Anaerobic Nitrate Respiration by Erwinia carotovora subsp. atroseptica during Potato Tuber Invasion.

The in planta induction of anaerobic nitrate respiration by Erwinia carotovora subsp. atroseptica in relation to the in situ oxygen status in soft rotting potato tubers has been investigated. In vitro experiments have shown that nitrate was required for the induction of respiratory nitrate reductase activity in E. carotovora. In addition, oxygen was found to repress this activity. Expression of respiratory nitrate reductase was found in E. carotovora cells extracted from soft rotting potato tuber tissue. However, the rate of nitrite production in these cells was approximately 70-fold lower than the rate recorded in fully induced anaerobic cultures. Oxygen measurements in soft rotting potato tubers indicated that the invading bacteria encounter the lowest oxygen concentration at the interphase between healthy and macerated tissue. Consequently, growth of bacteria present in this specific zone will be stimulated by nitrate which is present in sufficient amounts in tuber tissue. A high nitrate content of the tuber will most likely facilitate the proliferation of E. carotovora in the tuber tissue.

The role of different P-glycoproteins in hepatobiliary secretion of fluorescently labeled short-chain phospholipids.

Class III P-glycoproteins (Pgps) mediate biliary phosphatidylcholine (PC) secretion. Recent findings that class I P-glycoproteins are able to transport several short-chain phospholipid analogues raises questions about the role of these Pgps in physiological lipid transport. We investigated the biliary secretion of C6-7-nitro-2,1, 3-benzoxadiazol-4-yl (NBD)-labeled ceramide and its metabolites in Mdr1a/b and Mdr2 knockout mice compared to control mice. Biliary secretion of these NBD-lipids was unaffected in Mdr1a/b -/- mice. Thus neither Mdr1a nor Mdr1b Pgp mediates biliary secretion of these lipids. In contrast, secretion of all three NBD-labeled short-chain phospholipids was significantly reduced in Mdr2 -/- mice. As in vitro studies revealed that Mdr2 Pgp is not able to translocate these lipid analogues, we hypothesized that Mdr2 -/- mice had a reduced PC content of the exoplasmic canalicular membrane leaflet so that extraction of the short-chain lipid probes from this membrane by canalicular bile salts was impaired. To investigate this possibility we studied the bile salt-mediated extraction of natural sphingomyelin (SM) and NBD-labeled short-chain SM from small unilamellar vesicles of different lipid composition. Natural SM could be extracted by the bile salt tauroursodeoxycholate from vesicles containing PC, cholesterol (CHOL), and SM (1:2:2) but not from vesicles containing only SM and CHOL (3:2). NBD-labeled short-chain SM could be extracted from vesicles containing PC while its extraction from pure SM:CHOL vesicles was reduced by 65%. These data confirm that the efficiency of NBD-SM extraction depends on the lipid composition and suggest that the canalicular membrane outer leaflet of Mdr2 -/- mice has a reduced PC content.

Reversed-phase liquid chromatographic method for the determination of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labelled lipid analogues.

This paper reports the development of a dual column system for the simultaneous separation of fluorescent short-chain ceramide, 6-[(7-nitrobenz-2-oxa-1,3,-diazol-4-yl[NBD])amino]hexanoyl-sphingo sine and its metabolites, C6-NBD-sphingomyelin and C6-NBD-glucosylceramide, as well as the fluorescent derivatives of choline and serine phosphatides. The method enables the separation of these lipids in a single run on the basis of the polarity of their headgroups and hydrophobicity of their acyl backbone. The fluorescent properties of the NBD-label make it possible to quantitate small amounts of NBD-lipid analogues. The sensitivity of the presented method thus permits the use of small sample volumes and the determination of NBD-lipid analogues secreted into mouse bile directly, without prior extraction or concentration steps.