Estimation of Measurement Uncertainty in Quantitation of Benzoylecgonine (BZE) and 11-nor-Δ9-THC-9-carboxylic acid (THCA).

Calculating measurement uncertainty is a helpful exercise for identifying components or steps in a forensic analytical procedure that contribute to measurement variance. In this study, we used a bottom up approach to identify components in our procedures that contribute to measurement variance in our Department of Defense (DoD) Drug Demand Reduction Program (DDRP) Gas Chromatography Mass Spectroscopy (GCMS) analytical procedures for benzoylecgonine (BZE) and the THC metabolite, 11-nor-Δ9-THC-9-carboxylic acid (THCA) at 125% the DDRP concentration threshold (cutoff). Each assay was run 10 times over 30 days, each assay containing five calibrators and five samples (125%). Measurement uncertainty was estimated to be ±7.6 and ±0.6 ng/mL, for the BZE and THCA methods, respectively (α = 0.05). In both assays, method precision and the preparation of calibrator and samples were major contributors to measurement uncertainty. While this exercise will help with evaluating assay performance from a Quality Assurance perspective, these estimates should not be applied in interpreting DDRP test results. DDRP cut offs are already inherently conservative being above the Limit of Quantitation and were developed taking into consideration variability in assay performance across instruments and laboratories within the DDRP drug testing system.

An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism.

Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites.

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch-like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

Structural and functional characterization of MppR, an enduracididine biosynthetic enzyme from streptomyces hygroscopicus: functional diversity in the acetoacetate decarboxylase-like superfamily.

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus. Enduracididine is produced in Streptomyces hygroscopicus by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5'-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of "4-hydroxy-2-ketoarginine" shows the correct 4R enantiomer of "2-ketoenduracididine" bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function.

Genes expressed by the biological control bacterium Pseudomonas protegens Pf-5 on seed surfaces under the control of the global regulators GacA and RpoS.

Gene expression profiles of the biological control strain Pseudomonas protegens Pf-5 inhabiting pea seed surfaces were revealed using a whole-genome oligonucleotide microarray. We identified genes expressed by Pf-5 under the control of two global regulators (GacA and RpoS) known to influence biological control and secondary metabolism. Transcript levels of 897 genes, including many with unknown functions as well as those for biofilm formation, cyclic diguanylate (c-di-GMP) signalling, iron homeostasis and secondary metabolism, were influenced by one or both regulators, providing evidence for expression of these genes by Pf-5 on seed surfaces. Comparison of the GacA and RpoS transcriptomes defined for Pf-5 grown on seed versus in broth culture overlapped, but most genes were regulated by GacA or RpoS under only one condition, likely due to differing levels of expression in the two conditions. We quantified secondary metabolites produced by Pf-5 and gacA and rpoS mutants on seed and in culture, and found that production profiles corresponded generally with biosynthetic gene expression profiles. Future studies evaluating biological control mechanisms can now focus on genes expressed by Pf-5 on seed surfaces, the habitat where the bacterium interacts with seed-infecting pathogens to suppress seedling diseases.

Phloroglucinol mediates cross-talk between the pyoluteorin and 2,4-diacetylphloroglucinol biosynthetic pathways in Pseudomonas fluorescens Pf-5.

The antibiotics pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) contribute to the biological control of soilborne plant diseases by some strains of Pseudomonas fluorescens, including Pf-5. These secondary metabolites also have signalling functions with each compound reported to induce its own production and repress the other's production. The first step in DAPG biosynthesis is production of phloroglucinol (PG) by PhlD. In this study, we show that PG is required at nanomolar concentrations for pyoluteorin production in Pf-5. At higher concentrations, PG is responsible for the inhibition of pyoluteorin production previously attributed to DAPG. DAPG had no effect on pyoluteorin production, and monoacetylphloroglucinol showed both stimulatory and inhibitory activities but at concentrations 100-fold greater than the levels of PG required for similar effects. We also demonstrate that PG regulates pyoluteorin production in P. aeruginosa and that a phlD gene adjacent to the pyoluteorin biosynthetic gene cluster in P. aeruginosa strain LESB58 can restore pyoluteorin biosynthesis to a ΔphlD mutant of Pf-5. Bioinformatic analyses show that the dual role of PhlD in the biosynthesis of DAPG and the regulation of pyoluteorin production could have arisen within the pseudomonads during the assembly of these biosynthetic gene clusters from genes and gene subclusters of diverse origins.

Inactivation of the GacA response regulator in Pseudomonas fluorescens Pf-5 has far-reaching transcriptomic consequences.

The GacS/GacA signal transduction system is a central regulator in Pseudomonas spp., including the biological control strain P. fluorescens Pf-5, in which GacS/GacA controls the production of secondary metabolites and exoenzymes that suppress plant pathogens. A whole genome oligonucleotide microarray was developed for Pf-5 and used to assess the global transcriptomic consequences of a gacA mutation in P. fluorescens Pf-5. In cultures at the transition from exponential to stationary growth phase, GacA significantly influenced transcript levels of 635 genes, representing more than 10% of the 6147 annotated genes in the Pf-5 genome. Transcripts of genes involved in the production of hydrogen cyanide, the antibiotic pyoluteorin and the extracellular protease AprA were at a low level in the gacA mutant, whereas those functioning in siderophore production and other aspects of iron homeostasis were significantly higher in the gacA mutant than in wild-type Pf-5. Notable effects of gacA inactivation were also observed in the transcription of genes encoding components of a type VI secretion system and cytochrome c oxidase subunits. Two novel gene clusters expressed under the control of gacA were identified from transcriptome analysis, and we propose global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp.