SummaryThe genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic-di-GMP metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic-di-GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic-di-GMP with a K m of 0.054 μM, confirming it is a functional cyclic-di-GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic-di-GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic-di-GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic-di-GMP, B. burgdorferi motility and Lyme disease pathogenesis. A mechanism by which cyclic-di-GMP influences motility and infection is proposed.

The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [K d ], 1.25 M), consistent with K d values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID 50 ]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently, plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded.

Motility and chemotaxis are essential components of pathogenesis for many infectious bacteria, includingBorrelia burgdorferi, the causative agent of Lyme disease. Motility and chemotaxis genes comprise 5 to 6% of the genome of B. burgdorferi, yet the functions of most of those genes remain uncharacterized, mainly due to the paucity of a nonpolar gene inactivation system. In this communication, we describe the development of a novel gene inactivation methodology to target B. burgdorferi fliL, a putative periplasmic flagellar gene located in a large motility operon and transcribed by RNA polymerase containing 70 . Although the morphology of nonpolar fliL mutant cells was indistinguishable from that of wild-type cells, the mutant exhibited a defectivemotility phenotype. Cryo-electron tomography (cryo-ET) of intact organisms revealed that the periplasmic flagella in the fliL mutant were frequently tilted toward the cell pole instead of their normal orientation toward the cell body. These defects were corrected when the mutant was complemented in cis. Moreover, a comparative analysis of flagellar motors from the wild type and the mutant provides the first structural evidence that FliL is localized between the stator and rotor. Our results suggest that FliL is likely involved in coordinating or regulating the orientation of periplasmic flagella in B. burgdorferi.Borrelia burgdorferi is the causative agent of Lyme disease and belongs to a group of bacteria called spirochetes. B. burgdorferi cells have a characteristic flat-wave morphology and unique means of motility (9,10,19,35). As a result of its unique morphology and motility, B. burgdorferi is able to traverse viscous gel-like media in which most other flagellated bacteria slow down or stop (30). Consequently, B. burgdorferi may efficiently bore through host tissues, leading to pathogenesis in the joints, nervous system, and heart (19,30,54). The motility of B. burgdorferi results from the coordinated rotation of the periplasmic flagella residing between the outer membrane and the cell cylinder (9,10,12,20,33,35). The current swimming model suggests that a run occurs when the anterior periplasmic flagella rotate in one direction (e.g., counterclockwise [CCW]) and the posterior flagella rotate in the opposite direction (clockwise [CW]). Reversals occur when periplasmic flagella at both poles of the cell change their direction of rotation. During a nontranslational mode (flex), the periplasmic flagella at both cell poles rotate in the same direction (9,20,35).Bacterial flagella are composed of three major parts: the motor, hook, and filament. The motor can be divided into two functional units, the rotor and the stator. The stator is the torque generator consisting of the MotA-MotB complex. The rotor is composed, minimally, of the MS ring (FliF), the rod, and the switch complex (FliG, FliM, and FliN). In B. burgdorferi, the periplasm-localized flagellar filament consists of the core protein FlaB and the sheath protein FlaA (17, 46). There are 7 to 11 filaments conne...

HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K m of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

The P97 adhesin and P102 genes of Mycoplasma hyopneumoniae each have six paralogs in the genome. We tested whether these genes were expressed during infection. P102 is associated with the mycoplasma and with swine cilia. Further, most of the paralogs were transcribed in vivo in two gene transcriptional units.