Polyhydroxybutyrate synthesis from phenanthrene and waste glycerol by the marine bacterium Halomonas titanicae KHS3

ESCOBAR, M; HERRERA SEITZ, M.K.; RODRÍGUEZ, A.N.; REDERSDORFF, I.E.; STUDDERT, C.A.

Buenos Aires (virtual)

Congreso; LVI Reunión Anual de SAIB; 2020

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Halomonas titanicae KHS3 is a moderate halophilic bacterium isolated from hydrocarbon-contaminated seawater of Mar del Plata harbor based on its ability to grow using phenantrene as the sole carbon and energy source and besides to show chemotactic responses to this compound. The genomic sequence of H.titanicae KHS3 allowed the identification of genes related to polyhydroxybutyrate (PHB) metabolism.In this work the ability of H.titanicae to accumulate PHB under nitrogen (N) limiting conditions when grown on phenanthrene or waste glycerol was analyzed. PHB accumulation was determined by sodium hypochlorite turbidimetric assay, gravimetric determinations and microscopic observation of Nile Blue stained cells.Cultures grown with phenanthrene or glycerol as sole carbon source up to the exponential phase were split in two batches; one was changed to N limiting conditions and the other one remained in complete medium as a control. Whereas the level of PHB in nitrogen control conditions reached less than 20% of cell dry weight, nitrogen depletion caused PHB accumulation of up to 60% in both tested carbon sources. Clear PHB accumulation occurred at different incubation temperatures, agitation conditions and salt levels after nitrogen depletion. As part of studies attempting to understand the role of the two PHA synthases (PhaC1 and PhaC2) codified in the H. titanicae genomic sequence, PHB accumulation was evaluated in a H. titanicae mutant strain (SF18) carrying a plasmid insertion in thephaC1 gene. When grown on glycerol, the mutant SF18 behave very similar to the wild-type strain under nitrogen control conditions, but lost the ability to accumulate PHB upon nitrogen depletion. This result supports a leading role for PhaC1 enzyme in the PHB accumulation under this condition.In contrast, SF18 showed severe growth problems when phenanthrene was used as the carbon source. After several reinoculations in phenanthrene medium, SF18 recovered normal growth rate and the mutant was able to accumulate PHB in conditions of N depletion, similar to the wild-type strain. However, further PCR analysis of these cellsshowed that they had lost the insertion that blocked PhaC1 expression, thus reverting the mutation. These results suggest the presence of a functional PHB synthesis pathway, even in the presence of normal levels of nitrogen, is an essential feature for growth on phenantrene as the only carbon source. This observation highlights the role of the PHB biosynthetic pathway to counteract redox stress, and assigns a major role to the enzyme PhaC1 within the pathway.