals and changes on pH have a major effect on the biosorption of nonpolar HOCs.     

大肠杆菌对酸的抗性变化研究

由于大肠杆菌具有耐酸性,且乳酸可引起大肠杆菌的亚致死性损伤,故使其成为食品安全领域关注的重要问题之一.了解不同酸性条件下大肠杆菌的生存特点,有助于人们进一步认识其耐酸性的分子机制,从而为制定预防方案和措施提供理论依据.     

谷氨酸合成途径基因缺失对大肠杆菌发酵L-色氨酸的影响

降低谷氨酸的积累可提高L-色氨酸产量及糖酸转化率。敲除Escherichia coli TRTH中的谷氨酸脱氢酶及谷氨酸合成酶编码基因gdh A、glt B,构建TRTHA(TRTH,Δgdh A)、TRTHB(TRTH,Δglt B),考察gdh A、glt B缺失对L-色氨酸发酵的影响。结果表明,gdh A及glt B缺失能有效降低谷氨酸的积累,但会降低细胞生长及色氨酸合成;培养基中谷氨酸的添加可恢复TRTHA及TRTHB的生长及色氨酸合成能力。在含1 g/L谷氨酸培养基中,利用TRTHB发酵L-色氨酸,L-色氨酸产量(41.23 g/L)及糖酸转化率(15.45%)最高,较TRTH分别提高了10.92%和7.89%;谷氨酸生成量(5.72 g/L)及乙酸积累量(1.73 g/L)分别较TRTH降低了25.23%及提高了10.19%。TRTH和TRTHB代谢流分析结果表明,glt B缺失会降低谷氨酸合成代谢流并提高乙酸合成代谢流;TRTHB的色氨酸合成代谢流(11.4%)较TRTH提高了40.74%。     

Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanoate) polymers synthesized by Escherichia coli

The composition of medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) biopolymers is normally an uncontrollable random mixture of repeating units with differing side chain lengths. Attempts to generate MCL PHA homopolymers and control repeating unit composition have been published in native PHA-producing organisms but have limited ranges for the different sizes of repeating units that can be synthesized. In this study, a new Escherichia coli-based system that exhibits control over repeating unit composition for both MCL PHAs and short-chain-length (SCL) PHAs has been developed, covering an unprecedented range of repeating units. The fadB and fadJ genes from the β-oxidation pathway were eliminated from the chromosome of E. coli LS5218. The subsequent blockage in β-oxidation caused a buildup of enoyl-CoA intermediates, which were converted to PHAs by an (R)-specific enoyl-CoA hydratase (PhaJ4) and PHA synthase [PhaC1(STQK)] expressed from a plasmid DNA construct. Fatty acid substrates were converted to PHAs with repeating units equal in the number of carbon atoms to the fatty acid substrate. The broad substrate specificities of the PhaJ4 and PhaC1(STQK) enzymes allowed for the production of homopolymers with strict control over the repeating unit composition from substrates of four to twelve carbons in length. Polymers were purified and analyzed by GC, GC-MS, and NMR for structural composition and by DSC, TGA, and GPC for thermal and physical characteristics. This study marks the development of the first single biological system to achieve consistent repeating unit control over such a broad range of repeating units in PHAs.     

美国营养标签发展特征及其对我国食品营养标签制度的启示

美国是世界上营养标签起步早、类别多、制度比较完善的国家。本文基于营养事实标签以及心脏检查标志、指引星标签、NuVal评分标签、前面事实标签等包装正面(Front of package,FOP)标签概况总结美国营养标签发展特征发现,美国营养标签实施主体多元、适用性广,营养事实标签与FOP标签优势互补;营养标签电子信息化与国际化趋势显著。因此,结合我国营养标签实际情况,建议新增与营养健康密切相关的营养成分信息,为FOP标签设计提供支撑;加强中国居民膳食指南对营养标签的科普与使用推荐;完善生鲜农产品营养成分数据库,加快FOP标签在生鲜农产品的运用;通过政府监管与行业自律,构建企业FOP标签认证管理体系。     

大肠杆菌高效表达屎肠球菌纤维素结合域谷氨酸脱羧酶的条件优化

为了规避人肠杆菌(Escherichia coli)自身基因组表达的天然GAD的干扰,以再生无定形纤维素(regenerated amorphous cellulose,RAC)特异性吸附纤维素结合结构域谷氨酸脱羧酶(cellulose-binding domairr glutamate decarboxylase,CBD-GAD)制备的RAC-CBDGAD固定化酶作为评价指标,采用单因素和田口试验设计法对E.coli GDMCC60445高效表达CBD-GAD的条件进行了优化。结果表明,E.coli GDMCC60445表达CBD-GAD的适宜培养基为改良LB(Luria-Bertani)培养基,其组成为胰蛋白胨8 g/lL、酵母膏6 g/L、NaCl10g/L、pH 5.5;适宜的培养条件为温度37℃、摇床转速120 r/min、培养时间24 h。在该适宜条件下,RAC-CBDGAD活力为(419.79±10.37)U/g,与田口法预测值一致,较优化前提高了(30.28±3.22)%。     

Structural functions of taste in the sugar series: Taste properties of sugar alcohols and related compounds

The sweetness of simple polyhydric alcohols is examined in relation to conformation and configuration. Comparative studies of the relative sweetness of these and polyhydric glycosides are also carried out and further evidence is obtained regarding the importance of the glycosidic linkage in taste stimulation. Although the AH,B systems in these sugars are located at the polyhydric aglycones, the sugar moieties seem to exert a strong influence on their tastes.     

色氨酸转运系统改造对大肠杆菌产L-色氨酸的影响

近年来,转运系统改造已经成为氨基酸菌株菌种改良的重要手段。本研究以工业生产菌Escherichia coli MT-01/p Trp-01为出发菌株,首先利用Red重组技术,在菌株MT-01/p Trp-01基因组上敲除了色氨酸吸收基因mtr,发酵结果表明,敲除敲除突变菌的L-色氨酸产量达到35.87 g/L,与出发菌株E.coli MT-01/p Trp-01相比提高了32%;在此基础上,进一步考察了三种不同启动子（Pr,Ptac,Pser A）控制下L-色氨酸分泌基因ydd G的差异表达对菌体生长及菌株产L-色氨酸的影响。结果表明,当采用组成型启动子tac时,ydd G基因的过表达菌株L-色氨酸的产量为41.01 g/L,比mtr敲除菌株E.coli MT-11/p Trp-01的产量提高了14.3%,当采用温度诱导型启动子Pr调控ydd G基因表达时,L-色氨酸的产量与mtr敲除菌株E.coli MT-11/p Trp-01的产量相比提高了9.3%,L-色氨酸的产量达到了39.22 g/L;而采用基因ser A的天然启动子调控ydd G表达时,菌体的生长受到了明显抑制,L-色氨酸产量仅有27.1 g/L的色氨酸。综上,大肠杆菌基因mtr的敲除和基因ydd G的过表达均可以有效提高工程菌株生产色氨酸的能力。      

Novel system for controlled investigation of environmental partitioning of hydrophobic compounds in water

Partitioning behavior of hydrophobic and semivolatile chemicals (such as many POPs and PAHs) in water is key in controlling their environmental distribution and fate. A new equilibrium method is presented here which allows determination of the equilibrium partition coefficient of hexachlorobenzene with suspended particle (K(SPM)≈ 337 L gOC(-1)) in a complex bulk water sample by correcting for a number of sampling artifacts and for the presence of dissolved matter. The method provides simultaneous experimental determination of the fraction of chemical truly dissolved in water (representing in this case about 54% of the bulk water concentration) and that associated to DM (21%). The Henry's law constant was also experimentally determined during the K(SPM) measurements, providing information on the occurrence of partitioning equilibrium in the system for each single observation. Results showed that the high level of quality control and accuracy provided confidence intervals for the K(SPM) estimates within 1 order of magnitude.     

Occurrence of Escherichia coli O157:H7 and other enterohemorrhagic Escherichia coli in the environment

Enterohemorrhagic Escherichia coli (EHEC) (O157 and other serotypes) are zoonotic pathogens linked with severe human illnesses. The main virulence factors of EHEC are the Shiga toxins, among others. Most of the genes coding for these toxins are bacteriophage-encoded. Although ruminants are recognized as their main natural reservoir, water has also been documented as a way of transmission of EHEC. E. coli O157:H7 and other EHEC may contaminate waters (recreational, drinking or irrigation waters) through feces from humans and other animals. Indeed, the occurrence of EHEC carrying the stx2 gene in raw municipal sewage and animal wastewater from several origins has been widely documented. However, the evaluation of the persistence of naturally occurring EHEC in the environment is still difficult due to methodological problems. Methods proposed for the detection and isolation of stx-encoding bacteria, ranging from the classic culture-based methods to molecular approaches, and their application in the environment, are discussed here. Most virulence factors associated with these strains are linked to either plasmids or phages, and consequently they are likely to be subject to horizontal gene transfer between species or serotypes. Moreover, the presence of infectious stx-phages isolated as free particles in the environment and their high persistence in water systems suggest that they may contribute to the spread of stx genes, as they are directly involved in the emergence of new pathogenic strains, which might have important health consequences.     

The adherence of Escherichia coli to the intestinal epithelium of piglets

The ability of some pathogenic strains of E. coli to adhere to the intestinal epithelium significantly enhances their effectivity. This adhesion of particular pigs' strains is mediated by specific pili possessing the K 88 antigen found on the outer membrane of the bacterial cell. In in vitro experiments with isolated piglets' enterocytes, a considerable adherence of the E. coli K 88+ strain was found when compared with the same bacterial strain but lacking this plasmid-directed antigen. Comparable results were obtained in in vivo experiments with ligated intestinal loops as well as with monoassociated piglets. Furthermore, the adherence ability is most pronounced at the early postnatal period and is negligible in adult pigs.     

Electrochemical disinfection: an efficient treatment to inactivate Escherichia coli O157:H7 in process wash water containing organic matter

The efficacy of an electrochemical treatment in water disinfection, using boron-doped diamond electrodes, was studied and its suitability for the fresh-cut produce industry analyzed. Tap water (TW), and tap water supplemented with NaCl (NaClW) containing different levels of organic matter (Chemical Oxygen Demand (COD) around 60, 300, 550 ± 50 and 750 ± 50 mg/L) obtained from lettuce, were inoculated with a cocktail of Escherichia coli O157:H7 at 10⁵ cfu/mL. Changes in levels of E. coli O157:H7, free, combined and total chlorine, pH, oxidation–reduction potential, COD and temperature were monitored during the treatments. In NaClW, free chlorine was produced more rapidly than in TW and, as a consequence, reductions of 5 log units of E. coli O157:H7 were achieved faster (0.17, 4, 15 and 24 min for water with 60, 300, 500 and 750 mg/L of COD, respectively) than in TW alone (0.9, 25, 60 min and 90 min for water with 60, 300, 600 and 800 mg/L of COD, respectively). Nonetheless, the equipment showed potential for water disinfection and organic matter reduction even without adding NaCl. Additionally, different mathematical models were assessed to account for microbial inactivation curves obtained from the electrochemical treatments.     

Mathematical model of the acute inflammatory response to Escherichia coli in intramammary challenge

We constructed a mathematical model of the early response to Escherichia coli infection of the mammary gland and explored the roles and interactions between inflammatory cells and bacteria. The model incorporates 3 equations that describe the interactions among bacteria, milk somatic cells, and blood leukocyte densities. These 3 equations were fitted to cell densities observed during acute inflammatory responses in unvaccinated and vaccinated heifers inoculated with 10⁴ or 10⁶ cfu of E. coli. The rates computed for the cellular transit from the storage sites to the blood and from the blood to the milk were lower in cows receiving 10⁴ cfu but increased at approximately 6 × 10⁻⁶ and 30 × 10⁻⁶ μL/cfu per h in nonvaccinated or vaccinated cows inoculated with 10⁶ cfu, respectively. The cellular rates of bacterial killing were highest in unvaccinated cows (∼400 × 10⁻⁶ μL/cell per h) when compared with vaccinated cows (200 to 300 × 10⁻⁶ μL/cell per h). A critical density of milk somatic cells at which bacteria density is constant was computed from the model at 2 × 10⁶ cells/mL, and a one-way sensitivity analysis revealed that the changes in milk cellular densities were mostly sensitive to variations in the rate of bacterial killing and in the rate of production of inflammatory cells.     

丝氨酸吸收系统多基因敲除对大肠杆菌产L-丝氨酸的影响

sst T、cyc A、sda C和tdc C是大肠杆菌L-丝氨酸的四个吸收基因,本文在前期吸收基因单基因敲除菌的基础上,系统研究了四个吸收基因不同组合敲除对菌株生长及L-丝氨酸生产的影响。摇瓶发酵结果表明,在11个多基因敲除突变菌中,sst T·sda C双基因敲除菌,sst T·sda C·tdc C和sst T·cyc A·sda C三基因敲除菌的L-丝氨酸产量与对照相比有明显提升,分别达到271.11、312.58和322.57 mg/L;sst T·cyc A·sda C·tdc C四基因敲除菌的L-丝氨酸产量最高,达到了450.58 mg/L,是对照菌的6倍。在菌株生长方面,双基因敲除菌和三基因敲除菌的生长量优于对照菌或者与对照菌相近,而四基因敲除菌的生长量与对照菌相比则下降了28%。