Functionality of Purified ς ^N (ς ⁵⁴ ) and a NifA-Like Protein from the Hyperthermophile Aquifex aeolicus

Abstract

The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor N ( 54 , RpoN) and five potential N -dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative N -dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus N and the NifA homologue, AQ_218. Purified A. aeolicus N bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative N -dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus N supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspF⌬HTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the N enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and N have coevolved.The special form of bacterial RNA polymerase (RNAP) containing alternative sigma factor N or 54 ( N -RNAP), initiates transcription by a mechanism quite distinct from RNAP containing the major 70 sigma factor (35,39,48). Transcription initiation by N -RNAP requires the hydrolysis of nucleoside triphosphate, catalyzed by activator proteins bound to upstream activator sequences (enhancer elements). The mechanisms of regulation involving this form of RNAP are among the most sophisticated in bacteria (52).Since this novel form of RNAP was first recognized in enteric bacteria (25,29,47), N has been discovered in many other bacteria, including several proteobacteria, as well as in the gram-positive Bacillus subtilis (14) and in Planctomyces limnophila (36). Furthermore, N appears to be encoded by the genomes of the hyperthermophile Aquifex aeolicus (15), the spirochete Borrelia burgdorferi (18), and the obligate intracellular pathogens Chlamydia trachomatis (51) and Chlamydia pneumoniae (31).The N proteins from Klebsiella pneumoniae and Escherichia coli have been the subject of much genetic and biochemical analysis (e.g., see references 8, 20, and 23). Although considerable progress has been made towards understanding the mechanisms by which N performs its function, to date no high-resolution structural data are available for this system. Thermophilic homologues of mesophilic proteins have often been shown to be tractable for structural studies. For example, the crystal structures of the histidine kinase domain of CheA from Thermotoga maritima and the core RNAP from...

Regulation of Type VI Secretion Gene Clusters by σ 54 and Cognate Enhancer Binding Proteins

“…Our results from the reporter fusions show that the bEBP from several microorganisms, such as V. cholerae, A. hydrophila, Pectobacterium atrosepticum, and Marinomonas sp., adapt with the E. coli 54 -RNAP complex. Interactions of a bEBP and a 54 from two distinct microorganisms have already been demonstrated (50). This is made possible by the high degree of conservation between the 54 binding region of E. coli bEBPs and the bEBPs used in this study: the central domain, including the GAFTGA sequence, which mediates bEBP binding to 54 , is conserved among all the bEBPs used in this study (8,43,49,65) (see Fig.…”

“…Gel shift experiments were carried out with soluble bEBPs and with reconstituted E 54 . DNA binding activity of E 54 was measured as previously described (11,50,56 , and 3.5% PEG 6000) in the presence of sonicated salmon sperm DNA (10 g/ml) and bovine serum albumin (BSA; 50 g/ml). Controls include incubation with RNAP core enzyme or with doublestranded DNA competitors (consensus 54 and Fur binding boxes obtained by the annealing of two complementary oligonucleotides; Table 2).…”

Crystal structure of Aquifex aeolicus σ N bound to promoter DNA and the structure of σ N -holoenzyme

“…1 σ N supports promoter-specific transcription in combination with E. coli (Eco) RNAP and an Aae activator in vitro (16). Aae σ N lacks the nonessential region II (RII) (Fig.…”

Domain Architectures of σ54-Dependent Transcriptional Activators

“…YgaA (also known as NorR) has recently been shown to activate transcription of a nitric oxide detoxification system (21,28). The stimuli detected by AQ_218 and AQ_1792 are unknown, but AQ_218 was shown to bind upstream of the glnBA operon for nitrogen assimilation (65).…”