Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR

Abstract

The efficient expression of the tdc operon of Escherichia coli requires the products of two regulatory genes, tdcR and tdcA. We have identified the transcription site of tdcR by primer extension mapping and established the translation start site of TdcR by mutational analysis of its reading frame. In a tdcR tdcABC deletion strain, tdcR+ promoted high-level LacZ expression from a )dcAB-lacZ lysogen and mutations introduced in tdcR resulted in a greater than sixfold decrease in LacZ level. In-frame deletions of tdcA also reduced LacZ expression, and chromosomal and plasmid-borne tdcA+ increased the LacZ level in tdcA mutant lysogens.Interestingly, multicopy tdcA+ plasmids introduced into tdcR mutant strains completely restored tdc expression. In separate experiments we found that mutations in the tdc promoter DNA around positions -70, -140, and -175 greatly reduced tdc expression relative to that for the wild-type promoter and the tdcP mutation around -175 prevented multicopy tdcA+ from rescuing tdcR mutants. Furthermore, competition experiments revealed that a wild-type promoter fragment encompassing the -175 region cloned into a plasmid reduced tdc expression by titrating TdcA in vivo, and this effect was reversed with excess TdcA. These results suggest that in tdcR+ cells TdcR interacts with tdcP and/or TdcA to enhance tdc transcription whereas in tdcR mutant cells a new tdcP-TdcA complex around -175 in the native promoter bypasses the requirement for TdcR. On the basis of the accumulated data summarized here and elsewhere we propose that multiple transcription factors enhance tdc operon expression by bending and looping of the promoter DNA to form an active transcription complex.The inducible threonine dehydratase operon tdcABC of Escherichia coli (6) is implicated in transport and metabolism of threonine and serine during anaerobic growth to provide a source of metabolic energy. The operon contains two structural genes, tdcB and tdcC, encoding, respectively, the biodegradative threonine dehydratase (EC 4.2.1.16) catalyzing the dehydration of L-threonine and L-serine to ammonia and the corresponding ox-keto acids and a membrane-associated Lthreonine-L-serine permease (2, 6, 23). The regulatory gene tdcA, proximal to the tdc promoter, encodes a protein homologous to the LysR family of transcriptional activators and is required for maximal induction of the tdc operon (3, 5). Upstream of tdcABC and transcribed in the opposite orientation is tdcR, which specifies a small protein that is also required for efficient expression of the tdc genes (19). The entire 6.3-kb E. coli DNA fragment harboring the tdc genes and their flanking sequences has been sequenced (18) and mapped at min 68.3, or at kb 3330, on the E. coli chromosome with clockwise direction of transcription (17,20).In addition to the operon-specific transcriptional activators TdcR and TdcA, several other gene products also influence tdc operon expression in vivo. The cyclic-AMP-catabolite gene activator protein (CAP) complex and integration host factor (I...

The Fur regulon in anaerobically grown Salmonella enterica sv. Typhimurium: identification of new Fur targets

“…The genes in this operon ( tdcBCDEG ) are activated by tdcA [74]. TdcA is a member of the LysR family of transcriptional activators [75].…”

Differential Binding of Escherichia coli O157:H7 to Alfalfa, Human Epithelial Cells, and Plastic Is Mediated by a Variety of Surface Structures

“…It is known that TdcA is a transcriptional activator of the tdcABC operon involved in the transport and metabolism of threonine and serine during anaerobic growth (18). In contrast, the nature of the genes regulated by TdcA which are required for binding (or alternatively, inhibiting binding) to plant surfaces is unknown, and characterization of the TdcA regulatory network is a current interest of our laboratory.…”

Involvement of Fnr and ArcA in anaerobic expression of the tdc operon of Escherichia coli

“…Because tdcR gene product is required for high-level expression of tdcABC (10,33), it is possible to argue that lack of Fnr or ArcA could somehow lower the level of TdcR in the cell, thus reducing tdc expression. However, the following observations ruled out this possibility: (i) no significant difference in LacZ expression from the tdcR-lacZ fusion was found in fnr ϩ and fnr strains, and (ii) multicopy tdcA ϩ was unable to restore tdc expression in fnr and arcA mutants.…”

“…Genetic and biochemical experiments revealed that TdcA, IHF, and CAP occupy their unique binding sites on tdcP at Ϫ175, Ϫ104, and Ϫ41, respectively, with respect to the tdcP transcription start site at ϩ1 (Fig. 1B), and act in concert most likely by bending and looping the promoter DNA to form an active transcription complex (10,42). We continue to examine the tdc operon as a well-defined model system to investigate the basic mechanisms underlying anaerobic gene expression.…”

“…1A), is implicated in transport and metabolism of the amino acids during anaerobic growth to provide a source of metabolic energy (3,6,7,37). Upstream of tdcABC and transcribed in opposite orientation is tdcR, which specifies a small protein that is essential for tdc transcription (10,33). In addition to an anaerobic (microaerobic) environment and the two operonspecific transcription factors TdcA and TdcR, the efficient expression of the operon requires two global regulatory proteins, integration host factor (IHF) and the cyclic AMP (cAMP)-catabolite gene activator protein (CAP) complex (42,44).…”