GRADUATE STUDIES BULLETIN

ANNOUNCEMENT

Florida International University

Division of Graduate Studies

Master’s Thesis Defense

Abstract

USE OF luxA SEQUENCES FOR INVESTIGATION ON LUCIFERASES KINETICS AND CLASSIFICATION OF LUMINOUS BACTERIA

by

Rita Di Bonito

The species of luminous bacteria presently described have been assigned to four genera: Vibrio, Photobacterium, Shewanella, and Photorhabdus. The luminescent reaction is catalyzed by the enzyme luciferase, an heterodimer composed by the a and b polypeptides, encoded by two adjacent genes, luxA and luxB, situated in the lux operon. The a subunit is responsible for substrate binding and catalytic activities. The analysis of the enzyme kinetics has been used to classify the known isolates into the two groups: “slow luciferases” tipical of Vibrio harveyi and “fast luciferases” of Photobacterium sps. Luciferase kinetics intermediate between the “slow” and “fast” have been described in Shewanella woodyi and a few isolates from Oman. Object of this work is to test the hypothesis of existence of a class of “intermediate luciferases” characterized by conserved sets of amino acids on the polypeptide encoded by the luxA gene of Oman and Florida luminous bacteria. The secondary objective is the characterization of the isolates by phenotypic analysis (substrate utilization) and sequencing of 16S rRNA gene. A portion of luxA and 16S rRNA genes have been amplified by PCR, sequenced and compared to known luminous species. Phenotypic analysis has shown the presence of clusters distinct from the known species. The analysis of nucleotides sequences (luxA and 16S rRNA) and derived amino acids sequences of luxA, has shown the new strains clustering into 5 major clades corresponding to the known species. LuxA sequences of strains LO3 and Shewanella woodyi MS32, characterized by “intermediate” luciferases, don’t form a distinct clade, but are respectively in the clades with Vibrio harveyi and S. hanedai. Some of the new isolates form a clade distinct from the known luminous and related non luminous species and could be new taxa. Geographic location and fish habitat affected the distribution into clades (16S rRNA analysis).

Date: April 18th, 2002 Department: Biological Sciences

Time: 9:30 a.m. Major Professor: Dr. John Makemson

Place: University Park, WC 130