Date of Award

August 2016

Degree Type

Thesis

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Charles W. Wimpee

Committee Members

Steven Forst, Sandra Mclellan

Keywords

Bioluminescence, Lateral Gene Transfer, Molecular Evolution, Vibrio

Abstract

In a screening of bioluminescent bacteria isolated from the intestines of coral reef fish, two strains (designated D6 and M1) were identified that have a luxA gene sequence significantly different from those of other Vibrio species. Phylogenetic analysis of several housekeeping genes, as well as toxR, shows that D6 and M1 branch within a bioluminescent clade (designated the “D1 group,” isolated at the same time and place as D6 and M1) that is a close sister group to Vibrio harveyi. However, whereas the luxA genes of the D1 group are >98% identical to V. harveyi luxA, the luxA genes of D6 and M1 have a surprisingly low identity (86%) to the D1 group and to V. harveyi. Strain D6 and strain D1 (a representative of the D1 group) were chosen for further investigation. The lux operons (luxCDABEGH) and flanking regions of both strains were cloned into E. coli and sequenced by primer walking. Although distinguishable from Vibrio harveyi, and possibly representing a new species, strain D1 is clearly a close relative, and has the same genes flanking the lux operon as V. harveyi. However, in addition to a highly divergent lux operon, the flanking regions of D6 are completely different from those of D1 and V. harveyi. Based on differences in luxCDABEGH sequence and chromosomal context, we conclude that the lux operon of D6 was acquired by lateral gene transfer. PCR and Southern hybridizations show that D6 contains a single lux operon, so we conclude that this operon represents not simply a lateral transfer, but a lateral replacement of the original operon. We also show, in an E. coli expression system, that the lux operons of both D1 and D6 are up-regulated by the V. harveyi LuxR protein, indicating evolutionary conservation of lux gene regulation, despite the high degree of sequence dissimilarity between the two. These results show that we have not exhausted the diversity of bioluminescence genes in bacteria.

Available for download on Saturday, August 25, 2018

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