Construction of an Effective Protein Expression System Using the <Emphasis Type="Italic">tpl</Emphasis> Promoter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis.     

Artificial diet delivery system for Philaenus spumarius,the European vector of Xylella fastidiosa

Artificial diets represent an essential tool for investigations on the intimate relationship between plant pathogens and their vectors. Previous research failed in devising an artificial diet delivery system for the meadow spittlebug Philaenus spumarius, to date considered the most important vector of the bacterium Xylella fastidiosa in Europe. Here, we describe a new delivery “tube system” by which we succeeded in artificial feeding of P. spumarius with holidic diets (one sucrose diet and two amino acids diets). Spittlebug probing and feeding behaviour on either the tube system or a traditional “flat system” realized out of a small Petri dish filled with diet and covered with stretched Parafilm^® were observed in real time by video‐EPG (Electrical Penetration Graph), in order to assess the occurrence of ingestion and excretion. Moreover, we evaluated P. spumarius survival on either the tube system filled with the two holidic diets that gave the best EPG results or an empty tube system serving as control. Contrary to the flat system, where just brief stylet insertions through the Parafilm^® were recorded, the spittlebug ingested the artificial diets when delivered with the tube system. Survival on the diets provided with the tube system was significantly greater than the control, with no differences between the diets tested. Furthermore, the tube system was suitable also for another spittlebug species shown to be a competent vector of X. fastidiosa, that is Neophilaenus campestris. The tool we devised opens new perspectives for investigations on X. fastidiosa/spittlebugs interactions, as well as for the functional analysis of mutant X. fastidiosa strains in respect to insect colonization and transmission.     

嗜热链球菌CRISPR-Cas系统的检测

【目的】CRISPR-Cas系统为嗜热链球菌抵抗噬菌体等外源基因元件提供获得性免疫,分析NCBI中已公开发表全基因组序列的9株嗜热链球菌所含CRISPR-Cas系统的数目和类型,对实验室相应菌株的CRISPR-Cas系统进行检测。【方法】利用生物信息学方法对NCBI中9株已测序嗜热链球菌所含CRISPR-Cas系统进行分析,根据其Cas基因序列设计引物,对实验室嗜热链球菌菌株的Cas基因进行扩增、测序,分析实验室6株嗜热链球菌的CRISPR-Cas系统情况。【结果】9株标准菌株均含不同数目的CRISPR-Cas系统,其类型主要为Ⅱ-A型、Ⅲ-A型和Ⅰ-E型,各类型的标志Cas基因高度保守。6株供试菌中,S4仅含Cas9基因,其它5株均含有Cas9基因、Cas10基因和Cas9*基因,79和KLDS3.0207还含有Cas3基因。【结论】可根据标准菌株高度保守的Cas基因设计引物,预测未知嗜热链球菌所含CRISPRCas系统的数目和类型。S4仅含1个Ⅱ-A型CRISPR-Cas系统,其它5株均含有2个Ⅱ-A型CRISPR-Cas系统和1个Ⅲ-A型CRISPR-Cas系统,此外,79和KLDS3.0207均含有1个Ⅰ-E型CRISPR-Cas系统。     

The Fcu controlling-element system in maize

Summary The relationship between the Fcu controlling-element system and the spotted-dilute R system was investigated. The Fcu controlling-element system consists of the receptor element allele r-cu and the regulatory element Fcu. The equivalent components of the spotted-dilute R system are respectively R-r#2 (or R-r#2 Dil) and Spf. The R-r#2 allele of the latter system was shown to be responsive (mutable) to Fcu, provided that it has had an uninterrupted association with Dil or Dpf as evidenced by the color variegation of the aleurone tissue. The reverse test, in which the r-cu allele of the Fcu controlling element system was tested for its response to Spf, proved negative. This was surprising in view of the relationship and specificity between systems. The possibility was considered that maize controlling elements may have different sizes as is known for bacterial insertion sequences. —The variable dilute pigmenting capacity of the r-cu allele also was studied. A given level of r-cu-induced pigmentation, despite the wide range in pigmentation expression, was found to be generally non-heritable, as based on a test of correspondence between parent and progeny.Journal Paper No. J-9204 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1884Now with Funk's Seeds, Casalmorano (Cremona), Italy     

大肠杆菌的耐酸机制及其改造研究进展

微生物细胞在自然环境或工业应用中经常受到酸胁迫,严重制约细胞生长性能和产物合成效率。为了在各种酸性环境中生存,耐酸细菌发展出多种保护机制来维持细胞内pH稳态,如氢离子消耗、细胞膜保护、代谢修饰等。因此,深入研究耐酸机制、改进菌株耐酸能力对于利用微生物发酵合成高附加值产品具有重要意义。作为模式微生物,大肠杆菌耐酸机制的研究较为透彻,近年来其耐酸性改造也取得了重大进展。本文主要总结了大肠杆菌的氧化或葡萄糖抑制系统(acid resistance system 1, AR1)、谷氨酸依赖型耐酸系统(acid resistance system 2, AR2)、精氨酸依赖型耐酸系统(acid resistance system 3, AR3)、赖氨酸依赖型耐酸系统(acid resistance system 4, AR4)和鸟氨酸依赖型耐酸系统(acid resistance system 5, AR5)、细胞膜保护以及生物大分子修复等方面的耐酸机制,并概述了利用传统代谢工程、全局转录工程和适应性实验室进化等方法构建大肠杆菌耐酸菌株的研究进展,同时展望了大肠杆菌耐酸机制及其改造的后续研究方向...     

Development of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> expression system using the improved P<Emphasis Type="Italic">glv</Emphasis> promoter

Background  

B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter P _glv . The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the P _glv promoter system and enhance its expression strength.     

Specificity of Enzyme System Producing C6-Aldehyde in Tea Chloroplasts

The substrate specificity of enzyme system producing C₆-aldehyde in Thea chloroplasts was clarified with an entire series of synthesized positional isomers, in which the position of cis-1, cis-4-pentadiene system varies from C-3 to C-13 in C₁₈ fatty acid and geometrical isomers of linoleic acid. The structural requirement for the substrate of enzyme system producing C₆-aldehyde is the presence of cis-1, cis-4-pentadiene system between ω-6 and ω-10.     

A method for simultaneous determination of (I) the blood flows to the organs and (II) the cardiac output

A method is presented for the simultaneous determination of (i) the blood flow to the organs and (ii) the cardiac output. Part I of the paper deals with the analysis of ann compartment (organ) vascular system model. The data, employed in the analysis, consists of continuous monitoring of the amounts of indicatorM _i in the organs (or compartments). An analysis for determination of the cardiac output and the absolute flows to the organs is presented. Since it is difficult to isolate certain organ systems and measure the amounts of indicator in them exclusively, a more realistic model of then compartment vascular system is presented in Part II. Herein, the analysis has accounted for the finite transit time, of the indicator, from one organ system to another. Further, estimation theory is employed to make estimates of blood flow to different organs by taking note of (i) the measurement errors due to the detectors' monitoring (for an organ system) some combination ofM _i 's instead of theM _i for the particulari ^th organ and (ii) noise uncertainties introduced by the measuring instruments.     

Specific immunorecognition by histocompatibility markers: The original polymorphic system of immunoreactivity characteristic of all multicellular animals

The existence of two immunorecognition systems, theH system of cell-mediated immunity and theIg system of antibody-mediated immunity, is proposed. TheH system, based on polymorphism of cell-surface histocom-patibility markers, is postulated to have originated in multicellular invertebrates, probably beginning with coelenterates. Subsequent differentiation and diversification of immunocytes, associated with increasing physiological complexity in phylogeny, are postulated to have led to the addition of a regulatoryIg system at the level of primitive chordates or fishes. Progressive integration of theH andIg systems then provided T-B lymphocyte collaboration and the immunoregulatory network characteristic of higher vertebrates. Experimental tests are suggested for the hypothesis of a two-component system of specificH-marker immunorecognition, leading to nonspecific cytotoxic reactions mediated by nonantibody proteins.     

Physiological role of tyrosine transport systems in Halobacterium salinarium

The quantitative content of three transport systems for aromatic amino acids in cells of Halobacterium salinarium was measured: the common system (K _m is about 10^-6 M) and two tyrosine-specific systems with high and low affinity (K _m is about 10^-8 and 10^-5 M, respectively). To determine the activity of each of three systems separately, a method was developed based on the selective phenylalanine effect on these activities. When phenylalanine exeeds [¹⁴C]tyrosine by four to sixforld, it inhibits competitively the activity of the common system, and its 50- to 100-fold molar excess is inhibitory in a non-competitive way for the specific high affinity system (HAT system). The specific low affinity system (LAT system) is practically insensitive to phenylalanine. The activities of tyrosine-specific transport systems are slightly dependent on the culture age, and the observed decrease in transport activity during growth is due mainly to the decreased content of the common system. The HAT system formation is regulated by the repression type, and the effectors are aromatic amino acids especially tyrosine itself. The physiological sense of the tyrosine transport system's multiplicity in H. salinarium is discussed.     

The Ktr potassium transport system in Staphylococcus aureus and its role in cell physiology,antimicrobial resistance and pathogenesis

Potassium (K⁺) plays a vital role in bacterial physiology, including regulation of cytoplasmic pH, turgor pressure and transmembrane electrical potential. Here, we examine the Staphylococcus aureus Ktr system uniquely comprised of two ion‐conducting proteins (KtrB and KtrD) and only one regulator (KtrA). Growth of Ktr system mutants was severely inhibited under K⁺ limitation, yet detectable after an extended lag phase, indicating the presence of a secondary K⁺ transporter. Disruption of both ktrA and the Kdp‐ATPase system, important for K⁺ uptake in other organisms, eliminated regrowth in 0.1 mM K⁺, demonstrating a compensatory role for Kdp to the Ktr system. Consistent with K⁺ transport mutations, S. aureus devoid of the Ktr system became sensitive to hyperosmotic conditions, exhibited a hyperpolarized plasma membrane, and increased susceptibility to aminoglycoside antibiotics and cationic antimicrobials. In contrast to other organisms, the S. aureus Ktr system was shown to be important for low‐K⁺ growth under alkaline conditions, but played only a minor role in neutral and acidic conditions. In a mouse competitive index model of bacteraemia, the ktrA mutant was significantly outcompeted by the parental strain. Combined, these results demonstrate a primary mechanism of K⁺ uptake in S. aureus and a role for this system in pathogenesis.     

Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds

Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9‐cis‐epoxycarotenoid dioxygenase (NCED), a rate‐limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA‐stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE‐binding factor) expression in Arabidopsis Columbia‐0 seeds, which caused 9‐ to 73‐fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non‐dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre‐harvest sprouting during crop production, and therefore contributes to translational biology.     

Rapid one-step inactivation of single or multiple genes in Escherichia coli

Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid-based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose-inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β-D -1-thiogalactopyranoside-inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE–aspA and sfcA–aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.     

Production of Honbushi-like Flavor by the Fermentation of Nijiru

It takes about 2 months for the molding process of Katsuobushi (dried bonito) production. A model of a short-term system for the molding process was desirable for the efficient selection of useful fungi for Katsuobushi production by evaluating the flavor change in the model system.

A liquid culture system using Nijiru (waste fluid of Katsuobushi sterilization), which is obtained from the Kezuribushi (sliced Katsuobushi) manufacturing process and contains abundant phenolic compounds of smoke tar, was found to be suitable for the short-term evaluation of a 10 day culture.     

Complementation system for Helicobacter pylori

Previously Langford et al. (2006) developed the pIR203C04 complementation system for Helicobacter pylori, which can be used to complement and restore phenotypic effects in H. pylori mutant, and furthermore they used the complementation system in vivo experiments to animals without altering the ability of strain SSI to colonize mice. In their previous study, the pIR203C04 was able to transform 26695, SSI, J99, and 43504 H. pylori strains by an electroporation method. However, in the present study using a natural transformation the pIR203C04 transformed only 26695 H. pylori but not SSI, J99, 7.13, and G27 H. pylori strains. Since the useful complementation system has a limitation of narrow selection among H. pylori strains, we redesigned the complementation system for the improvement. The same intergenic chromosomal site between hp0203 and hp0204 was utilized for the new complementation system because the insertion at the intergenic site didn’t show any polar effects and disruption of other H. pylori genes. The genome sequence analysis showed that the intergenic regions among H. pylori strains may have too low homology to each others to do a homologous recombination. Thus, in addition to the short intergenic region, the fragments of the new complementation system included 3′ conserved parts of hp0203 and hp0204 coding regions. Between the fragments there are a chloramphenicol acetyltransferase cassette and multicloning sites, resulting in pKJMSH. DNA fragment of the interest can be cloned into the multicloning sites of pKJMSH and the fragment can be integrated at the intergenic region of H. pylori chromosome by the homologous recombination. Indeed, by the natural transformation, pKJMSH was able to transform all five H. pylori strains of 26695, SSI, J99, 7.13, and G27, which are common for the investigation of molecular pathogenesis. Thus, the new pKJMSH complementation system is applicable to most H. pylori wild-type stains.     

Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods

We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella enterica prpBCDE promoter (P_prpB) and compared it to that from the strongest IPTG-inducible promoter, P_T7. In contrast to our previous study showing slightly higher production of green fluorescent protein (GFP) from the pPro system compared to that from the T7 system, production of two plant proteins CMT and NCS from P_prpB was 2- to 4-fold higher than that from P_T7. Unlike P_T7, expression from P_prpB did not reduce cell growth even when highly induced, indicating that this propionate-inducible system is more efficient for overproduction of proteins that result in growth inhibition. In an auto-induction experiment, which does not require monitoring the culture or adding inducer during cell growth, the pPro system exhibited much higher protein production than the T7 system. These results strongly indicate that the pPro system is well-suited for overproduction of recombinant proteins.     

乳酸乳球菌双组分系统调控有氧呼吸的研究

【背景】乳酸乳球菌作为食品行业的代表性菌株,如何通过双组分系统响应环境因子与代谢调控的分子机制研究,对发酵食品产业和益生菌制剂行业有着重要的意义。【目的】探究乳酸乳球菌双组分系统对有氧呼吸代谢调控的相关网络,为乳酸菌适应性代谢研究提供新思路。【方法】采用生物信息学方法,系统性地分析乳酸乳球菌双组分系统组氨酸激酶和反应调节因子的结构域组成及预测双组分系统功能,筛选出与有氧呼吸有潜在联系的双组分,并进一步通过基因转录表达和非靶向代谢组学验证。【结果】以乳酸乳球菌的代表菌株NZ9000为例构建相互作用蛋白网络,显示双组分系统与丙酮酸代谢网络关键连接点为丙酮酸铁氧还蛋白氧化还原酶(nifJ)。在不同的生长时期,Lactococcus lactis NZ9000双组分转录表达在延滞期变化显著。与厌氧培养相比,有氧培养和有氧呼吸培养的菌体双组分呈现下调趋势。双组分系统参与乳酸菌氧化应激和血红素胁迫过程。【结论】明确乳酸乳球菌参与有氧呼吸的双组分系统以及代谢通路,有助于提高发酵剂、益生菌剂的存活率和竞争力。     

Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

Purification and Characterization of an Alkaliphilic Choline Oxidase of Fusarium oxysporum

Uptake of cesium, potassium, and rubidium by Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 followed Michaelis-Menten saturation kinetics. The K_m’s for uptake of these monovalent cations by R. erythropolis CS98 and Rhodococcus sp. strain CS402 were 136 and 436μM for Cs⁺, 65 and 101μM for K⁺, and 102 and 113μM for Rb⁺, respectively. These values were significantly lower than those of Rhodobacter capsulatus and the Kup system in Escherichia coli. Potassium was a competitive inhibitor of cesium uptake by these strains, suggesting that cesium was accumulated by the potassium transport system. Although an uncoupler, FCCP, inhibited the cesium transport system, this system was not repressed by high concentrations of potassium in both Rhodococcus strains. However, the specificity in both Rhodococcus strains was different from the Trk system. These results suggest that the potassium transport system which can transport cesium in both Rhodococcus strains may be novel.