Date of Award

2011

Document Type

Campus Access Dissertation

Department

Environmental Health Sciences

First Advisor

R. Sean Norman

Abstract

Bacteria and other microbes play critical roles in the world by influencing important biochemical processes in the biosphere. However, the bacterial realm remains one of the largest unexplored biological reservoirs on earth. Microbial communities carry out biochemical processes, interactively, by functioning as social units and ultimately define the overall functionality of ecosystems. Bacterial communities in all ecosystems likely utilize signaling pathways to adapt to localized changes in environmental conditions. One such pathway is called quorum sensing (QS), a cell-density dependent phenomenon involving the production, release, and detection of small diffusible signaling molecules referred to as autoinducers. This pathway ultimately coordinates gene expression among the bacterial community leading to synchronized behaviors in a multicellular fashion. Quorum sensing is known to be controlled by synthases of autoinducers and transcriptional regulatory proteins. Among them are LuxI and LuxR homologues; well characterized proteins in several bacterial isolates including Gram-negative and Gram-positive bacteria in laboratory settings under standard conditions. Recent studies have also reported the QS phenomenon in the archaeal domain. Despite the important role quorum sensing plays in community gene regulation, there is little knowledge about its occurrence and relevance in natural ecosystems. This study used functional metagenomic techniques to identify quorum sensing genes in microbial mat communities obtained from

Salt Pond on San Salvador Island, The Bahamas. We first extracted high molecular weight metagenomic DNA (> 48 kb) from the microbial mats and used this DNA in the construction of a fosmid-based metagenomic library. Using an approach based on E. coli (JLD271) pEAL01 QS biosensor, we screened 15,000 clones for putative QS activity. Initial screening of the 15, 000 clones showed putative QS activity in 2,975 clones. Following further screening, 302 clones showed consistent putative QS activity. Using 454-sequencing technology, we have sequenced the fosmid inserts of 11 clones, which demonstrated strong biosensor activation. Among a host of hypothetical proteins, we have identified LuxO and LysR; known to be transcriptional regulatory proteins. We also identified genes involved in the synthesis of S-adenosylmethionine (SAM) transferases. S-adenosylmethionine is the precursor for the production of acyl-homoserine lactones (AHL) or autoinducer-2 (AI-2) depending on the biosynthetic pathway. This suggests that the synthesis of QS signaling molecules and the QS phenomenon may be occurring in hypersaline microbial mats. These results also suggest that functional metagenomics can be used to study QS in the natural environment to better understand the role of QS in shaping the structure and functionality of different natural ecosystems.

Rights

© 2011, Benjamin Senyo Bey

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