A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans

MmsR (33.3 kDa) is a putative LysR-type transcriptional activator of Pseudomonas denitrificans. With the help of 3-hydroxypropionic acid (3-HP), an important platform chemical, MmsR positively regulates the expression of mmsA, which encodes methylmalonylsemialdehyde dehydrogenase, the enzyme involved in valine degradation. In the present study, the cellular function of MmsR and its binding to the regulatory DNA sequence of mmsA expression were investigated both in vivo and in vitro. Transcription of the mmsA was enhanced >140-fold in the presence of 3-HP. In the MmsR-responsive promoter region, two operators showing dyad symmetry, designated O-1 and O-2 and centered at the -79 and -28 positions, respectively, were present upstream of the mmsA transcription start site. An electrophoretic mobility shift assay indicated that MmsR binds to both operator sites for transcription activation, probably in cooperative manner. When either O-1 or O-2 or both regions were mutated, the inducibility by the MmsR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcription activation. A 3-HP sensor was developed by connecting the activation of MmsR to a green fluorescent readout. A more than 50-fold induction by 25 mM 3-HP was observed

A 96-well DNase I footprinting screen for drug–DNA interactions

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture

Development of a Simple Web-based GIS System for Sustainable Housing Projects VisualizatioN

This paper presents a simple web-based GIS system for visualization of Baan Mankong, as known as sustainable housing projects for communities of low-incomers in Thailand. The Baan Mankong project has been so successful that numbers of projects initialized have been increasing during the past couple of years. The projects information is gathered in a huge database, but there is a lack of visualization tool to help the officers and executives to have a global view of the projects spatial distribution and to have a quick access to project information. GIS is proposed as a tool to be developed on the web for the visualization of this project. The Baan Mankongis work flow and project information are studied in order to select only necessary information to be stored in the system. Then, the geographic visualization tool development process is presented

A Novel Antibiotic Mechanism of l-Cyclopropylalanine Blocking the Biosynthetic Pathway of Essential Amino Acid l-Leucine

The unusual amino acid l-cyclopropylalanine was isolated from the mushroom Amanita virgineoides after detection in an anti-fungal screening test. l-Cyclopropylalanine was found to exhibit broad-spectrum inhibition against fungi and bacteria. The anti-fungal activity was found to be abolished in the presence of the amino acid l-leucine, but not any other amino acids, indicating that l-cyclopropylalanine may block the biosynthesis of the essential amino acid l-leucine, thereby inhibiting fungal and bacteria growth. Further biochemical studies found l-cyclopropylalanine indeed inhibits α-isopropylmalate synthase (α-IMPS), the enzyme that catalyzes the rate-limiting step in the biosynthetic pathway of l-leucine. Inhibition of essential l-leucine synthesis in fungal and bacteria organisms, a pathway absent in host organisms such as humans, may represent a novel antibiotic mechanism to counter the ever-increasing problem of drug resistance to existing antibiotics