Screening overlay-assay

Screening based on halo formation is not only suited for the identification of single enzyme activities but may also be used to screen for whole metabolic pathways. Aarons et al. recently set up a system to screen for a functional enzyme cascade that is involved in the production of substances that act as antibiotics towards an indicator bacteria strain but not the producer strain. Using the Bacillus overlay-assay [50] they were able to isolate genes capable of complementing P. fluorescens FI 13 gacS and gacA mutants [51]. 

Transposon-based approaches have also been applied to myxobacteria (Figure 3). Mutants from transposon libraries can be analyzed chemically by comparison of their secondary metabolite profiles with the wild-type strain, or screened for altered bioactivity, for example, by overlay assays. This strategy was successfully used to identify the biosynthetic gene clusters responsible for the known metabolites tubulysin, disorazol, aurachin, and DKxanthene in the genomes of their respective producer strains, and has the potential to detect novel, as yet unidentified compounds and their corresponding biosynthetic pathways and regulatory elements (see Section 2.07.5). 

Figure 2. Agar-overlay assay used to screen for hydantoinase-producing microorganisms.

Other overlays demonstrate a very fast rate of conversion indicated by the onset of a predominant product peak in a very short time. Some assay conditions were deemed optimal for a particular application because they provided the desired interchange of decreasing substrate concentration and increasing product concentration in a desired time. Once conditions are optimized, an assay can be easily adapted for screening via a /iPLC system. 

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