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

Abstract

The expression pattern of the genome in Escherichia coli is controlled by regulating the utilization of a limited number of RNA polymerases between a total of 4600 genes on its genome. The distribution pattern of RNA polymerase on the genome changes after two steps of protein-protein interaction with seven sigma subunits and about 300 transcription factors (TFs). Based on a systematic search for the regulation target promoters recognized by each TF, we propose two novel concepts: each TF regulates a number of target promoters; and each promoter is regulated by many TFs. In parallel, attempts have been made to determine the intracellular concentrations of all TFs using two systems: quantitative immunoblot analysis using TF-specific antibodies; and reporter assay of TF promoter activities. The direct measurement of TF protein level has so far been published for a set of 60 regulators with known functions. This study describes the determination of growth phase-dependent expression levels of 90 TFs using the reporter assay system. The translational fusion vector was constructed from the TF promoter sequence including an N-terminal proximal TF segment and the reporter GFP. At the beginning of cell growth, highlevel expression was observed only for a small number of TFs. In the exponential phase, approximately 80 % TFs are expressed, but the expressed TF species change upon transfer to the stationary phase. Significant changes in the pattern of TF expression were observed between aerobic and anaerobic conditions. The list of intracellular levels of TFs provides further understanding to the transcription regulation of the E. coli genome under various stressful conditions. INTRODUCTIONSingle-cell bacteria are directly exposed to frequently changing environments in nature and thus carry sophisticated genetic systems for adaptation to environmental changes (Ishihama, 2010(Ishihama, , 2012Yamamoto, 2014). On the basis of the complete genome sequence of several model Escherichia coli strains, the whole set of about 4600 genes has been predicted to exist in E. coli K-12 (Hayashi et al., 2006;Riley et al., 2006). In growing E. coli cells under laboratory culture conditions, only one-quarter to one-third of the genes on its genome are expressed, the others remaining silent. The majority of these uncharacterized silent genes must be expressed and utilized for adaptation and survival of E. coli under stressful conditions. Thus, even for this well-characterized model organism, the gene functions remain unidentified or unpredicted for approximately one-quarter because expression conditions of these silent genes have not yet been established.As the total number of RNA polymerases (RNAPs) in E. coli K-12 is less than the total number of genes on its genome (Ishihama, 2000), we proposed a model in which the change in genome transcription pattern takes place mainly through controlling the utilization of this limited number of transcription apparatuses between 4600 genes on the genome (Ishihama, 2010(Ishihama, , 2012. Two groups of reg...

Expanding theKlebsiella pneumoniaevolatile metabolome using advanced analytical instrumentation for the detection of novel metabolites

“…Previous studies involving E. coli have demonstrated important transcriptomic differences between mid-exponential and stationary growth phase cultures (Yamamoto et al 2014). These transcriptomic differences likely alter the bacterial proteome, which in turn could alter both the soluble and volatile metabolomes, accounting for the growth phase-specific appearance, and subsequent disappearance, of a subset of molecules.…”

Cooperative regulation of the common target genes between H2O2-sensing YedVW and Cu2+-sensing CusSR in Escherichia coli

“…If the functional forms of both TFs exist at high concentrations, they should compete with each other, but both belong to the low-abundance TF group in WT E. coli Yamamoto et al, 2014). Under steady-state growth in the absence of external Cu 2+ and H 2 O 2 , both should remain as nonphosphorylated non-functional forms.…”

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli

“…Excluding the high-abundance nucleoid proteins, the estimated concentrations of regulatory proteins range between v50 and *1500 molecules per genome in exponentially growing cells . Measurements of protein-GFP fusions suggest a concentration range of three orders of magnitude for different regulatory proteins in growing cells (Yamamoto et al, 2014). Note that NsrR concentration was not measured in these studies, and we have been unable to detect NsrR by Western blotting (using a strain with a GTG start codon), which is consistent with a low cellular abundance.…”

“…The seven proteins with very low concentrations (v50 molecules per genome) have between one and five regulatory targets . In this paper, we show with a translational fusion [a technique similar to that used by Yamamoto et al (2014)] that the abundance of NsrR is below the detection limit of the reporter enzyme assay. NsrR appears to be unusual in being a low-abundance regulatory protein with a relatively large number (w10) of target promoters.…”

“…Yet, until recently, measurements of concentrations were available for only a very small number of proteins. This knowledge gap has now been partially filled for E. coli, by high-throughput measurements of regulator abundances using immunological and fluorescence-based (Yamamoto et al, 2014) methods. Excluding the high-abundance nucleoid proteins, the estimated concentrations of regulatory proteins range between v50 and *1500 molecules per genome in exponentially growing cells .…”