Figure 1.
Gene disruption and confirmation.
A) Gene disruption strategy. The infectious B. burgdorferi strain B31, clone 5A4 (B31-5A4) was transformed with a knockout plasmid carrying a one kb gentamicin cassette (blue) that replaced the central portion of the target gene (yellow) as described in Materials and Methods. The two possible outcomes of recombination events with the target gene are shown: allelic exchange would result in gene disruption while integrative recombination of the knockout plasmid would result in merodiploid formation. The position of PCR primers used for construct verification are shown by arrows on the schematic. B) Construct verification of the mutL disruption by PCR. Each gene disruption was subjected to four PCR analyses. 1) The presence of the gentamicin resistance cassette was confirmed as shown in lanes 1 and 2. The shuttle vector pBSV2G [71] served as the positive control c+ for amplification of the gent cassette (lane 3.) 2) The portion of mutL expected to be deleted in a gene disruption was not detected in either mutL1 or 2 (lanes 5 and 6); however, it was detected in the positive control (c+), which contained wild-type B31-5A4 DNA as a template in lane 7. 3) The size of the target gene was compared in mutL1 and 2 genotypes. The expected 2.1 kb products for a gene disruption were observed (lanes 9 and 10) in comparison to the 1.5 kb product from the mutL+ genotype (lane 11). Lanes 4, 8 and 12 are negative controls (c−) that lacked DNA template. 4) Confirmation of the correct insertion site was performed using combinations of the target gene primers and primers internal to the gentamicin cassette to amplify the boundaries. The left boundary in both mutL clones gave the expected 0.55 kb product (lanes 13 and 15). The right boundary in both clones gave the expected product of approximately 1.3 kb (lanes 14 and 16). A 100bp ladder on the left side, relevant to the two left panels, and a 1kb ladder on the right side, which applies to the two right panels, were the molecular weight markers (M) used.
Table 1.
Gene disruption targets and knockout plasmid attributes.
Figure 2.
Restriction fragment length polymorphism assay for switching at vlsE.
A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. PCR reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with HphI and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2/1 DNA templates are found in lanes 5 & 6, and 7 & 8 respectively. M denotes a 100bp molecular weight marker.
Table 2.
Effect of DNA repair and replication mutants on B. burgdorferi inefction and switching at vlsE in C3H/HeN mice.
Table 3.
Effect of DNA repair and replication mutations on B. burgdorferi infection in SCID C3H/HeN mice.
Figure 3.
Number of switched vlsE clones in SCID C3H/HeN mice.
Sequencing of the cloned PCR product of the vlsE variable regions using primer pJET1.2/forward was performed on 10 clones from each tissue type culture for each genotype (see Materials and Methods). The y-axis denotes the number of clones out of ten that contained templated nucleotide changes in variable regions 1–6 (switches) and the x-axis denotes the tissue type. The P-values above the bars indicate the level of significance of the difference between the wild-type and mutant samples, calculated using Fisher's Exact test.