Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector

Abstract

When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300-to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.The RNA polymerase of Escherichia coli is composed of the core enzyme (subunit composition, ␣ 2 ␤␤Ј) with RNA polymerization catalytic activity and one of seven different species of the sigma subunit, each of which participates in transcription of a specific set of genes (10, 15). The intracellular concentration of RNA polymerase in the steady state of growing E. coli W3350 cells is maintained at a constant level characteristic of the rate of cell growth (16). The total number of core enzymes is not more than the total number of genes on the E. coli genome (1). Thus, the distribution pattern for RNA polymerase genes among about 4,000 genes in the genome should vary depending on the culture conditions (15). This finding accentuates the importance of the need for the RNA polymerase to choose which genes to transcribe and how often they are transcribed. The replacement of one core enzyme-associated sigma subunit by another sigma subunit is the most efficient way to alter the promoter recognition specificity of the transcription apparatus and is thus believed to be the major mechanism for switching of the transcription pattern. Thus, the competition between available sigma subunits should be a key determinant of which group genes are transcribed (8,33). In addition to sigma subunit replacement, the activity and specificity of RNA polymerase is also modulated by interaction with about 300 molecular species of transcription factors (14,15). Most of these accessory transcription factors are DNA-binding proteins and interact with RNA polymerase when ...

The transcription regulator AllR senses both allantoin and glyoxylate and controls a set of genes for degradation and reutilization of purines

“…In brief, GFP was expressed under the control of a test promoter while red fluorescent protein (RFP) was under the control of a reference promoter. The promoter assay vector pGRP carries two types of fluorescent protein genes, one for RFP under the control of reference promoter lacUV5 and the other for GFP under the control of a test promoter (Makinoshima et al, 2002;Shimada et al, 2004). The gcl promoter sequence upstream from the initiation codon was amplified by PCR using the genomic DNA from E. coli KP7600 as a template and a pair of primers, H026S (59-TGGGCAGAGATCTGCCACTGCATGCTTCCGGT-39) and H026T (59-TCATTTTATGCATTTTATTCCTACCCTA-39).…”

“…Promoter strength was determined as described previously (Makinoshima et al, 2002;Shimada et al, 2004). In brief, GFP was expressed under the control of a test promoter while red fluorescent protein (RFP) was under the control of a reference promoter.…”

Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families

“…The first report of results using the quantitative immunoblot system has been published, which describes the intracellular concentration of 60 species of TFs with known regulatory functions (Ishihama et al, 2014). As a quick and accurate means of monitoring the expression level of a number of TFs in E. coli cells under various growth conditions, we employed in this study the second method, in which the expression level of TFs was determined using a modified version of the promoter assay vector (Shimada et al, 2004). As the synthesis of regulatory proteins is often controlled at the level of translation (for instance see Gottesman, 2004), we constructed the TF promoter assay vector as translational fusions between the reporter GFP and the test TF sequences from the entire promoter segment (about 500 bp sequence upstream of the initiation codon) down to the N-terminal proximal short segment (approx.…”

“…DNA sequences of each TF gene between approximately 2500 and +150 with respect to the initiation codon, including the promoters and the N-terminal proximal TF-coding sequences, were PCR-amplified using pairs of the respective primers containing BglII and EcoT22I sites. PCR-amplified sequences were treated with BglII and EcoT22I and inserted into pGRP vector, the promoter assay vector of the GFP reporter (Shimada et al, 2004), between BglII and EcoT22I (Fig. 1).…”

Sulfate assimilation pathway intermediate phosphoadenosine 5′-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli

“…5a), we investigated whether the Congo red and calcofluor phenotype of the MG1655DcysH mutant could also be dependent on this regulator. In addition, we considered a possible role for the alternative sigma factor s S , which, in addition to being necessary for csgD transcription (Römling et al, 1998b), also controls expression of the slp gene (Shimada et al, 2004) and of several genes responsible for biosynthesis of c-di-GMP (Sommerfeldt et al, 2009) -a signal molecule playing a pivotal role in regulation of cell-surface-associated structures (Tamayo et al, 2007). Finally, we tested the possibility that Cbl, the regulator of indicates the exclusive presence of a protein in one sample.…”