Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control

Abstract

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the Ipp Thus, topological studies of lipoprotein distribution in the outer membrane and of lipoprotein attachment to the peptidoglycan layer might be useful in improving our understanding of cell wall morphology and biosynthesis. For such studies (29), it is necessary to regulate the lipoprotein levels of the cell and to detect the newly inserted and linked molecules by immunoelectron microscopy.In this paper we approach both of these problems. The first condition was met by using E. coli JA221 Ipp/F' lacIP(pKEN125), which has a deletion in the chromosomal Ipp gene, whereas plasmid pKEN125 encodes lipoprotein under lacUV5 promoter control (21). The MATERIALS AND METHODS Organisms, growth, and radiolabeling conditions. E. coli JA221 lppIF' lacIq and E. coli JA221 IppIF' lacIP(pKEN125) were grown on M9 medium (20) supplemented with Ltryptophan (100 pug/ml) and L-leucine (100 ,ug/ml), with 0.5% (wt/vol) glucose or 0.5% (wt/vol) lactose as the carbon source. The strain harboring plasmid pKEN125 was grown in the presence of ampicillin (50 p.g/ml). E. coli JE5513, which lacks lipoprotein (5), was grown on Vogel and Bonner E salts medium (27) with 0.5% (wt/vol) glucose as the carbon source. All strains were grown with shaking (200 rpm) at 37°C. Cells containing plasmid pKEN125 growing exponentially on glucose were induced for lipoprotein with 2 mM IPTG (isopropyl-3-D-thiogalactopyranoside; Calbiochem-

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain

“…First, lipoprotein is not detectable at the cell surface because it is deeply buried in the inner leaflet of the outer membrane (9,22). Treatment of the cells with Tris-EDTA, which results in destabilization of the outer metmbrane structure (14,27,33), can be used to make lipoprotein accessible to externally added antibodies.…”

“…In a previous study, we adapted the technique described above for LamB to lipoprotein (22). First, lipoprotein is not detectable at the cell surface because it is deeply buried in the inner leaflet of the outer membrane (9,22).…”

“…It was shown that our cleared antilipoprotein serum is highly specific for lipoprotein and does .not react with LPS and other outer membrane proteins (22). The colloidal goldprotein A particles used for immunoelectron microscopy were prepared as described before (53).…”

“…The peptidoglycan sacculi were suspended in 1 ml of a 2% SDS solution. Large aggregates were removed by low-speed centrifugation (10 min, 2,000 x g) before application of sacculi (equivalent to 5 x 107 cells) to a perforated Parlodion film as described before (22 (19), and replication is stringently controlled. Table 1 shows the lipoprotein levels and the extent to which they were detected at the cell surface of the strains examined.…”

“…Gold-protein A particles that did not bind to the cell surface were removed by extensive washing. The cells were prepared for electron microscopy as described before (22).…”