Use of QuEChERS as a manual and automated high-throughput protocol for investigating environmental matrices.

Environmental pollution has strong links to adverse human health outcomes with risks of pollution through production, use, ineffective wastewater (WW) remediation, and/or leachate from landfill. 'Fit-for-purpose' monitoring approaches are critical for better pollution control and mitigation of harm, with current sample preparation methods for complex environmental matrices typically time-consuming and labour intensive, unsuitable for high-throughput screening. This study has shown that a modified 'Quick Easy Cheap Effective Rugged and Safe' (QuEChERS) sample preparation is a viable alternative for selected environmental matrices required for pollution monitoring (e.g. WW effluent, treated sludge cake and homogenised biota tissue). As a manual approach, reduced extraction times (hours to ∼20 min/sample) with largely reproducible (albeit lower) recoveries of a range of pharmaceuticals and biocidal surfactants have been reported. Its application has shown clear differentiation of matrices via chemometrics, and the measurement of pollutants of interest to the UK WW industry at concentrations significantly above suggested instrument detection limits (IDL) for sludge, indicating insufficient removal and/or bioaccumulation during WW treatment. Furthermore, new pollutant candidates of emerging concern were identified - these included detergents, polymers and pharmaceuticals, with quaternary ammonium compound (QAC) biocides observed at 2.3-70.4 mg/kg, and above levels associated with priority substances for environmental quality regulation (EQSD). Finally, the QuEChERS protocol was adapted to function as a fully automated workflow, further reducing the resource to complete both the preparation and analysis to <40 min. This operated with improved recovery for soil and biota (>62%), and when applied to a largely un-investigated clay matrix, acceptable recovery (88.0-131.1%) and precision (≤10.3% RSD) for the tested pharmaceuticals and biocides was maintained. Therefore, this preliminary study has shown the successful application of a high-throughput QuEChERS protocol across a range of environmental solids for potential deployment in a regulated laboratory.

An investigation of the utility of QuEChERS for extracting acid, base, neutral and amphiphilic species from example environmental and clinical matrices.

Accurate measurement of the composition of complex samples is key for the safety and efficacy of a range of products used in daily life, with sample preparation a critical step in this workflow. QuEChERS is one such method, however published protocols do not explicitly address acidic, basic, neutral, and amphiphilic species in a single protocol and often use extra steps or an alternative preparation to recover the breadth of chemical types. Our work addresses this need by investigating the use of QuEChERS for monitoring this wide range of chemistries within environmental solids and blood plasma, using a protocol that can accommodate both milliliter and microliter sample volumes. While published methods can require significant resource and time, our approach offers a reduction in preparation time (for environmental samples), with the "micro-QuEChERS" protocol offering a further reduction in cost. The analytical performance of these methods were assessed using reversed-phase LC-MS and showed good accuracy, precision, and sensitivity for the expected concentrations in the tested applications. Target analytes of variable lipophilicity/acidity were extracted and isolated from soil, with largely repeatable matrix effects < 15%RSD and recoveries of 39-100%. An initial "proof-of-concept" investigation using the "micro-QuEChERS" protocol showed reduced matrix enhancement (median value of 90%ME) for soil, and improved matrix effects and recovery (>65%) for blood plasma. This novel sample preparation method can therefore offer an improved approach with wider applicability providing "cleaner" extracts than other methods used for high-throughput clinical analysis.

Production of specialized metabolites by Streptomyces coelicolor A3(2).

The actinomycetes are well-known bioactive natural product producers, comprising the Streptomycetes, the richest drug-prolific family in all kingdoms, producing therapeutic compounds for the areas of infection, cancer, circulation, and immunity. Completion and annotation of many actinomycete genomes has highlighted further how proficient these bacteria are in specialized metabolism, which have been largely underexploited in traditional screening programs. The genome sequence of the model strain Streptomyces coelicolor A3(2), and subsequent development of genomics-driven approaches to understand its large specialized metabolome, has been key in unlocking the high potential of specialized metabolites for natural product genomics-based drug discovery. This review discusses systematically the biochemistry and genetics of each of the specialized metabolites of S. coelicolor and describes metabolite transport processes for excretion and complex regulatory patterns controlling biosynthesis.

Draft Genome Sequence of Rhodococcus rhodnii Strain LMG5362, a Symbiont of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the Principle Vector of Trypanosoma cruzi.

We report the 4,385,577-bp high-quality draft assembly of the bacterial symbiont Rhodococcus rhodnii strain LMG5362, isolated from the gut of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the principle vector of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This sequence might provide useful information for subsequent studies of the symbiotic relationship between Rd. prolixus and Rc. rhodnii, while also providing a starting point for the development of biotechnological applications for the control of Rd. prolixus.

Negatively-marked MCQ assessments that reward partial knowledge do not introduce gender bias yet increase student performance and satisfaction and reduce anxiety.

Multiple-choice question (MCQ) examinations are increasingly used as the assessment method of theoretical knowledge in large class-size modules in many life science degrees. MCQ-tests can be used to objectively measure factual knowledge, ability and high-level learning outcomes, but may also introduce gender bias in performance dependent on topic, instruction, scoring and difficulty. The 'Single Answer' (SA) test is often used in which students choose one correct answer, in which they are unable to demonstrate partial knowledge. Negatively marking eliminates the chance element of guessing but may be considered unfair. Elimination testing (ET) is an alternative form of MCQ, which discriminates between all levels of knowledge, while rewarding demonstration of partial knowledge. Comparisons of performance and gender bias in negatively marked SA and ET tests have not yet been performed in the life sciences. Our results show that life science students were significantly advantaged by answering the MCQ test in elimination format compared to single answer format under negative marking conditions by rewarding partial knowledge of topics. Importantly, we found no significant difference in performance between genders in either cohort for either MCQ test under negative marking conditions. Surveys showed that students generally preferred ET-style MCQ testing over SA-style testing. Students reported feeling more relaxed taking ET MCQ and more stressed when sitting SA tests, while disagreeing with being distracted by thinking about best tactics for scoring high. Students agreed ET testing improved their critical thinking skills. We conclude that appropriately-designed MCQ tests do not systematically discriminate between genders. We recommend careful consideration in choosing the type of MCQ test, and propose to apply negative scoring conditions to each test type to avoid the introduction of gender bias. The student experience could be improved through the incorporation of the elimination answering methods in MCQ tests via rewarding partial and full knowledge.

A laterally acquired galactose oxidase-like gene is required for aerial development during osmotic stress in Streptomyces coelicolor.

Phylogenetic reconstruction revealed that most Actinobacterial orthologs of S. coelicolor SCO2837, encoding a metal-dependent galactose oxidase-like protein, are found within Streptomyces and were probably acquired by horizontal gene transfer from fungi. Disruption of SCO2837 (glxA) caused a conditional bld phenotype that could not be reversed by extracellular complementation. Studies aimed at characterising the regulation of expression of glxA showed that it is not a target for other bld genes. We provide evidence that glxA is required for osmotic adaptation, although independently from the known osmotic stress response element SigB. glxA has been predicted to be part of an operon with the transcription unit comprising the upstream cslA gene and glxA. However, both phenotypic and expression studies indicate that it is also expressed from an independent promoter region internal to cslA. GlxA displays an in situ localisation pattern similar to that one observed for CslA at hyphal tips, but localisation of the former is independent of the latter. The functional role of GlxA in relation to CslA is discussed.

The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases.

Streptomyces coelicolor A3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all three narGHJI operons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triple narGHJI mutant, the moaA mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in S. coelicolor are also molybdenum-dependent. Analysis of S. coelicolor growth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-type S. coelicolor spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the moaA and triple nar mutants were chlorate-resistant. Complementation of the triple nar mutant with the individual narGHJI operons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nar1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.

Antibiotic overproduction in Streptomyces coelicolor A3 2 mediated by phosphofructokinase deletion.

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

The obligate aerobic actinomycete Streptomyces coelicolor A3(2) survives extended periods of anaerobic stress.

The actinomycete Streptomyces coelicolor is an obligate aerobe that is found in soil and aqueous habitats. The levels of oxygen in these environments can vary considerably, which raises the question of how these bacteria survive during periods of anaerobiosis. Although S. coelicolor cannot grow in the complete absence of oxygen, we demonstrate here that it is capable of microaerobic growth and maintaining viability through several weeks of strict anaerobiosis. Both resting and germinated spores are able to survive abrupt exposure to anaerobiosis, which contrasts the situation with Mycobacterium species where gradual oxygen depletion is required to establish a latent state in which the bacterium is able to survive extended periods of anaerobiosis. Growth of S. coelicolor resumes immediately upon re-introduction of oxygen. Taken together these findings indicate that survival is not restricted to spores and suggest that the bacterium has evolved a mechanism to maintain viability and a membrane potential in the hyphal state. Furthermore, although we demonstrate that several members of the genus also survive long periods of anaerobic stress, one species, Streptomyces avermitilis, does not have this capacity and might represent a naturally occurring variant that is unable to adopt this survival strategy.

Gas vesicles in actinomycetes: old buoys in novel habitats?

Gas vesicles are gas-filled prokaryotic organelles that function as flotation devices. This enables planktonic cyanobacteria and halophilic archaea to position themselves within the water column to make optimal use of light and nutrients. Few terrestrial microbes are known to contain gas vesicles. Genome sequences that have become available recently for many bacteria from non-planktonic habitats reveal gas vesicle gene clusters in members of the actinomycete genera Streptomyces, Frankia and Rhodococcus, which typically live in soils and sediments. Remarkably, there is an additional level of complexity in cluster number and gene content. Here, we discuss whether putative gas vesicle proteins in these actinomycetes might actually be involved in flotation or whether they might fulfil other cellular functions.

Deletion of the yiaMNO transporter genes affects the growth characteristics of Escherichia coli K-12.

Binding-protein-dependent secondary transporters make up a unique transport protein family. They use a solute-binding protein in proton-motive-force-driven transport. Only a few systems have been functionally analysed. The yiaMNO genes of Escherichia coli K-12 encode one family member that transports the rare pentose l-xylulose. Its physiological role is unknown, since wild-type E. coli K-12 does not utilize l-xylulose as sole carbon source. Deletion of the yiaMNO genes in E. coli K-12 strain MC4100 resulted in remarkable changes in the transition from exponential growth to the stationary phase, high-salt survival and biofilm formation.

Improved method for the isolation of RNA from (standing liquid cultures of) Streptomycetes.

Streptomycetes are complex soil bacteria capable of producing aerial reproductive mycelium and secondary metabolites. We observed novel phenomena such as an extended life cycle including flotation and anaerobiosis using standing liquid cultures. This paper describes an improved method for isolating good quality RNA from standing liquid cultures of S. coelicolor via excellent cell lysis.

Improved recovery of DNA from polyacrylamide gels after in situ DNA footprinting.

Methods used to date for the isolation of DNA from polyacrylamide gels are elution based, time-consuming and with low yield in DNA. This paper describes an improved system employing polyacrylamide gels made of a meltable matrix. The new system was successfully applied to in situ DNA footprinting following gel retardation assays.

Analysis of DNA binding and transcriptional activation by the LysR-type transcriptional regulator CbbR of Xanthobacter flavus.

The LysR-type transcriptional regulator CbbR controls the expression of the cbb and gap-pgk operons in Xanthobacter flavus, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO2 fixation. The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and cbb promoter activity were used to show that the potential CbbR binding sequences are functional. Inverted repeat IR1 is a high-affinity CbbR binding site. The main function of this repeat is to recruit CbbR to the cbb operon promoter. In addition, it is required for negative autoregulation of cbbR expression. IR3 represents the main low-affinity binding site of CbbR. Binding to IR3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR1 also prevent binding to the low-affinity site. Although mutations in IR3 have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity. This is most likely due to steric hindrance of RNA polymerase by CbbR since IR3 partially overlaps with the -35 region of the cbb operon promoter. Mutations in IR2 do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the cbb operon promoter. This IR2 binding site is therefore critical for transcriptional activation by CbbR.

Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor.

Streptomyces coelicolor differentiates on solid agar media by forming aerial hyphae that septate into spores. We here show that differentiation also occurs in standing liquid minimal media. After a period of submerged growth, hyphae migrate to the air interface, where they become fixed by a rigid reflecting film. Colonies that result from these hyphae form sporulating aerial hyphae. In addition, submerged hyphae in the liquid minimal medium may attach to the surface. Liquid standing cultures easily become anoxic only 1 to 2 mm below the surface. Yet, biomass increases, implying the existence of metabolic pathways supporting anaerobic growth.

Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface.

The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (DeltardlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the DeltardlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.

Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism.

The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate on medium supplemented with gluconate or succinate. The regulation of the gap-pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1.1 kb ApaI fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism.