Purification and properties of the plasmid maintenance proteins from the Borrelia burgdorferi linear plasmid lp17

Abstract

The Lyme disease spirochete Borrelia burgdorferi carries more plasmids than any other bacterium, many of which are linear with covalently closed hairpin ends. These plasmids have also been referred to as mini-chromosomes and essential genetic elements and are integral components of its segmented genome. We have investigated two plasmid maintenance proteins, BBD14 (the replication initiator) and BBD21 (a presumptive ParA orthologue), encoded by the linear plasmid lp17; these proteins are representatives of paralogous families 62 and 32, respectively. We have purified recombinant 6-his-BBD21 and shown it possesses an ATPase activity. 6-his-BBD14 initially could not be overexpressed in Escherichia coli by itself. It was only effectively overproduced in recombinant form through coexpression with other B. burgdorferi proteins and codon optimization. Although the mechanism for increased production through coexpression is not clear, this method holds promise for expression and purification of other B. burgdorferi proteins, a number of which have remained recalcitrant to purification from E. coli. Finally, we present evidence for the physical interaction of BBD14 and BBD21, a feature suggesting that BBD21 and the paralogous family 32 proteins are more likely involved in DNA replication than functioning as simple ParA orthologues as previously surmised based upon sequence homology. Such a role would not preclude a function in plasmid partitioning through interaction with the replication initiator.

FIG. 1.

Putative B. burgdorferi plasmid replication proteins. The 12 linear and 10 circular plasmids of Borrelia burgdorferi strain B31 are shown (8, 17, 32). The five paralogous gene families encoding plasmid maintenance proteins are denoted as colored circles (8). The four lp28 plasmids have been grouped by a bracket. This figure was adapted from reference with permission of the publisher.

FIG. 2.

Alignment of BBD21 with two related protein families. The proposed ATPase domain of four members of the ParA family and four members of the cobrynic acid synthase family found through BLAST searches were aligned with BBD21 using ClustalW (11), followed by manual adjustment of the sequences. Completely conserved residues are shaded in orange, and similar residues (as defined by the Blossom62 matrix [25]) are yellow. The threshold for shading was 67% identity/similarity. Known ParA/ATPase motifs (19) are indicated above the sequence. Numbering on the bottom of the alignment corresponds to that of BBD21. White-on-blue residues are those that were mutated in this study. Similarity extends throughout the length of the proteins but is not shown here. The GenBank accession numbers of the aligned proteins are as follows: Soj protein in Rickettsia prowazekii, NP_220452; Spo0A activation inhibitor in Clostridium perfringens, NP_563568; MinD family ATPase Soj in Clostridium sp., NP_350310; Soj protein in Rickettsia conorii, NP_359723; cobyrinicacid-a,c-diamidesynthase of Desulfovibrio vulgaris, YP_965488.1; hypotheticalprotein RcanM of Rickettsia canadensis, ZP_01347170.1; cobyrinicacid-a,c-diamidesynthase of Methylophilalesbacterium sp. strain HTCC2181, ZP_01551553.1; cobyrinicacid-a,c-diamidesynthase of Acidobacteriabacterium ellin, ABF39046.1.

FIG. 3.

Induction and purification of 6-his-BBD21. A Coomassie blue-stained 15% SDS-polyacrylamide gel is shown. Lanes: 1 and 6, molecular mass markers; 2, 10 μl of whole-cell extract of uninduced Rosetta cells containing pCB55 for overexpression of 6-his-BBD21; 3, 10 μl of whole-cell extract of Rosetta cells containing pCB55, induced by 1 mM IPTG and grown for 45 min at 37°C; 4, 10 μg of 6-his-BBD21 after purification with a Ni-NTA column; 5, 10 μg of Ni-NTA-purified 6-his-BBD21 after subsequent purification by glycerol gradient centrifugation.

FIG. 4.

Glycerol gradient purification and ATPase activity of 6-his-BBD21. (A) Glycerol gradient purification of wild-type 6-his-BBD21 protein was performed subsequent to the Ni-NTA step. The ATPase activity, as well as the protein concentrations from fractions obtained from the glycerol gradient, was determined as described in Materials and Methods. (B) Glycerol gradient purification of 6-his-BBD21Δ19. Further details are as for panel A. (C) ATPase activities of 6-his-BBD21 wild-type protein and various mutant proteins are shown. All proteins were purified by Ni-NTA column chromatography followed by glycerol gradient centrifugation as shown in panels A and B. The fraction with the highest concentration of 6-his-BBD21 protein was assayed for ATPase activity as described in Materials and Methods. ATPase activity per pmol of protein is shown relative to wild-type 6-his-BBD21.

FIG. 5.

Construct for coexpression of BBD21 and 6-his-BBD14. A map of the coexpression plasmid pJD204, which expresses both wild-type bbd21 and His-tagged bbd14 from the same inducible T7 promoter is shown. The construct is derived from the pET15b vector (see Materials and Methods and Table 1). The sequence of the cistronic linker connecting bbd21 and 6-his-bbd14 is shown in blue below the map. Only the BamHI and NdeI sites used in the cloning are shown. The construct carries a codon-optimized synthetic 6-his-bbd14 gene (see Materials and Methods).

FIG. 6.

Coelution of BBD21 from Ni-NTA with His-tagged BBD14. (A) Wild-type BBD21 and His-tagged BBD14 were coexpressed from a single promoter on pJD204 (Fig. 5). A Coomassie blue-stained 15% SDS-polyacrylamide gel of the coelution of the two proteins from Ni-NTA is shown. A size marker is shown in the left-most lane. Lanes 7 to 19 (fraction numbers) contained 20 μl of the eluate from the Ni-NTA purification step (see Materials and Methods). (B) Wild-type BBD21 was expressed in the absence of His-tagged BBD14 from pJD164 (Table 1). A Coomassie blue-stained 15% SDS-polyacrylamide gel is shown as for panel A. The lane labeled FT contains 20 μl of the flowthrough applied to the Ni-NTA column, showing that BBD21 did not bind to the column in the absence of His-tagged BBD14. (C) Wild-type BBK21 (the family 32 paralogue from lp36) and His-tagged BBD14 were coexpressed from a single promoter on pJD223 (Table 1). A Coomassie blue-stained 15% SDS-polyacrylamide gel of the elution profile from Ni-NTA is shown. M denotes the marker lane with purified BBK21.