FIG 4.

Complementation of pigmentation occurs using exogenous sources of A-LPS. (A and B) Complementation of pigmentation with ultrapurified P. gingivalis LPS. Two different dilutions of 33277 wzy, 1:4 (A) and 1:9 (B), were spread onto BHI-T BA plates. Square pieces of blotting paper were then placed on top, and 20-μl volumes of ultrapurified LPS from P. gingivalis were applied (1, 20 μg; 2, 2 μg; 3, 0.2 μg; 4, 20 ng; 5, 2 ng). Plates were anaerobically incubated for 15 days at 37°C. Pigmentation is observed surrounding blotting paper 1 on both plates. (C and D) Complementation of an A-LPS biosynthetic mutant using purified OMVs from 381 porU. (C) The control patch was 10 μl of strain 33277 wzy mixed with 10 μl of control sterile water and spread onto either BBA or BHI-T BA plates. M1, M2, M3, and M4 are from mixes (10 μl-10 μl) of the wzy strain with serial 2-fold dilutions of purified filtered OMVs from the 381 porU mutant diluted in sterile water starting at 200 μg/μl. Control and mixes were applied to plates and incubated anaerobically for 4 days. (D) Whole-cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunostained with anti-A-LPS. Lane 1, WT 33277; lane 2, WT 381. Lanes 3 and 4 are equivalent to starting amounts of 381 porU OMVs; lanes 5 to 14 are whole-cell lysates from cells scraped from growth media shown in panel C. Because cell pellets were resuspended to the same wet weight per milliliter, lanes 3 and 4 represent the minimum and maximum starting amounts of porU OMVs in the mixed lysates (M1, M2, M3, and M4). The resulting image of the Western blot is produced by two exposures of the same blot perfectly overlapped and cropped to reveal the prestained protein ladder.