Sally M. McFall

Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA)-cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate. In addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR mutants. DNase-I footprinting, DNA bending and in vitro transcription analyses with RNA polymerase mutants indicate that CatR and ClcR activate transcription via a similar mechanism which involves interaction with the C-terminal domain of the alpha-subunit (alpha-CTD) of RNA polymerase. In vitro transcription assays with different regions of the clc promoter indicate that the ClcR dimer bound to the promoter proximal site (the activation binding site) interacts with the alpha-CTD. Gel shift assays and DNase-I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of inducer and an additional binding site within the catB structural gene called the internal binding site (IBS). CatR binds the IBS with low intrinsic affinity that is increased by cooperativity in presence of the two promoter binding sites. Site-directed mutations in the IBS indicate a probable cis-acting repressor function for the IBS. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies and phasing studies suggest that the IBS participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA. Although the core transcriptional activation mechanisms of CatR and ClcR have been conserved, nature has provided some flexibility to respond to different environmental signals in addition to the presence of inducer. Transcriptional fusion studies demonstrate that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate or fumarate and that this repression is ClcR-dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA-cycle intermediate, fumarate, directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as activator on either the cat or clc template. Since both the catechol and the chlorocatechol pathways feed into the TCA cycle, but only the chlorocatechol pathway is inhibited by fumarate, there is a subtle difference in the regulation of these two pathways where intracellular sensing of a TCA-cycle intermediate leads to a reduction of chloroaromatic degradation.

Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid

Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.

Rapid, Point-of-Care Extraction of Human Immunodeficiency Virus Type 1 Proviral DNA from Whole Blood for Detection by Real-Time PCR

ABSTRACT PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 μl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%.

DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism

In Pseudomonas putida, benzoate and 3‐chlorobenzoate are converted to catechol and 3‐chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA‐bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2‐chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the α‐subunit truncation mutant (α‐235) of RNA polymerase was sharply reduced, indicating that the α‐subunit C‐terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

Microbial degradation of toxic environmental pollutants: Ecological and evolutionary considerations

Microorganisms are the major scavengers in nature, responsible for recycling most natural waste materials into harmless compounds. When faced with an increasing array of synthetic compounds, such as various chlorinated compounds manufactured by the chemical industry for use as herbicides/pesticides, industrial solvents, refrigerants, etc., the microorganisms attempt to evolve new genes, particularly for simple lowly chlorinated compounds, encoding enzymes that use the chlorinated compounds as their primary substrates. An understanding of how new biodegradative genes evolve in nature is therefore of the utmost importance to enhance the rate and the range of biodegradative processes. The various parameters that influence both natural as well as selective evolutionary processes are discussed.

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.

BACKGROUND Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution. OBJECTIVE To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture. METHODS Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded. RESULTS One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay. CONCLUSION The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting.

XtracTB Assay, a Mycobacterium tuberculosis molecular screening test with sensitivity approaching culture

Nucleic acid amplification tests are increasingly used to diagnose tuberculosis (TB) due to their speed and sensitivity compared to sputum smear microscopy. However, these tests fail to equal culture’s sensitivity with sputum smear microscopy negative specimens and therefore cannot be used to rule out TB disease. For molecular tests to match culture’s sensitivity, they must detect ≤10 genomic copies of Mycobacterium tuberculosis (MTB) DNA, the limit of detection of culture, process ≥1 ml of sputum ensuring sufficient number of MTB are in the reaction, and efficiently remove sputum associated inhibitors from this large sample. Here we report the preliminary characterization of XtracTB Assay, a MTB testing protocol designed for inclusion in either an integrated point-of-care platform or a high throughput automated central laboratory system. The test combines DNA sequence specific sample prep to reduce the co-extraction of qPCR inhibitors with the amplification of two MTB specific loci (IS6110 and senX3-regX3) to increase test sensitivity and minimize the likelihood of false negatives. The analytical sensitivity of the XtracTB Assay was 5 genomic copies/ml of sputum rivaling that of culture. Furthermore, 142 valid test results yield clinical sensitivity of 94.9% (95% CI: 90.1–99.9) and specificity of 100% (95% CI: 90.0–100.0).

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

Commercial products to preserve specimens for tuberculosis diagnosis: a systematic review.

SETTING Eliminating tuberculosis in high-burden settings requires improved diagnostic capacity. Important tests such as Xpert® MTB/RIF and culture are often performed at centralised laboratories that are geographically distant from the point of specimen collection. Preserving specimen integrity during transportation, which could affect test performance, is challenging. OBJECTIVE To conduct a systematic review of commercial products for specimen preservation for a World Health Organization technical consultation. DESIGN Databases were searched up to January 2018. Methodological quality was assessed using Quality Assessment of Technical Studies, a new technical study quality-appraisal tool, and Quality Assessment of Diagnostic Accuracy Studies-2. Studies were analysed descriptively in terms of the different products, study designs and diagnostic strategies used. RESULTS Four products were identified from 16 studies: PrimeStore-Molecular-Transport-Medium (PS-MTM), FTA card, GENO•CARD (all for nucleic acid amplification tests [NAATs]) and OMNIgene•SPUTUM (OMS; culture, NAATs). PS-MTM, but not FTA card or GENO•CARD, rendered Mycobacterium tuberculosis non-culturable. OMS reduced Löwenstein-Jensen but not MGIT™ 960™ contamination, led to delayed MGIT time-to-positivity, resulted in Xpert performance similar to cold chain-transported untreated specimens, and obviated the need for N-acetyl-L-cysteine-sodium hydroxide decontamination. Data from paucibacillary specimens were limited. Evidence that a cold chain improves culture was mixed and absent for Xpert. The effect of the product alone could be discerned in only four studies. CONCLUSION Limited evidence suggests that transport products result in test performance comparable to that seen in cold chain-transported specimens.