Omp85 _Tt from Thermus thermophilus HB27: an Ancestral Type of the Omp85 Protein Family

Abstract

Proteins belonging to the Omp85 family are involved in the assembly of ␤-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria, and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered, including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, Omp85 Tt , representing an ancestral type of this family. We overexpressed Omp85 Tt in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85 Tt existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly ␤-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85 Tt revealed ring-like structures with a central cavity of ϳ1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85 Tt forms ion channels with two different conducting states, characterized by conductances of ϳ0.4 nS and ϳ0.65 nS, respectively.

Homogeneous incorporation of secondary cell wall polysaccharides to the cell wall of Thermus thermophilus HB27

“…This single SLH domain of SlpA is required in vivo for the attachment of the OML envelope to the cell wall (Olabarria et al 1996), suggesting that it constitutes a primitive form to attach ancestral OMs to the cells. The functional similarity of the OML from T. thermophilus to the OM from Proteobacteria has been recently reinforced by the discovery of a homologue of the Omp85 protein family implicated in the insertion of betabarrel proteins like porins in the OM (Nesper et al 2008).…”

Crystal Structure of a Major Outer Membrane Protein from Thermus thermophilus HB27

“…Native TtOmp85 was purified from T. thermophilus HB27∷nar pMKE2-TtOmp85 cells as described previously. 10 TtoA was purified together with TtOmp85; separation was achieved by gel-filtration chromatography. For crystallization, His-TtoA was expressed in T. thermophilus HB8 harboring plasmid pMK18-HisTTC0834, which expresses TtoA from its own promoter, with its signal sequence and a His tag followed by two Ala residues inserted after the cleavage site of the signal peptidase at position 2 of the mature sequence.…”

“…10 For the identification of proteins, OM preparations of strain HB27 were separated by SDS-PAGE 36 and stained with Coomassie G250 (Serva). Protein bands were cut out and subjected to mass spectrometric analysis (Proteome Factory, Berlin, Germany).…”

“…9 We previously characterized TtOmp85, an Omp85 family protein of HB27, as a ring-like, poreforming and primarily monomeric OMP. 10 Apart from TtOmp85, no further putative β-barrel OMP was predicted on the basis of sequence homology in the genome sequences of HB27 and HB8. 11,12 Prediction of β-barrel OMPs from the primary sequence is still difficult because of the presence of short transmembrane stretches.…”

Expression, crystallization and preliminary X-ray analysis of an outer membrane protein fromThermus thermophilusHB27

“…They are involved either in the translocation of proteins across the OM or in the insertion of -barrel proteins into the OM (Schleiff & Soll, 2005). To date, we have characterized TtOmp85 in vitro as a monomeric stable protein that forms ion channels (Nesper et al, 2008). In order to obtain evidence of whether TtOmp85 is involved in the biogenesis of OM proteins in T. thermophilus, we first had to identify putative -barrel OM proteins.…”