Analysis of reading frame and expressional regulation of randomly selected promoter-proximal genes in Escherichia coli (original) (raw)
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Applied and Environmental Microbiology
A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designed specifically for the start and stop regions of E. coli genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830-1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175: . The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation in E. coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coli global gene regulation in response to additional environmental stimuli.
Journal of Bacteriology, 2005
The S (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, S is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of S and that S should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a S consensus promoter in silico. Moreover, our results suggest that S-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect S and/or 70 selectivity of many promoters. These observations indicate that certain modules of the S-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with 70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.
Biotechnology and Bioengineering, 2003
Bacteria develop a number of devices for sensing, responding, and adapting to different environmental conditions. Understanding within a genomic perspective how the transcriptional machinery of bacteria is modulated, as a response to changing conditions, is a major challenge for biologists. Knowledge of which genes are turned on or turned off under specific conditions is essential for our understanding of cell behavior. In this study we describe how the information pertaining to gene expression and associated growth conditions (even with very little knowledge of the associated regulatory mechanisms) is gathered from the literature and incorporated into Regulon-DB, a database on transcriptional regulation and operon organization in E. coli. The link between growth conditions, signal transduction, and transcriptional regulation is modeled in the database in a simple format that highlights biological relevant information. As far as we know, there is no other database that explicitly clarifies the effect of environmental conditions on gene transcription. We discuss how this knowledge constitutes a benchmark that will impact future research aimed at integration of regulatory responses in the cell; for instance, analysis of microarrays, predicting culture behavior in biotechnological processes, and comprehension of dynamics of regulatory networks. This integrated knowledge will contribute to the future goal of modeling the behavior of E. coli as an entire cell. The RegulonDB database can be accessed on the web at the
Heterologous Gene Expression in an Escherichia coli Population Under Starvation Stress Conditions
Journal of Molecular Evolution, 1998
The occurrence of promoter-generating mutations allowing the transcription of heterologous genes has been studied in a system based on the plasmid-mediated conjugal transfer of histidine biosynthetic genes from a donor bacterium (Azospirillum brasilense) into a heterologous Escherichia coli mutant population lacking histidine biosynthetic ability and initially unable to recognize the transcriptional signal of the introgressed gene(s). Under selective stressful conditions, His + revertants accumulated in the E. coli His − culture. The number of His + colonies was dependent on the time of incubation under selective conditions, the strength of selective pressure, and on the crowding of cells plated; moreover, it was independent of the physiological status of the cell (i.e. the growth phase). Sequence analysis of plasmid DNA extracted from E. coli His + revertants revealed that single base substitutions in the region upstream of the A. brasilense his operon resulted in an adjustment of the pre-existing sequence that was rendered similar to the E. coli −10 promoter sequence and transcriptable by the host RNA-polymerase. One particular transition (C → T) was predominant in the His + revertants. Data presented here indicated that the barriers to the expression of horizontally transferred heterologous genes or operons may be overcome in a short time scale and at high frequency, and supported the selfish operon model on the origin and evolution of gene clusters. 2001 Éditions scientifiques et médicales Elsevier SAS histidine biosynthesis / promoter-generating mutations / operon evolution / horizontal gene transfer / selfish operon * Correspondence and reprints.
PloS one, 2014
The fast adaptation of Escherichia coli to stressful environments includes the regulation of gene expression rates, mainly of transcription, by specific and global stress-response mechanisms. To study the effects of mechanisms acting on a global level, we observed with single molecule sensitivity the effects of mild acidic shift and oxidative stress on the in vivo transcription dynamics of a probe gene encoding an RNA target for MS2d-GFP, under the control of a synthetic promoter. After showing that this promoter is uninvolved in fast stress-response pathways, we compared its kinetics of transcript production under stress and in optimal conditions. We find that, following the application of either stress, the mean rates of transcription activation and of subsequent RNA production of the probe gene are reduced, particularly under oxidative stress. Meanwhile, the noise in RNA production decreases under oxidative stress, but not under acidic shift. From distributions of intervals betwe...
F1000 - Post-publication peer review of the biomedical literature, 2014
The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.
Generic transcriptional response of E. coli to stress
2015
Bacteria are often exposed to various stressors with inter-linked effects leading to cross-resistance. In order to study the common molecular response to stress in bacteria, we collated and compared transcriptional response of E. coli under a variety of biotic and abiotic stresses. Bacterial genome-wide gene expression data were retrieved from the NCBI Gene Expression Omnibus (GEO) database and interrogated to identify differentially expressed genes. One hundred and sixty eight genes displayed distinct transcriptional response of E. coli to stress, with simultaneous down-regulation of flagellar assembly pathway and up-regulation of the global regulator rpoS. This computational analysis summarizes stress responsive genes in E. coli and their inter-relationships.
Physiological genomics, 2003
The involvement of heat-inducible genes, including the heat-shock genes, in the acute response to temperature stress is well established. However, their importance in genetic adaptation to long-term temperature stress is less clear. Here we use high-density arrays to examine changes in expression for 35 heat-inducible genes in three independent lines of Escherichia coli that evolved at high temperature (41.5 degrees C) for 2,000 generations. These lines exhibited significant changes in heat-inducible gene expression relative to their ancestor, including parallel changes in fkpA, gapA, and hslT. As a group, the heat-inducible genes were significantly more likely than noncandidate genes to have evolved changes in expression. Genes encoding molecular chaperones and ATP-dependent proteases, key components of the cytoplasmic stress response, exhibit relatively little expression change; whereas genes with periplasmic functions exhibit significant expression changes suggesting a key role f...
International Journal of Food Microbiology, 2005
Heat shock proteins and RNA polymerase sigma factor play an important role in protecting cells against environmental stresses, including starvation, osmotic and oxidative stresses, and cold shock. In this study, the effect of environmental stresses on activity of the auto-fluorescent Escherichia coli O157:H7 generated by the fusion of gfp uv to E. coli uspA, grpE and rpoS promoters were examined. Osmotic shock caused about a 4-fold increase in green fluorescence of E. coli O157:H7 harboring uspADgfp uv or rpoSDgfp uv at 37 8C and room temperature whereas osmotic shock at 5 8C did not induce green fluorescence. When starved, E. coli O157:H7 possessing grpEDgfp uv was more sensitive for evaluating stress at low temperature while uspADgfp uv was better suited for detecting the stress response at higher temperature. The uspA, grpE and rpoS promoters were up-regulated to varying degrees by stresses commonly encountered during food processing.
Physiological and Biochemical Zoology, 2005
Transcription profiling (quantitative analysis of RNA abundance) can provide a genome-wide picture of gene expression changes that accompany organismal adaptation to a new environment. Here, we used DNA microarrays to characterize genome-wide changes in transcript abundance in three replicate lines of the bacterium E. coli grown for 2,000 generations at a stressful high temperature (41.5ЊC). Across these lines, 12% of genes significantly changed expression during high-temperature adaptation; the majority of these changes (55%) were less than twofold increments or decrements. Thirty-nine genes, four times the number expected by chance alone, exhibited moderately or highly replicated expression changes across lines. Expression changes within a priori defined functional categories showed an even greater level of replication than did individual genes. Expression changes in the phenotypically defined stress genes and adaptation functional categories were important in evolutionary adaptation to high temperature.