Fatty acid degradation in Caulobacter crescentus

Abstract

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membranemediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid 13-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, 3-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in 1-oxidation by 2-to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the 13-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. TnS insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had 1-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the 1-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case in fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.The cell cycle of the gram-negative bacterium Caulobacter crescentus exhibits a series of unicellular differentiation events. Several of these events involve changes at the cell surface and include the biogenesis of a polar flagellum, chemosensory receptors, phage receptors, and pili (23). The synthesis of many of the membrane proteins involved in these structures and functions is temporally regulated. Upon synthesis these proteins are localized to specific portions of the cell and segregate to only one daughter cell upon division (23). Thus, some type of spatial control mechanism must contribute to their positioning in the cell membrane. To help define the role that the cell membrane might play in these processes, we investigated the composition and metabolism of the membrane phospholipids.The negatively charged phospholipids, phosphatidylglycerol and cardiolipin, are the major components, and neither phosphatidylethanolamine nor phosphatidylserine can be detected in C. crescentus membranes (6). The enzymatic pathway of phospholipid synthesis in C. crescentus is the same as the comparable portions of the pathway in Escherichia coli (5). The fatty acid components of the phospholipids consist of both saturated and unsaturated 14-, 16-, and 18-carbon fatty acids (4, 16), and these ...

Purification and characterization of fatty acid beta-oxidation enzymes from Caulobacter crescentus

“…It remains to be determined whether C. crescentus uses enzymes besides 13-ketoacylCoA thiolase and acetoacetyl-CoA thiolase for the long-and short-chain fatty acid substrates. If more than one enzyme is present for different-chain-length fatty acids at specific steps of the P-oxidation pathway, it may explain our difficulty in obtaining mutants in the p-oxidation pathway (22). The substrate specificities of these enzymes might overlap and mask a lesion in a specific enzyme.…”

“…The C. crescentus 3-oxidation enzymes, acyl-CoA synthetase, acyl-CoA dehydrogenase, crotonase, 3-hydroxyacyl-CoA dehydrogenase, and ,B-ketoacyl-CoA thiolase, have been identified, and their initial characterization has been described previously (22). The enzymes are constitutively expressed in C. crescentus and are not induced by oleic acid.…”

“…Long-chain fatty acids are transported into the cell and degraded by constitutive p-oxidation enzymes. Five enzymes in the long-chain fatty acid p-oxidation pathway have been identified in C. crescentus (22). These (1), with palmityl-CoA as the substrate.…”

“…Some of these mutants are auxotrophic for glycerol-3-phosphate and lack glycerol-3-phosphate dehydrogenase activity (3); others require oleic acid for growth (10, 11). A common feature of these mutants is that they all block membrane lipid synthesis in the absence of supplement, coincident with the disruption of surface differentiation events.To determine how membrane lipid turnover might contribute to the expression of cell surface differentiation events in C. crescentus, we have begun an investigation of both longand short-chain fatty acid catabolism in this organism (22 3-hydroxyacyl-CoA dehydrogenase, and P-ketoacyl-CoA thiolase. Long-chain fatty acids are degraded by these five p-oxidation enzymes in E. coli (Fig.…”

“…To determine how membrane lipid turnover might contribute to the expression of cell surface differentiation events in C. crescentus, we have begun an investigation of both longand short-chain fatty acid catabolism in this organism (22 3-hydroxyacyl-CoA dehydrogenase, and P-ketoacyl-CoA thiolase. Long-chain fatty acids are degraded by these five p-oxidation enzymes in E. coli (Fig.…”