Sannino, Filomena (2016) NOVEL BIOACTIVE PRODUCTS FROM ANTARCTIC BACTERIA. [Tesi di dottorato]

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Marine bacteria have considerable importance as sources of biologically active products. Marine microorganisms that live in cold regions have been largely underexplored, and may be endowed with interesting chemical repertoire. The microorganisms that thrive in these cold environments are referred to as psychrophiles or cold-adapted bacteria and are able to produce a large number of bioactive compounds, such as antimicrobial, anti-fouling and various pharmaceutically-relevant activities. In this contest, the aim of my PhD project was the research of new bioactive compounds of biotechnological interest from Polar marine bacteria. In particular, I focused my attention on three classes of molecules: I. Antimicrobial volatile organic compounds (VOCs); II. Anti-biofilm molecules; III. Cryoprotectant compounds. In order to explore the Pseudoalteromonas haloplanktis TAC125 (P.haloplanktis TAC125) chemical diversity as source of bioactive compounds, a suitable synthetic growth medium was developed, containing D-gluconate and L-glutamate as carbon, nitrogen and energy sources (GG medium). The definition of a synthetic medium is necessary for the scale up of P. haloplanktis TAC125 growth in automatic bioreactors. Moreover, a defined “minimum” medium could enhance the secondary metabolites production, and it surely makes their purification easier. Preliminary studies demonstrated that some Antarctic marine bacteria are able to produce volatile organic compounds (VOCs) that specifically inhibit the growth of Burkholderia cepacia complex (Bcc) strains. Amongst the tested Antarctic marine bacteria, P.haloplanktis TAC125 was further investigated. It is known that the P.haloplanktis TAC125 production of VOCs changes with growth medium composition. With the aim to identify the anti-Bcc VOCs, a suitable capture trap for volatile compounds was developed. A bioactive compound was identified, the methylamine, and its anti-Bcc activity was demonstrated by defining the Minimum Volatile Inhibitory Concentration (MVIC) on a panel of Bcc strains. Anti-biofilm molecules may have interesting biomedical applications in targeting adhesive properties of several insidious human pathogens. Previous results showed that the cell-free supernatant of P.haloplanktis TAC125 grown in static condition strongly inhibited bacterial adhesion. In particular, Staphylococcus epidermidis showed the highest susceptibility to the treatment. During this part of my PhD project the best conditions in which P.haloplanktis TAC125 produces the anti-biofilm compound/s were searched and a preliminary purification scheme was set up. In particular, the effect of growth mode, culture medium composition, growth phase and temperature was explored. The best production conditions were set as a benchmark for the scale-up of P.haloplanktis TAC125 anti-biofilm molecule/s in bioreactor. Marine cold-adapted microorganisms may be also source of another interesting class of chemical compounds, known as cryoprotectors, as they are able to avoid ice crystal formation inside living cells. Freeze-thaw cycles are quite common in the cold regions, especially in Polar one. Cold-adapted microorganisms are accustomed to being frozen within their habitats. Such organisms are also expected to have evolved adaptations to survive repeated freezing and thawing, as these processes tend to damage living cells and attenuate cell viability. The cold-adapted bacterium Colwellia psychrerythraea strain 34H (C. psychrerythraea 34H), attracted particular attention because it was reported to physically interact with sea ice crystals and secrete cryoprotectants of polysaccharidic nature in culture medium as a survival strategy. During my project, it was demonstrated that C. psychrerythraea 34H cells are covered by a capsula: the determination of chemical composition of purified capsular material revealed a novel polysaccharidic structure. Indeed the capsula was made by a linear tetrasaccharide repeating unit containing two amino sugars and two uronic acid, one of which is amidated by a threonine. The presence of an amminoacidic decoration of the capsular polysaccharide is quite uncommon in marine bacteria, but more intriguing is the decoration with Thr residues, as glycosilated Thr residues are essential for the interaction of anti-freeze glycoproteins (AFGPs) with ice crystals. In line with this indirect observation, in vitro assays demonstrated that the C. psychrerythraea 34H capsular polysaccharide is endowed with ice re-crystallization inhibition activity.

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