Identification and Localization of Myxococcus xanthus Porins and Lipoproteins

Swapna Bhat,Ricky P Patel,Xiang Zhu,Ron Orlando,Lawrence J Shimkets,Roy Martin Roop

doi:10.1371/journal.pone.0027475

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.

Highlights

The life cycle of Myxococcus xanthus involves a vegetative stage, in which cells feed on bacteria and organic detritus, and a developmental stage in which thousands of cells aggregate to form a multicellular fruiting body containing spores
B-barrel prediction in M. xanthus proteome Integral outer membrane (OM) proteins are synthesized with an N-terminal signal sequence
Proteins with at least two putative transmembrane helices using the TMHMM program were classified as inner membrane (IM) proteins [13]. 560/2068 (27%) proteins with putative signal peptides were identified as IM proteins

Summary

Introduction

The life cycle of Myxococcus xanthus involves a vegetative stage, in which cells feed on bacteria and organic detritus, and a developmental stage in which thousands of cells aggregate to form a multicellular fruiting body containing spores. Fruiting body development involves intercellular communication with at least six extracellular signals [1]. Identification of outer membrane (OM) proteins in M. xanthus may reveal components of these signaling pathways that are used to export or import signals. The OM acts as a selective barrier that allows the passage of nutrients, water and chemical signals through pores formed by porin proteins. Active diffusion of specific nutrients through porins is carried out by TonB systems, which utilize energy provided by the inner membrane (IM) to mediate solute passage through the OM [4]. Chaperones in the periplasm facilitate protein folding and insertion into the OM using the Omp machinery [7]

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