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1.
Fig. 5.

Fig. 5. From: CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate.

Western blot analysis of motility and chemotaxis proteins among mutants. Western blot analysis using B. burgdorferi monoclonal anti-FlaB, -FlaA, and -DnaK and E. coli polyclonal anti-CheA antibodies. Cell lysates (10 μg protein) from wild-type and cheY3 and cheY1 cheY2 cheY3 mutant (cheY1Y2Y3) cells were probed with the indicated antibodies. For FlaB, 3 μg was loaded in each lane, and for Dnak, 2 μg of lysate was loaded in each lane.

M. A. Motaleb, et al. J Bacteriol. 2011 Jul;193(13):3332-3341.
2.
Fig. 6.

Fig. 6. From: CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate.

Transfer of phosphate from CheA1-32P (top panels) and CheA2-32P (bottom panels) to CheY3. CheA1 and CheA2 were autophosphorylated with [32P]ATP, and unincorporated [32P]ATP was removed by centrifugation on Bio-Spin6 columns. Recovered CheA1-32P and CheA2-32P (2 μM each) were incubated with 14.8 μM CheY3 for the indicated periods of time. Reactions were stopped, and products were analyzed as described in the text. Phosphor images are shown in the left panels, and relative intensities (PI “volumes”) are shown in the right panels. CheA1-32P and CheA2-32P are represented as solid lines and squares, and CheY3-P is represented as circles and dashed lines.

M. A. Motaleb, et al. J Bacteriol. 2011 Jul;193(13):3332-3341.
3.
Fig. 4.

Fig. 4. From: CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate.

Chemotaxis assays of cheY mutants. (a and b) Capillary tube chemotaxis assays coupled with flow cytometry were performed with wild-type (WT) and mutant strains. The results are expressed as n-fold increases in the number of cells entering capillary tubes containing glucosamine attractant relative to the number of cells entering tubes containing a no-attractant control (buffer alone). A 2-fold increase (horizontal line) compared to the buffer control is considered significant (). Results are expressed as the mean ± standard error from three independent experiments.

M. A. Motaleb, et al. J Bacteriol. 2011 Jul;193(13):3332-3341.
4.
Fig. 1.

Fig. 1. From: CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate.

Amino acid sequence alignments of B. burgdorferi CheY proteins with E. coli and B. subtilis CheY using T-Coffee (). Conserved residues that are essential for function in E. coli are indicated by solid circles. The nonconserved active-site residues of E. coli CheY, N59 and E89, that are conserved in B. burgdorferi CheY2, but not in CheY1 or CheY3, are identified by arrowheads. T81 of CheY3, which is involved in CheX dephosphorylation of CheY3-P and is also conserved in B. burgdorferi CheY1 and in B. subtilis CheY, is identified by a diamond. Secondary structure elements as determined for E. coli CheY and predicted for B. burgdorferi CheY proteins are shown above the sequence alignment. The E. coli and B. subtilis CheY proteins are identified as Ec-CheY and Bsu-CheY, respectively. Amino acid residue numbers are indicated on the left. The last amino acid residue number of each CheY sequence is shown on the right.

M. A. Motaleb, et al. J Bacteriol. 2011 Jul;193(13):3332-3341.
5.
Fig. 3.

Fig. 3. From: CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate.

Western blot analyses of CheY1, CheY2, and CheY3 in wild-type and cheY mutant B. burgdorferi cells. Approximately 10 μg protein of cell lysates from the wild-type strain and the indicated cheY mutant strains was subjected to Western blotting and probed with antibodies specific to the CheY proteins (αCheY1, αCheY2, and αCheY3). Samples containing 3 μg lysates were used for the loading control (αDnaK, panel a, rightmost). The approximate molecular masses of CheY1 (11 kDa), CheY2 (10 kDa), CheY3 (14 kDa), and DnaK (72 kDa) were determined based on the masses of marker proteins (not shown). In panel a, lysates from wild-type; cheY1, cheY2, or cheY3 single mutant; and complemented cheY3+ B. burgdorferi cells were probed with the indicated CheY antisera. In panel b, lysates from wild-type, cheY1 cheY2 double mutant (cheY1Y2), and cheY1 cheY2 cheY3 triple mutant B. burgdorferi cells (cheY1Y2Y3) were probed with CheY1, CheY2, and CheY3 antisera.

M. A. Motaleb, et al. J Bacteriol. 2011 Jul;193(13):3332-3341.
6.
Fig. 2.

Fig. 2. From: CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate.

cheY1 (bb0551) operon structure. (a) Schematic representation of the cheY1 operon. Arrowheads indicate specific primer pairs that amplify regions between genes. (b) Agarose gel showing the RT-PCR products. The number above each lane represents a number between arrowheads in panel a. Lane 1 did not amplify a product, as cheY1 and bb0552 are divergently transcribed. A no-RT control reaction is represented by a minus sign. (c) DNA sequencing chromatogram of an RLM-RACE analysis that resulted in the identification of the TSS of the cheY1 operon (right-angled arrow). The cheY1 translation start TTG codon is boxed. A horizontal line with arrowheads represents the 5′ RACE adapter sequence (provided in the kit). (d) Predicted −35 and −10 promoter sequences with the TSS (right-angled arrow) of cheY1 operon (top) and a typical σ70 promoter sequence (bottom) are shown ().

M. A. Motaleb, et al. J Bacteriol. 2011 Jul;193(13):3332-3341.

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