| dc.description | Acyl carrier protein (ACP) is a multifunctional fatty acid carrier which interacts with several different enzymes during the synthesis of fatty acids, phospholipids, and other specialized molecules in bacteria. Luminescent marine bacteria such as Vibrio harveyi also require 14:0-ACP for the synthesis of the aldehyde substrate of luciferase. To characterize V. harveyi ACP and elucidate its role in the metabolism of exogenous fatty acid, V. harveyi ACP was purified to homogeneity. A degenerate oligonucleotide probe based on partial protein sequence data was used to isolate a 1.4 kb EcoR1 fragment in pUC18 encoding the N-terminal two-thirds of V. harveyi ACP. This was subsequently used to clone an overlapping 3 kb HindIII fragment encoding the C-terminal region. The predicted amino acid sequence of V. harveyi ACP (76 residues; Mr = 8,734; pI = 4.0) is 86% identical to that of E. coli ACP, while the DNA sequence is 79% identical. Sequencing of upstream regions indicated an organization of V. harveyi fatty acid biosynthetic genes similar to that found in E. coli (fabD-fabG-AcpP). V. harveyi acyl-ACP synthetase was used to enzymatically prepare fatty acylated derivatives of E. coli and V. harveyi ACP (chain lengths C$\sb6$ to C$\sb )$. Both ACPs migrated anomalously as 20 kDa bands on SDS-polyacrylamide gel electrophoresis, but they exhibited different behaviour upon fatty acylation. Immunoprecipitation with anti-V. harveyi ACP serum was used to demonstrate that V. harveyi acyl-ACP can be labeled in vivo with exogenous (9,10$\sp{-3}$H) myristic acid; chain length-dependent resolution of acyl-ACPs on urea gels revealed that most of the label was associated with chain lengths (C$\sb8$ to C$\sb )$ shorter than the added fatty acid. However, different labeling profiles were observed after incubation with ($\sp3$H) acetate, indicating that acyl-ACP labeling with ($\sp3$H) 14:0 is not due to total degradation of ($\sp3$H) 14:0 to ($\sp3$H) acetyl-coenzyme A followed by resynthesis. Additional bands which comigrated with acyl-ACP on SDS gels were identified as lipopolysaccharide. Thus, in contrast to E. coli, exogenous fatty acids can be activated to form acyl-ACP intermediates after partial degradation in V. harveyi and can effectively label products (i.e. lipid A) that require ACP as an acyl donor. The transport mechanism of exogenous long chain fatty acid in V. harveyi was also explored by using mutagenesis and different screening methods. The results indicated that a protein-mediated transport system similar to E. coli may not be required for uptake of 14:0 in V. harveyi. Despite overall similarities in fatty acid and phospholipid composition, therefore, fatty acid metabolism in the luminescent bacterium V. harveyi exhibits several key differences with that in E. coli. | en_US |
