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1.
Figure 6

Figure 6. Detection of B. burgdorferi chemoreceptor arrays by cryo-ET. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

The cryo-ET analysis was carried out as previously described (). Six strains were included: (A) B31A, (B) ΔW2, (C) & (D) ΔW3 and its complemented strain ΔW3+, and (E) & (F) ΔW1 and its complemented strain ΔW1+. Arrows point to chemoreceptor arrays. OM: outer membrane; CM: cytoplasmic membrane; A/W: the basal plate composed of CheA and CheW.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.
2.
Figure 3

Figure 3. Immunoblot analysis of the three cheW mutants and their complemented strains. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

(A) Immunoblot analysis of the cheW1 mutant (ΔW1) and its complemented strain (ΔW1+) using αCheW1. (B) Immunoblot analysis of the cheW2 mutant (ΔW2) using αCheW2. (C) Immunoblot analysis of the cheW3 mutant (ΔW3), its complemented strain (ΔW3+), and the mutant complemented with the N-terminal CheW domain (aa 1–210) of CheW3 (ΔW3N+) using αCheW3. The predicted molecular weights of CheW1, CheW2, CheW3, and the N-terminal CheW domain of CheW3 are approximately 20 kDa, 21 kDa, 53 kDa, and 24 kDa, respectively.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.
3.
Figure 7

Figure 7. Detecting the interactions between two CheAs and three CheWs of B. burgdorferi by co-IP. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

Pull down of the CheWs using αCheA1 (right panel) or αCheA2 (left panel). Precipitated proteins were probed with αCheW1 (A), αCheW2 (B), or αCheW3 (C). A previously constructed cheA1A2 double-deletion mutant (ΔA1A2) of B. burgdorferi () was used as a negative control for the co-IP. (D) Pull down of CheA2 using αCheW1 (left panel) and αCheW3 (right panel). Precipitated proteins were probed with CheA2. Extracts from the ΔW1 or ΔW3 mutants were used as negative controls for the co-IP.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.
4.
Figure 5

Figure 5. Localization of B. burgdorferi chemoreceptor arrays using IFA. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

The wild-type (A), the ΔW1 (B), ΔW2 (C), and ΔW3 (D) mutant cells were fixed with methanol, stained with anti-MCP3 antibody, and counterstained with anti-rat Texas red antibody as previously described (;). The micrographs were taken under DIC light microcopy or fluorescence microscopy with a tetramethylrhodamine isothiocyanate (TRITC) emission filter, and the resultant images were merged. Arrows point to the location of the chemoreceptor arrays within cells.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.
5.
Figure 2

Figure 2. Homology modeling of B. burgdorferi CheWs. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

(A) Structure alignment of CheW1 (yellow), CheW3 (red), E. coli CheW (green), and T. maritima CheW (blue). (B) Structure alignment of CheW2 (orange), E. coli CheW, and T. maritima CheW. The N-terminal regions ahead of β strand were removed for better visualization. T. maritima CheW ()(Protein Data Bank ID: 1K0S) was selected as the basis for structural modeling using the program Modeller 9v7 (). All structures were analyzed and visualized in PyMol. The numbers represent the highly variable regions (HVR) identified.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.
6.
Figure 1

Figure 1. Sequence comparison between E. coli CheW and the three CheWs of B. burgdorferi. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

The numbers show the positions of residues in E. coli CheW and B. burgdorferi CheW1, CheW2, and the CheW domain of CheW3. Dots represent functionally important residues identified in E. coli CheW (;;;). The black dots represent residues conserved in all four CheWs, and grey dots represent residues that are different in one or more of the three CheWs of B. burgdorferi. The boxes represent conserved residues of CheWs. The alignments were performed using the program MacVector 10.6.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.
7.
Figure 4

Figure 4. B. burgdorferi cheW1 and cheW3 mutants are non-chemotactic. From: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi.

Swim plate (A) and capillary (D) assays of the cheW1 mutant (ΔW1) and its complemented strain (ΔW1+). Swim plate (B) and capillary (E) assays of the cheW2 mutant (ΔW2). Swim plate (C) and capillary (F) assays of the cheW3 mutant (ΔW3) and its complemented strains (ΔW3+ and ΔW3N+). The swim plate and capillary assays were carried out as previously described (;;). For the swim-plate assay, ΔflaB, a previously constructed non-motile mutant (), was used as a control to determine the size of non-spreading colonies on the plates. For the capillary assay, N-acetyl-D-glucosamine (GlcNAc) was used as an attractant. Results are expressed as the means ± SEM from five plates or capillary tubes. * represents a P value < 0.01.

Kai Zhang, et al. Mol Microbiol. ;85(4):782-794.

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