JB Accepted Manuscript Posted Online 7 December 2015 J. Bacteriol. doi:10.1128/JB.00877-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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A hypothetical protein BB0569 is essential for chemotaxis of the Lyme disease spirochete Borrelia

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burgdorferi

3 Kai Zhang1, Jun Liu3, Nyles W. Charon4, and Chunhao Li1,2*

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University of New York at Buffalo, New York 14214

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Houston, Texas 770302

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Department of Oral Biology, and 2Department of Microbiology and Immunology, The State

Department of Pathology and Laboratory Medicine, University of Texas Medical School at

Departments of Microbiology, Immunology, and Cell Biology, Health Sciences Center, West

Virginia University, Morgantown, West Virginia 26506-9177

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Running title: Chemoreceptor of Borrelia burgdorferi

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Key words (Lyme disease /Borrelia burgdorferi/ Chemotaxis/ Chemoreceptor)

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*

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Mailing address: Department of Oral Biology, SUNY at Buffalo, 3435 Main St., Buffalo, NY 14214-

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3092

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Electronic mail address: [email protected]; phone: (716) 829-6014; Fax: (716) 829-3942

Corresponding author

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ABSTRACT

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The Lyme disease spirochete Borrelia burgdorferi has five putative methyl-accepting chemotaxis

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proteins (MCPs). In this report, we provide evidence that a hypothetical protein BB0569 is essential

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for the chemotaxis of B. burgdorferi. While BB0569 lacks significant homology to the canonical

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MCPs, it contains a conserved domain (spanning residues 110 - 170) that is often evident in

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membrane-bound MCPs such as Tar and Tsr of Escherichia coli. Unlike Tar and Tsr, BB0569 lacks

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transmembrane regions, recognizable HAMP and methylation domains, and is similar to TlpC, a

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cytoplasmic chemoreceptor of Rhodobacter sphaeroides. An isogenic mutant of BB0569 constantly

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runs in one direction and fails to respond to attractants, indicating that BB0569 is essential for

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chemotaxis. Immunofluorescence, GFP-fusion, and cryo-electron tomography analyses demonstrate

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that BB0569 localizes at the cell poles and is required for chemoreceptor clustering at the cell poles.

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Protein cross-linking studies reveal that BB0569 forms large protein complexes with MCP3,

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indicative of its interactions with other MCP proteins. Interestingly, analysis of B. burgdorferi mcp

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mutants shows that inactivation of either mcp2 or mcp3 has reduced the level of BB0569

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substantially and that such a reduction is caused by protein turnover. Collectively, these results

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demonstrate that the domain composition and function of BB0569 are similar in some respects to

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TlpC, but different in their cellular locations, further highlighting that the chemotaxis of B.

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burgdorferi is unique and different to the Escherichia coli and Salmonella enterica paradigm.

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IMPORTANCE

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Spirochete chemotaxis differs substantially from the Escherichia coli and Salmonella enterica

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paradigm, and the basis for controlling the rotation of the bundles of periplasmic flagella at each

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end cell is unknown. In recent years, Borrelia burgdorferi, the causative agent of Lyme disease, has

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been used as a model organism to understand spirochete chemotaxis and its role in infectious

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processes of the diseases. In this report, BB0569, a hypothetical protein of B. burgdorferi, has been

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investigated by using an approach of genetics, biochemistry, and cryo-electron tomography. The

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results indicate that BB0569 has a distinct role in chemotaxis that may be unique to spirochetes and

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represents a novel paradigm.

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INTRODUCTION

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Chemotaxis allows bacteria to swim toward favorable environments or away from harmful ones by

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modulation of their swimming behavior (1,2). The molecular mechanisms involved in bacterial

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chemotaxis have been extensively studied in two prototype organisms, Escherichia coli and

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Salmonella enterica [for recent reviews see (3-5)]. The chemotaxis signaling apparatus works as a

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supramolecular unit that is composed of three major components: methyl-accepting chemotaxis

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proteins (MCPs), the histidine kinase CheA, and the response regulator CheY (6,7). MCPs sense

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various environmental and intracellular signals and control the activity of CheA. Activated CheA

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phosphorylates CheY, which then interacts with the motor switch complex to increase the

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probability of flagellar clockwise (CW) rotation and destabilize counterclockwise (CCW) rotation.

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CCW rotation results in smooth swimming (a run), and CW rotation leads to chaotic movement (a

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tumble). Cells showing a positive response have longer runs and suppress the intervals spent

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tumbling. Cells deleted for cheA and cheY continuously rotate their flagella CCW and consequently

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fail to tumble (8,9).

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MCPs form clusters that reside at the cell poles (10-12). They typically consist of an extracellular

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ligand-binding domain (sensor) and a cytoplasmic signaling domain (13). These two domains are

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connected by a HAMP-domain (14,15). The sensor domain recognizes and binds to specific

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chemicals (i.e., attractants). The signaling domain interacts with CheW/CheA and communicates

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with the flagellar motor apparatus by modulating the flux of phosphoryl groups from CheA to CheY

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(3,15). There are five different MCP-like proteins in E. coli, and each senses different signals (e.g.,

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Tar senses aspartate and maltose) (16,17). Mutants lacking a specific receptor fail to respond to the

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corresponding attractants. Recently, novel cytoplasmic chemoreceptors, also known as transducer-

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like proteins (Tlps), were found in Rhodobacter sphaeroides (18,19). These MCP-like proteins lack 4

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transmembrane regions and recognizable HAMP and methylation domains. They localize to a

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discrete region in the cytoplasm. Unlike E. coli MCPs, some of these proteins such as TlpC are

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essential for chemotaxis of R. sphaeroides (18).

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E. coli and S. enterica contain only one copy of each of their six chemotaxis genes (cheA, cheW,

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cheY, cheR, cheB, and cheZ) (5,9,20). In contrast, the Lyme disease spirochete Borrelia burgdorferi

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has multiple homologs of its chemotaxis genes, including two cheA (cheA1 and cheA2), three cheY

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(cheY1, cheY2 and cheY3), three cheW (cheW1, cheW2 and cheW3), two cheB (cheB1 and cheB2), and

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two cheR (cheR1 and cheR2) (21-23). Many of these chemotaxis genes are located within the flaA

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operon or the cheW2 operon (24,25). The flaA operon contains flaA, cheA2, cheW3, cheX, and cheY3.

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The cheW2 operon contains cheW2, BB0566 (a hypothetical protein), cheA1, cheB2, BB0569, and

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cheY2. Recent studies revealed that all of the chemotaxis genes in the flaA operon that have been

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examined are essential for chemotaxis of B. burgdorferi (e.g., cheA2, cheW3, and cheY3 mutants

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never reverse, and the cheX mutant constantly flexes). All of these mutants are non-chemotactic to

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attractants (25-28). In contrast to the flaA operon, most genes studied to date in the cheW2 operon

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are not required for the chemotaxis (e.g., cheA1, cheW2, and cheY2 mutants have similar swimming

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behaviors as the parental wild-type strain) (27,28). Within the cheW2 operon, BB0569 encodes a

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hypothetical protein (23). The function of this gene remains unknown. In this report, we provide the

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first experimental evidence that BB0569 is a TlpC-like protein that is essential for B. burgdorferi

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chemotaxis.

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MATERIAL AND METHODS

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Bacterial strains and growth conditions. A high-passage Borrelia burgdorferi sensu stricto strain,

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B31A (wild type) (29), and its isogenic mutants were grown either in BSK-II liquid medium 5

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supplemented with 6% rabbit serum or on semi-solid agar plates in a humidified incubator at 34°C

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in the presence of 3~5% CO2, as previously described (25).

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Constructing a BB0569 deletion mutant and its complemented strain. A previously described

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method was used to construct a BB0569 deletion mutant (25). Briefly, a part of BB0569 gene (1,559

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bp) was PCR amplified with primers P1/P2, and the resultant PCR product was cloned into the

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pGEM-T Easy vector (Promega, Madison, WI). A 216 bp HindIII DNA fragment within the

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BB0569 gene was deleted and replaced by a kanamycin resistance cassette (kan) that was amplified

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by primers P3/P4. The final construct (BB0569::kan, FIG. 1A) was linearized and electroporated

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into B31A competent cells to inactivate the targeted gene via allelic exchange. Transformants were

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selected on semisolid agar plates containing kanamycin (300 µg/ml). To complement the BB0569

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mutant, the flgB promoter (flgBp) of B. burgdorferi (30) was PCR amplified (primers P5/P6) with

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engineered BamHI and NdeI cut sites at its 5’ and 3’ ends, respectively. Then, the full-length

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BB0569 gene was PCR amplified (primers P7/P8) with engineered NdeI and PstI cut sites at its 5'

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and 3' ends, respectively. The two fragments were fused together at the site of NdeI, and the

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resultant fragment (flgBp-BB0569) was confirmed by DNA sequencing. The BamHI-PstI-digested

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flgBp-BB0569 fragment was cloned into pKFSS1, a shuttle vector of B. burgdorferi (31), yielding

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pKBB0569 (FIG. 1B). To complement the mutant, pKBB0569 was electroporated into BB0569

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mutant cells, and transformants were selected on semisolid agars containing both kanamycin (300

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µg/ml) and streptomycin (50 µg/ml). All of the primers used are shown in Table S1.

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Preparing BB0569 recombinant protein and antibody. To over-express BB0569, the full-length

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gene was PCR amplified (primers P9/P10), and the obtained PCR product was cloned into the

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pGEM-T Easy vector. The resulting insert was further subcloned into the pQE30 expression vector 6

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(Qiagen, Valencia, CA), which codes for an amino-terminal histidine tag. The expression of

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BB0569 was induced using 0.1 M isopropyl-β-D-thiogalactoside (IPTG), and the recombinant

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protein was purified using nickel-nitrilotriacetic acid beads (Qiagen) under denaturing conditions

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using 8 M urea. The purified protein was dialyzed in a buffer containing 10 mM Tris-HCl at 4°C

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overnight. To produce antiserum against BB0569, rats were first immunized with 1 mg of the

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recombinant protein during a one-month period and then boosted (100 μg per rat) twice at weeks 6

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and 7 (antiserum was produced by General Bioscience Corporation, Brisbane, CA).

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Constructing a plasmid that expresses BB0569-GFP fusion protein. A previously described

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method (32) was used to construct a vector that expresses a BB0569-GFP fusion protein. Briefly,

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the cheW2 promoter (PW2) (25), gfp, and BB0569 gene were each amplified by PCR. For DNA

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cloning, BamHI, NdeI, NruΙ, and PstΙ cut sites were engineered into the respective primers (Table

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S1). The PCR products were then cloned into the pGEM-T Easy vector. PW2 and the BB0569 gene

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were fused at its NdeI cut site, and then gfp was inserted in frame at the 3’ end of BB0569 at NruΙ

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and PstΙ sites. The PW2BB0569gfp fragment was then subcloned into the shuttle vector pKFSS1 at

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its BamHI and PstI sites, generating BB0569gfp/pKFSS1 (FIG. S1A). The primers (P11 to P16) used

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in the construction of this vector are enclosed in Table S1. To express BB0569-GFP in B.

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burgdorferi, the plasmid was transformed into B31A competent cells by electroporation. The

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transformants were selected and confirmed as described above. The expression of BB0569-GFP

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was detected by immunoblotting using GFP monoclonal antibody (αGFP) and BB0569 antiserum

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(αBB0569) (FIG. S1B).

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Fluorescence microscopy and cryo-electron tomography (cryo-ET). An immunofluorescence

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assay (IFA) and cryo-ET were conducted to localize BB0569 and MCPs in B. burgdorferi as 7

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previously described (28,32). For the IFA, the spirochetes were incubated with either 1:100 diluted

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αBB0569 (1:100 dilution) or MCP3 antibody (αMCP3; 1:500), or MCP5 antibody (αMCP5; 1:500)

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for 1 hour at room temperature. The resultant samples were incubated with secondary goat anti-rat

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Texas red antibody (Invitrogen) for one hour at room temperature, washed with PBS, and mounted

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in 40% glycerol for imaging. Fluorescence images were taken using a Zeiss Axiostar plus

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microscope at a wavelength of 480 nm. Texas red images were taken using a Zeiss Axioimager Z1

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Axiophot wide-field microscope with an excitation filter (541-569 nm) and an emission filter (581-

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654 nm). The images were captured and processed using the program Axiovision (Zeiss, Germany).

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For the cryo-ET analysis, freshly prepared B. burgdorferi cells were deposited onto a glow-

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discharged holey carbon EM grid, blotted, and rapidly frozen in liquid ethane. The frozen-hydrated

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specimens were imaged at -170°C using a Polara G2 electron microscope (FEI Company, Hillsboro,

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OR) equipped with a field emission gun and a Direct Detection Camera (Gatan K Summit). The

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microscope was operated at 300 kV with a magnification of 15,500×. Serial EM (33) was used to

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collect images from each bacterium at -6 µm defocus with a cumulative dose of ~60 e-/Å2

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distributed over 41 images with an angular increment of 3°, covering a range from -60° to +60°.

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The tilt series images were aligned and reconstructed using the IMOD software package (34). Cryo-

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tomograms of B31A (30 cells) and the BB0569 isogenic mutant (30 cells) were and visualized using

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IMOD.

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Swim plate, motion tracking, and capillary assays. Swim plate assays were performed as

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previously described (25,35). Approximately 1 x 106 cells in a 5 μl volume were inoculated into

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0.35% agarose plates containing BSK-II medium diluted 1:10 in PBS and incubated for 4 days. For

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motion-tracking analysis, B. burgdorferi cells were first pelleted at 2,500 x g and then resuspended

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in motility buffer containing 1% bovine serum albumin (BSA) and 1% methylcellulose (400 mesh, 8

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Sigma-Aldrich, St. Louis, MO). Cells were videotaped under a dark-field microscopy (Zeiss) and

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tracked using the program of Volocity™ (Perkin Elmer, Waltham, MA). For each strain, at least 10

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individual cells were recorded for approximately 1 minute. Capillary tube assays using flow

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cytometry to enumerate bacterial cells were carried out as reported previously (36). A positive

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chemotaxis response was defined as at least twice the number of cells entering the attractant-filled

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tubes as compared to buffer-filled tubes.

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Protein turnover assay. This assay was carried out as previously described (37,38). Briefly, B.

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burgdorferi strains were grown in BSK-II medium at 34°C. After the cell density reached 2 x 108

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cells per ml, 2 ml cultures were added to 50 ml of fresh BSK-II medium containing spectinomycin

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(100 µg/ml) and incubated at 34°C. Samples (5 ml) were harvested and processed for

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immunoblotting at the indicated time points. Immunoblots were developed using horseradish

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peroxidase labeled secondary antibodies using the Pierce ECL Western Blotting Substrate Kit

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(Thermo Scientific, Rockford, IL). Densitometry of immunoreactive proteins in the blots was used

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to determine the relative amounts of proteins as previously described (38). Densitometry was

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measured using the Molecular Imager® ChemiDoc™ XRS Imaging system (Bio-Rad, Hercules,

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CA).

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Protein cross-linking assay. Cross-linking was carried out as previously described (39,40). Briefly,

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late log phase B. burgdorferi cells were freshly harvested in PBS and then incubated with or without

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50 mM formaldehyde for up to 2 hrs at room temperature with gentle shaking. The reactions were

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terminated by adding an equal volume of 2 x SDS Laemmli sample buffer. Cross-linked samples

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were either incubated at 65 °C for 5 min or boiled for 10 min prior to 10% SDS PAGE analysis. The

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targeted proteins were detected on immunoblots probed with specific antibodies. 9

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Bioinformatics and statistical analyses. Protein sequence alignment was conducted using the

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program MacVector 10.6. Predictions of membrane spanning regions and orientation were carried

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out using the TMpred server (http://www.ch.embnet.org/software/TMPRED_form.html). The data

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were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison at p
250 kDa)

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containing MCP3 were detected in wild-type cells treated with formaldehyde (lane 5 and 6), but not

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in wild type cells not treated with formaldehyde (lane 1), nor in CL569 cells with or without

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formaldehyde (lane 3 and 4). A similar pattern was observed when the samples were probed with

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αBB0569 (FIG.8B). These results indicate that BB0569 interacts with other MCPs (i.e., MCP3) to

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large molecular complexes.

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DISCUSSION

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In general, chemotaxis proteins are well conserved among different bacterial species (41). The

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genome of B. burgdorferi encodes at least 19 chemotaxis proteins (21,23). The genes encoding

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these chemotaxis proteins are predominantly within two gene clusters: the flaA operon (flaA-cheA2-

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cheW3-cheX-cheY3) and the cheW2 operon (cheW2-cheA1-BB0566-cheB2-BB0569-cheY2) (21).

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Among these genes, BB0566 and BB0569 are annotated as hypothetical proteins. They reside in the

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chemotaxis gene cluster but lack obvious sequence similarity to bacterial chemotaxis proteins,

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which has led us to study their functions. The experiments undertaken in this report attempted to

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investigate the role of BB0569. The results shown here demonstrate that BB0569 plays a critical

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role in the chemotaxis of B. burgdorferi. First, the sequence alignment shows that BB0569 harbors

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a conserved sequence that is often present in the family of MCP proteins (FIG.2). The sequence is

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located within a highly conserved domain (HCD) found in all MCP classes (41), indicating that

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BB0569 may play a role in chemotaxis. Accordingly, inactivation of BB0569 produced a phenotype

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that was similar to other chemotaxis mutants of B. burgdorferi: the BB0569 mutant swims in only

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one direction and is non-chemotactic to several attractants (FIG. 3). Second, IFA, GFP-fusion, and

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cryo-ET studies showed that BB0569 localizes at the cell poles and that deletion of BB0569 15

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abolishes the polar clustering of MCPs (FIG. 4 & 5). Quantitative immunoblots revealed that the

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deletion of BB0569 had no impact on the expression levels of MCPs and other chemotaxis proteins

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that are essential for the polar clustering of MCPs in B. burgdorferi (28,32), including MCP3,

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MCP5, and CheW3 (data not shown). This finding ruled out the possibility that BB0569 indirectly

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affects the polar clustering of MCPs by impairing expression of other chemotaxis genes.

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Collectively, these results indicate that BB0569 may be required for clustering chemoreceptors at

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the cell poles. Finally, inactivation of either mcp2 or mcp3 substantially impaired the stability of

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BB0569 (FIG. 6), and the lack of BB0569 abolished the cross-linking of MCP proteins (FIG. 8),

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suggesting that there might be a physical interaction between BB0569 and the chemoreceptor

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proteins. Based on these results, we propose: 1) that BB0569 is an MCP-like protein or an MCP-

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binding protein that is required for the polar clustering of MCPs; and 2) that the absence of BB0569

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disrupts the clustering of chemoreceptors, which in turn impairs chemotaxis.

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A canonical MCP protein consists of an extracellular ligand-binding domain, a cytoplasmic

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signaling domain, and an adaptation domain (13,41,48). However, all of these domains are absent in

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BB0569. Thus, it is unlikely that BB0569 functions as a chemoreceptor. Also, BB0569 lacks

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homology to other chemotaxis proteins such as CheA and CheW. An intriguing question is how

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BB0569 integrates into the chemosensory pathways and exerts its role on the chemotaxis of B.

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burgdorferi. Our initial hypothesis was that BB0569 functions like TlpC and FrzCD, cytoplasmic

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chemoreceptor proteins from R. sphaeroides and Myxococcus xanthus (18,49,50), respectively.

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However, the results from the IFA, GFP-fusion and cryo-ET experiments do not support this

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hypothesis. In R. sphaeroides, TlpC interacts with other chemotaxis proteins and forms cytoplasmic

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clusters (18,51). In M. xanthus, FrzCD forms cytoplasmic clusters that appear helically arranged

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and extend along the entire cell (50). In contrast, BB0569 localizes at the cell poles, and its presence 16

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is essential for the polar clustering to MCPs in B. burgdorferi (FIG. 5). In addition, immunoblot

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analysis showed that BB0569, along with MCP3 and MCP5, were detected only in the membrane

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fractions of B. burgdorferi and that no trace of BB0569 was detected in the soluble cytoplasmic

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fractions (FIG. 7). These results rule out the possibility that BB0569 functions as a cytoplasmic

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chemoreceptor like TlpC.

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As mentioned above, the conserved region identified in BB0569 (FIG. 2) shares some

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homology to the highly conserve domain (HCD) of chemoreceptors. A large-scale comparative

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genomic analysis showed that the HCD is found in all MCP classes (41). Genetic and structural

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analyses of E. coli Tsr and the Thermotoga maritima MCP TM1143 suggest that the HCD

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contributes to interactions between MCPs as well as interactions of MCPs with CheW and CheA

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(13,52-54). Therefore, it is possible that BB0569 interacts with the MCPs and/or with CheW and

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CheA to hold the MCP clusters together or to facilitate the interactions between the MCPs and

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CheW and CheA. This interpretation is strengthened by the fact that BB0569 turns over in the mcp2

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and mcp3 mutants (FIG. 6) and that the presence of BB0569 is required for cross-linking of MCP3

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(FIG. 8). B. burgdorferi has three CheWs. Our previous studies have shown that CheW3 is essential

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for both chemotaxis and the polar clustering of the MCPs (28). The C-terminus of CheW3 contains a

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CheR-like domain, a combination that is found only in some spirochete species (28,55). It is likely

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that this unique domain architecture may require an additional partner, such as BB0569, to facilitate

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its physical interactions with the MCPs. With this in mind, we tried different approaches (e.g., co-

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immunoprecipitation and affinity chromatography) to identify potential proteins that interact with

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BB0569. However, we found that the native BB0569 protein could be only detected in insoluble

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fractions of B. burgdorferi cells (FIG. 7). Furthermore, the recombinant BB0569 protein formed

409

inclusion bodies in E. coli and could not be purified under native conditions. We are currently 17

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applying different expression systems, such as Bacillus, to over-express and prepare active BB0569

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recombinant protein. Success in these experiments will allow us to determine whether BB0569

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interacts with other chemotaxis proteins of B. burgdorferi.

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In summary, the results reported here demonstrate that the domain composition and function of

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BB0569 are unusual -- similar in some respects to TlpC but different in their cellular localization.

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Our findings provide further evidence that chemotaxis in B. burgdorferi represents a new paradigm

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that is different from that of prototype bacteria like E. coli, S. enterica, and B. subtilis. This

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conclusion is consistent with the very different control of swimming behavior that is required to

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accomplish chemotaxis in spirochetes, which must reverse swimming direction rather than run and

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tumble to generate the biased three-dimensional random walk that is essential for bacterial

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chemotaxis.

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ACKNOWLEDGEMENT

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This research was supported by Public Health Service grants (AI078958) to C. Li., and DE023431

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to N. Charon. J. Liu was supported in part by grants AI087946 from NIAID and AU-1714 from the

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Welch Foundation.

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Figure Legends:

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FIG. 1. Constructing an isogenic mutant of BB0569 (CL569) and its complemented strain

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(CL569c). The diagrams illustrate the construction of the vectors for inactivation of BB0569 and the

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complementation of the CL569 mutant. The vector BB0569::kan (A) was used to construct CL569,

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and the plasmid pKBB0569 (B) was used to complement the mutant. Arrows indicate the relative

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positions of PCR primers for constructing these vectors. The sequences of these primers are listed in

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Table S1. “Δ” shows the DNA fragment (216 bp) deleted from the gene. Detection of BB0569 by

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immunoblots (C). Similar amounts of whole-cell lysates from the wild-type (WT), CL569, and

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CL569c strains were analyzed by SDS-PAGE and then probed with a specific antibody against

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BB0569 (αBB0569).

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FIG. 2. Multiple sequence alignment of BB0569 and the MCP proteins. The numbers represent

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the positions of amino acids in BB0569, the Tar and Tsr proteins of E. coli, and the McpG protein

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of R. sphaeroides. GenBank accession numbers for the aligned proteins are: BB0569 (NP_212703),

449

Tar (NP_416400), Tsr (NP_418775), and McpG (WP_012641054). The alignment was carried out

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using the program MacVector 10.6. Conserved residues are boxed.

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FIG. 3. The CL569 mutant is non-chemotactic. Swim plate (A) and capillary (B) assays of the

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CL569 mutant and its complemented strain CL569c. For the swim plate assay, ΔflaB, a previously

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constructed non-motile mutant (35), was used as a control to determine the size of non-spreading

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colonies on the plates. The averaged ring sizes are: WT (21.9 ± 0.5 mm, n=5), CL569 (9.4 ± 0.74

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mm, n=5), CL569c (21.8 ± 0.76 mm, n=5), and ΔflaB (8.55 ± 0.37 mm, n=5). For the capillary

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assay, N-acetyl-D-glucosamine (NAG) was used as an attractant; and cheA2-, a previously 19

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constructed non-chemotactic mutant (25,36), was used as a negative control. Results are expressed

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as the means ± SEM from five capillary tubes.

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FIG. 4. BB0569 localizes at the cell poles of B. burgdorferi. (A) & (B) Localization of BB0569

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using IFA. The bacterial cells were fixed with methanol, stained with αBB0569, and counter-

463

stained with anti-rat Texas red antibody, as previously described (28,32). The micrographs were

464

taken under DIC light and fluorescence microscopy with a tetramethylrhodamine isothiocyanate

465

(TRITC) emission filter, and the resultant images were merged. (C) & (D) Localization of BB0569-

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GFP fusion protein. B31BB569GFP represents a strain that expresses BB0569-GFP fusion protein;

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B31GFP is a strain that expresses only GFP. The micrographs were taken under fluorescence

468

microscopy (40 x) with a fluorescein isothiocyanate (FITC) emission filter.

469 470

FIG. 5. BB0569 is essential for the polar localization of MCPs. Determining MCP localization in

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the wild type (A) and the CL569 mutant (B) using IFA with antibodies against B. burgdorferi MCP3

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or MCP5. Only the results of using anti-MCP5 are shown here. The assay and micrographic

473

processes were conducted as described in FIG.4. Cryo-ET was utilized to visualize chemoreceptor

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arrays in the wild-type (C) and CL569 mutant cells (D), as previously described (28,32). Arrow

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points to the chemoreceptor arrays. A total of 30 mutant cells were examined, and no array-like

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structures were observed. OM: outer membrane; CM: cytoplasmic membrane.

477 478

FIG. 6. BB0569 is turned over in mcp2 and mcp3 mutants. Detecting BB0569 in B. burgdorferi’s

479

five mcp mutants by quantitative immunoblotting (A). The mcp mutants (Drs. Charon, Li, and

480

Motaleb, unpublished data) were constructed by targeted mutagenesis, as previously described

481

(25,35). CheA1 (the gene coding this protein is co-transcribed with BB0569) (25) was used as an 20

482

internal loading control. Detecting the stability of BB0569 in the wild type (B), and Δmcp2 (C), and

483

Δmcp3 (D) mutants. The assay was carried out as previously described (38). Translation of B.

484

burgdorferi was arrested by adding spectinomycin (100 µg/ml) to the cultures. Samples were

485

withdrawn at the indicated time points and analyzed by immunoblotting. The results are expressed

486

as the percent of protein density at zero time compared to the protein density at subsequent time

487

points. Gels were loaded with the whole cell lysates of wild-type (10 µg) and mutant cells (50 µg)

488

and then transferred to PVDF membranes, which were probed with αBB0569 and αDnaK

489

antibodies. DnaK was used as a loading control.

490 491

FIG. 7. BB0569 fractionates with insoluble membrane fractions. The cells were treated and

492

fractionated as previously documented (56). The presence of BB0569, MCP3, and MCP5 in three

493

different fractions was detected by immunoblotting. WC: whole-cell lysates, S: soluble fractions,

494

and P: insoluble fractions.

495 496

FIG. 8. Protein cross-linking assays. Late-log-phase B. burgdorferi cells were harvested and

497

treated with (lanes 3-6) or without (lanes 1-2) formaldehyde, as described in Materials and

498

Methods. The targeted proteins were detected by immunoblotting with specific antibodies as

499

labeled. Lane 1, WT; lane 2, Δmcp3; lane 3, CL569 treated with formaldehyde for 90 minutes; lane

500

4, CL569 treated with formaldehyde for 60 minutes; lane 5, WT treated with formaldehyde for 90

501

minutes; lane 6, WT treated with formaldehyde for 60 minutes. Black arrows point to cross-linked

502

protein complexes, and gray arrows point to protein monomers.

503 504 505 21

506

Table 1. Reversal frequency and cell velocity of the CL569 mutant. Strains

Reversal frequency

Velocity

(reversal/min) Wild type CL569 CL569c

(µm/sec)

19 ± 4

13.20 ± 2.0

0

11.30 ± 2.4

21 ± 3

12.25 ± 2.9

507

Bacterial cells were videotaped under a dark-field microscopy and tracked using the program of

508

Volocity™. For each strain, at least 10 individual cells were recorded for approximately 1 minute.

509

The data are expressed as average reversal frequency (reversal/min) and velocity (µm/sec) ±

510

standard deviations.

511 512

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