NIOO, Netherlands Institute of Ecology

Certain bacterial species produce antimicrobial compounds only in the presence of a competing species. However, little is known on the frequency of interaction-mediated induction of antibiotic compound production in natural communities of soil bacteria. Here we developed a high-throughput method to screen for the production of antimicrobial activity by monocultures and pair-wise combinations of 146 phylogenetically different bacteria isolated from similar soil habitats. Growth responses of two human pathogenic model organisms, Escherichia coli WA321 and Staphylococcus aureus 533R4, were used to monitor antimicrobial activity. From all isolates, 33% showed antimicrobial activity only in monoculture and 42% showed activity only when tested in interactions. More bacterial isolates were active against S. aureus than against E. coli. The frequency of interaction-mediated induction of antimicrobial activity was 6% (154 interactions out of 2798) indicating that only a limited set of species combinations showed such activity. The screening revealed also interaction-mediated suppression of antimicrobial activity for 22% of all combinations tested. Whereas all patterns of antimicrobial activity (non-induced production, induced production and suppression) were seen for various bacterial classes, interaction-mediated induction of antimicrobial activity was more frequent for combinations of Flavobacteria and alpha- Proteobacteria. The results of our study give a first indication on the frequency of interference competitive interactions in natural soil bacterial communities which may forms a basis for selection of bacterial groups that are promising for the discovery of novel, cryptic antibiotics.

The importance of volatile organic compounds for functioning of microbes is receiving increased research attention. However, to date very little is known on how inter-specific bacterial interactions effect volatiles production as most studies have been focused on volatiles produced by monocultures of well-described bacterial genera. In this study we aimed to understand how inter-specific bacterial interactions affect the composition, production and activity of volatiles. Four phylogenetically different bacterial species namely: Chryseobacterium, Dyella, Janthinobacterium, and Tsukamurella were selected. Earlier results had shown that pairwise combinations of these bacteria induced antimicrobial activity in agar media whereas this was not the case for monocultures. In the current study, we examined if these observations were also reflected by the production of antimicrobial volatiles. Thus, the identity and antimicrobial activity of volatiles produced by the bacteria were determined in monoculture as well in pairwise combinations. Antimicrobial activity of the volatiles was assessed against fungal, oomycetal, and bacterial model organisms. Our results revealed that inter-specific bacterial interactions affected volatiles blend composition. Fungi and oomycetes showed high sensitivity to bacterial volatiles whereas the effect of volatiles on bacteria varied between no effects, growth inhibition to growth promotion depending on the volatile blend composition. In total 35 volatile compounds were detected most of which were sulfur-containing compounds. Two commonly produced sulfur-containing volatile compounds (dimethyl disulfide and dimethyl trisulfide) were tested for their effect on three target bacteria. Here, we display the importance of inter-specific interactions on bacterial volatiles production and their antimicrobial activities.

Recent studies indicated that the production of secondary
metabolites by soil bacteria can be triggered
by interspecific interactions. However, little is known
to date about interspecific interactions between
Gram-positive and Gram-negative bacteria. In this
study, we aimed to understand how the interspecific
interaction between the Gram-positive Paenibacillus
sp. AD87 and the Gram-negative Burkholderia sp.
AD24 affects the fitness, gene expression and the
production of soluble and volatile secondary
metabolites of both bacteria. To obtain better insight
into this interaction, transcriptome and metabolome
analyses were performed. Our results revealed that
the interaction between the two bacteria affected
their fitness, gene expression and the production of
secondary metabolites. During interaction, the
growth of Paenibacillus was not affected, whereas
the growth of Burkholderia was inhibited at 48 and
72 h. Transcriptome analysis revealed that the interaction
between Burkholderia and Paenibacillus
caused significant transcriptional changes in both
bacteria as compared to the monocultures. The
metabolomic analysis revealed that the interaction
increased the production of specific volatile and soluble
antimicrobial compounds such as 2,5-bis(1-
methylethyl)-pyrazine and an unknown Pederin-like
compound. The pyrazine volatile compound produced
by Paenibacillus was subjected to bioassays
and showed strong inhibitory activity against
Burkholderia and a range of plant and human pathogens.
Moreover, strong additive antimicrobial effects
were observed when soluble extracts from the interacting
bacteria were combined with the pure 2,5-bis
(1-methylethyl)-pyrazine. The results obtained in this
study highlight the importance to explore bacterial
interspecific interactions to discover novel secondary
metabolites and to perform simultaneously
metabolomics of both, soluble and volatile compounds.