Deletion of lolB, encoding an outer membrane lipoprotein, is lethal for Escherichia coli and causes accumulation of lipoprotein localization intermediates in the periplasm

Abstract

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.

FIG. 1

Effect of Lpp on the growth of the ΔlolB::kan mutant. KT5 (A and B) and KT6 (C and D) were grown at 30°C (open circles) or 42°C (closed circles) for the indicated times by inoculating portions of the cultures into fresh medium. The culture turbidity at 660 nm was monitored and plotted after correction for the culture dilution (A and C). The number of viable cells (B and D) during growth at 30°C and 42°C was determined at the specified times by plating aliquots of the cultures onto L broth containing kanamycin at 25 μg/ml, followed by overnight incubation at 30°C.

FIG. 2

Depletion of LolB in the ΔlolB::kan mutant at 42°C. KT5 (A) and KT6 (B) grown at 30°C on L broth containing kanamycin at 25 μg/ml were transferred to 42°C and then cultured for the indicated times. Aliquots of the cultures were withdrawn and treated with Laemmli sample buffer at 100°C for 5 min. Cellular proteins (100 μg) were analyzed by SDS-PAGE and immunoblotting with anti-LolB antibodies. As controls (C), JE5505 (lanes 2 and 4) and JE5506 (lanes 1 and 3) grown for 10 h at 30°C (lanes 1 and 2) and 42°C (lanes 3 and 4) were also analyzed.

FIG. 3

Accumulation of Pal and Lpp in the periplasm of LolB-depleted cells. (A) KT5, KT1, and FS1576 were grown on M63 minimal medium supplemented with 0.2% maltose at 30°C or 42°C and then labeled with Tran³⁵S-label for 30 s. The labeling at 42°C was started immediately before growth arrest occurred and stopped by the addition of cold methionine and cysteine on ice. Periplasm fractions were prepared from these cells as described in Materials and Methods and then subjected to immunoprecipitation with antibodies raised against Pal, Lpp, and MBP. The precipitates were analyzed by SDS-PAGE and fluorography. (B) The periplasm fraction prepared from KT1 cells grown on L broth at 42°C was immunoprecipitated with nonimmune (lanes 1 and 3), anti-LolA (lane 2), and anti-Lpp (lane 4) antibodies and then analyzed by SDS-PAGE and immunoblotting with anti-Lpp (lanes 1 and 2) and anti-LolA (lanes 3 and 4) antibodies.

FIG. 4

Accumulation of Pal in the inner membrane of LolB-depleted cells. KT5/pTAN20 and JE5505/pTAN20 were grown at 42°C and then labeled with Tran³⁵S-label as described in the legend to Fig. 3. Envelope fractions were prepared from the labeled cells and subjected to sucrose density gradient centrifugation, followed by fractionation into 13 fractions from the bottom to the top of the gradient. Each fraction was analyzed by SDS-PAGE and fluorography. OmpA and Pal were identified on the gel by their molecular masses and migration positions. OM and IM represent the outer and inner membrane fractions, respectively.