The Effects Of Cell Density And Biofilm Formation On Melanin Production In A Novel pseudomonas Species

P. L. Lawrence, L. R. Aaronson

Author address: 

Utica Coll., Utica, NY

Abstract: 

Pseudomonas sp. UC17F4 is a novel bacterial species that was isolated from the cutaneous flora of red-backed salamanders on the basis of its antifungal activity. One of the most interesting characteristics of this species is its ability to produce two different forms of the brown pigment, melanin. Eumelanin is a high MW molecule that is contained within the cytoplasm of the bacterial cell, while pyomelanin is a low MW secreted form. Studies in our lab have shown that melanin production in this bacterium is regulated by light exposure, and by the availability of iron and suitable carbon sources. We have recently observed that melanogenesis is dependent on high cell density and the development of biofilms. Cultures of UC17F4 were grown in either tryptic soy-yeast extract (TSYE) broth or in citrate-enriched Lawrence Minimal Medium (LMM). Eumelanin content was assayed by spectrophotometric analysis of cell lysates in 1% SDS. As culture densities increased, so did cellular melanin content, increasing 6-fold from the onset of stationary phase. Cultures inoculated at higher cell density also exhibited higher melanin content. Biofilm-forming cultures produced 5-fold higher levels of eumelanin than did planktonic broth cultures. Biofilm cultures were grown in 35 mm plates in both TSYE and LMM broth, and exhibited density-dependent melanin content. Similar results were observed in biofilms formed on nitrocellulose filters placed on LMM and TSYE agar plates. To further study the dependence of UC17F4 biofilm formation of melanogenesis, NaCl was added to cultures to inhibit the development of biofilms. Bacterial eumelanin content in the biofilm mass declined by 90% as NaCl concentration increased to 1% (w/v) compared to untreated controls. Treatment of cultures with Furanone 56, an inhibitor of quorum signaling in Pseudomonads, resulted in a 30% reduction in eumelanin content. The quorum signaling compound C12-homoserine lactone stimulates only a modest increase in melanogenesis. Supplementation of early exponential-phase cultures with cleared supernatants from 48 hr biofilm cultures resulted in up to a 2-fold increase in cellular melanin content. These data suggest that melanogenesis in Pseudomonas sp. UC17F4 is under the control of one or more signaling compounds. Ongoing studies are being conducted to isolate and characterize melanogenic signal compounds, and regulatory pathways in the bacterium.
2016

abstract No: 

FRIDAY-770

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)