The Preliminary Classification and Identification of Janthinobacterium sp. Hyper Violacein-Producing Mutants as Effective Anti-Microbial Agents

A pigmented aquatic microbial isolate was obtained from the Esopus Creek, a stream in the Hudson Valley Region. This isolate was cultured, biochemically characterized, and preliminary 16S rRNA sequencing resulted in a high similarity to Janthinobacterium. A Gram-negative aquatic bacterium, Janthinobacterium sp. produces a biofilm at the air-liquid interface while also producing a violet pigment known as violacein (Pantanella et al., 2007) (Lopes et al., 2009). It has been previously demonstrated that the production of violacein occurs when there is a high bacterial cell density, a process known as quorum sensing, which is a form of cell to cell communication. Violacein has also been shown to be a useful antimicrobial agent in high enough concentrations (Harris et al., 2009) (Lopes et al., 2009) (Becker et al., 2009) (Brucker et al., 2008) (Hakvag et al., 2009) (De Azevedo et al., 2000). We were interested in characterizing the genes and gene products required for the initial surface attachment, biofilm formation, and pigment production of our isolate of Janthinobacterium sp. We utilized random transposon mutagenesis to create a library of mutant strains in order to identify candidate genes responsible for biofilm formation, as well as to identify candidate genes responsible for violacein production. From this library, several hyper-pigmented mutants were identified. In order to accurately identify locations of transposon insertion, the genome of the original isolate is currently being sequenced. In parallel, extraction of violacein and the use of thin layer chromatography are being utilized to identify the components of the pigmented fraction. These assays are being followed up with LC-mass spectrometry in order to verify the chemical identity of the pigment. Preliminary extraction results show a higher concentration of violacein production in the mutant strains than in comparison to the wild type strain, and are able to inhibit fungal growth and growth of Staphylococcus aureus. We are currently setting up trials to examine the efficacy of these violacein hyper-producing strains in chytrid fungal killing assays. Use of these hyper-pigment producing strains may prove to be an effective therapeutic agent in fungal outbreaks in amphibious populations