Marina L. Sartakova

Asymmetrical flagellar rotation in Borrelia burgdorferi nonchemotactic mutants

The Lyme disease spirochete Borrelia burgdorferi has bundles of periplasmic flagella subpolarly located at each cell end. These bundles rotate in opposite directions during translational motility. When not translating, they rotate in the same direction, and the cells flex. Here, we present evidence that asymmetrical rotation of the bundles during translation does not depend upon the chemotaxis signal transduction system. The histidine kinase CheA is known to be an essential component in the signaling pathway for bacterial chemotaxis. Mutants of cheA in flagellated bacteria continually rotate their flagella in one direction. B. burgdorferi has two copies of cheA designated cheA1 and cheA2. Both genes were found to be expressed in growing cells. We reasoned that if chemotaxis were essential for asymmetrical rotation of the flagellar bundles, and if the flagellar motors at both cell ends were identical, inactivation of the two cheA genes should result in cells that constantly flex. To test this hypothesis, the signaling pathway was completely blocked by constructing the double mutant cheA1∷kan cheA2∷ermC. This double mutant was deficient in chemotaxis. Rather than flexing, it failed to reverse, and it continually translated only in one direction. Video microscopy of mutant cells indicated that both bundles actively rotated. The results indicate that asymmetrical rotation of the flagellar bundles of spirochetes does not depend upon the chemotaxis system but rather upon differences between the two flagellar bundles. We propose that certain factors within the spirochete localize at the flagellar motors at one end of the cell to effect this asymmetry.

Erythromycin Resistance in Borrelia burgdorferi

ABSTRACT Susceptibility testing of laboratory strains and clinical isolates of Borrelia burgdorferi indicates that resistance to erythromycin is present in them. Evaluation of the MICs, minimal bactericidal concentrations, and kinetics of bacterial killing of erythromycin suggests that this resistance is increased by preexposure to the antibiotic, is dependent on inoculum size, and may be the result of selection of subpopulations of bacterial cells with increased resistance.

Complementation of a Nonmotile flaB Mutant of Borrelia burgdorferi by Chromosomal Integration of a Plasmid Containing a Wild-Type flaB Allele

With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.

Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells.

ABSTRACT Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.

Genetic manipulation of spirochetes – light at the end of the tunnel

Abstract Spirochetes cause many important diseases in humans and animals, including syphilis, relapsing fever, leptospirosis and Lyme disease. Recent advances in molecular genetics have allowed Kochs molecular postulates to be applied to the study of the virulence factors in these bacteria. Such advances include the use of electroporation, the identification of several antibiotic resistance phenotypes as useful genetic markers, the development of extrachromosomal cloning vectors and the isolation of knockout mutants and their complementation.

Characterization of the Stringent Response and rel Bbu Expression in Borrelia burgdorferi

The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (rel(Bbu)) was produced by B31 as by N40 during in vitro growth (2 +/- 0.5 and 4 +/- 0.8 fg of rel(Bbu) mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 rel(Bbu) mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in rel(Bbu) mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel(Bsu) were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel(Bbu) promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel(Bbu) ATG start codon and identical sigma(70)-like promoters were identified by primer extension and sequencing analysis. rel(Bbu) complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel(Bbu) mRNA. These results indicate that rel(Bbu) is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control.