Identification and localization of Myxococcus xanthus porins and lipoproteins

Abstract

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site.

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.

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 7^th position (highlighted by a solid box). (B) 8/12 predicted IM lipoproteins have lysine at the 2^nd 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).

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.