Abstract

Forming the interface between the bacterial cell and the host, the outer membrane of Borrelia is known to play a key role in pathogenicity. Although Borrelia burgdorferi sensu lato are considered to be Gram-negative, their outer membrane is unique, lacking liposaccharides and phosphatidylethanolamine. It contains a variety of glycolipids, surface exposed lipoproteins and a number of membrane-spanning β-barrels. BAPKO_0422, BB_0562 and BG_0408 are membrane proteins, theoretically predicted to form 8-stranded, membrane-spanning β-barrels. The aim of this work is to produce recombinant versions of these proteins, and determine molecular envelopes by small-angle X-ray scattering (SAXS). The β-barrel model can then be tested by comparing the experimental molecular envelope with the theoretical predictions. Three Borrelia proteins (BAPKO_0422, BB_0562 and BG_0408) were recombinantly expressed in the E. coli expression system using the pET-47 expression vector. The putative membrane proteins were purified by immobilised metal affinity chromatography (IMAC). The His tag of BAPKO_0422 was enzymatically removed to produce a native protein, and to allow for a visual comparison of the protein both with and without the His tag. SAXS data for each protein were collected and the overall shape was determined using ab initio methods. The pair-distance distribution function (P(r) function) of BAPKO_0422 with the His tag indicated a particle overlap potentially caused by the flexible 6-His tag at the N-terminus. Kratky plots of BAPKO_0422, BB_0562 and BG_0408 revealed the parabolic convergence for a folded particle. The low-resolution molecular envelopes of BAPKO_0422, BB_0562 and BG_0408 are consistent with the structure of an 8-stranded β-barrel. The filtered envelopes are in agreement with the shape and size of the E. coli homologue OmpX. The likely orientation of the protein within the outer membrane can be deduced by comparing molecular envelopes with and without the N-terminal His tag. The data suggest that BAPKO_0422, BB_0562 and BG_0408 are single-domain cylindrical-shaped molecules with no evidence of an internal pore. Several questions remain to be answered, such as the oligomeric state of BG_0408 and BAPKO_0422. The function of these 8-stranded β-barrels in the Borrelial outer membrane remains to be investigated.

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