Microbial Enzyme and Signaling Profiling

Pectine depolymerase and cellulase activities were assessed as described by (Andro et al., 1984 (link)). Briefly, for polygalacturonase/pectate lyase assay, isolates were incubated on a solid M63 medium (Miller, 1972 ) containing polygalacturonic acid. After 48 h incubation at 30°C, plates were stained by flooding with 10% (w/v) copper acetate, which forms a blue complex with the polymer, leaving clear haloes around colonies that produce pectolytic enzymes. Cellulase activity was assessed on a medium containing 0.1% (w/v) carboxymethylcellulose (CMC). After 48 h of incubation at 30°C, the plates were stained with an aqueous 0.1% (w/v) Congo red solution for 1 h at room temperature and washed with 1 M NaCl. Cellulase-producing colonies formed “halo” zones. Protease, lipase and oligo-1,6-glucosidase activities were assessed on skimmed milk, egg yolk agar and starch agar, respectively (Tindall et al., 2014 (link)). Siderophore production was assessed with chrome azurol S (CAS) agar plate assay (Himpsl and Mobley, 2019 (link)). The diameter of “halo” zones around bacterial colonies was measured in each case. Experiments were performed in two repetitions and the means were calculated for each strain. Gelatinase production was assayed with the standard gelatin stab method (Tindall et al., 2014 (link)). Liquefication of the medium was assessed after one week of incubation. Malonate utilization as a sole carbon source was evaluated using the malonate broth (Tindall et al., 2014 (link)). A shift in the pH indicator color from green to dark violet indicated malonate utilization.
The capability of the strains to produce N-acyl homoserine lactone (AHL) was assessed using Chromobacterium violaceum CV026 as a biosensor as described by (Ravn et al., 2001 (link)). Briefly, tested strains were streaked on the LA plates and then incubated overnight at 30°C. After that time, C. violaceum CV2026 was streaked on the plates in parallel. Plates were then once again incubated at 30°C overnight. After that time, the plates were evaluated for the purple violacein pigment produced by C. violaceum in the presence of AHLs.