Rory M. Watt

Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis

The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg2+-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol. The enzymatic cyclization had a kcat of 6.2 ± 0.5 × 10−3 s−1 and a Km for farnesyl diphosphate of 62 ± 8 nM. Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower kcat of 3.2 ± 0.4 × 10−3 s−1 and a twofold greater km of 115 ± 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.

Identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics.

Abstract The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected more than 8,000 people across 29 countries and caused more than 900 fatalities. Based on the concept of chemical genetics, we screened 50,240 structurally diverse small molecules from which we identified 104 compounds with anti-SARS-CoV activity. Of these 104 compounds, 2 target the SARS-CoV main protease (Mpro), 7 target helicase (Hel), and 18 target spike (S) protein-angiotensin-converting enzyme 2 (ACE2)-mediated viral entry. The EC50 of the majority of the 104 compounds determined by SARS-CoV plaque reduction assay were found to be at low micromolar range. Three selected compounds, MP576, HE602, and VE607, validated to be inhibitors of SARS-CoV Mpro, Hel, and viral entry, respectively, exhibited potent antiviral activity (EC50 < 10 μM) and comparable inhibitory activities in target-specific in vitro assays.

The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5′ to 3′ Viral Helicases

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5′ single-stranded overhang, indicating a 5′o 3′ polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.

The adamantane-derived bananins are potent inhibitors of the helicase activities and replication of SARS coronavirus

Summary Bananins are a class of antiviral compounds with a unique structural signature incorporating a trioxa-adamantane moiety covalently bound to a pyridoxal derivative. Six members of this class of compounds: bananin, iodobananin, vanillinbananin, ansabananin, eubananin, and adeninobananin were synthesized and tested as inhibitors of the SARS Coronavirus (SCV) helicase. Bananin, iodobananin, vanillinbananin, and eubananin were effective inhibitors of the ATPase activity of the SCV helicase with IC50 values in the range 0.5–3 μM. A similar trend, though at slightly higher inhibitor concentrations, was observed for inhibition of the helicase activities, using a FRET-based fluorescent assay. In a cell culture system of SCV, bananin exhibited an EC50 of less than 10 μM and a CC50 of over 300 μM. Kinetics of inhibition are consistent with bananin inhibiting an intracellular process or processes involved in SCV replication.

Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development

BackgroundElucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.ResultsA significant reduction in colony forming units (CFU) of C. parapsilosis (90 min), C. albicans and C. tropicalis (90 min, 24 h and 48 h), C. dubliniensis and C. glabrata, (24 h and 48 h) was noted when co-cultured with P. aeruginosa in comparison to their monospecies counterparts (P < 0.05). A simultaneous significant reduction in P. aeruginosa numbers grown with C. albicans (90 min and 48 h), C. krusei (90 min, 24 h and 48 h),C. glabrata, (24 h and 48 h), and an elevation of P. aeruginosa numbers co-cultured with C. tropicalis (48 h) was noted (P < 0.05). When data from all Candida spp. and P. aeruginosa were pooled, highly significant mutual inhibition of biofilm formation was noted (Candida P < 0.001, P. aeruginosa P < 0.01). Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses confirmed scanty architecture in dual species biofilm in spite of dense colonization in monospecies counterparts.ConclusionsP. aeruginosa and Candida in a dual species environment mutually suppress biofilm development, both quantitatively and qualitatively. These findings provide a foundation to clarify the molecular basis of bacterial-fungal interactions, and to understand the pathobiology of mixed bacterial-fungal infections.

Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli

BackgroundThe low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).ResultsIn this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.ConclusionGcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

Significance Aptamers are oligonucleotides selected and evolved to bind tightly and specifically to molecular targets. Aptamers have promise as diagnostic tools and therapeutic agents, but little is known about how they recognize or discriminate their targets. In this study, X-ray crystallography together with several other biophysical techniques reveal how a new DNA aptamer recognizes and discriminates Plasmodium lactate dehydrogenase, a protein marker that is a diagnostic indicator of infection with the malaria parasite. We also demonstrate application of the aptamer in target detection. This study broadens our understanding of aptamer-mediated molecular recognition and provides a DNA aptamer that could underpin new innovative approaches for point-of-care malaria diagnosis. DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.

The involvement of replication in single stranded oligonucleotide-mediated gene repair

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the ‘annealing-integration’ model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a λ-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.

Aptamer-Mediated Inhibition of Mycobacterium tuberculosis Polyphosphate Kinase 2

Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzymes catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC(50) of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC(50) of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2.

Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation

Demystification of microbial behaviour in mixed biofilms could have a major impact on our understanding of infectious diseases. The objectives of this study were to evaluate in vitro the interactions of six different Candida species and a Gram-negative coliform, Escherichia coli, in dual-species biofilms, and to assess the effect of E. coli LPS on Candida biofilm formation. A single isolate of E. coli ATCC 25922 and six different species of Candida, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA-646, were studied using a standard biofilm assay. Each Candida species was co-cultured with E. coli on a polystyrene surface and biofilm formation was quantified by a c.f.u. assay. The biofilm was then analysed by Live/Dead staining and fluorescence microscopy (confocal laser-scanning microscopy, CLSM), whilst scanning electron microscopy (SEM) was employed to visualize the biofilm architecture. The effect of E. coli LPS on Candida biofilm cell activity at defined time intervals was assessed with an XTT reduction assay. A significant quantitative reduction in c.f.u. counts of C. tropicalis (after 90 min), C. parapsilosis (after 90 min and 24 h), C. krusei (after 24 h) and C. dubliniensis (after 24 and 48 h) was noted on incubation with E. coli in comparison with their monospecies biofilm counterparts (P <0.05). On the other hand, a simultaneous and significant reduction in E. coli cell numbers occurred on co-culture with C. albicans (after 90 min), and an elevation of E. coli cell numbers followed co-culture with C. tropicalis (after 24 h) and C. dubliniensis (after 24 h and 48 h) (P <0.05). All quantitative findings were confirmed by SEM and CLSM analyses. By SEM observation, dual-species biofilms demonstrated scanty architecture with reduced visible cell counts at all stages of biofilm development, despite profuse growth and dense colonization in their single-species counterparts. Significantly elevated metabolic activity, as assessed by XTT readings, was observed in E. coli LPS-treated C. tropicalis and C. parapsilosis biofilms (after 48 h), whilst this had the opposite effect for C. dubliniensis (after 24 h) (P <0.05). These data indicate that E. coli and Candida species in a mixed-species environment mutually modulate biofilm development, both quantitatively and qualitatively, and that E. coli LPS appears to be a key component in mediating these outcomes.