Sathish Rajamani

My Story:

Thesis title:
Small molecule signaling and detection systems in protists and bacteria

Abstract:
In a mechanism called quorum sensing (QS) Gram-negative bacteria use small molecular weight signaling molecules such as acylated homoserine lactones (AHLs) and 4,5-dihydroxy-2,3-pentanedione derivatives (AI-2) to regulate gene expression in a population denisty-dependent manner. We report here the identification of QS mimic production by the unicellular green alga Chlamydomonas reinhardtii and other microalgae. Using ethyl acetate partitioning of culture filtrates, coupled with HPLC fractionation and bacterial reporter strain bioassays, we demonstrated the production of AHL class QS mimics by these algae. Purified AHL mimic activities from Chlamydomonas were further analyzed using liquid chromatography coupled to mass spectrometry. The bacterial enzyme AHL lactonase, capable of inactivating AHLs when expressed in Chlamydomonas, substantially lowered the algal QS mimic activities of these algae. To develop a reliable detection system for monitoring the boron derivative of AI-2 (BAI-2) QS signals, we developed a fluorescence resonance energy transfer (FRET) based biosensor consisting of a tandem N- to C-terminal cyan fluorescent protein (CFP)-BAI-2 receptor LuxP-yellow fluorescent protein (YFP) fusion protein. Following binding of BAI-2, the LuxP protein changes its conformation, resulting in a decreased YFP/CFP ratio. Importantly, addition of BAI-2 precursors or the introduction of BAI-2 binding site mutations in the LuxP protein blocked the FRET changes. The apparent binding affinity of the LuxP receptor protein for BAI-2 was determined. The utility of this biosensor system was demonstrated by the quantification of BAI-2 signals from the Vibrio harveyi bacterial cultures. The BAI-2-insensitive LuxP mutant biosensor was used for demonstrating QS mediated exoprotease secretion from V. harveyi. Extending our FRET studies, we also generated an in vivo algal-biosensor for quantification of bioavailable heavy metals in aqueous environments. Similar to the LuxP-FRET biosensor, the heavy metal biosensor has an N- to C-terminal fusion of CFP-metallothionein II (MTII)-YFP. In its unliganded state, MTII adopts an unfolded, random-coil structure. Following heavy metal binding, MTII folds into a compact dumbbell shaped protein resulting in increased FRET between the donor (CFP) acceptor (YFP) pair. Transgenic algae, expressing the MTII-biosensor demonstrated differences in the cytoplasmic bioavailability for different heavy metals tested.