Application of Biolog FF MicroPlate for substrate utilization and metabolite profiling of closely related fungi.

Filamentous fungi are known to have unique biochemical pathways to assimilate a vast array of simple and complex nutrients available to them and to produce a variety of metabolites. Morphological and biochemical uniqueness of these organisms are commonly used for their identification, but differentiation of closely related cultures requires extensive phenotypic and genomic investigations. The Biolog FF MicroPlate was recently introduced for rapid identification of common filamentous fungi based on their abilities to utilize 95 discrete substrates. We used the FF MicroPlate for substrate utilization, growth, secondary metabolite and antimicrobial profiles of some fungal cultures important to our microbial drug discovery program. Culture growth was monitored by change in absorbance in each well, and the presence of secondary metabolites and their corresponding bioactivities was detected by LCMS analyses and antimicrobial assays of the extracts of each well, respectively. Fingerprints were created with Spotfire visualization software, and data were analyzed in various ways. The substrate utilization fingerprints were useful in selecting media components for media optimization of secondary metabolite production for the various cultures. In general, a strong correlation was found among substrate utilization, growth, antimicrobial activity and presence of the responsible secondary metabolites. The method was used for dereplication of isolated fungi and in the differentiation of closely related variants within one species.

A simple, rapid, sensitive method detecting homoserine lactone (HSL)-related compounds in microbial extracts.

A simple, rapid, sensitive microtiter plate method detecting N-acyl homoserine lactone (HSL)-related compounds was established using an Agrobacterium tumefaciens strain harboring a traG::lacZ/traR reporter gene responsive to HSLs. This strain did not produce its own HSL, but the traG::lacZ reporter gene was induced only when its transcription activator TraR detected a cognate exogenous HSL. Therefore, the assay was expected to be highly specific for HSL-related compounds. Induction of the reporter gene, leading to production of beta-galactosidase enzyme, was measured by using two different beta-galactosidase substrates, X-gal and Galacton-Star, for colorimetric and chemiluminometric detection, respectively. The screen was validated in both the 96-well and 384-well plate formats, and extracts derived from 696 different microbial isolates, mostly unidentified actinomycetes isolated from diverse locations, were tested. Crude extracts of 81 (11.64%) cultures tested positive for HSL-related compounds, and an additional 34 (4.8%) crude extracts showed a moderate to weak signal for HSLs. Data from the fractionated samples, however, suggested a much higher prevalence of HSL signals in these extracts. Of 144 crude extracts fractionated into 10 individual samples at a 10x concentration, 72 (50%) cultures tested positive for HSLs. Six cultures were active only in the crude extract, 18 were active both in crude and one or more of their fractions, and an additional 48 were active in just one or more of their fractions. This finding may be the first to suggest such a high prevalence of HSL-signals found in nature, and a large number of actinomycetes in our collection appeared to produce HSL-related compounds.

Chemiluminometric biochemical induction assay (CBIA) for the detection of DNA-damaging agents.

A microbroth chemiluminometric version of the biochemical induction assay (BIA) was developed using a chemiluminescent substrate widely used to detect beta-galactosidase in high-throughput screening (HTS) laboratories. The assay was run in both 96-well and 384-well plate formats using the Zymark RapidPlate liquid handling system to transfer samples and reagents. Chemiluminescence was read using the Victor-2 multilabel counter. The new microbroth chemiluminometric method, the CBIA, allowed rapid screening of samples, crude extracts, and pure compounds for their DNA-damaging effects in bacteria. In screening a small subset of our natural products library samples by the agar plate BIA and the CBIA, the latter yielded a higher hit rate, suggesting it is more sensitive than the agar plate assay. The CBIA was unaffected by the colored samples often encountered during screening of crude natural products extracts.