Construction, characterization, and colonization of Vibrio fischeri amino acid auxotrophs. (Environmental Poster Session 10:00 AM-11:00 AM).
Vibrio fischeri is a symbiotic bacterium associated with the Hawaiian bobtail squid, Euprymna scolopes. It has been shown, through the work of Joerg Graf on amino-acid auxotrophs of V. fischeri strain ESR1, that the host squid provides certain amino acids to support the bacteria, and auxotrophy for amino acids in the bacteria can lower the level of its colonization of the squid light organ. We created similar auxotrophs in a different strain background and hope to explain any differences. The original ESR1 auxotrophs were created by transposon mutagenesis and assigned inferred genotypes based on their ability to grow on certain metabolic intermediates. The construction scheme for the ES114 auxotrophs involved a genomic approach--targeting specific genes in the biosynthetic pathways for lysine, cysteine, serine, methionine, and threonine and amplifying with PCR. The resulting fragments were sub-cloned into V. fischeri vector pEVS179 to undergo transposon mutagenesis and ultimately antibiotic marker exchange, placing mutants back into the ES114 chromosome. The resulting putative auxotrophs will be characterized, sequenced, and confirmed by Southern blotting. Colonization assays were performed with two lysine mutants and one methionine mutant, and Initial assays showed lower than wild-type levels of colonization of the squid light organ. One lysine mutant was characterized as an auxotroph on selective media, and the transposon-inserted gene fragment confirmed as incorporated into the chromosome by a Southern blot (N=1).
AMBER G. POLLACK POLLACKAG@HIRAM.EDU AND CHERYL WHISTLER (1); (PRUDENCE HALL HALLPJ@HIRAM.EDU) HIRAM COLLEGE, HIRAM 44234, (1) KEWALO MARINE LABORATORY
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|Author:||Pollack, Amber G.; Whistler, Cheryl|
|Publication:||The Ohio Journal of Science|
|Date:||Mar 1, 2003|
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