Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions

Abstract

RNA polymerase holoenzyme of Escherichia coli consists of the core enzyme with the subunit composition of ␣ 2 ␤␤Ј and a subunit, which directs the core enzyme to initiate transcription at specific promoter sites on DNA (4). The major factor, 70 (the rpoD gene product), is responsible for transcription of most genes expressed during the exponential cell growth (12, 17). Besides 70 , six different molecular species of alternative subunits in E. coli have been identified. 54 (the rpoN gene product) is concerned with expression of a wide variety of genes including those involved in nitrogen metabolism (27,28). Temperature upshift increases transcription of the genes under the control of two heat shock subunits, 32 (the rpoH gene product) (8, 41) and 24 (the rpoE gene product) (7). Holoenzyme containing 32 transcribes the heat shock genes including those encoding chaperons and proteases (42), while the regulons under the control of 24 are known to be involved in extracytoplasmic functions (6,35). 38 (the rpoS gene product) is a key factor in the stress response during the transition from the exponential growth phase to the stationary growth phase (13,25). 28 (the rpoF gene product) governs transcription of the genes for flagellar formation and chemotaxis (3, 31). FecI involved in the ferric citrate transport system is now identified as a member of a new subfamily of subunits for extracytoplasmic functions (2).The switch of gene expression pattern upon sudden exposure to various stresses is thought to take place by replacement of the subunit on RNA polymerase. The level of each form of holoenzyme is thought to be determined by the concentration of each subunit and its affinity to core RNA polymerase. Until recently, however, little was known of the intracellular concentrations of individual subunits except for the major subunit, 70 (17). We then initiated a systematic determination of the intracellular concentrations of the subunits, and in a previous study (19), we reported the levels of 38 in E. coli MC4100 growing under various conditions. As an extension of this line of research, we determined in this study the intracellular levels of four subunits in E. coli growing under steadystate conditions or various stress conditions by the same quantitative Western blot (immunoblot) method employed in the previous study (19). Since the MC4100 strain lacks 28 for flagellar formation (39), we analyzed another strain, W3110, in order to understand the possible influence of the lack of one subunit on the levels of other subunits. MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in this study were E. coli W3110 and MC4100. Cells were grown at 30 or 37ЊC under aeration in Luria broth (LB). Growth was monitored by measuring the turbidity with a Klett-Summerson photometer. The culture conditions were fixed as follows. A few colonies from a culture grown overnight on a LB agar plate were inoculated onto 5 ml of fresh LB medium. At the cell density of 30 Klett units, the culture was diluted 2...

Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival

“…The intracellular concentrations of all seven species of the subunit were determined at various phases of cell growth. The levels of H , E and FecI are almost negligible under the culture conditions employed (Jishage et al 1996;Maeda et al, manuscript in preparation). Some unused subunits are conserved as complexes with anti-factors.…”

“…The determination of intracellular contents was later extended to include the remaining five subunits, N , F , H , E and FecI (Jishage et al 1996;H. Maeda et al, manuscript in preparation).…”

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

“…CbpA (curved DNAbinding protein A), CbpB (curved DNA-binding protein B; or Rob, right origin binding protein), DnaA (DNA replication protein A or initiator protein), Hfq (host factor for phage Q b ), IciA (inhibitor of chromosome initiation A), Lrp (leucine-responsive regulatory protein), and StpA (suppresser of Td À phenotype) . The intracellular concentrations have also been determined for RNA polymerase core enzyme subunits (Ishihama 1981), all seven s subunits, and two anti-s subunits (Jishage & Ishihama 1995Jishage et al 1996;Maeda et al 2000). Among the nucleoid components, the intracellular concentration in E. coli W3110 is the most abundant for Fis, HU and Hfq in the exponential growth phase, but only a single species, Dps, predominates in stationary-phase cells ; also reviewed in Ishihama 1999Ishihama , 2000.…”

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

“…The intracellular concentrations of all seven sigma factors in growing cells of E. coli W3110 have been determined (Jishage & Ishihama, 1995;Jishage et al, 1996;Maeda et al, 2000). Since then we have been involved in determination of the intracellular concentrations of the 300 TFs using two approaches: (1) quantitative immunoblot analysis and (2) reporter assay of promoters of the genes encoding TFs.…”

“…Moreover, for the accurate estimation of intracellular levels of the regulatory proteins for transcription, all the test proteins must be measured using the same cultures of the same bacterial strains and the same experimental systems. We then systematically measured intracellular concentrations of sigma subunits and TFs using two approaches: quantitative immunoblot analyses using anti-sigma (Jishage & Ishihama, 1995;Jishage et al, 1996;Maeda et al, 2000) or anti-TF antibodies (Ishihama, et al, 2014); and quantification of promoter activity using the promoter assay vector of the fluorescent protein reporter system (Shimada et al, 2005). In this paper, we describe the determination of intracellular levels of 90 TFs of six TF families by using the TF promoter-GFP translation fusion vectors.…”