Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

Abstract

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

Conditionally Redundant Bacteriocin Targeting byPseudomonas syringae

“…Bacteriocin samples were isolated as described 43,77 . Briefly, overnight KB broth cultures of Psy B728a and derived mutants were diluted 1/100 into fresh KB medium and allowed to incubate for 2-4 hrs with shaking, after which mitomycin C was amended to the culture (0.5 μg/ml final concentration).…”

Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots

“…Killing activity of UV lysates was gauged using a soft-agar overlay assay similar to one previously reported (37). Overnight cultures of P. putida KT2440 (and the strains listed in Table 1) were diluted to an OD 620 of 0.05 and grown at 28°C with shaking (200 rpm) until the culture reached an OD 620 of 0.4 to 0.5.…”

LlpB represents a second subclass of lectin‐like bacteriocins

“…Antagonistic activity of the LlpBs was evaluated via spot assay against a panel of pseudomonads, including several Pseudomonas reference strains. Ten‐μl drops of recombinant protein (concentration 1 mg ml −1 ) were applied onto bacterial cell lawns, incubated overnight, and scored for the presence of zones of growth inhibition the following day (Hockett and Baltrus, ). For both LlpBs, eight out of 49 strains in the Pseudomonas test panel proved susceptible (Table ), confirming the bactericidal function of these proteins.…”