Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases

Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC–MS/MS). Steady-state kinetics experiments showed similar k_cat and K_M values for both enzymes. In addition, similar IC₅₀ values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.     

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3^∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3^∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3^∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.     

A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis

Cofactor biosynthetic pathways represent a rich source of potential antibiotic targets. The second step in biotin biosynthesis is performed by BioA, a pyridoxal 5′-phosphate (PLP)-dependent enzyme. This enzyme has been confirmed as a candidate target in Mycobacterium tuberculosis; however, the current bioassay used to measure BioA activity is cumbersome and low throughput. Here we describe the design, development, and optimization of a continuous coupled fluorescence displacement assay to measure BioA activity. In this coupled assay, BioD converts the product of the BioA-catalyzed reaction into dethiobiotin, which is subsequently detected by displacement of a fluorescently labeled dethiobiotin probe from streptavidin. The assay was further adapted to a high-throughput screening format and validated against the LOPAC¹²⁸⁰ library.     

An Improved Screening Method for Inhibitors of Nucleic Acid Synthesis

A screening method for the antibiotics capable of inhibiting synthesis of nucleic acid in bacterial cells or mammalian tumor cells was investigated. The DNA and RNA syntheses in Bacillus subtilis 168 thymine^?, indole^? were studied by the assay of incorporations of ³H-thymine and ¹⁴C-uracil into the cells, respectively. With known antibiotics against nucleic acid synthesis, the adequacy of the method was examined, and the result proved that this method is more sensitive and specific than the conventional assay methods.

It was found as a new fact that cellocidin is a potent and specific inhibitor to the thymine incorporation into DNA.

By an almost similar procedure, an inhibitory effect of several antibiotics on the incorporations of ³H-thymidine and ¹⁴C-uridme into Ehrlich ascites carcinoma cells was also studied.     

Selection of Brassica rapa genotypes for tolerance to boron toxicity

Although boron (B) is a micronutrient essential for the growth of vascular plants, it reduces growth and seed yield when present in excessive amounts. A hydroponic assay of nineteen Brassica rapa genotypes resulted in the identification of two tolerant genotypes, WWY Sarson and Local at a range of boron concentrations (15–165 μM). The most tolerant and sensitive genotypes were assessed for shoot boron concentrations in a soil assay with 4, 29 and 54 mg B kg⁻¹ soil. The soil assay confirmed the results of the hydroponic screening. Shoot boron uptake was at least three times lower and shoot boron concentrations about 10 times lower in the tolerant than sensitive genotypes, indicating that boron tolerance involved boron exclusion from the shoot.     

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNA^Asp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNA^Asp with a K_d value that is comparable to their IC₅₀ value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.     

Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites

We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodium berghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1αa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC₅₀ values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1αa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.     

Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs

A total of 124 samples were collected from the intestine of broiler chickens, piglet faeces, fermented foods, soils and Chinese herbs. More than 750 strains of aerobic, spore-forming bacteria were isolated from these samples. The inhibitory activity of these spore-forming strains against Escherichia coli K88, E. coli K99, Salmonella typhimurium and Staphylococcus aureus was assessed using a disc plate diffusion assay. The six bacilli with the largest inhibition zones against the four indicator bacteria were chosen and assessed for their resistance to unfavorable conditions within simulated gut environments. The strain Bacillus subtilis MA139 showed full resistance to pH 2, 0.3% bile salts and exhibited the highest antimicrobial activity. Based on these results, B. subtilis MA139 was selected as a potential probiotic and fed to piglets at concentrations of 2.2 × 10⁵, 2.2 × 10⁶ or 2.2 × 10⁷ CFU/g of feed during a 28-day feeding trial. A negative control consisting of the basal diet with no additives and a positive control consisting of the basal diet supplemented with 16 g/ton flavomycin were also included. Ninety piglets between 35 and 40 days old were used in the in vivo animal trials. B. subtilis MA139 enhanced daily gain (P = 0.10) and feed conversion (P = 0.03) compared with the negative control. The performance of pigs fed B. subtilis MA139 supplemented diets did not differ from that of pigs fed the antibiotic diet. There was a significant increase in Lactobacilli cell counts (P = 0.02) and a numerical decrease in E. coli counts (P = 0.05) in the faecal samples of pigs fed B. subtilis MA139 with 2.2 × 10⁵ CFU/g of feed. The overall results of this study show that the use of initial co-culture with indicator pathogens, a disc plate diffusion assay and simulated gut environment tolerance tests is one of effective methods of screening Bacillus for probiotic use and that B. subtilis MA139 is a promising alternative to antibiotics for use as a feed additive in piglet diets.     

Detection of Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Summary Environmental monitoring is important to enable effective resource management and public health protection as well as rapid and accurate identification of Vibrio cholerae in drinking-water sources. Traditional methods employed in identification are laborious, time-consuming and practically not viable for screening of a large number of samples. In this study, a direct cell duplex PCR assay for the detection of viable toxigenic V. cholerae in environmental water samples was developed. In the PCR assay, two gene sequences were amplified together, one of outer membrane protein (ompW), which is species-specific and another of cholera toxin (ctxAB). The detection limit of duplex PCR was 5 × 10⁴ V. cholerae/reaction. Different environmental water samples were artificially spiked with V. cholerae O1 cells and filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly in the duplex PCR assay. The PCR procedure coupled with enrichment could detect as few as 1.2 c.f.u./ml in ground water, 1.2 × 10² c.f.u. ml⁻¹ in sewer water and 1.2 × 10³c.f.u. ml⁻¹ in tap water. The assay was successfully applied directly for screening of environmental potable water samples collected from a cholera-affected area. The proposed method is simple and can be used for environmental monitoring of toxigenic as well as non-toxigenic V. cholerae.     

Two-site automated chemiluminescent assay for measurement of immunoreactive renin

Measurement of renin is important for the clinical assessment of hypertensive patients and for the screening for primary aldosteronism. The aim of this study was to evaluate the performances of an automated immunoassay for measurement of immunoreactive renin. Functional sensitivity, in vitro stability, and reference values were determined. Method comparison with the plasma renin activity assay was also performed. Our results demonstrate that the Liaison^® direct renin assay may assist the clinician in the assessment of hypertensive patients and in the screening for primary aldosteronism.     

A qauntitative in vitro assay for the evaluation of phototoxic potential of topically applied materials

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2–500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.Abbreviations C centigrade - cm centimeter - cm² square centimeter - ml milliliters - mm millimeters - 8-MOP 8-methoxy psoralen - mW milliwatt - nm nanometer - UVA ultraviolet radiation, 320–400 nm The first in a series of research papers on alternatives to animal toxicity studies.     

Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta)

A stable transformation system for theexpression of foreign genes in theunicellular marine green alga Dunaliella salina was established. Amongfive antibiotics, 60 g mL^-1chloramphenicol completely inhibitedgrowth. Of five promoters tested, theubiquitin-$Ohgr; promoter yielded thehighest -glucuronidase (GUS) activity.The hepatitis B surface antigen (HBsAg)gene was introduced into the cells by usingelectroporation. PCR and Southern blotanalysis amd it was shown that the gene wasintegrated into the genome. The stableexpression of HBsAg protein was confirmedby enzyme-linked immunosorbent assay(ELISA) and Western blot analysis. Theintroduced DNA and HBsAg expression weremaintained stable for at least 60generations in medium devoid ofchloramphenicol. This is an important steptoward the production of useful foreignproteins in the alga.     

Identifi cation of antibiotics that are effective 2 in eliminatingAgrobacterium tumefaciens

An assay was performed to identify the antibiotics that are most effective againstAgrobacterium tumefaciens strains EHA101 and LBA4404, and to determine if these antibiotics inhibited tobacco callus and shoot formation. We tested ten antibiotics: cefotaxime, carbenecillin, erythromycin, spectinomycin, polymixin B, chloramphenicol, methicillin, Augmentin 500, Augmentin 250, and moxalactam. The effectiveness of each antibiotic against the two strains was determined by measuring the zones of inhibition of bacterial growth in a disk-diffusion assay. The five antibiotics that generated the largest zones of inhibition for each strain were assayed to determine their effects on callus formation. Cefotaxime was the most active antibiotic tested against strain LBA4404, and moxalactam was the most effective antibiotic against EHA101. Both cefotaxime and moxalactam had little or no effect on callus development.     

Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles

A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01–0.2 mg ml⁻¹). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.     

Effect of Some Antibiotics on the In Vitro Morphogenetic Response from Callus Cultures of Coryphantha Elephantidens

The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm^–3 kanamycin, which was under study.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Rapid screening of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mutants for overproduction of lovastatin

Summary A novel rapid screening method is demonstrated for isolating lovastatin-overproducing strains of Aspergillus terreus. The screening methodology, based on the activity of lovastatin against the yeast Candida albicans, is nearly three times as fast as the selection methods used earlier. The new 6-h assay shows a linear correlation between the quantity of lovastatin generated by A. terreus isolates and the inhibition zones obtained on plates of C. albicans. The new technique is less expensive and requires less labour.     

A non-PCR-based Invader<Superscript>®</Superscript> assay quantitatively detects single-copy genes in complex plant genomes

An accurate determination of gene copy number is critical to the success of a molecular breeding program involving both transgenic and non-transgenic plants. In this paper, we have described the application of a non-PCR-based technology, Invader^®*, for determination of gene copy number and zygosity in plants. A biplex assay format detected both a target gene and an endogenous reference gene simultaneously from the genomic DNA. The ratio between the signals of the two genes in relation to known copy number standards of the same target gene allowed copy number determination. The linear range of the Invader assay was 1–4 copies per genome, but it can be accurate over a larger copy number range depending on the assay conditions. This technique was utilized for screening plants carrying low transgene copy numbers from a large number of events generated by plant transformation, and shown to produce results comparable to that of Southern blots. We have also utilized this technique to screen thousands of field-grown plants for zygosity determinations and obtained data that was over 98% accurate, thus proving that this assay can be used to improve the efficiency of a breeding program. Overall, the Invader assays proved to be reproducible, specific, applicable to any gene sequence and amenable to high-throughput screening.     

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.     

A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

Threat of global warming due to carbon dioxide (CO₂) emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO₂ to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by the following: C[mmol C (g dry algae)^?1?h^?1]?=?0.064?×?(dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on S. platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina that were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)^?1?h^?1, respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool.