A rapid screening method for the detection of specialised metabolites from bacteria: Induction and suppression of metabolites from Burkholderia species

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.

Kill and cure: genomic phylogeny and bioactivity of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles

“…HPLC analysis was used to quantify and identify known B. gladioli metabolites as follows. Specialized metabolites were produced by growing on BSM-G medium (as above) and extracted directly from a 20 mm agar disc cut from the plates as described elsewhere [ 44 ]. Extracts (20 µl injection volume) were analysed on a Waters AutoPurification HPLC System fitted with a reverse-phase analytical column (Waters XSelect CSH C18, 4.6×100 mm, 5 µm) and a C18 SecurityGuard cartridge (Phenomenex) in series.…”

Burkholderia gladioli CGB10: A Novel Strain Biocontrolling the Sugarcane Smut Disease

“…We then reconstructed the phylogeny of the 22 bacterial species based on these 763 single-copy core genes, and the results showed that CGB10 was closest to B. gladioli, and together they clustered with B. glumae (Figure 1A). We further confirmed the taxonomic status of CGB10 by calculating the average nucleotide identity (ANI) [28] based on the whole genome of the CGB10 strain in comparison with another six B. gladioli strains, including established rice pathogen BSR3 [24], strains isolated from healthy plants and displaying antifungal activity [29][30][31], and strains isolated from a cystic fibrosis patient [32], as well as a B. glume strain BGR1 [24], which is also known as a rice pathogen (information of selected strains was detailed in Table 1). The identity between CGB10 and the six selected B. gladioli strains was all higher than 98%, yet the identity between CGB10 and the B. glumae strain BGR1 was 88.12%.…”

Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development

“…The B . ambifaria BCC1105 and P. phytofirmans PsJN hosts containing the cloned cepacin and caryoynecin BGCs also gained new antagonistic activity against S. aureus (Figure 2C ), a bacterium specifically susceptible to polyynes (Webster et al, 2020 ).…”

“…20 mm discs were excised from each agar plate and metabolites were extracted from the agar piece by incubation in 0.5 ml extraction solvent for 2 h with gentle shaking. Ethyl acetate (EtOAc) was used for the extraction of cepacin, whilst dichloromethane (DCM) was used for caryoynencin (Webster et al, 2020 ). HPLC analysis was conducted as previously described (Webster et al, 2020 ).…”

“…Ethyl acetate (EtOAc) was used for the extraction of cepacin, whilst dichloromethane (DCM) was used for caryoynencin (Webster et al, 2020 ). HPLC analysis was conducted as previously described (Webster et al, 2020 ). Polyynes such as caryoynencin are known to be unstable (Ross et al, 2014 ).…”