(627d) Vibrio fischeri Aspartate 1-Decarboxylase Revealed By Model-Enabled Gene Search

Vibrio fischeri strain ES114 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. In this study, we developed model-enabled gene search (MEGS), which is an integrated computational and experimental approach, and identified that VF_0892 encodes the V. fischeri aspartate 1-decarboxylase (PanP). We also compared in vitro kinetic properties of PanP with Escherichia coli aspartate 1-decarboxlyase (PanD) and the V. fischeri glutamate decarboxylase.

Using the MEGS approach, PanP was first identified as missing from the genome annotation when discrepancies between metabolic model predictions and experimental culturing data arose. Then a selection strategy for PanP was designed, which consisted of a glucose minimal medium condition and an E. coli ∆panD strain whose growth in this medium was dependent on PanP. Finally, a genomic library of V. fischeri was transformed into E. coli ∆panD, and growth selection identified the VF_0892 as encoding the missing PanP enzyme.

VF_0892 is currently annotated in NCBI as a glutamate decarboxylase (E.C. 4.1.1.15). To compare the activities and substrate specificities of the VF_0892 and E. coli PanD enzymes towards the two potential substrates (aspartate and glutamate), decarboxylase activities were evaluated using the decarboxylase, phosphoenolpyruvate carboxylase, and malate dehydrogenase -linked assays. The VF_0892 enzyme was around 10-fold more active at 28°C compared to 37°C, which is consistent with the optimal growth of V. fischeriat 28°C and its intolerance to higher temperatures. Both PanD and the VF_0892 enzyme showed a much higher reaction rate when aspartate, rather than glutamate, was used as the substrate (PanD: 10-fold; VF_0892: 5-fold at 37°C, and 26-fold at 28°C). Using the same assays, we verified the VF_1064, annotated as a glutamate decarboxylase, truly encodes a decarboxylase with a strong substrate preference of glutamate over aspartate.

In addition to V. fischeri PanP, we have successfully used this integrated MEGS approach to determine the functions of ten metabolic genes in V. fischeri, Gluconacetobacter xylinus, and Zymomonas mobilis, which demonstrates the versatility of this method.