Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 microg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 microg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 microg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 microg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).     

微重力诱变大肠杆菌快速生长突变株特性

利用旋转培养装置处理大肠杆菌,筛选生长曲线发生变化、提前进入对数期的突变菌株,对菌株进行基因芯片的表达谱分析和质谱分析,研究微重力条件下微生物的生理代谢变化和对微重力条件的适应机制。结果发现突变菌株有114个差异表达基因,其中99个基因表达上调。表达上调基因主要集中在ABC转运系统、糖代谢、三羧酸代谢、磷酸转移酶系统、核酸代谢、脂类代谢等方面。质谱分析从蛋白水平上验证了这个结果。表明经过微重力处理可以筛选到生长加快的菌株,生长加快是菌株相关代谢水平上调的结果。空间微重力通过对微生物生长代谢相关基因的影响来使菌株适应空间环境。     

Contrapuntal networks of gene expression during Arabidopsis seed filling

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, approximately 35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid-regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO(2) refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, <1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

牛磺胆酸作用下副溶血弧菌全基因组比较转录谱的表达分析

目的利用DNA芯片技术研究副溶血弧菌对牛磺胆酸刺激反应的全局性基因转录变化概况,找出其中的表达调控变化规律,为副溶血弧菌基因转录调控网络的构建提供实验和理论依据。方法副溶血弧菌分别在正常和添加了50mmol／L牛磺胆酸的培养基中孵育至对数中期,收集菌体,提取RNA,利用全基因组DNA芯片分析比较两者基因转录变化。并应用聚类分析比较其中的变化规律。结果比较转录谱分析证实一共有255个基因的转录表达发生显著性变化,和对照组相比,上调的基因明显占主导优势。而在这些变化的基因中,关于蛋白合成和硫代谢以及谷氨酸合成相关的基因均呈现明显的转录上调变化。结论我们利用DNA芯片技术描绘出了副溶血弧菌在添加牛磺胆酸后全部基因转录水平变化的概图,并发现了蛋白合成,硫代谢和谷氨酸合成相关的基因的变化规律,这给我们下一步的转录调控网络研究提供了良好的靶标。     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

高果糖引起的脂肪肝大鼠肾脏脂质合成相关基因和蛋白的表达

大量研究表明,高果糖可引起脂肪肝,但对肾脏脂质代谢的影响尚不清楚。该实验研究给予10％果糖水5周后诱导的脂肪肝大鼠肾脏的脂质代谢情况,并探讨其可能机制。将16只雄性SD大鼠随机分为正常组（con）和果糖组（fru）,果糖组给予10％（W／V）果糖水,第5N末称体重、取血、处死,检测血浆GLU、TG、TC和INSULIN含量。取肾脏、肝脏和白色脂肪称重,采用形态学方法观察肝脏和肾脏脂质沉积情况,酶法测其TG、TC含量,以Real time—PCR检测肾脏、肝脏中脂质合成和脂质氧化相关基因水平,以Westemblot检测肾、肝细胞核脂质合成转录因子的蛋白表达。结果显示,果糖组大鼠血浆TG、INSULIN明显升高,并出现肥胖体征,肝脏脂质沉积严重,其调控脂质合成的两个关键的转录因子ChREBP和SREBPlcmRNA和核蛋白表达都明显升高,并且它们靶向的脂质合成相关酶FAS、ACCl、SCDlmRNA表达也显著增加。但是,在肾脏中,高果糖没有引起TG含量的变化,调控脂质重新合成的基因和蛋白的表达也未发生变化。因此,与果糖致脂肪肝不同,高果糖饮食并没有造成肾脏的脂质沉积和脂质合成相关基因、蛋白的变化。     

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.     

低能离子束辐照下水稻中与ABA相关基因的差异表达

本文检测了用基因芯片筛选出水稻种子被低能N＋辐照后引起的差异表达的ABA代谢和信号途径相关基因。结果显示,与ABA合成相关的ZDS、Lyc-β、ZEP、NCED、SDR这五种酶的基因表达量均为上调;受ABA调控的的H＋-ATPase、NR、Rubisco的基因表达变化显著;ABA依赖的逆境应答蛋白DREB和ASR的表达量上调;受ABA信号转导调控的蛋白LEA的表达量下调,GAD和P5CS的表达量上调。这些结果表明,6×1017N＋·cm-2剂量的辐照可能促进了ABA的合成和幼苗气孔的开放,同时促进了ABA信号系统并激活或抑制了一些相关基因的表达。     

Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).     

Insulin-stimulated protein synthesis in submandibular acinar cells: interactions with adrenergic and cholinergic agonists

The effects of insulin and secretory agonists on amino acid incorporation into submandibular gland proteins were studied using isolated acinar cell aggregates. Insulin stimulated the incorporation of 3H-leucine into TCA-precipitable proteins in a rapid, dose-dependent manner (half-maximal response at 1 nM). Isoproterenol, a beta-adrenergic agonist, also stimulated amino acid incorporation, and this effect was mimicked by both dibutyryl cAMP and IBMX, a phosphodiesterase inhibitor. Although insulin further stimulated incorporation in the presence of isoproterenol and IBMX, no additional increase in the rate of synthesis was observed after stimulation by dibutyryl cAMP. High concentrations of carbamylcholine, a cholinergic agonist, inhibited both basal and insulin-stimulated incorporation. At low concentrations, however, carbamylcholine stimulated synthesis, and the effects of insulin and carbamylcholine were additive. A23187, a calcium ionophore, also inhibited 3H-leucine incorporation and insulin stimulation, but in contrast to carbamylcholine, low concentrations of A23187 neither inhibited nor enhanced the rate of synthesis. Thus, protein synthesis in the rat submandibular gland is regulated by both insulin and neurotransmitters. Whereas beta-adrenergic stimulation appears to be mediated through cAMP, the intracellular signals mediating the actions of insulin and cholinergic agonists remain to be elucidated.