CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834, USA.
Journal of bacteriology (Impact Factor: 2.81). 07/2011; 193(13):3332-41. DOI: 10.1128/JB.00362-11
Source: PubMed


Spirochetes have a unique cell structure: These bacteria have internal periplasmic flagella subterminally attached at each cell end. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis is poorly understood. In other bacteria, modulation of flagellar rotation is essential for chemotaxis, and phosphorylation-dephosphorylation of the response regulator CheY plays a key role in regulating this rotary motion. The genome of the Lyme disease spirochete Borrelia burgdorferi contains multiple homologues of chemotaxis genes, including three copies of cheY, referred to as cheY1, cheY2, and cheY3. To investigate the function of these genes, we targeted them separately or in combination by allelic exchange mutagenesis. Whereas wild-type cells ran, paused (flexed), and reversed, cells of all single, double, and triple mutants that contained an inactivated cheY3 gene constantly ran. Capillary tube chemotaxis assays indicated that only those strains with a mutation in cheY3 were deficient in chemotaxis, and cheY3 complementation restored chemotactic ability. In vitro phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by CheA2 than by CheA1, and the CheY3-P intermediate generated was considerably more stable than the CheY-P proteins found in most other bacteria. The results point toward CheY3 being the key response regulator essential for chemotaxis in B. burgdorferi. In addition, the stability of CheY3-P may be critical for coordination of the rotation of the periplasmic flagella.

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Available from: Chunhao Li, May 15, 2015
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    • "In contrast to pdeA, inactivation of pdeB resulted in cells that exhibited a swimming pattern similar to wild-type except they flexed significantly more, suggesting that c-di-GMP may play a role in chemotaxis (Sultan et al., 2011; Kulasekara et al., 2013; Russell et al., 2013). Increased flexing would be expected to be a result of overexpression of the chemotaxis response regulator, CheY3, inhibition of the activity of CheX (the CheY-P phosphatase), or decreased expression of CheX (Motaleb et al., 2005; Pazy et al., 2010; Motaleb et al., 2011). However, Western blot analysis showed that both proteins are expressed at wild-type levels in the pdeB mutant, eliminating the possibility of a receptor-c-di-GMP complex directly regulating the expression of chemotaxis proteins (Sultan et al., 2011). "
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    ABSTRACT: In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.
    Frontiers in Cellular and Infection Microbiology 05/2014; 4:56. DOI:10.3389/fcimb.2014.00056 · 3.72 Impact Factor
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    • "Non-chemotactic mutants often show altered swimming behaviours, e.g. the cheA2 and cheY3 mutants of B. burgdorferi fail to reverse and constantly run (Li et al., 2002; Motaleb et al., 2011b). The tracking analysis using a computer-assisted cell tracker coupled with video microscopy disclosed that the DW2 mutant had swimming behaviour indistinguishable from the wild type (Videos S1 and S2, Table 1), whereas the DW1 and DW3 mutants had altered swimming behaviours. "
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    ABSTRACT: In the model organism Escherichia coli, the coupling protein CheW, which bridges the chemoreceptors and histidine kinase CheA, is essential for chemotaxis. Unlike the situation in E. coli, Borrelia burgdorferi, the causative agent of Lyme disease, has three cheW homologues (cheW(1) , cheW(2) and cheW(3) ). Here, a comprehensive approach is utilized to investigate the roles of the three cheWs in chemotaxis of B. burgdorferi. First, genetic studies indicated that both the cheW(1) and cheW(3) genes are essential for chemotaxis, as the mutants had altered swimming behaviours and were non-chemotactic. Second, immunofluorescence and cryo-electron tomography studies suggested that both CheW(1) and CheW(3) are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW(1) and cheW(3) , cheW(2) is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW(1) and CheW(3) interact with CheA(2) whereas CheW(2) binds to CheA(1) . Collectively, our results indicate that CheW(1) and CheW(3) are incorporated into one chemosensory pathway that is essential for B. burgdorferi chemotaxis. Although many bacteria have more than one homologue of CheW, to our knowledge, this report provides the first experimental evidence that two CheW proteins coexist in one chemosensory pathway and that both are essential for chemotaxis.
    Molecular Microbiology 07/2012; 85(4):782-94. DOI:10.1111/j.1365-2958.2012.08139.x · 4.42 Impact Factor
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    • "This spirochete lacks homologs of FliA and FlgM, and there is no σ 28 promoter consensus sequence evident in its genome (Fraser et al., 1997;Charon and Goldstein, 2002). In addition, all of the motility and chemotaxis genes analyzed to date are controlled by σ 70 , a house-keeping transcription factor (Charon and Goldstein, 2002;Ge and Charon, 1997;Ge et al., 1997;Yang and Li, 2009;Motaleb et al., 2011) . In contrast to other bacteria, recent studies of specific B. burgdorferi mutants indicate that this spirochete regulates flagellar synthesis by a posttranscriptional mechanism rather than via a transcriptional cascade (Motaleb et al., 2004;Ge et al., 1998;Sal et al., 2008). "
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    ABSTRACT: The Lyme disease spirochete Borrelia burgdorferi lacks the transcriptional cascade control of flagellar protein synthesis common to other bacteria. Instead, it relies on a post-transcriptional mechanism to control its flagellar synthesis. The underlying mechanism of this control remains elusive. A recent study reported that the increased level of BB0184 (CsrA(Bb); a homologue of carbon storage regulator A) substantially inhibited the accumulation of FlaB, the major flagellin protein of B. burgdorferi. In this report, we deciphered the regulatory role of CsrA(Bb) on FlaB synthesis and the mechanism involved by analysing two mutants, csrA(Bb)(-) (a deletion mutant of csrA(Bb)) and csrA(Bb)(+) (a mutant conditionally overexpressing csrA(Bb)). We found that FlaB accumulation was significantly inhibited in csrA(Bb)(+) but was substantially increased in csrA(Bb)(-) . In contrast, the levels of other flagellar proteins remained unchanged. Cryo-electron tomography and immuno-fluorescence microscopic analyses revealed that the altered synthesis of CsrA(Bb) in these two mutants specifically affected flagellar filament length. The leader sequence of flaB transcript contains two conserved CsrA-binding sites, with one of these sites overlapping the Shine-Dalgarno sequence. We found that CsrA(Bb) bound to the flaB transcripts via these two binding sites, and this binding inhibited the synthesis of FlaB at the translational level. Taken together, our results indicate that CsrA(Bb) specifically regulates the periplasmic flagellar synthesis by inhibiting translation initiation of the flaB transcript.
    Molecular Microbiology 11/2011; 82(4):851-64. DOI:10.1111/j.1365-2958.2011.07853.x · 4.42 Impact Factor
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