Discovering the Function of a Vibrio Fischeri Metabolic Gene Using a Newly Constructed Genome-Scale Model and Genomic Library

Vibrio
fischeri is a bioluminescent marine bacterium that forms a symbiotic
relationship in the light-emitting organ of the Hawaiian bobtail squid, Euprymna
scolopes. Its metabolic capabilities are representative of many other
marine bacteria, including both beneficial and pathogenic members of the genus Vibrio
and, thus, are of great interest to researchers. To better understand the metabolism
of V. fischeri and, in particular, to search for metabolic genes without
any experimentally characterized functions, we constructed a V. fischeri
genome-scale metabolic model ­and developed a novel gene-finding method using genomic
libraries.

Our V.
fischeri model is a structured systems-level representation of most known V.
fischeri biochemical reactions and their associated genes. It is built upon
both the annotated genome and literature information, and is parameterized by the
biochemical composition of V. fischeri, as well as its growth rates and metabolite
consumption and production rates. The current V. fischeri model consists
of 1725 reactions, 1019 unique metabolites, and 822 metabolic genes. To validate
the model, we tested the growth phenotypes of V. fischeri under 181 different
sole carbon-source growth conditions. The model correctly predicted 86.2% of
the experimentally validated growth phenotypes. It also accurately categorized
83.4% of the 822 metabolic genes into either essential or non-essential genes for
growth in complex-nutrient medium, as based on a previous report [1].

We
also developed a method to search for genes that were missing from the model. In
our experiment, V. fischeri was able to grow in a minimal medium without
β-alanine, a precursor to the synthesis of the required metabolite coenzyme
A. However, V. fischeri is missing an aspartate decarboxylase gene (encoded
by panD in E. coli), which converts aspartate to β-alanine
and is required for growth in minimal medium. However, no ortholog to the E.
coli panD could be identified in V. fischeri. We located the V.
fischeri aspartate decarboxylase gene by complementing an E. coli DpanD mutant (an auxotroph for
β-alanine) with a plasmid library of V. fischeri genomic DNA. The
library had an average insert size of 2.4 Kb, and a total size of 53 Gb. Sequencing
a plasmid that rescued growth of the E. coli DpanD mutant found the V. fischeri aspartate
decarboxylase gene. We cloned just the coding region of the gene into a new
plasmid vector, and this plasmid was again able to rescue E. coli panD
mutant in minimal medium. These results strongly suggest that the gene
functions as an aspartate decarboxylase in V. fischeri, despite the
sequence dissimilarity between the gene and panD from E. coli. We
are currently doing in vitro enzymatic assays and phylogenetic analyses to
further characterize this gene.

[1] J.
F. Brooks, M. C. Gyllborg, D. C. Cronin, S. J. Quillin, C. a Mallama, R.
Foxall, C. Whistler, A. L. Goodman, and M. J. Mandel, ?Global discovery of
colonization determinants in the squid symbiont Vibrio fischeri.,? Proc.
Natl. Acad. Sci. U. S. A., 111: 17284-89, Nov. 2014.