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Journal of Bacteriology, October 2007, p. 7145-7150, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00892-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Flavobacterium johnsoniae SprA Is a Cell Surface Protein Involved in Gliding Motility{triangledown}

Shawn S. Nelson, Padden P. Glocka, Sarika Agarwal, David P. Grimm, and Mark J. McBride*

Department of Biological Sciences, University of Wisconsin—Milwaukee, P. O. Box 413, Milwaukee, Wisconsin 53201

Received 7 June 2007/ Accepted 13 July 2007


   ABSTRACT
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Flavobacterium johnsoniae cells glide rapidly over surfaces by an unknown mechanism. Transposon-induced sprA mutants formed nonspreading colonies on agar, and the cells examined in wet mounts were deficient in attachment to surfaces and were almost completely nonmotile. Exposure of intact cells to proteinase K cleaved the 270-kDa SprA into several large peptides, suggesting that it is partially exposed on the cell surface.


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Flavobacterium johnsoniae cells move rapidly over surfaces in a process called gliding motility (19). Rapid gliding motility is common in the large and diverse phylum of bacteria known as the Bacteroidetes, of which F. johnsoniae is a member. F. johnsoniae cells typically move at speeds of approximately 5 µm/s over wet glass surfaces. They also adsorb latex spheres and propel them rapidly around the cell along multiple paths (27). As a result of the gliding motility of cells, colonies of F. johnsoniae have thin spreading edges. Several models have been proposed to explain this type of gliding motility, but the mechanism of cell movement remains unknown (6, 17, 19, 26, 27).

Genetic analyses have identified the genes required for F. johnsoniae motility (22). gldA, gldF, and gldG encode proteins that appear to form an ATP-binding cassette transporter that is required for gliding (1, 13). Eight other gld genes (gldB, -D, -H, -I, -J, -K, -L, and -M) are also required for movement (4, 5, 14, 15, 20, 21). The disruption of any of these genes results in a complete loss of motility. The mutants form nonspreading colonies, and individual cells exhibit no movement on agar or glass. Genetic analyses suggest that few genes that are required for gliding remain to be identified (4).

All of the Gld proteins localize to the cell envelope, but surprisingly, none appear to be exposed on the cell surface (4, 5, 13-15, 20, 21). Surface-exposed or extracellular structures play critical roles in most other forms of bacterial surface translocation (3), and it is difficult to explain F. johnsoniae gliding without including proteins exposed on the cell surface. In an attempt to identify additional motility proteins, we analyzed Tn4351-, HimarEm1-, and HimarEm2-induced mutants with less severe motility defects than those of the gld mutants. These mutants formed nonspreading colonies on agar that were indistinguishable from those of completely nonmotile gld mutants, but individual cells retained some ability to glide on glass in wet mounts. Analysis of these "motile nonspreading" (MNS) mutants resulted in the identification of sprA, which encodes a large protein required for colony spreading and efficient cell movement.

Bacterial and bacteriophage strains, plasmids, and growth conditions. F. johnsoniae MM101, UW101, and FJ1 were the wild-type strains used in this study. Each of these strains are descendants of F. johnsoniae ATCC 17061, but they have slight differences as a result of propagation in different laboratories (20). The 37 spontaneous and chemically induced MNS mutants of F. johnsoniae UW101 were obtained from J. Pate and are designated UW102-1, -2, -3, -18, -24, -37, -43, -45, -46, -50, -51, -67, -73, -88, -91, -93, -106, -128, -133, -135, -136, -142, -143, -148, -149, -150, -155, -156, -157, -158, -168, -171, -172, -176, -298, -344, and -345 (7, 31). F. johnsoniae strains were grown in Casitone-yeast extract (CYE) medium at 30°C, as previously described (22). To observe colony spreading, F. johnsoniae was grown on PY2 agar medium (1) at 25°C. The following antibiotics were used at the indicated concentrations when needed: ampicillin, 100 µg/ml; chloramphenicol, 25 µg/ml; erythromycin, 100 µg/ml; kanamycin, 50 µg/ml; and tetracycline, 20 µg/ml. The plasmids used in this study are listed in Table 1.


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  		TABLE 1. Plasmids used in this study

 
Transposon mutagenesis and identification of sprA. Tn4351, HimarEm1, and HimarEm2 were introduced into wild-type F. johnsoniae, and 294 mutants that formed nonspreading colonies were isolated as previously described (4, 13-15). The mutants were screened for motility defects in wet mounts and were divided into three groups. Nineteen of the mutants exhibited cell division defects resulting in the production of filamentous cells. These were not considered further in this study. Another 51 mutants were completely nonmotile and had normal cell morphology. These mutants each had mutations in the gld genes described previously (1, 4, 5, 13-15, 20, 21). The remaining 224 mutants exhibited the MNS phenotype and retained some ability to move in wet mounts. The sites of transposon insertions were determined for 37 randomly selected mutants essentially as described previously (4, 13, 16, 25). Six mutants (CJ693, CJ983, CJ984, CJ1366, CJ1381, and FJ118) had insertions in a 7.2-kbp gene, which we named sprA (Fig. 1). These sprA mutants had the most severe motility defects of the 37 MNS mutants studied. CJ693, CJ983, CJ984, CJ1366, and CJ1381 were derived from F. johnsoniae MM101, whereas FJ118 was derived from F. johnsoniae FJ1. CJ693, CJ983, and CJ984 each had a Tn4351 insertion; CJ1366 and CJ1381 each had a HimarEm1 insertion; and FJ118 had a HimarEm2 insertion in sprA.


Figure 1
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  		FIG. 1. Map of the sprA region of F. johnsoniae. The numbers below the map indicate the kilobase pairs of the sequence. The sites of the Tn4351, HimarEm1, and HimarEm2 insertions are indicated by triangles, open circles, and closed circles, respectively. The region of DNA carried by the complementing plasmid pPG1 is indicated beneath the map. maeB, ruvA, and gcsH encode predicted malate dehydrogenase, Holliday junction DNA helicase, and glycine cleavage system protein, respectively.

 
sprA encodes a predicted product of 2,403 amino acids. It appears to have an amino-terminal hydrophobic signal peptide, but the bulk of the protein is predicted to be hydrophilic. Comparisons to database sequences using the BLAST (2) and FASTA (28) algorithms revealed that SprA does not exhibit sequence similarity to proteins of known function, but it is similar in sequence to large hypothetical proteins from other members of the phylum Bacteroidetes. These include gliding bacteria such as Cytophaga hutchinsonii (32) and nonmotile bacteria such as Porphyromonas gingivalis W83 (23). Homologs of sprA are not universal among the Bacteroidetes, as they are absent from some nonmotile members of the phylum, such as Bacteroides thetaiotaomicron (33).

SprA may be an atypical outer membrane protein. Although SprA is not predicted to have an extensive ß-sheet structure, PSORTb analysis (12) suggested that it is an outer membrane protein (prediction score of 9.98 out of 10). This prediction was based on the detection of an outer membrane protein motif (enterobacterial virulence outer membrane protein signature 2; PROSITE accession number PS00695) and the results of the Support Vector Machines learning-based classifiers analysis of the PSORTb program.

Analysis of the region surrounding sprA revealed that the stop codon of the DNA helicase gene, ruvA, lies 23 bp upstream of the sprA start codon, and the coding region of the glycine cleavage system gene, gcsH, begins 84 bp downstream of the sprA stop codon. There is no evidence linking the expression or the function of ruvA or gcsH with sprA. Each of these three genes has a close match to the –7 regions of Flavobacterium promoters sequence (TAXXTTTG) (8, 9) within 70 bp upstream of its start codon.

Cloning of sprA and complementation of sprA mutants. A library of wild-type DNA partially digested with Sau3aI was constructed in Lambda Gem-11 (Promega, Madison, WI) as previously described (16). Clones containing sprA were detected by hybridization with radiolabeled DNA prepared using a fragment of chromosomal DNA adjacent to the transposon insert in CJ693 and the Prime-a-Gene labeling kit (Promega). One lambda clone spanned the entire sprA gene. The 12-kb SacI fragment of this lambda clone was inserted into pBCKS+ to generate pSA64. pSA64 was digested with XhoI to release a 7.8-kb fragment containing sprA and gcsH. This fragment was treated with a DNA polymerase Klenow fragment to make the ends blunt and inserted into the SmaI-digested shuttle vector pCP23 to generate pPG1 (Fig. 1). To construct pPG3, which has sprA inserted into pCP23 in the opposite orientation, sprA was excised from pSA64 using XhoI, carrying with it a BamHI restriction site from the vector. The XhoI fragment was inserted into SalI-digested pSPORT1 to generate pPG2. sprA was cut out of pPG2 using BamHI and inserted into pCP23 to generate pPG3. pPG1 and pPG3 were introduced into the sprA mutants CJ693 and CJ984 by triparental conjugation as previously described (15, 22). The introduction of either plasmid resulted in the complementation of each of the sprA mutants as evidenced by the formation of spreading colonies. To determine if gcsH was needed for complementation, we inserted the SmaI fragment of pHP45{Omega}Tc, containing the tetracycline resistance gene, into the EcoRV site of pPG1 in gcsH, generating pSN48. The introduction of pSN48 into each of the sprA mutants (CJ693, CJ983, CJ984, CJ1366, CJ1381, and FJ118) resulted in the restoration of colony spreading, demonstrating that the defect in the sprA mutants was not the result of a polar effect on gcsH (Fig. 2). In addition to the transposon-induced MNS mutants described above, we introduced pSN48 into 37 spontaneous and chemically induced MNS mutants that were described by Pate and coworkers (7, 31) and identified 5 (UW102-3, UW102-43, UW102-106, UW102-142, and UW102-150) that were complemented.


Figure 2
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  		FIG. 2. Photomicrographs of F. johnsoniae colonies. The colonies were grown for 24 h at 25°C on PY2 agar media. Photomicrographs were taken with a Kodak DC290 digital camera mounted on an Olympus IMT-2 inverted microscope. Bar, 0.5 mm. (A) Wild-type F. johnsoniae FJ1; (B) sprA mutant FJ118; (C) FJ118 complemented with pSN48, which carries sprA.

 
Microscopic observations of cell attachment and movement. Wild-type and mutant cells of F. johnsoniae were examined for movement over glass by phase-contrast microscopy at 25°C. The cells were grown in motility medium (MM), which consisted of 3.3 g Casitone per liter, 1.7 g yeast extract per liter, and 3.3 mM Tris (pH 7.5). Five milliliters of MM in a 125-ml flask was inoculated with cells and incubated overnight at 25°C without shaking until a density of approximately 5 x 108 cells/ml was reached. The cells in MM were examined for motility on glass and agar and for their ability to propel polystyrene latex spheres as previously described (14, 15). The wild-type cells in wet mounts attached readily to the glass slide and the oxygen-permeable Teflon membrane (Yellow Springs Instrument Co., Inc., Yellow Springs, OH) that was used as a coverslip and displayed rapid motility (approximately 5 µm/s) over both surfaces. Under the conditions used, over 95% of cells that were in contact with the glass or coverslip exhibited gliding movements during a 2-minute period. In addition to gliding movements, the cells that attached to a surface by a single pole often rotated in place at frequencies of about 2 revolutions/s. Polystyrene latex spheres (0.4 µm) readily attached to and were propelled by wild-type cells, as previously described (27). The sprA mutants exhibited very limited motility. Most cells of the sprA mutants failed to attach to the glass or Teflon surfaces. The cells that did attach usually displayed no movement, but extended observation revealed a few cells that exhibited occasional slight movements. Typically less than 1 cell out of 1,000 would move. Even in these rare cells, movement was limited and sporadic, in contrast to the more continuous movements of the wild-type cells. The cells of the sprA mutants failed to bind or propel latex spheres under the conditions tested. The introduction of pSN48 into any of the sprA mutants restored wild-type motility and the ability to propel polystyrene latex spheres. The defect in attachment associated with the sprA mutations was also examined in the more controlled environment of a Petroff-Hausser counting chamber to provide uniform volume and concentration of cells. The cells were grown in MM overnight at 25°C to a density of 2.5 x 108 cells/ml and incubated in the chamber for 2 min at 25°C, and photomicrographs were taken in the plane of the glass coverslip. The wild-type cells readily attached to the coverslip, whereas few cells of the sprA mutant FJ118 attached (Fig. 3). The introduction of pSN48 into FJ118 restored the ability to attach to the glass. The average number of cells attached per field (from 10 random fields per strain) was 73 (standard deviation [SD], 15), 7.4 (SD, 2.2), and 77 (SD, 17) for cells of the wild type (FJ1), the sprA mutant (FJ118), and the complemented strain (FJ118 with pSN48), respectively.


Figure 3
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  		FIG. 3. Attachment of F. johnsoniae cells to glass. Cells grown in MM to a density of 2.5 x 108 cells/ml were introduced into a Petroff-Hauser counting chamber and incubated for 2 min at 25°C. To observe the attached cells, photomicrographs in the plane of the glass coverslip were taken with a Photometrics CoolSNAPcf2 camera mounted on an Olympus BH-2 phase-contrast microscope. The phase-dark cells (in focus) were attached to the coverslip, whereas the bright cells (out of focus) were suspended and unattached. (A) Wild-type F. johnsoniae FJ1; (B) sprA mutant FJ118; (C) FJ118 complemented with pSN48, which carries sprA. Bar, 20 µm.

 
sprA mutants are partially defective in chitin utilization. Wild-type cells of F. johnsoniae digest chitin (29), whereas all nonmotile mutants fail to utilize this insoluble polysaccharide (4, 5, 7, 20, 21). The connection between chitin utilization and gliding is not understood. Chitin utilization may involve transport of long chitin oligomers across the outer membrane by using some of the gliding motility machinery (5, 20, 21). The effect of a mutation in sprA on chitin utilization was determined in MYA medium supplemented with chitin as the primary carbon, energy, and nitrogen source, as previously described (20). The cells of the sprA mutant FJ118 digested chitin more slowly than the wild-type cells (Fig. 4). Complementation with pSN48 restored the ability to digest chitin to wild-type levels in addition to restoring gliding motility.


Figure 4
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  		FIG. 4. Effect of mutation in sprA on ability to utilize chitin. Approximately 4 x 107 cells of wild-type F. johnsoniae FJ1 (A), sprA mutant FJ118 (B), and FJ118 complemented with pSN48, which carries sprA (C), were spotted on MYA-chitin medium and incubated for 3 days at 25°C.

 
Bacteriophage resistance of sprA mutants. Nonmotile mutants of F. johnsoniae are resistant to infection by all known F. johnsoniae bacteriophages, including {phi}Cj1, {phi}Cj13, {phi}Cj23, {phi}Cj28, {phi}Cj29, {phi}Cj42, {phi}Cj48, and {phi}Cj54 (1, 4, 5, 13-15, 20, 21, 30). The connection between motility and bacteriophage susceptibility is not understood, but it is suggested that the movement of cell surface components is necessary to allow productive interaction of bacteriophages with cells. Sensitivity to F. johnsoniae bacteriophages was determined as previously described by spotting 3 µl of phage lysates (109 PFU/ml) onto lawns of cells in CYE overlay agar (15). The plates were incubated for 24 h at 25°C to observe lysis. Wild-type F. johnsoniae showed complete lysis by all of the bacteriophages described above. The sprA mutant FJ118 was completely resistant to {phi}Cj1, {phi}Cj13, {phi}Cj23, and {phi}Cj29, partially resistant to {phi}Cj42, {phi}Cj48, and {phi}Cj54, and sensitive to {phi}Cj28 (Fig. 5). The introduction of pSN48 into FJ118 resulted in restoration of sensitivity to each of the bacteriophages in addition to restoration of wild-type motility.


Figure 5
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  		FIG. 5. Effect of mutation in sprA on bacteriophage resistance. Bacteriophages (3 µl of lysates containing approximately 109 PFU/ml) were spotted onto lawns of cells in CYE overlay agar. The plates were incubated at 25°C for 24 h to observe lysis. Bacteriophages were spotted in the following order from left to right: top row, {phi}Cj1, {phi}Cj13, and {phi}Cj23; middle row, {phi}Cj28, {phi}Cj29, and {phi}Cj42; and bottom row, {phi}Cj48 and {phi}Cj54. (A) Wild-type F. johnsoniae FJ1; (B) sprA mutant FJ118; (C) FJ118 complemented with pSN48, which carries sprA.

 
Protein expression and antibody production. pPG1 was digested with BglII and HindIII to generate a 3,950-bp fragment encoding the C-terminal 1,170 amino acids of SprA. The fragment was ligated into the BamHI and HindIII sites of pMalC2 to generate pSN93. pSN93 was introduced into Escherichia coli Rosetta 2(DE3) (Novagen), which expresses seven rare tRNAs required for efficient expression of sprA. To isolate recombinant SprA, the cells were grown to mid-log phase at 37°C in rich medium plus glucose (10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 g glucose/liter), induced by the addition of 0.3 mM IPTG (isopropyl-ß-D-thiogalactopyranoside), and incubated for an additional 48 h. The cells were disrupted using a French press, and inclusion bodies containing recombinant SprA were isolated by centrifugation at 6,000 x g for 10 min. The inclusion bodies were suspended in bacterial protein extraction reagent (B-PER; Pierce) containing 200 µg/ml lysozyme and incubated for 5 min at 24°C. The inclusions were collected by centrifugation at 27,000 x g for 15 min and washed with B-PER. The SprA inclusions were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and purified by SDS-PAGE. Recombinant SprA was visualized by CuCl2 staining (18), the band was cut from the gel and destained in 0.25 M Tris (pH 9.0)-0.25 M EDTA, and the protein was electroeluted at 60 mA for 5 h into 25 mM Tris, 192 mM glycine, and 0.1% SDS using a model 422 electroeluter (Bio-Rad). Polyclonal antibodies against recombinant SprA were produced and affinity purified using the recombinant protein by Proteintech Group, Inc. (Chicago, IL).

Detection and localization of SprA. Antisera to SprA were used to detect SprA in cell extracts. Overnight cultures of F. johnsoniae were grown in MM at 25°C without shaking. The cells were pelleted at 4,000 x g for 15 min and suspended in 20 mM sodium phosphate (pH 7.5) containing 10 mM EDTA. The cells were disrupted with a French press and fractionated into soluble and membrane fractions as described previously (13), except that Halt protease inhibitor (Pierce, Rockford, IL) was added to the cell extracts. Proteins were separated on 5% acrylamide gels, and Western blot analyses were performed as described previously (15). Antibodies against SprA were detected using goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate and SuperSignal West Pico chemiluminescent substrate (Pierce). SprA migrated at approximately 270 kDa, which is close to its predicted size (Fig. 6A). PSORTb analysis (12) indicated that SprA was likely to be an outer membrane protein. As expected, SprA in cell extracts sedimented with the cell membranes during ultracentrifugation at 226,000 x g for 60 min (Fig. 6B). To determine whether SprA was exposed on the cell surface, we incubated intact cells in 20 mM sodium phosphate-10 mM MgCl2 (pH 7.5) with 100 µg/ml proteinase K at 25°C. At various times, 10 mM phenylmethylsulfonyl fluoride was added to stop the reaction, the samples were boiled for 1 min, and the cells were collected by centrifugation, suspended in SDS-PAGE loading buffer, and boiled for 3 min. Equal volumes were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes, and proteins were detected with the appropriate antisera. Exposure of the cells to proteinase K resulted in the cleavage of SprA into several large peptides that were stable to further digestion (Fig. 7A, lanes 7, 8, and 9). The addition of 0.1% Triton X-100 to solubilize the membrane resulted in a dramatic increase in susceptibility to proteinase K (Fig. 7A, lanes 11, 12, and 13). These results indicate that part of SprA is exposed on the cell surface but that much of the protein lies beneath the surface of the outer membrane. GldJ, an outer membrane lipoprotein that is not exposed on the cell surface (5), was not susceptible to proteinase K treatment unless Triton X-100 was added (Fig. 7B), and there was also no major difference between the total protein profiles of the cells before and after proteinase K treatment in the absence of detergent (Fig. 7C), indicating that the cells were intact and that the outer membrane was not damaged.


Figure 6
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  		FIG. 6. Immunodetection of SprA. (A) Whole cells were examined for SprA by Western blot analysis. Lane 1, wild-type F. johnsoniae FJ1; lane 2, sprA mutant FJ118; lane 3, FJ118 with pSN48, which carries sprA. Eighty micrograms of protein was loaded in each lane. (B) Cell fractions of wild-type cells were examined for SprA by Western blot analysis. Lane 1, whole cells; lane 2, soluble fraction; lane 3, membrane fraction. Eighty micrograms of protein was loaded in each lane.

 

Figure 7
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  		FIG. 7. Effect of proteinase K treatment of cells on SprA and GldJ. (A) An immunoblot was probed with antibodies against SprA. Lane 1, cells of sprA mutant FJ118; lanes 2 to 13, cells of wild-type FJ1 incubated for the times indicated in 20 mM sodium phosphate buffer-10 mM MgCl2 (pH 7.4) with or without proteinase K (100 µg/ml) or Triton X-100 (0.1%). One hundred sixty micrograms of protein was loaded in each lane. The arrow indicates full-length SprA. (B) An immunoblot was probed with antibodies against GldJ. Lane 1, cells of sprA mutant FJ118; lane 2, cells of gldJ mutant FJ123; lanes 3 to 9, cells of wild-type FJ1 incubated for the times indicated in 20 mM sodium phosphate buffer-10 mM MgCl2 (pH 7.4) with or without proteinase K (100 µg/ml) or Triton X-100 (0.1%). Eighty micrograms of protein was loaded in each lane. The arrow indicates full-length GldJ. (C) Coomassie brilliant blue-stained SDS-PAGE gel of samples shown in panel A. The cells of wild-type FJ1 were incubated for the times indicated in 20 mM sodium phosphate buffer-10 mM MgCl2 (pH 7.4) with or without proteinase K (100 µg/ml) or Triton X-100 (0.1%). Eighty micrograms of protein was loaded in each lane.

 
Disruption of sprA does not alter GldJ protein levels. The previously described secDF mutants have low levels of GldJ protein, which is thought to explain their motility defects (24). Western blots of wild-type and sprA mutant cells revealed that disruption of sprA had no effect on GldJ levels (Fig. 7B, lanes 1 and 3) and eliminated decreased levels of GldJ protein as an explanation for the motility phenotype of sprA mutants.

SprA appears to be involved in gliding motility, but its exact role in this process remains uncertain. sprA mutants form nonspreading colonies and most cells fail to move on agar or in wet mounts on glass. However, extended observations revealed that a few cells exhibited slight gliding movements on glass, indicating that parts of the motility apparatus are functional in the absence of SprA. SprA is the first protein associated with F. johnsoniae gliding to be identified that is at least partially exposed on the cell surface. As such, it may provide a link between the gliding motor, presumably composed of Gld proteins, and adhesive components on the cell surface that interact with the substratum during cell movement. The reduction in adhesion of the cells of sprA mutants to glass, polystyrene, and Teflon surfaces is consistent with this possibility. Future studies of MNS mutants may identify additional cell surface components of the motility apparatus and help determine the mechanisms of adhesion and cell movement.

Nucleotide sequence accession number. The sequence reported in this paper has been deposited in the GenBank database (accession number AY850225).


   ACKNOWLEDGMENTS
 
This research was supported by grants MCB-0130967 and MCB-0641366 from the National Science Foundation.

We thank D. Saffarini for the careful reading of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, 181 Lapham Hall, University of Wisconsin—Milwaukee, 3209 N. Maryland Ave., Milwaukee, WI 53211. Phone: (414) 229-5844. Fax: (414) 229-3926. E-mail: mcbride{at}uwm.edu Back

{triangledown} Published ahead of print on 20 July 2007. Back


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Journal of Bacteriology, October 2007, p. 7145-7150, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00892-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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