K-078. Expression of Functional hydA from Anerobic Enterobacter cloacae into Aerobic Azotobacter vinelandii

L. Pulakat, N. Gavini;
Mississippi State Univ., Mississippi State, MS.

The most efficient H2 producing hydrogenases are the Fe-hydrogenases that shows almost 100 times more efficient H2 production when compared to any other hydrogenases. However these enzymes are normally expressed by anaerobes. The smallest of these enzymes is found in Enterobacter cloacae IIT-BT 08, a gram negative anerobic bacterium. This enzyme is encoded by the gene hydA. A. vinelandii is a diazotroph with one of the highest respiratory rates known among living organisms. In spite of being an aerobe, this organism maintains an internal environment that supports efficient function of oxygen sensitive nitrogenase. Therefore we tested the ability of this organism to overproduce the smallest hydrogenase encoded by hydA and maintain its functional properties. The broad-host range vector pBHR1 was used for this study. Transcription from the nifH promoter is upregulated by 100 fold when the cells are starved for N2. Therefore we generated a gene construct in which the nifH promoter was fused to the open reading frame of the hydA gene of E. cloacae and a His-Tag. The construct was cloned into pBHR1 to generate pMS 1000 and was introduced into A. vinelandii. Cells carrying pMS1000 over expressed hydA when grown in nitrogen minus medium. Over expressed protein was visualized by Western blot analysis and its ability to produce hydrogen was assessed by S121 H2 Sensor. Our results suggest that the functional hydA can be over expressed in A.. vinelandii.