Robert E. Drew

Crystal structure and induction mechanism of AmiC–AmiR: a ligand‐regulated transcription antitermination complex

Inducible expression of the aliphatic amidase operon in Pseudomonas aeruginosa is controlled by an antitermination mechanism which allows production of the full‐length transcript only in the presence of small‐molecule inducers, such as acetamide. Ligand‐regulated antitermination is provided by AmiC, the ligand‐sensitive negative regulator, and AmiR, the RNA‐binding positive regulator. Under non‐inducing or repressing growth conditions, AmiC and AmiR form a complex in which the activity of AmiR is silenced. The crystal structure of the AmiC–AmiR complex identifies AmiR as a new and highly unusual member of the response‐regulator family of bacterial signal transduction proteins, regulated by sequestration rather than phosphorylation. Comparison with the structure of free AmiC reveals the subtle mechanism of ligand‐induced release of AmiR.

Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon.

In vivo titration experiments have demonstrated a direct interaction between the Pseudomonas aeruginosa transcription antiterminator, AmiR, and the mRNA leader sequence of the amidase operon. A region of 39 nucleotides has been identified which is sufficient to partially titrate out the AmiR available for antitermination. Site‐directed mutagenesis has shown that the leader open reading frame has no role in the antitermination reaction, and has identified two critical elements at the 5′ and 3′ ends of the proposed AmiR binding site which are independently essential for antitermination. A T7 promoter/RNA polymerase‐driven system shows AmiR‐mediated antitermination, demonstrating a lack of promoter/polymerase specificity. Using the operon negative regulator, AmiC, immobilized on a solid support and gel filtration chromatography, an AmiC‐AmiR complex has been identified and isolated. Complex stability and molecular weight assayed by gel filtration alter depending on the type of amide bound to AmiC. AmiC‐AmiR‐anti‐inducer is a stable dimer‐dimer complex and the addition of the inducer, acetamide, causes a conformational change which alters the complex stability and either this new configuration or dissociated AmiR interacts with the leader mRNA to cause antitermination.

Crystal structure of AmiC: the controller of transcription antitermination in the amidase operon of Pseudomonas aeruginosa.

The crystal structure for the negative regulator (AmiC) of the amidase operon from Pseudomonas aeruginosa has been solved at a resolution of 2.1 A. AmiC is the amide sensor protein in the amidase operon and regulates the activity of the transcription antitermination factor AmiR, which in turn regulates amidase expression. The AmiC structure consists of two domains with an alternating beta‐alpha‐beta topology. The two domains are separated by a central cleft and the amide binding site is positioned in this cleft at the interface of the domains. The overall fold for AmiC is extremely similar to that for the leucine‐isoleucine‐valine binding protein (LivJ) of Escherichia coli despite only 17% sequence identity, however, the two domains of AmiC are substantially closed compared with LivJ. The closed structure of AmiC is stabilized significantly by the bound acetamide, suggesting a molecular mechanism for the process of amide induction. The amide binding site is extremely specific for acetamide and would not allow a closed conformation in the presence of the anti‐inducer molecule butyramide.

The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa.

The nucleotide sequence of the amiE gene, encoding the aliphatic amidase of Pseudomonas aeruginosa, has been determined. The sequence of 1038 nucleotides shows a strong bias in favour of codons with G or C in the third position, and only 44 different codons are utilised.

Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein.

Amide‐inducible expression of the aliphatic amidase system of Pseudomonas aeruginosa can be reconstituted in Escherichia coli with only the amidase structural gene amiE, the negative regulator amiC and the positive regulator amiR, a transcription antitermination factor. Complementation experiments in E. coli suggest that negative control of amidase expression by AmiC is mediated by a protein‐protein interaction with AmiR. Purified AmiC binds acetamide with a KD of 3.7 microM in equilibrium dialysis studies, and therefore AmiC appears to be the sensory partner of the AmiC/AmiR pair of regulatory proteins, responding to the presence of amides. Sequence analysis techniques suggest that AmiC is a member of the structural family of periplasmic binding proteins, but has a distinct and novel cytoplasmic role.

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa

SummaryThe amidase genes of Pseudomonas aeruginosa were inserted into a λ replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant λami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with λami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of λami were made by introducing Q-, S- mutations. Cultures of E. coli infected with λamiQ-S- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificity, thermal stability and immunological crossreaction.

Positive control of Pseudomonas aeruginosa amidase synthesis is mediated by a transcription anti-termination mechanism

The DNA sequence of the region upstream from the amidase structural gene (amiE) of Pseudomonas aeruginosa indicates that amidase (EC 3.5.1.4) is transcribed from an Escherichia coli-like promoter located 150 bp before the amiE translation initiation codon. The sequence between the promoter and the coding sequence includes a single open reading frame followed by an E. coli-like rho-independent transcription terminator. A deletion within the presumed terminator region which disrupts the potential stem/loop formation leads to high constitutive amidase expression which is independent of the product of the regulator gene (amiR). It is proposed that the catabolic aliphatic amidase of P. aeruginosa is regulated by a transcription anti-termination mechanism. The magnoconstitutive mutant PAC433 has promoter and terminator sequences identical to the wild-type PAC1 but contains a single base pair change in the amiE gene ribosome-binding site.

An experiment in enzyme evolution studies withPseudomonas aeruginosa amidase

The regulation of amidase synthesis inP. aeruginosa is under positive control. This review describes the experimental evolution of amidase and its regulator protein for the hydrolysis of novel substrates and experiments to elucidate the mechanism of the control system.

Steric Hindrance Regulation of the Pseudomonas aeruginosa Amidase Operon

Expression of the amidase operon ofPseudomonas aeruginosa is controlled by AmiC, the ligand sensor and negative regulator, and AmiR the transcription antitermination factor activator. We have titrated out AmiC repression activity in vivo by increased AmiR production intrans and shown AmiC regulation of the antitermination activity of AmiR by a steric hindrance mechanism. In the presence of the co-repressor butyramide we have isolated a stable AmiC·AmiR complex. Addition of the inducing ligand acetamide to the complex trips the molecular switch, causing complex dissociation and release of AmiR. The AmiC·AmiR butyramide complex exhibits acetamide-dependent, sequence-specific RNA binding activity and a K d of 1.0 nm has been calculated for the AmiR·RNA interaction. The results show that amidase operon expression is controlled by a novel type of signal transduction system in which activity of a site-specific RNA binding activator is regulated via a sequestration mechanism.

The Amidase Regulatory Gene (amiR) of Pseudomonas aeruginosa

Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing subcloned DNA fragments, the amiR gene was located within a 1 kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.