Date of Award

1-1-2012

Document Type

Campus Access Thesis

Department

Environmental Health Sciences

First Advisor

Robert S Norman

Abstract

Bacteria communicate with each other by producing and releasing chemical and protein signals called autoinducers in order to control and regulate specific gene expression. The process of cell-to-cell communication is termed quorum sensing (QS). According to cell density, if a specific threshold concentration of autoinducers is reached, the neighboring cells are able to detect and respond to the signal. The phenomenon of QS can control different biochemical processes (e.g. the formation of biofilms, virulence, or the production of antibiotics) within several bacteria species and has been well characterized in both Gram-negative and Gram-positive bacteria. However, to our knowledge there are only a few studies investigating the QS phenomenon in archaea. In this study, a halophilic archaea belonging to the strain Halococcus hamelinensis sp. nov. 100NA1 was analyzed for putative QS activity using a functional genomic approach based on fosmid expression and biosensor (E. coli JLD271, Lindsay and Ahmer, 2005) screening as well as liquid chromatography coupled to mass spectrometry (LCMS/MS). From the cell media, the putative signal was extracted, separated thru a silica column, and further analyzed via biosensor and LC-MS/MS. Hcc. hamelinensis genomic high molecular weight DNA was extracted, purified, sheared into ~40 kb fragments and ligated into CopyControl fosmid vectors. The vectors were packaged into a lambda phage and used to infect E.

coli EPI300-T1R. Based on blue/white colony screening, clones containing genomic inserts were further analyzed for the production of autoinducers. The fosmid clones were screened for QS activity using the E. coli JLD271 biosensor co-culture assay, which generates an observable phenotype in the presence of autoinducers. If a clone demonstrated putative QS activity by activation of the biosensor it was verified by extracting the putative signal with methanol and chloroform. The organic extract was analyzed for acyl-homoserine lactones (AHL) using LC-MS/MS. Two peaks that were not observed in the negative control showed the same fragmentation pattern as the used AHL standards however there was a mobility shift of the peaks in the LC chromatogram. It is hypothesized that the putative signal produced by Hcc. hamelinensis is a carboxylated AHL.

Pacific Biosciences sequencing of genomic fosmids resulted in 36,079 bp for fosmid 17 and 34,966 bp for fosmid 18, and subsequent annotation of 30 and 18 predicted ORFs that might play a role in quorum sensing. When comparing both fosmid sequences 5 gene annotations were similar. One of those annotations was a glycosyl transferase which was recently found to be important for polysaccharide production and might be quorum sensing-regulated as Hcc. hamelinensis produces EPS towards the stationary phase during the development of biofilms. An annotation for a kinase might be involved in the AI-2 and AI-1 mechanism of quorum sensing, hence a putative luxI homolog. The annotation for transcriptional regulator could be a luxR homolog since LuxR protein is a transcriptional regulator. Future experiments will determine the actual luxI homologs, the structure of the signal, and the genetic regulation of this genetic system.

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