Abstract
BackgroundThe glutamate synthase operon (gltBDF) contributes to one of the two main pathways of ammonia assimilation in Escherichia coli. Of the seven most-global regulators, together affecting expression of about half of all E. coli genes, two were previously shown to exert direct, positive control on gltBDF transcription: Lrp and IHF. The involvement of Lrp is unusual in two respects: first, it is insensitive to the usual coregulator leucine, and second, Lrp binds more than 150 bp upstream of the transcription starting point. There was indirect evidence for involvement of a third global regulator, Crp. Given the physiological importance of gltBDF, and the potential opportunity to learn about integration of global regulatory signals, a combination of in vivo and in vitro approaches was used to investigate the involvement of additional regulatory proteins, and to determine their relative binding positions and potential interactions with one another and with RNA polymerase (RNAP).ResultsCrp and a more local regulator, ArgR, directly control gltBDF transcription, both acting negatively. Crp-cAMP binds a sequence centered at -65.5 relative to the transcript start. Mutation of conserved nucleotides in the Crp binding site abolishes the Crp-dependent repression. ArgR also binds to the gltBDF promoter region, upstream of the Lrp binding sites, and decreases transcription. RNAP only yields a defined DNAse I footprint under two tested conditions: in the presence of both Lrp and IHF, or in the presence of Crp-cAMP. The DNAse I footprint of RNAP in the presence of Lrp and IHF is altered by ArgR.ConclusionThe involvement of nearly half of E. coli's most-global regulatory proteins in the control of gltBDF transcription is striking, but seems consistent with the central metabolic role of this operon. Determining the mechanisms of activation and repression for gltBDF was beyond the scope of this study. However the results are consistent with a model in which IHF bends the DNA to allow stabilizing contacts between Lrp and RNAP, ArgR interferes with such contacts, and Crp introduces an interfering bend in the DNA and/or stabilizes RNAP in a poised but inactive state.
Highlights
The glutamate synthase operon contributes to one of the two main pathways of ammonia assimilation in Escherichia coli
Effects of Lrp and IHF on RNA polymerase (RNAP) binding to the gltBDF promoter region We have previously shown that Lrp and IHF bind simultaneously to gltBDF promoter (PgltB) DNA, yielding a band with intermediate gel shift mobility between the IHFDNA and Lrp-DNA complexes (Fig. 3C in [14])
Since the absence of either Lrp or IHF leads to a >30-fold reduction in gltBDF transcription [5,14], we investigated whether these two proteins influence RNAP binding to PgltB
Summary
The glutamate synthase operon (gltBDF) contributes to one of the two main pathways of ammonia assimilation in Escherichia coli. Of the seven most-global regulators, together affecting expression of about half of all E. coli genes, two were previously shown to exert direct, positive control on gltBDF transcription: Lrp and IHF. There was indirect evidence for involvement of a third global regulator, Crp. Given the physiological importance of gltBDF, and the potential opportunity to learn about integration of global regulatory signals, a combination of in vivo and in vitro approaches was used to investigate the involvement of additional regulatory proteins, and to determine their relative binding positions and potential interactions with one another and with RNA polymerase (RNAP). Just seven proteins (ArcA, Crp, FIS, Fnr, IHF, Lrp, and NarL) directly control expression of about half of all genes in E. coli [1]. There are two main pathways for assimilating ammonia into glutamate in Escherichia coli [2] (see Fig. 1). In the presence of high ammonia concentrations and limited carbon/energy, glutamate dehydrogenase (GdhA) produces glutamate from ammonia, α-ketoglutarate, and NADPH [equation 1, and left side of Fig. 1].
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