Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rodshaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.spirochete ͉ Lyme disease ͉ allelic exchange ͉ morphology S pirochetes have a unique position among the bacteria. These motile bacteria are one of the few bacterial phyla that can be identified by both 16S ribosomal RNA sequence analysis and morphology (1). Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella. A given periplasmic flagellum is attached subterminally at only one end of the cell cylinder, and it resides within the periplasmic space (2). Depending on the species, the cell morphology is either a helix, a flat wave, or an irregularly shaped helix (2-4). The size of the spirochete, the number of periplasmic flagella attached at each end, and whether the filaments overlap at the center of the cell varies from species to species (2, 5). This phylum contains many medically important bacteria including Treponema pallidum (syphilis), several Borrelia species (relapsing fever), Borrelia burgdorferi (Lyme disease), Leptospira interrogans (leptospirosis), Brachyspira sp. (human diarrheal disease, swine dysentery), and oral treponemes associated with periodontal disease (5-8).The periplasmic flagella of spirochetes have been characterized in detail. Genetic evidence, including targeted mutagenesis studies in Treponema denticola and Brachyspira hyodysenteriae, have shown that these organelles are directly involved in motility (9, 10) (C. Li and N.W.C., unpublished observations). By analyzing protruding periplasmic flagella from certain motility mutants of T. phagedenis, and from stationary-phase cells of several spirochete species, these organelles have been shown to rotate in a manner similar to th...

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; rel Bbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is maintained in nature via an enzootic cycle that comprises a tick vector and a vertebrate host. Transmission from the tick to the mammal, acquisition from the mammal back to the tick, and adaptation to the two disparate environments require sensing signals and responding by regulating programs of gene expression. The molecular mechanisms utilized to effect these lifestyle changes have begun to be elucidated and feature an alternative sigma factor cascade in which RpoN (σ(54)) and RpoS (σ(S)) globally control the genes required for the different phases of the enzootic cycle. The RpoN-RpoS pathway is surprisingly complex, entailing Rrp2, an unusual enhancer-binding protein and two-component regulatory system response regulator activated by acetyl phosphate; BosR, an unorthodox DNA-binding protein; DsrA(Bb), a small noncoding RNA; and Hfq and CsrA, two RNA-binding proteins. B. burgdorferi also has a c-di-GMP signaling system that regulates the tick side of the enzootic cycle and whose function is only beginning to be appreciated.

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SummaryThe alternative sigma factor RpoS (s 38 or s S ) plays a central role in the reciprocal regulation of the virulence-associated major outer surface proteins OspC and OspA in Borrelia burgdorferi, the Lyme disease spirochete. Temperature is one of the key environmental signals controlling RpoS, but the molecular mechanism by which the signal is transduced remains unknown. Herein, we identify and describe a small non-coding RNA, DsrABb, that regulates the temperature-induced increase in RpoS. A novel 5Ј end of the rpoS mRNA was identified and DsrABb has the potential to extensively base-pair with the upstream region of this rpoS transcript. We demonstrate that B. burgdorferi strains lacking DsrABb do not upregulate RpoS and OspC in response to an increase in temperature, but do regulate RpoS and OspC in response to changes in pH and cell density. Analyses of the rpoS and ospC steady-state mRNA levels in the dsrABb mutant indicate that DsrABb regulates RpoS post-transcriptionally. The 5Ј and 3Ј ends of DsrABb were mapped, demonstrating that at least four species exist with sizes ranging from 213 to 352 nucleotides. We hypothesize that DsrABb binds to the upstream region of the rpoS mRNA and stimulates translation by releasing the Shine-Dalgarno sequence and start site from a stable secondary structure. Therefore, we postulate that DsrABb is a molecular thermometer regulating RpoS in Borrelia burgdorferi.

Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete 11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.

In Lyme disease spirochetes, the ospC gene encodes a 22.7-kDa protein referred to as either the pC or the OspC protein. Using a variety of electrophoretic approaches followed by Southern blotting and probing with oligonucleotide probes, we mapped the ospC gene to a circular 26-kb plasmid. The ospC gene represents the first gene to be mapped to a circular plasmid in Lyme disease spirochetes. The occurrence of this gene in isolates belonging to each of the three Lyme disease-associated species, Borrelia burgdorferi, Borrelia garinii, and the VS461 group, was evaluated. The ospC gene was found to occur in all 21 isolates tested from each of the three species. Differential hybridization with a series of ospC probes in both Northern (RNA) and Southern blot analyses demonstrated that there is sequence variability in the ospC gene among isolates. While the gene was found to be present in all isolates, not all actively transcribed the gene. Transcriptional start site analyses suggest that the gene may be under the control of multiple promoters that are highly similar in nucleotide sequence.

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.