Antimicrobial Activities of Secondary Metabolites from Model Mosses

Abstract

Plants synthetize a large spectrum of secondary metabolites with substantial structural and functional diversity, making them a rich reservoir of new biologically active compounds. Among different plant lineages, the evolutionarily ancient branch of non-vascular plants (Bryophytes) is of particular interest as these organisms produce many unique biologically active compounds with highly promising antibacterial properties. Here, we characterized antibacterial activity of metabolites produced by different ecotypes (strains) of the model mosses Physcomitrium patens and Sphagnum fallax. Ethanol and hexane moss extracts harbor moderate but unstable antibacterial activity, representing polar and non-polar intracellular moss metabolites, respectively. In contrast, high antibacterial activity that was relatively stable was detected in soluble exudate fractions of P. patens moss. Antibacterial activity levels in P. patens exudates significantly increased over four weeks of moss cultivation in liquid culture. Interestingly, secreted moss metabolites are only active against a number of Gram-positive, but not Gram-negative, bacteria. Size fractionation, thermostability and sensitivity to proteinase K assays indicated that the secreted bioactive compounds are relatively small (less than <10 kDa). Further analysis and molecular identification of antibacterial exudate components, combined with bioinformatic analysis of model moss genomes, will be instrumental in the identification of specific genes involved in the bioactive metabolite biosynthesis.

Identification and Analysis of Antimicrobial Activities from a Model Moss Ceratodon purpureus

“…C. purpureus R40 (male) and GG1 (female) strains (a gift from Dr. Stuart McDaniel, University of Florida) were propagated on Petri plates with BCD nutrient agar medium as described before [ 20 , 27 ]. Moss was propagated weekly by homogenizing tissue with the IKA Ultra-Turrax T10 basic tissue dispenser, followed by plating the homogenized samples onto cellophane disks on top of solid BCD nutrient medium in Petri dishes.…”

“…For secreted metabolite analysis, C. purpureus strains were grown in 250 mL flasks with 100 mL of the BCD liquid nutrient medium on the orbital shaker (at 150 rpm) at 22 °C, 65% humidity, 880 lux light intensity, and 12/12 h light/dark conditions. Exudate was collected and processed as described previously [ 20 ]. Frozen exudate samples were dried using a lyophilizer (model 7382021, Labconco, Kansas City, MO, USA) and stored at −80 °C until needed.…”

“…Next, 17.5 mg of moss exudate was dissolved in BCD medium and added onto sterile Whatman disks (disk diameter = 7 mm). Disks soaked with the same amount of sterile liquid BCD medium were used as the negative control and disks soaked with carbenicillin or chloramphenicol antibiotics were used as the positive control, as described previously [ 20 ]. Disks were placed on top of inoculated plates and incubated at 35 °C for 18 h. The diameter of the bacterial growth inhibition zone (halo) around each disk was then measured in mm and plotted.…”

“…The model liverwort Marchantia polymorpha , for example, secretes specialized “oil bodies” containing sesquiterpenes and cyclic bisbibenzyls, which serve as a defense against arthropod herbivores [ 17 , 18 ]. The model moss Physcomitrium patens secretes a number of extracellular compounds and peptides with antibacterial properties [ 19 , 20 ]. For potential future biotechnological applications, such secreted bioactive metabolites may offer several technical advantages over intracellular compounds, as they are usually water-soluble and can be easier to biochemically isolate and purify.…”

Antimicrobial Natural Products

“…Bryophytes are important resources with the potential to produce unique natural compounds with antimicrobial properties. Valeeva and co-workers evaluated and characterized the antibacterial activity of intracellular and extracellular metabolites produced by the model mosses Physcomitrium patens and Sphagnum fallax [ 3 ]. Both polar (methanol-based) and non-polar (hexane-based) extracts of the two mosses inhibited the growth of Gram-negative P. syringae DC3000 bacteria.…”