OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

Roland Benz,Ulrich Kleinekathöfer,Elke Maier,Friedrich Lottspeich,Michael D Jones,Niraj Modi,John Smit,Reinhard Mentele,Farhan Younas,Eric Cascales

doi:10.1371/journal.pone.0143557

Abstract

Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.

Highlights

The cell-envelope of Gram-negative bacteria consists of different layers
Crude membranes from C. crescentus CB15A NA1000 353F cells that were disrupted by sonication and membrane material released by phosphate-buffered saline (PBS)/EDTA treatment were analyzed by SDS-PAGE
We demonstrated the presence of a channel in the enriched outer membranes of the alpha proteobacterium C. crescentus

Summary

Introduction

The cell-envelope of Gram-negative bacteria consists of different layers. The inner or cytoplasmic membrane contains the respiration chain, proteins for the transport of nutrients and proteins involved in the synthesis of phospholipids, peptidoglycan and lipopolysaccharides [1, 2]. The periplasmic space between the membranes is an aqueous compartment iso-osmolar to the cytoplasm [3]. It contains the peptidoglycan and a large number of different proteins. In addition to constitutive porins, an outer membrane may contain porins that are induced under special growth conditions [3]. They often form solute-specific channels and contain binding sites for neutral substrates such as carbohydrates [5, 6], or nucleosides [7] and phosphate [8, 9]. Many of the specific porins are part of uptake and degradation systems, such as the maltose uptake system of Escherichia coli [10]

Methods

Results

Conclusion