Gene expression profiling of the pH response in Shigella flexneri 2a

The pH response of Shigella flexneri 2a 301 was identified by gene expression profiling. Gene expression profiles of cells grown in pH 4.5 or 8.6 were compared with the profiles of cells grown at pH 7.0. Differential expression was observed for 307 genes: 97 were acid up-regulated, 102 were acid down-regulated, 91 were base up-regulated, and 86 were base down-regulated. Twenty-seven genes were found to be both acid and base up-regulated, and 29 genes were both acid and base down-regulated. This study showed that (1) the most pH-dependent genes regulate energy metabolism; (2) the RpoS-dependent acid-resistance system is induced, while the glutamate-dependent acid resistance system is not; (3) high pH up-regulates some virulence genes, while low pH down-regulates them, consistent with Shigella infection of the low gut; and (4) several cross-stress response genes are induced by pH changes. These results also illustrate that many unknown genes are significantly regulated under acid or basic conditions, providing researchers with important information to characterize their function.     

Gene expression analysis of the response by Escherichia coli to seawater

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3000) of the 4228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genome-wide expression profiling in Escherichia coli K-12.

We have established high resolution methods for global monitoring of gene expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655). Glass- and nylon-based arrays of PCR products were prepared and used to assess global changes in gene expression. Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-beta-D-thiogalactopyranoside (IPTG). Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon. Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.     

Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli

In Escherichia coli, the two-component Cpx system comprising the CpxA sensor kinase and the CpxR response regulator modulates gene expression in response to a variety of stresses including membrane-protein damage, starvation, and high osmolarity. To date, the few known CpxR-P target operons were mostly identified by genetic screens. To facilitate the discovery of all target operons, we derived a 15-bp weighted matrix for CpxR-P recognition that takes into account the relative base frequency at each nucleotide position. This matrix essentially consists of two tandem 5'-GTAAA-3' motifs separated by a 5-bp linker. All of the 15-bp stretches on both strands of the E. coli MG1655 genome were then scored for their degree of matching with the matrix and classified in statistical deviation groups. The effectiveness of this screening is indicated by the identification of eight new target operons (ung, ompC, psd, mviA, aroK, rpoErseABC, secA, and aer) among eleven candidates tested. Moreover, the matrix score correlates with the likelihood that a site is a true target and with the relative site affinity for CpxR-P in vitro. Our data indicate that some 100 operons are under direct CpxR-P control and that the signal transduction pathway interacts with several other control circuits in manners hitherto unanticipated.     

Gene expression in a temperature-sensitive gyrB mutant of Escherichia coli.

The effect of gyrase inactivation on gene expression was studied by examining the activities of different promoters in a temperature-sensitive gyrB mutant of Escherichia coli. The relative activities of promoters affected by cAMP-binding protein (CAP), e.g., the lac promoter, are not reduced by gyrase inactivation but can, on the contrary, be enhanced. This stimulation depends on the promoter location or its structure. The tnaA promoter is activated when located near the origin of replication, suggesting a differential effect of gyrase inactivation on various chromosomal domains. Only silent or mutant promoters such as the non-functional wild-type bgl or the lacIq can be activated. No differential effect of gyrase inactivation on the lambda pL and the trp promoters carried by the phage can be detected.     

Gene expression profiling to define host response to baculoviral transduction in the brain

Recombinant baculoviral vectors efficiently transduce several types of cells in the brain. To characterize host responses to virus challenge, thus verifying the suitability of using baculovirus for the development of gene therapy strategies in the central nervous system, we used cDNA microarray technology to examine in vitro and in vivo global cellular gene expression profiles in the rat brain, cultured human astrocytes and human neuronal cells after viral transduction. We demonstrated that the transduction induced host antiviral responses as a major reaction in all three types of samples profiled. The related genes were mainly those associated with innate immunity, including several of the genes involved in Toll-like receptor signaling pathway and cytokine-cytokine receptor interaction. These findings should be useful in understanding the molecular basis for neural cell response to baculoviral transduction and in guiding rational therapeutic applications of baculoviral vectors in the central nervous systems.     

luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic expression profiling

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Gene expression profiling in response to the histone deacetylase inhibitor BL1521 in neuroblastoma

Neuroblastoma is a childhood tumor with a poor survival in advanced stage disease despite intensive chemotherapeutic regimes. The new histone deacetylase (HDAC) inhibitor BL1521 has shown promising results in neuroblastoma. Inhibition of HDAC resulted in a decrease in proliferation and metabolic activity, induction of apoptosis and differentiation of neuroblastoma cells. In order to elucidate the mechanism mediating the effects of BL1521 on neuroblastoma cells, we investigated the gene expression profile of an MYCN single copy (SKNAS) and an MYCN amplified (IMR32) neuroblastoma cell line after treatment with BL1521 using the Affymetrix oligonucleotide array U133A. An altered expression of 255 genes was observed in both neuroblastoma cell lines. The majority of these genes were involved in gene expression, cellular metabolism, and cell signaling. We observed changes in the expression of vital genes belonging to the cell cycle (cyclin D1 and CDK4) and apoptosis (BNIP3, BID, and BCL2) pathway in response to BL1521. The expression of 37 genes was altered by both BL1521 and Trichostatin A, which could indicate a common gene set regulated by different HDAC inhibitors. BL1521 treatment changed the expression of a number of MYCN-associated genes. Several genes in the Wnt and the Delta/Notch pathways were changed in response to BL1521 treatment, suggesting that BL1521 is able to induce the differentiation of neuroblastoma cells into a more mature phenotype.     

大肠埃希菌蛋白指纹图谱的建立

目的建立大肠埃希菌（Escherichia coli,E．coli）蛋白指纹图谱,为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株,提取细菌DNA,PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白,采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本,评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰,其中19个蛋白峰构成E．coli特征性蛋白指纹图谱,各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱,为快速诊断E.coli感染提供了新思路。