Advice to consider when developing a CRISPR-Cas assay for single species detection using eDNA

Development of simple and rapid techniques to monitor species of conservation importance is vital to further the capabilities of environmental DNA. Conventional methods for eDNA detection pose a logistical challenge for on-site monitoring due to the need for high temperatures and thermal cycling. To circumvent this, we recently adapted an isothermal CRISPR-Cas based detection assay for single-species assessment of Salmo salar as a route to a simple, cost-effective biosensor device (Williams et al., 2019). CRISPR-Cas for detection (rather than genome editing) was first developed for clinical diagnostic applications. The variety of Cas nucleases allow detection of either RNA or DNA with attomolar sensitivity (Chen et al., 2018; Gootenberg et al., 2017). This detection approach is versatile and has recently been adopted for the detection of SARS-CoV-2 (Broughton et al., 2020). The CRISPR-Cas detection system consists of two main elements; a guide RNA specific to the target and an effector Cas12a nuclease. The Cas12a nuclease will only cleave at the target site when a specific protospacer adjacent motif (PAM) is present downstream. The requirement to recognise two separate sequences supports a highly specific recognition system that can distinguish closely related species. However, although its use is expanding rapidly for the detection of pathogens, it is yet to be fully embraced for eDNA detection. The RPA-CRISPR-Cas methodology we have developed utilises the isothermal recombinase polymerase amplification and CRISPR-Cas12a detection, leading to four unique sequence recognition elements, which require stringent design and in-lab testing to ensure assay specificity. Development of our published S. salar CRISPR-Cas assay (Williams et al., 2019), and subsequent assays for Salmo trutta and Salvelinus alpinus, highlighted critical steps to consider and pitfalls to avoid when designing such isothermal assays. 1) Only the target sequence should contain the required PAM site. In version 1 of our assay, both S. salar and S. trutta contained the PAM site; we were unable to distinguish them. In version 1 of our assay, both S. salar and S. trutta contained the PAM site; we were unable to distinguish them. 2) An RPA primer screen is essential. Multiple forward and reverse primers are screened up-/down-stream of the gRNA binding region to select the optimum primer pair. Multiple forward and reverse primers are screened up-/down-stream of the gRNA binding region to select the optimum primer pair. 3) Specificity tests should be carried out on tissue from the target species and other species present in the sampling environment. In silico design is not sufficient to ensure assay specificity. In silico design is not sufficient to ensure assay specificity. References Broughton, J. P., Deng, X., Yu, G., Fasching, C. L., Servellita, V., Singh, J., … Chiu, C. Y. (2020). CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology, 38(7). https://doi.org/10.1038/s41587-020-0513-4 Chen, J. S., Ma, E., Harrington, L. B., Costa, M. Da, Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436–439. https://doi.org/10.1126/SCIENCE.AAR6245 Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … Zhang, F. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356(6336), 438–442. https://doi.org/10.1126/science.aam9321 Williams, M. A., O’Grady, J., Ball, B., Carlsson, J., de Eyto, E., McGinnity, P., … Parle-McDermott, A. (2019). The application of CRISPR-Cas for single species identification from environmental DNA. Molecular Ecology Resources, 19(5). https://doi.org/10.1111/1755-0998.13045

Application of Response Surface Methodology for Optimization of Siderophore Production

Background: In the present study a statistical model (Response Surface Methodology) was proposed for optimization of siderophore production by using Enterobacter hormaechei.Methods: The rhizospheric soil was used for isolation and isolates were screened for siderophore production by chrome-azurol S (CAS) assay. One potent isolate producing maximum siderophore was selected and characterized by 16S rRNA gene sequencing. The culture conditions were optimized for maximum siderophore production by using Central Composite Design. The response surface curves were used to predict the optimum levels of the factors affecting the yield of siderophore. Result: By using rhizospheric soil,eight isolates were obtained and one potent organism was identified as Enterobacter hormaechei subsp. oharae (Accession No. MT 775835) by BLAST. The maximum siderophore production (98%) was obtained in succinate medium and the optimum values of variables were found as pH 7, time 60 hrs, temp. 28°C and succinic acid conc. 0.40%. RSM was used to analyze the data by developing 3D surface plots and the residuals plots. ANOVA was used to determine regression coefficients.

Discovery and biosynthesis of gladiochelins: unusual lipodepsipeptide siderophores from Burkholderia gladioli

Burkholderia is a genus of diverse Gram-negative bacteria that includes several opportunistic pathogens. Siderophores, which transport iron from the environment into microbial cells, are important virulence factors in most pathogenic Burkholderia species. However, it is widely believed that Burkholderia gladioli, which can infect the lungs of cystic fibrosis (CF) sufferers, does not produce siderophores. B. gladioli BCC0238, isolated from the lung of a CF patient, produces two novel metabolites in a minimal medium containing glycerol and ribose as carbon sources. HPLC purification, followed by detailed spectroscopic analyses, identified these metabolites as unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The absolute configurations of the amino acid residues in the two metabolites was elucidated using Marfey’s method and the gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of most B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S (CAS) assay showed the metabolites bind ferric iron and that this supresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes. Together, these observations suggest that these metabolites likely function as siderophores in B. gladioli.

The Serratia marcescens Siderophore Serratiochelin Is Necessary for Full Virulence during Bloodstream Infection

ABSTRACT Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.

EFFECT ON MICROBIAL PRODUCTS ON CAESIUM ELUTION BEHAVIOUR FROM CLAY MINERALS

Some microorganisms in the environment make siderophores, which are low molecular chelators, to take up minerals from soil. Eleven bacteria were separated from the root of white clover by chlome azrol S (CAS) assay. Each bacterium was incubated in casamino acid (CAA) culture, and siderophores in CAA culture were purified. These extractions were applied to biotite or vermiculite spiked with Cs. From each clay mineral, 57.1–72.8% (5100 ppm), 55.6–63.8% (920 ppm) and 48.6–54.3% (2300 ppm), 31.6–34.4% (520 ppm) was eluted, respectively. To understand elution behaviour, Cs desorption ratio of each clay was measured every 30 min. The results indicate Cs elution was occurred quickly.

Putative Iron Acquisition Systems in Stenotrophomonas maltophilia

Iron has been shown to regulate biofilm formation, oxidative stress response and several pathogenic mechanisms in Stenotrophomonas maltophilia. Thus, the present study is aimed at identifying various iron acquisition systems and iron sources utilized during iron starvation in S. maltophilia. The annotations of the complete genome of strains K279a, R551-3, D457 and JV3 through Rapid Annotations using Subsystems Technology (RAST) revealed two putative subsystems to be involved in iron acquisition: the iron siderophore sensor and receptor system and the heme, hemin uptake and utilization systems/hemin transport system. Screening for these acquisition systems in S. maltophilia showed the presence of all tested functional genes in clinical isolates, but only a few in environmental isolates. NanoString nCounter Elements technology, applied to determine the expression pattern of the genes under iron-depleted condition, showed significant expression for FeSR (6.15-fold), HmuT (12.21-fold), Hup (5.46-fold), ETFb (2.28-fold), TonB (2.03-fold) and Fur (3.30-fold). The isolates, when further screened for the production and chemical nature of siderophores using CAS agar diffusion (CASAD) and Arnows’s colorimetric assay, revealed S. maltophilia to produce catechol-type siderophore. Siderophore production was also tested through liquid CAS assay and was found to be greater in the clinical isolate (30.8%) compared to environmental isolates (4%). Both clinical and environmental isolates utilized hemoglobin, hemin, transferrin and lactoferrin as iron sources. All data put together indicates that S. maltophilia utilizes siderophore-mediated and heme-mediated systems for iron acquisition during iron starvation. These data need to be further confirmed through several knockout studies.

Revisiting the Chrome Azurol S Assay for Various Metal Ions

Siderophores play an important role in the solubilisation and mobilization of iron (III) and various metal ions. To have a useful method to test siderophores in culture supernatants for their metal binding affinity, we redesigned and optimized the liquid CAS-assay for selected metal ions. CAS-assay solutions were calibrated with desferrioxamine B in different concentrations to calculate DFOB-equivalents to get a semi-quantitative evaluation. With these assay solutions, we were able to test siderophores in culture supernatants for their ability to chelate with Fe, Al, Ga, Cu, V and As.

Thermochelin, a Hydroxamate Siderophore from Thermocrispum agreste DSM 44070

Siderophores are produced by microorganisms in iron-deficient environments. They are classified by structure as hydroxamate, catecholate, carboxylate or mixed type siderophores. These differences are also reflected in the selectivity for other valuable elements than iron, which allows designating them as “metallophores”, and makes them of interest for several industrial and medical applications. Thus, it is essential to understand the biosynthesis of these molecules to increase the set of available metallophores that are stable and suited for the respective applications. The probable structure of the metallophore from T. agreste DSM 44070 was predicted by similarity search and gene annotation. An N-hydroxylating monooxygenase (NMO: TheA) of T. agreste DSM 44070 that catalyzes an initial step was synthesized and characterized in detail. The respective metallophore was synthesized, purified and studied. The structure prediction suggested a hydroxamate-type (Erythrochelin-like) metallophore that contains L-N5-hydroxyornithine. This precursor is synthesized by TheA. The siderophore designated as “Thermochelin” is produced, extracted and purified successfully. Complexation was confirmed by CAS-assay. In this study, we expanded the scope of siderophores and the knowledge towards their biosynthetic pathways. Thermochelin is the second siderophore, which was purified from a thermophilic organism, and TheA is the first NMO, which was characterized from an extremophile.