Total RNA was isolated from multiple tissues covering the major stages of mouse-tick infection cycle and bb0323 transcripts were measured using quantitative RT-PCR and presented as copies of bb0323 transcript per copy of flaB transcript. Shaded area denotes duration of tick feeding. Error bars represent the mean ± SEM from four quantitative RT-PCR analyses of two independent murine-tick infection experiments.

A, Construction of DNA construct (pXLF14301-pbb0323) for chromosomal integration of the bb0323. The open reading frame of bb0323 and its native promoter together with streptomycin resistance gene (aadA) was cloned into pXLF14301, which contains B. burgdorferi inserts for integration of the complemented gene in spirochete chromosome. B, RT-PCR analysis of the bb0323 transcripts. Total RNA was isolated from the wild type (WT), bb0323 mutant (bb0323⁻) or bb0323-complemented B. burgdorferi (bb0323 Com), converted to cDNA and subjected to PCR analysis. C, Reproduction of BB0323 protein by the complemented B. burgdorferi. Lysates of B. burgdorferi were separated on a SDS-PAGE gel, which was either stained with Coomassie blue (upper panel) or immunoblotted with specific antibodies (lower panels). D, Complementation of bb0323 mutant with bb0323 gene restores ultrastructural defects of mutant cells. B. burgdorferi were grown at a density of 5×10⁷/ml in BSK medium and processed for transmission electron microscopy. Arrows indicate enhanced shedding of vesicular structures or blebs, possibly derived from the outer membrane, in the mutants. Inset shows higher magnification images of a representative single cell indicating altered organization of outer membrane in the bb0323 mutant. Bar = 500 nm (inset Bar = 100 nm). E, Complementation restores spirochete infectivity in mice. Spirochete burden in groups of infected mice (5 animals/group) was analyzed by quantitative RT-PCR at days 7, 14, 21 or 28 by measuring copies of the flaB gene and normalized against mouse β-actin levels. Bars represent the mean ± SEM of relative tissue levels of pathogen from three independent animal infection experiments. F, bb0323-complemented B. burgdorferi are acquired by ticks. Mice were inoculated with spirochetes (5 mice/group) and following two weeks of infection, naïve I. scapularis nymphs (25 ticks/mice) were allowed to feed on mice. Spirochete burdens were assessed in ticks by qRT-PCR, by measuring copies of the B. burgdorferi flaB gene and normalized against tick β-actin levels. Bars represent the mean ± SEM from three independent experiments. G, bb0323-complemented B. burgdorferi persisted through larval-nymphal molt and transmit from feeding ticks to mice. B. burgdorferi-infected nymphs were generated by feeding larva on infected mice, which were allowed to feed to repletion on naïve mice (3 tick/mouse, 5 animal/group). Murine tissues were isolated at day 14 after repletion and the spirochete burdens were assessed by qRT-PCR. Bars represent the mean ± SEM from two independent experiments.

A, bb0323 mutants were unable to persist in unfed Ixodes nymphs. Nymphal ticks were microinjected with B. burgdorferi (WT), bb0323 mutant (bb0323⁻) or bb0323-complemented B. burgdorferi (bb0323 Com), and spirochete burdens in the gut of unfed ticks were analyzed at 14 days after injection. The spirochetes (arrow) were labeled with FITC-labeled goat anti-B. burgdorferi antibody (shown in green), and the nuclei of the gut cells were stained with propidium iodide (shown in red). Images were obtained using a confocal immunofluorescence microscope at 400x magnification, and presented as merge image for clarity. B, Decreased burdens of microinjected bb0323 mutants in feeding ticks. Nymphal ticks were microinjected with B. burgdorferi, as described in figure 5A, and placed on naïve mice 48 hours after injection. Spirochete burden was analyzed by quantitative PCR analysis of whole ticks at 24 hours and dissected gut or salivary glands at 48 hours of feeding by measuring copies of the B. burgdorferi flaB gene, normalized against tick β-actin levels. Bars represent the mean ± SEM of relative tissue levels of B. burgdorferi from three independent animal infection experiments. Differences between bb0323 mutant burdens and those with complemented or wild type isolates were significant at all time points and tissues (P < 0.02 or lower). C, bb0323 mutants were unable to transmit to mice. Nymphal ticks were microinjected as described in figure 5B, and placed on naïve mice following 48 hours after injection. Ticks were allowed to engorge and transmission of B. burgdorferi was assessed by measuring copies of the flaB gene, normalized against mouse β-actin levels, in indicated murine tissues after 14 days of tick feeding. Bars represent the mean ± SEM of relative tissue levels of B. burgdorferi from three independent animal infection experiments.

A, Position and amino acid sequence alignment of putative LysM-like domain of B. burgdorferi BB0323. The upper panel represents BB0323 protein indicating an amino terminal signal peptide (encompassing the first 20 amino acids) and a carboxyl terminal LysM domain. Lower panel shows amino acid sequence alignment of LysM-like domain of BB0323 to a designated LysM domain (Pfam accession number PF01476) or to orthologs in major B. burgdorferi sensu lato isolates and representative non-spirochete proteins with closest homologies. Dashes indicate gaps introduced to optimize alignments and identical amino acids are shaded. The numbers on the left are the positions of the amino acid residues in the proteins, for which corresponding species and annotations are indicated in the supplementary text. B, BB0323 is detectable in the outer membrane. Outer membrane vesicles were separated by sucrose density gradient centrifugation, separated by SDS-PAGE, and stained with SYPRO Ruby or immunoblotted with BB0323, OspA and FlaB antibodies. C, BB0323 is partially sensitive to proteinase K-mediated degradation of B. burgdorferi surface proteins. Viable B. burgdorferi were incubated with (+) or without (−) proteinase K for the degradation of protease-sensitive surface proteins and processed for immunoblot analysis using BB0323 antibodies. B. burgdorferi OspA and FlaB antibodies were utilized as controls for surface-exposed and sub-surface proteins, respectively. D, Schematic drawings of the wild type (WT) and the bb0323 mutant (bb0323⁻) isolates at the bb0323 locus. Genes bb0319 - bb0326 (white box arrows) and the kanamycin-resistance cassette driven by the B. burgdorferi flaB promoter (flaB-Kan, black box arrow) are indicated. Primers P1–P4 (black arrow-heads) were used to amplify 5′ and 3′ arms for homologous recombination, and regions flanking up- and down-stream of the bb0323 locus were ligated on either side of the flaB-Kan cassette as detailed in the text. E, Integration of the mutagenic construct, flab-Kan, in the intended genomic locus. Primers 5–10 (gray arrow-heads, positions indicated in figure 2D) were used for PCR analysis using isolated DNA from wild type (WT) or mutant B. burgdorferi (bb0323⁻) and subjected to gel electrophoresis. The combination of primers used for PCR is indicated at the top, and migration of the DNA ladder is shown on the left. F, RT-PCR assessment of bb0323 ablation and the polar effects of mutagenesis. Total RNA was isolated from wild type B. burgdorferi (WT) or bb0323 mutant (bb0323⁻) converted to cDNA and used to amplify regions within bb0323, flaB, kanamycin and genes surrounding bb0323 locus (bb0322 and bb0324) and visualized on a gel. G, Protein analysis of wild type and mutant isolates. Equal amounts of protein were by SDS-PAGE gel, and either stained with Coomassie blue (left panel) or immunoblotted with BB0323 and FlaB antibodies (right panel). Migration of protein standards is shown to the left in kDa.

A, Severity of joint swelling in B. burgdorferi-infected mice. Groups of mice (5 animals/group) were infected with the wild type (WT), bb0323 mutant (bb0323⁻) or bb0323-complemented B. burgdorferi (bb0323 Com), and assessed for joint swelling. Bars represent the mean ± SEM from three independent infection experiments. Differences in the joint swelling between groups of mice infected with bb0323 mutant and those with the bb0323-complemented isolates were highly significant (*P < 0.01) at all time points, except for day 7. B, Representative demonstration of joint histology in mice infected with wild type or genetically-manipulated B. burgdorferi isolates. Twenty-one days following spirochete infection, tibiotarsal joints were analyzed for histopathology. The left panel indicates low-resolution (40x, bar = 400 μm) joint images. Higher-resolution (200x, bar = 80 μm) images of selected areas from corresponding sections (marked by box) are shown in the right panels.