Figure 1.
Scheme to identify OM proteins utilizing bioinformatic programs.
The Signal P program identified 2493 signal peptide containing proteins among the putative 7331 member M. xanthus proteome. Of these 425 lipoproteins were identified using Lipo P. Of the 2068 proteins without a lipoprotein signal 560 were integral IM proteins identified using TMHMM. The non-IM proteins include periplasmic proteins, secreted proteins and OM proteins. Finally, integral OM proteins containing a β-barrel domain were identified using TMBETA-SVM plus TMBETADISC-RBF.
Table 1.
M. xanthus β-barrel domain proteins obtained from the Pfam database.
Table 2.
M. xanthus IM, periplasmic and ECM proteins1.
Table 3.
OM β-barrel proteins identified by LC-MS/MS.
Table 4.
OM lipoproteins identified by LC-MS/MS.
Figure 2.
Role of oar in cell signaling.
(A) Extracellular complementation of oar (LS2453) cells with ΔcsgA (LS2441) and esg (JD300). WT (DK1622) cells were used as a control. Bar is 1 mm (B) Western blot analysis of vegetative cells and 24 h developing cells using anti-CsgA primary antibody. (C) Morphology of oar cells during development. The first panel represents 24 h developing WT cells while the subsequent panels represent various oar cell shapes as they ultimately transform into spheroplast (extreme right panel). Bar is 1 µm.
Figure 3.
Bioinformatic analysis of first eight amino acids of N-terminus of M. xanthus lipoproteins.
(A) Multiple sequence alignment of ECM proteins using WebLogo. The lipobox (highlighted by a box made of dashed lines) and the following seven amino acids of the N-terminal region of mature lipoproteins were aligned using WebLogo. Seven ECM lipoproteins have alanine at the 7th position (highlighted by a solid box). (B) 8/12 predicted IM lipoproteins have lysine at the 2nd position (highlighted by a solid box). (C) Western blot analysis of 18 h developing cells using Mab2105 primary antibody. Strains used include LS2760 (WT FibA), LS2208 (ΔfibA), LS2761 (N22K FibA), LS2764 (A27D FibA).
Table 5.
Putative lipoproteins identified by LC-MS/MS from IM fraction.
Figure 4.
Identification of M. xanthus lipoprotein sorting signals.
(A) Immuno transmission electron microscopy of developing cells using monoclonal antibody Mab2105. M. xanthus cells were allowed to form a biofilm on a formvar-carbon-coated nickel grid for 3 h. The cells were probed with Mab2105, which reacts primarily with FibA followed by anti-mouse antibodies conjugated with 10 nm colloidal gold particles. Bar is 500 nm. (B) Western blot analysis of membrane fractions purified from 7–8 h developing cells.
Figure 5.
Pie chart classifying 228 OM β-barrel proteins according to function.
Most M. xanthus OM proteins have no known function. The second major class of proteins includes TonBs, which are required for transport of a specific substrate. Transport of small molecules are carried out by transporters and OmpA, membrane efflux proteins are required for export of toxins and secretins form a large OM pore that allow export by Type II secretion system.
Table 6.
Bacterial strains, plasmids and primers used in this study.