Requirements for Borrelia burgdorferi plasmid maintenance

Abstract

Borrelia burgdorferi has multiple linear and circular plasmids that are faithfully replicated and partitioned as the bacterium grows and divides. The low copy number of these replicons implies that active partitioning contributes to plasmid stability. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated by the complexity of the genome and the possibility that products may act in trans. Consequently, we have studied the replication-partition region (bbb10-13) of the B. burgdorferi 26kb circular plasmid (cp26) in Escherichia coli, by fusion with a partition-defective miniF plasmid. Our analysis demonstrated that bbb10, bbb11, and bbb13 are required for stable miniF maintenance, whereas bbb12 is dispensable. To validate these results, we attempted to inactivate two of these genes in B. burgdorferi. bbb12 mutants were obtained at a typical frequency, suggesting that the bbb12 product is dispensable for cp26 maintenance as well. We could not directly measure cp26 stability in the bbb12 mutant, because cp26 carries essential genes, and bacteria that have lost cp26 are inviable. Conversely, we were unable to inactivate bbb10 on cp26 of B. burgdorferi. Our results suggest that bbb12 is dispensable for cp26 maintenance, whereas bbb10, bbb11, and bbb13 play crucial roles in that process.

Figure 1

A. cp26 region capable of conferring autonomous replication in B. burgdorferi. Potential promoter upstream of bbb10-13 denoted as P. Paralogous gene family (PF) names listed above. Potential functions, based on sequence homology (ParA) and ubiquitous presence on plasmids (Rep) indicated below. Putative parS is indicated by two arrowheads. Potential ori region downstream of bbb13 indicated. B. Map of miniF26, in which the autonomous replication region from cp26 is cloned into the miniF plasmid pDAG203. cam, gene conferring chloramphenicol resistance. ori2, miniF replication origin. C. Region upstream of bbb10-13 included in miniF26. Potential -35 and -10 regions of a promoter (predicted by the program BPROM) in bold type. Other potential -35 regions underlined. bbb10 potential ribosome binding site (RBS) and start codon boxed in gray. D. Sequence of the putative parS. Direct and inverted repeats indicated by arrows. Stop codon of bbb12 and start codon of bbb13 are boxed in gray, as is the presumed RBS. Bases in bold type are identical to the B. burgdorferi chromosomal parS (see section 3.2). E. Potential replication origin downstream of bbb13. Direct and inverted repeats indicated by colored arrows. ScaI site used in constructing bbb13 deletion is boxed in gray, as is stop codon of bbb13.

Figure 2

miniF plasmids containing various portions of the bbb1O-13 region used in this study. Positions of deleted sequences are indicated (Δ). ScaI and MfeI restriction enzyme sites used for mutant construction are indicated. P indicates position of putative promoter. Black triangles indicate repeats comprising the putative parS.

Figure 3

Stability assessment of pDAG203 (no insert) versus miniF26 (bbb10-13) and pDAG203 with a control insert (flgB_p-aacC1). Cultures grown with chloramphenicol were diluted into medium lacking antibiotic and plated at several times after dilution. Plasmid retention was assessed by replica plating to medium with and without chloramphenicol. Cultures derived from frozen stocks of HB101/pDAG203 (no insert) or HB101 carrying miniF with the control insert often exhibited an intermediate stability phenotype, between those of HB101/miniF26 (bbb1O-13) and that of a fresh transformant with pDAG203 (no insert, fresh transformant). In contrast, cultures derived from both fresh and frozen stocks of HB101/miniF26 exhibited similar stability.

Figure 4

Stability assessment of various miniF26 derivatives compared to those of pDAG203 (no insert) and miniF26 (bbb1O-13). A. Stability of plasmids miniF26Δbbb10, miniF26Δbbb11, and miniF26Δbbb10-11, all of which are less stable than miniF26. B. Stability of miniF26Δbbb13, which is less stable than miniF26. C. Stability of miniF26Δbbb12, which exhibits similar stabilty to miniF26. D. Comparison of plasmid stability of miniF26Δbbb11 and miniF26 with a chain termination mutation in bbb11(bbb11*). Data shown are from single experiments with duplicate cultures; all stability tests were performed at least twice and usually three or more times, with comparable results. Numbers at late time points represent calculated upper limits of plasmid retention, when zero plasmid-containing cells were obtained in the presence of antibiotic and detection limits varied, depending on the number of colonies obtained in the absence of antibiotic.

Figure 5

Lack of stabilization of miniF derivative plasmids by bbb10 and bbb11 integrated into the E. coli chromosome. Stability of pDAG203 (no insert), miniF26 (bbb10-13), miniF26Δbbb10, miniF26Δbbbll, and miniF26Δbbb10-11 in HB101 in which the bbb10 and bbb11 genes, with their own promoter region, were integrated into the E. coli chromosome.

Figure 6

Levels of bbb10 and bbb12 transcript in E. coli HB101 containing various miniF derivatives. Transcripts per 1000 copies of dxs (a chromosomal E. coli gene used as a normalization standard) detected in HB101 containing pDAG203 (no insert), miniF26 (bbb1O-13), miniF26Δbbb10, miniF26Δbbb11, miniF26Δbbb12, and miniF26Δbbb13. Transcript levels were determined by TaqMan analysis of cDNA, with primer/probe sets specific for bbb10 and bbb12 (Table 1). Data shown are averages of two experiments with triplicate samples in each experiment. Omitting the reerse transcription reaction led to a reduction of approximately 70-1000-fold, depending on the sample and primer/probe set.

Fig. 7

Assessing relative copy number of various miniF plasmids. Copy number was assessed by TaqMan analysis of total DNA preparations from HB101 containing pDAG203 (no insert), miniF26 (bbb10-13), miniF26Δbbb10, miniF26Δbbb11, miniF26Δbbb12, and miniF26Δbbb13. Each symbol represents the average of three assessments of miniF copies per dxs (E. coli chromosomal gene) copy. For pDAG203, miniF26, and miniF26Δbbb1O, three technical replicates of two biological replicates were performed, and for miniF26Δbbb11, miniF26Δbbb12, and miniF26Δbbb13, three technical replicates were performed.