Abstract
Screening microbial cultures for specialised metabolites is essential for the discovery of new biologically active compounds. A novel, cost-effective and rapid screening method is described for extracting specialised metabolites from bacteria grown on agar plates, coupled with HPLC for basic identification of known and potentially novel metabolites. The method allows the screening of culture collections to identify optimal production strains and metabolite induction conditions. The protocol was optimised on two Burkholderia species known to produce the antibiotics, enacyloxin IIa (B. ambifaria) and gladiolin (B. gladioli), respectively; it was then applied to strains of each species to identify high antibiotic producers. B. ambifaria AMMD and B. gladioli BCC0238 produced the highest concentrations of the respective antibiotic under the conditions tested. To induce expression of silent biosynthetic gene clusters, the addition of low concentrations of antibiotics to growth media was evaluated as known elicitors of Burkholderia specialised metabolites. Subinhibitory concentrations of trimethoprim and other clinically therapeutic antibiotics were evaluated and screened against a panel of B. gladioli and B. ambifaria. To enhance rapid strain screening with more antibiotic elicitors, antimicrobial susceptibility testing discs were included within the induction medium. Low concentrations of trimethoprim suppressed the production of specialised metabolites in B. gladioli, including the toxins, toxoflavin and bongkrekic acid. However, the addition of trimethoprim significantly improved enacylocin IIa concentrations in B. ambifaria AMMD. Rifampicin and ceftazidime significantly improved the yield of gladiolin and caryoynencin by B. gladioli BCC0238, respectively, and cepacin increased 2-fold with tobramycin in B. ambifaria BCC0191. Potentially novel metabolites were also induced by subinhibitory concentrations of tobramycin and chloramphenicol in B. ambifaria. In contrast to previous findings that low concentrations of antibiotic elicit Burkholderia metabolite production, we found they acted as both inducers or suppressors dependent on the metabolite and the strains producing them. In conclusion, the screening protocol enabled rapid characterization of Burkholderia metabolites, the identification of suitable producer strains, potentially novel natural products and an understanding of metabolite regulation in the presence of inducing or suppressing conditions.
Highlights
Microbial specialised metabolites continue to be a source of new biologically active molecules for use in medicine and agriculture (Bérdy 2005; Rutledge and Challis 2015)
Optimization of the rapid screening method was carried out using Burkholderia species identified as producers of the bioactive polyketides, enacyloxin IIa (Mahenthiralingam et al 2011) and gladiolin (Song et al 2017), from Burkholderia ambifaria strain AMMD and B. gladioli strain BCC0238, respectively
After growth of the bacteria for 72 h and removal of biomass, initial experiments evaluated the use of different volumes of extraction solvent (5, 2, 1, 0.5 ml) and injection volumes for high-performance liquid chromatography (HPLC) analysis (2, 5, 10, 15, 20 μl); dichloromethane was used as the initial solvent to optimise the method, with acetonitrile and ethylacetate evaluated subsequently
Summary
Microbial specialised ( known as secondary) metabolites continue to be a source of new biologically active molecules for use in medicine and agriculture (Bérdy 2005; Rutledge and Challis 2015) Their production, extraction and identification from bacterial growth medium can be complicated, labour intensive, and time consuming. The production of microbial compounds are frequently influenced by different cultivation parameters (e.g. nutrients, light, temperature, pH, and aeration) (Bode et al 2002, Pettit 2011, Begani et al 2018) and identifying optimum growth conditions can require labour intensive screening This has prompted the investigation of alternative approaches to identifying novel metabolites, such as high throughput screening (HTS) of synthetic compound libraries and fragment-based design (Payne et al 2006; Doak et al 2016). Inhibition of the target protein by the tested compounds is assessed outside the context of the cell, and
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
