The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing

The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor.

Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways.

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

▪ Abstract The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xylUWCAMBN for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylXYZLTEGFJQKIH, which encodes the enzymes for further conversion of these compounds into Krebs cycle intermediates; and xylS and xylR, which are involved in transcriptional control. The XylS and XylR proteins are members of the XylS/AraC and NtrC families, respectively, of transcriptional regulators. The xylS gene is constitutively expressed at a low level from the Ps2 promoter. The XylS protein is activated by interaction with alkylbenzoates, and this active form stimulates transcription from Pm by σ70- or σS-containing RNA polymerase (the meta loop). The xylR gene is also expressed constitutively. The XylR protein, which in the absence of effectors binds in a nonactive form to target DNA sequences, is activated by aromatic hydrocarbons and ATP; it...

Transcriptional control of the pseudomonas tol plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators

Transcription is the key control point for regulation of numerous cellular activities. Bacteria regulate levels of gene expression by using transcription factors that modulate the recruitment of RNA polymerase (RNAP) to promoter elements in the DNA. Many bacteria also control gene expression by using a second class of transcription factor that uses energy from nucleotide hydrolysis to actively promote transcription initiation.

The sigma (σ) subunit is required for promoter recognition and initiation of transcription by the bacterial RNAP. Typically a bacterial cell may contain several alternative σ subunits with differing sequence specificities that direct the RNAP holoenzyme to different sets of promoters. The σ54 subunit (also known as RpoN, NtrA, and σN) is unique (42) in that it shares no detectable homology with any of the other known sigma factors (e.g., σ70 and σ28). σ54-RNAP binds to specific promoter sites, with the consensus DNA sequence YTGGCACGrNNNTTGCW (6), to form a transcriptionally inactive closed complex consisting of holoenzyme bound to double-stranded DNA. In contrast to σ70-RNAP bound at its cognate promoter sites, σ54-RNAP is unable to spontaneously isomerize from a closed complex to a transcriptionally competent open complex (11, 72). To proceed with initiation of transcription, the closed complex must participate in an interaction with a transcriptional activator, involving nucleotide hydrolysis. This transcriptional activator is usually bound at least 100 bp upstream of the promoter site, and DNA looping is required for the activator to contact the closed complex and catalyze formation of the open promoter complex. In this respect the activator resembles the enhancer-binding proteins (EBPs) found in eukaryotic systems, and for this reason such activators are known as bacterial EBPs.

From a protein structural point of view, EBPs share in common a σ54 interaction module (Pfam accession number PF00158) but typically have at least one additional domain (Fig. ​(Fig.1).1). In nearly all of those investigated so far, there is a DNA-binding domain containing a helix-turn-helix sequence motif, enabling the protein to bind to specific DNA enhancer elements upstream of σ54-dependent promoters (44, 47, 52, 56, 72). One exception to this scenario was recently reported (30), where Pseudomonas aeruginosa FleQ can activate transcription while bound in the downstream vicinity of the promoter.



FIG. 1.

Major domain architectures of bacterial EBPs. Examples of each of the known domain organizations found in bacterial EBPs are given. Sequences are identified by SwissProt/trEMBL accession numbers, except for XAC3643, TTE0180, and {"type":"entrez-protein","attrs":{"text":"CPE23358","term_id":"896862659","term_text":"CPE23358"}} ...



Although the physiological advantages conferred by the σ54-EBP mode of transcription are not yet clear, activation of σ54-dependent transcription is highly regulated by environmental cues through regulatory modules in the EBPs and in some cases by interactions with other regulatory proteins. Sensory modules in EBPs include CheY-like response regulator domains, PAS domains, GAF domains, PRD modules, and V4R domains, often represented within an N-terminal region (Fig. ​(Fig.1).1). These sequence features are described in more detail later in this article. Intriguingly, recent complete genome sequences have revealed some unusual EBPs containing regions of homology to other signal transduction domains and enzymes. With the large number of complete bacterial genomes now available, and with the importance of accurate annotation of future sequence data, we feel that it is timely to survey the variety of domain architectures found in these important proteins.

Domain architectures of σ54-dependent transcriptional activators

Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ(54). We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ(54) for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ(54)-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ(54) to the bacterial cell and its unique role in regulating transcription.

The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of σ54-Dependent Transcription

Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator.

Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54). DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes. Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54. Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date. Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD. DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate. Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD. DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate. These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.

https://www.pnas.org/content/pnas/92/21/9702.full.pdf

Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.

Summary
Rhizobium meliloti DctD is believed to have three functional domains: an N-terminal, two-component receiver domain; and like other σ54-dependent activators, C-terminal and central domains for DNA binding and transcription activation. We have characterized a progressive series of M-terminal deletions of R meliloti DctD. The N-terminal domain was not needed for binding the dctA upstream activation sequence. Only 25% of the C-terminal end of the receiver domain was needed to significantly inhibit the central domain, and proteins lacking up to 60% of the N-terminal end of the receiver domain were‘inducible’in R. meliloti cells. We hypothesize that the W-terminal two-thirds of the DctD receiver domain augments and controls an adjacent subdomain for inhibiting the central domain.

Rhizobium meliloti DctD, a σ54‐dependent transcriptional activator, may be negatively controlled by a subdomain in the C‐terminal end of its two‐component receiver module

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between σ54-RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with σ54-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for σ54-holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in σ54-dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to σ54 and the β-subunit of RNA polymerase, suggesting that they bound normally to σ54-holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for σ54 and the β-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact σ54-holoenzyme during open complex formation.

Joon H. Lee

Papers

Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator.

Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.

Rhizobium meliloti DctD, a σ54‐dependent transcriptional activator, may be negatively controlled by a subdomain in the C‐terminal end of its two‐component receiver module

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation