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Journal of Bacteriology, June 2006, p. 4312-4320, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.01975-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Proteomic Analysis of Campylobacter jejuni 11168 Biofilms Reveals a Role for the Motility Complex in Biofilm Formation

Martin Kalmokoff,1,{dagger} Patricia Lanthier,2 Tammy-Lynn Tremblay,2 Mary Foss,2 Peter C. Lau ,2,{ddagger} Greg Sanders,1 John Austin,1 John Kelly,2 and Christine M. Szymanski2*

Health Canada Bureau of Microbial Hazards, Ottawa, Ontario K1A 0L2,1 NRC Institute for Biological Sciences, Ottawa, Ontario K1A 0R6, Canada2

Received 23 December 2005/ Accepted 23 March 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated that C. jejuni 11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellate flhA mutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA, flaA, flaB, flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment of C. jejuni 11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide (1) and the primary cause of bacterial food-borne infection in Canada, outnumbering reported cases of Salmonella, Shigella, Listeria, and Escherichia coli infections combined (43). In addition, C. jejuni is the most frequent antecedent to Guillain-Barré syndrome, the main cause of paralysis since the elimination of poliomyelitis. The primary source of human infection with campylobacters is poultry, with nearly 90% of retail-ready products reported to be contaminated, and yet chicks hatched in poultry houses are not colonized with campylobacters (45). Furthermore, campylobacter-positive flocks are not detected until 4 to 8 weeks of age (55), after which nearly 100% of chicks become colonized. These findings, together with low levels of campylobacter carryover from flock-to-flock, suggest that there must be additional reservoirs for contamination within these poultry operations (48). Campylobacters have been isolated from a variety of sources (34, 39, 46, 56, 63), including raw sewage and surface waters containing wastes from poultry, wild birds, and dairy herds (37, 52, 54, 56). Thus, water and surfaces may be an important source of campylobacter contamination in poultry houses (39).

Campylobacter survival in the environment is not well understood. Although considered a fastidious pathogen that normally only grows well under in vivo-like conditions, transmission of C. jejuni to a new host often involves a period of exposure to a hostile external environment. Therefore, it is important to understand the natural ecology of C. jejuni and the influence of environmental factors on C. jejuni survival. Biofilm formation may play a significant role in campylobacter survival in the environment.

More than 99.9% of bacteria in aquatic ecosystems have been demonstrated to exist in biofilms on a wide variety of surfaces. Biofilm formation is critical not only for environmental survival but also for successful infection by numerous pathogens, including the chronic colonization by Pseudomonas aeruginosa of the lungs of cystic fibrosis patients, the persistence of oral bacteria associated with human periodontal diseases, and the common occurrence of nosocomial infections resulting from biofilms formed on medical devices. The U.S. National Institutes of Health estimates that >80% of human infections involve biofilms. It is now widely accepted that bacteria exist in two different modes of growth, the first being as single planktonic cells and the second as structured, multicellular communities known as biofilms (10). A biofilm is a multicellular layer of bacteria attached to surfaces, interfaces, or other cells and embedded within a matrix of extracellular polymeric substances (EPS) (14). Biofilm formation occurs in a number of distinct steps, including adsorption to the target surface, adherence that generally results from the production of surface polysaccharides, and finally differentiation into a multicellular community surrounded by EPS. Growth in a biofilm provides many advantages for bacteria, including enhanced resistance to environmental stresses, such as desiccation and antimicrobials, as well as an increased resistance to host defenses (12, 35).

For the first time, we demonstrate that campylobacters can form multicellular biofilms. Proteomic analysis of these biofilms revealed differences in protein expression in comparison to planktonic cells, particularly in the expression of proteins involved in motility and stress response. Flagellar components were found to be important for both the initiation of biofilm formation on solid surfaces and pellicle formation in liquid cultures. The continued expression of the flagellar apparatus in mature biofilms suggests a role for flagella in the biofilm phenotype.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and growth conditions. C. jejuni 11168 (V26) (8) was routinely grown in Mueller-Hinton (MH) (Difco) media under microaerobic conditions (10% CO2, 5% O2, 85% N2) at 37°C. E. coli DH10B (Invitrogen) was grown on S-Gal/LB agar (Sigma) or MH agar at 37°C. Plasmid pPCR-Script Amp (Stratagene) was used as the cloning vector. When appropriate, antibiotics were added to the following final concentrations: kanamycin, 30 µg/ml; ampicillin, 150 µg/ml.

Construction and characterization of insertional mutations. The construction of the fliA and flhA mutants (8) and of the flaA and flaB mutants (27) was previously described. For the flaC::Km mutant, a 2,119-bp fragment was amplified with flaC-F2 (5'-CAAAATCATTACTCATCCCC-3') and flaC-R2 (5'-CAAAAAGAACCCAAAGAGG-3') and the kanamycin resistance (Km) cassette was cloned into the unique BsgI site of flaC. For the flaG::Km mutant, a 1,631-bp fragment was amplified with flaG-F2 (5'-TTTTGGGGGTAAAGCAGG-3') and flaG-R2 (5'-CCGCATAGAAACTTATCGCAC-3') and the Km cassette was cloned into the unique XbaI site of flaG. All C. jejuni antibiotic-resistant transformants were characterized by PCR to confirm that the incoming plasmid DNA had integrated by a double-crossover event. The orientation of the Km cassettes in the mutants was confirmed to be nonpolar by sequencing with the ckanB primer (5'-CCTGGGTTTCAAGCATTAG-3').

Motility assay. The centers of duplicate MH plates containing 0.4% agar were stabbed with 5 µl of each test strain adjusted to a final optical density at 600 nm (OD600) of 1.00. The plates were then incubated under microaerobic conditions at 37°C. After 52 h, the diameter of the zone of spread was measured with a ruler for each strain and the experiment was repeated to obtain duplicate measurements. The wild-type strain was always included for comparison.

Biofilm growth on inert surfaces. Biofilms of C. jejuni 11168 were grown on the surface of sterile nitrocellulose membranes (pore size, 0.45 µm; Millipore), 2- by 2-cm food grade stainless steel coupons (type 304, no. 4 finish), and 2-cm glass fiber filters (Whatman). Briefly, test surfaces were placed upright in 7 ml of MH broth contained within 12-well polystyrene tissue culture plates (Corning Inc.), with each well inoculated with 10 µl from an overnight liquid culture. The plates were incubated under stationary conditions with reduced oxygen at 37°C, representing the temperature of the human gastrointestinal tract. Test surfaces were aseptically transferred into fresh medium on 3 consecutive days, after approximately 24 h of growth at 37°C. Following the completion of growth, test surfaces were rinsed by repeated immersion of the test surface in sterile phosphate-buffered saline (PBS). Planktonic or unattached cells for proteomic analysis were collected from the wells used for incubation of the surfaces from the final transfer (24 h).

Extract preparation for comparative proteomic studies. Pellets of C. jejuni 11168 planktonic cells were washed twice with ice-cold 20 mM HEPES buffer, pH 7.5, or PBS buffer, pH 7.4. The cells were resuspended directly in lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 1% dithioerythritol, and 0.2% BioLytes and sonicated at 4°C with a Fisher Scientific micro-tip model 550 sonicator, six times for 30s each. Biofilm-grown cells attached to the surface of glass fiber filters were immersed directly into solubilization buffer following rinsing of the surface in PBS. Following sonication, Benzonase (150 U/ml; Sigma-Aldrich) was added and the proteins were solubilized by incubation of the lysate on ice for 1 h. Unlysed cells, cell debris, and glass fibers were removed by centrifugation at 14,000 x g for 10 min. On occasion, the supernatants were also subjected to ultracentrifugation at 100,000 x g for 1 h and stored at –20°C. Additional salt removal was done by using the 2D cleanup kit (Amersham Biosciences) or by overnight dialysis with a dialysis cup and a 3.5-kDa MWCO dialysis membrane (Fisherbrand). Extract protein concentrations were assayed with a modified Bradford assay (44).

Two-dimensional gel electrophoresis. For two-dimensional gel electrophoresis, aliquots of 125 to 200 µg of protein from both biofilm and planktonic extracts, from four separate experiments, were applied to duplicate pH 4 to 7 (Bio-Rad) or pH 6 to 11 (Amersham Biosciences) 11-cm IPG strips for first-dimension separation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in an 8 to 16% gradient precast acrylamide gel (Bio-Rad) or a 12% acrylamide gel. Gels were first stained with Sypro Ruby fluorescent protein stain (Bio-Rad) for quantitative gel comparison with PDQuest software (Bio-Rad). Then, the gels were silver stained for spot visualization prior to gel excision and overnight in-gel digestion with trypsin.

Protein identification. The in-gel digests were analyzed by nano-liquid chromatography-tandem mass spectrometry (MS/MS) with a CapLC capillary liquid chromatography system (Waters) coupled to a Q-TOF Ultima hybrid quadrupole time-of-flight mass spectrometer (Waters). The peptide extracts were injected onto a 75-µm (inner diameter) by 150-mm PepMap C18 nanocolumn (Dionex/LC-Packings) and resolved by gradient elution (5 to 75% acetonitrile-0.2% formic acid in 30 min at 350 nl/min). The mass spectrometer was set to operate in automatic MS/MS acquisition mode (6 s of acquisition time per precursor ion). MS/MS spectra were acquired on doubly, triply, and quadruply charged ions. Proteins were identified by matching the sequences derived from peptide MS/MS spectra with sequences in the C. jejuni NCTC11168 protein sequence database using Mascot Daemon database searching software (Matrix Science) and Nemesis, an algorithm generated in-house to extract and tabulate the significant sequence matches from multiple Mascot result files.

Pellicle assay. All bacteria were adjusted to an OD600 of 1, and 4 µl was inoculated into duplicate wells of 24-well plates containing 2 ml of MH broth. The first two wells contained only MH broth and served as negative controls. Plates were incubated for up to 5 days under stationary microaerobic conditions at 37°C. Pellicle formation at the air-liquid interface was photographed on days 3 and 5 with an AlphaImager 3400 (Alpha Innotech). Each strain was tested two to four times.

Electron microscopy. For examination of the flagellar architecture of the mutants, 1% (wt/vol) ammonium molybdate-stained grids were examined as previously described (8).

Biofilms grown on the various test surfaces (stainless steel, nitrocellulose, and glass fibers) were rinsed by immersion in Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and then fixed with 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.0. Samples for scanning electron microscopy (SEM) were prepared as previously described (3, 13). Mounted specimens were sputter-coated with 30 nm of platinum and viewed with a Hitachi S 3000 N scanning electron microscope at an accelerating voltage of 5.0 kV.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SEM examination of test surfaces following three successive transfers into fresh medium demonstrated that C. jejuni 11168 adhered and formed multicellular layers consisting of filamentous cells (Fig. 1). In contrast, examination of surfaces present for one complete day (24 h) consisted only of single attached cells (results not shown). Examination of the adherent layers indicated the presence of fiber-like extracellular material associated with mature biofilms (Fig. 1A insert), as well as larger regions of amorphous material within which the cells appeared to be imbedded (Fig. 1C). This material likely represents condensed EPS, as observed in biofilms of various other bacterial species (14); fiber-like extracellular material is indicated by arrows in Fig. 1A (inset). There were also marked differences in the densities of cell growth depending on the growth surface, with the resulting biofilms being much thicker on both nitrocellulose and glass fibers (Fig. 1B to D) than were found with food grade stainless steel (Fig. 1A). For proteomic analyses, the thick biofilms grown on glass fibers were compared with the corresponding stationary-phase planktonic cells taken from the liquid phase of wells from the last glass fiber transfer.


Figure 1
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  		FIG. 1. SEM of C. jejuni 11168 attached to various surfaces. (A) Biofilm formed on stainless steel, with the inset demonstrating extracellular fiber-like material. (B) Biofilm formed on nitrocellulose. (C) Larger image of biofilm formed on nitrocellulose, demonstrating extracellular material covering the cells. (D) Biofilm formed on glass fiber filters. (E) Glass fiber filter negative control. (F) flhA mutant demonstrating a loss in the ability to attach to the glass fiber filter, in comparison to the image in panel E. Bars, 10 µm.

 
Comparison of the two-dimensional proteome maps from C. jejuni planktonic and biofilm-grown cells demonstrated differences in protein expression levels in both the pH 4 to 7 (Fig. 2A versus C) and pH 6 to 11 (Fig. 2B versus D) ranges. Proteins that demonstrated a twofold or more increase in intensity in biofilm-grown cells were selected (indicated by arrows in Fig. 2) and identified. Proteins identified in the pH 4 to 7 gels (Fig. 2 and enlargement shown in Fig. 3) consisted predominantly of those involved in various motility functions. These included the flagellins (FlaA and FlaB), the filament-associated protein (FlaG), the putative hook-associated protein (FlgK), the filament cap (FliD), two proteins that may be associated with the basal body (FlgG2 and FlgG), and the chemotaxis protein CheA (Table 1). Two proteins having no known function (Cj1450 and Cj0391c), but under the control of the alternative sigma factor FliA ({sigma}28) (8), also demonstrated enhanced expression in biofilm-grown cells. Additional proteins having known functions with enhanced expression in the biofilm cells included the heat stress proteins GroEL and GroES; the serine peptidase ClpP; the adhesin FlaC; the elongation factor EF-G; two proteins involved in the oxidative stress response, alkyl-hydroperoxide reductase (Ahp) (4) and thiol-peroxidase (Tpx) (60); a putative bacterioferritin (Bfr) (49); a scaffolding protein involved in the synthesis of Fe-S proteins (NifU) (16); and finally PurL, involved in the de novo synthesis of purines. The remaining six identified proteins have no known function (Table 1).


Figure 2
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  		FIG. 2. Two-dimensional gel images of C. jejuni planktonic (A and B) and biofilm (C and D) cell lysates at two pH ranges. Proteins overexpressed in biofilm cells, indicated by arrows, were sequenced and identified by MS/MS (see Tables 1 and 2).

 

Figure 3
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  		FIG. 3. Expansion of a second set of two-dimensional gel images of C. jejuni planktonic (A) and biofilm (B) cell lysates showing additional flagellar proteins in the enlarged pH 4 to 6 range. Proteins overexpressed in biofilm cells, indicated by arrows, were sequenced and identified by MS/MS (see Table 1).

 

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  		TABLE 1. Identification of overexpressed proteins isolated from C. jejuni biofilm-grown cells, pH 4 to 7

 
Twelve proteins in the pH 6 to 11 range with enhanced expression in the biofilm-grown cells were identified (Fig. 2; Table 2). Those with known or previously characterized functions included the C. jejuni adhesin Peb1 (40) and the immunogenic protein Peb4 (41); three proteins having biosynthetic functions (nucleotide di-phosphate kinase, the ß chain of riboflavin synthase, and ribosome recycling factor) (24); a protein similar to the transthyretin-like periplasmic protein, which in bacteria may function in purine catabolism (32); and finally the {alpha} chain of the tricarboxylic acid cycle enzyme succinyl-coenzyme A (CoA) synthetase. The remaining proteins with enhanced expression had no known function, although several may be involved in yet-to-be-characterized signal transduction pathways (Table 2).


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  		TABLE 2. Identification of overexpressed proteins isolated from C. jejuni biofilm-grown cells, pH 6 to 11

 
Proteomic analysis indicated a role for the flagellar motility complex in biofilm formation. In order to investigate this more fully, we examined the biofilm-forming capability of the flhA knock-out mutant, which we previously demonstrated to regulate both virulence and motility in this strain (8). This mutant, which lacks flagella (Fig. 4A), was unable to attach to glass fiber surfaces and form a biofilm (Fig. 1F). Recent work has indicated that biofilm formation in a variety of bacteria correlates with the ability to form pellicles in liquid culture (5). Similarly, the flhA mutant was unable to form a pellicle (Fig. 5C) despite the finding that the growth rates of the wild type and the flhA mutant were the same (8).


Figure 4
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  		FIG. 4. Transmission electron microscopic analyses of C. jejuni flagellar mutants. (A) Aflagellate flhA mutant. (B and C) Stubby flagella produced by fliA and flaA mutants, respectively. (D and E) Normal-length flagella produced by flaB and flaC mutants, respectively. (F) Extremely long flagellar filaments produced by the flaG mutant. Bars, 1 µm.

 

Figure 5
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  		FIG. 5. Day-three pellicle formation in C. jejuni 11168. (A) Blank. (B) Wild type. (C) flhA mutant. (D) fliA mutant. (E) flaA mutant. (F) flaB mutant. (G) flaC mutant. (H) flaG mutant.

 
To further examine the importance of flagellar components in C. jejuni pellicle formation, specific insertion mutants were constructed in genes encoding several of the up-regulated proteins identified in the proteome comparisons (FlaA, FlaB, FlaC, and FlaG), as well as {sigma}28 (FliA), previously shown to be involved in flagellar biosynthesis (8). The effects of flaA, flaB, flaC, and flaG mutations on motility and flagellar filament production were determined and are summarized in Table 3 and Fig. 4. Both the fliA (Fig. 4B) and flaA (Fig. 4C) insertion mutants produced stubby flagellar filaments and were nonmotile. Inactivation of flaB (Fig. 4D) and flaC (Fig. 4E) resulted in flagellar filaments that were morphologically identical to those found in the wild type, whereas inactivation of flaG (Fig. 4F) resulted in very long flagellar filaments, as has been previously noted for Pseudomonas fluorescens (7). All three of the latter mutants retained motility similar to that of the wild type. Each mutant was compared against the wild type in terms of pellicle-forming ability. Following 3 days of incubation, all of the flagellar mutants and flaC were unable to form pellicles at the air-liquid interface of the cultures (Fig. 5). However, increased incubation (5 days) did eventually result in pellicle formation in the fliA, flaA, flaB, and flaC mutants but not in the flaG mutant (results not shown). Transmission electron microscopic examination of cells removed from the 3-day pellicle formed by the wild type retained both the spiral morphology and the flagellar filaments (not shown).


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  		TABLE 3. Summary of C. jejuni flagellar mutants examined in this study

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Biofilms are multicellular communities encased in a self-produced extracellular polymeric matrix that exhibit an altered phenotype with respect to growth and gene transcription (11, 14). Bacteria contained within a biofilm are more resistant to environmental stresses such as desiccation and antimicrobials and exhibit an increased resistance to host defenses (12, 35). While the majority of studies have concentrated on biofilm development on solid surfaces, other surfaces also support biofilm formation, including the air-liquid interface (5, 9, 15, 47, 53, 61) and the direct cell-to-cell interactions found in the floc associated with wastewater treatment processes (11). Paradoxically, campylobacters, which neither grow aerobically nor can be spread from human to human, remain one of the leading causes of diarrheal disease worldwide. It is possible that biofilm formation is important to campylobacter persistence in hostile environments and in colonization of the gastrointestinal tracts of humans and chickens.

Previous work with campylobacters has demonstrated that these organisms can attach to various solid matrices as single cells or can be found in association with the biofilms formed by other species of bacteria (58, 59). To date, there has been no microscopic evidence demonstrating that C. jejuni forms architecturally complex biofilms similar to those described for many other bacteria. However, in this study, repeated transfer of C. jejuni-inoculated surfaces into fresh medium did lead to the development of more-complex adherent cell layers. Furthermore, the cells within these adherent layers primarily retained the spiral morphology rather than the viable but nonculturable coccal form. Examination of cells within the adherent layers demonstrated the presence of extracellular material. This material likely represents EPS, which, through dehydration during processing for SEM, results in the collapse and condensation of EPS around the cells (14). Together, these findings indicate that these adherent cell layers represent a mature biofilm. Marked differences were found in the thicknesses of the biofilms depending on the type of surface utilized, with nitrocellulose and glass fiber filters providing better surfaces for thick biofilm formation than did stainless steel. This may be due to differences in hydrophobicity, texture, or surface area.

Proteomic comparison of biofilm-grown and planktonic cells demonstrated differences in protein expression between the two states of growth. Overall, there were similarities with other biofilm-grown bacteria (22, 23, 31) in terms of the types of proteins having enhanced levels of expression. These included proteins involved in energy generation (succinyl-CoA synthase), biosynthesis (PurL, NifU, EF-G, riboflavin synthase, ribosomal release factor), and catabolic functions (transthyretin-like protein). Enhanced expression of these proteins indicates that the biofilm-grown cells remain in an active growth state and correlates with the retention of the spiral cell morphology. However, many of the proteins identified with enhanced expression in the campylobacter proteome currently have no known function. Two groups of identified proteins were unusual (motility and stress) and are not generally features reported for other biofilm-grown bacteria.

The single largest group of proteins having enhanced expression in biofilms was related to the motility complex and included the flagellin structural proteins, hook and basal body proteins, filament cap protein, the chemotaxis protein CheA, and two unknown proteins regulated through the class III flagellar sigma factor FliA. While it is well known that flagella are important in the initial attachment phase of biofilm formation (28, 62), previous studies have indicated that the motility operons are generally repressed following the initiation of biofilm formation. In fact, it has recently been suggested that repression of motility genes may be a common feature of all biofilms (31). The enhanced expression of the flagellins, as well as other motility proteins, in mature biofilms of C. jejuni 11168 is unusual.

A second group of proteins having enhanced expression in the biofilms are involved in the oxidative (60) and general (21) stress responses. The oxidative stress response allows aerobic survival by the removal of damaging reactive oxygen species, such as superoxide or various peroxides generated during aerobic metabolism (60). In contrast, the general stress response is induced by many environmental stresses (21), and while cells within a biofilm have been shown to undergo a stress response following antibiotic treatment (64) or other stresses (19), up-regulation under what would be considered unstressed growth conditions has previously been observed only in the proteomic comparison of L. monocytogenes biofilm-grown cells (22, 23). Two additional campylobacter stress-related proteins with enhanced expression included EF-G, which also functions as a chaperone in E. coli following stress (6), and the ClpP homolog (Cj0192c). ClpP is an ATP-dependent serine protease associated with either of two ATPases (ClpA or ClpX) to form the Clp protein complex (42), which is involved in the regulation of various cell processes ranging from flagellin synthesis (57) to the induction of type III secretion systems (25, 26) and degradation of denatured or incomplete proteins (42, 65). ClpP proteins are normally present at low levels but are induced under stress conditions in other species of bacteria (20, 29, 33). Inactivation of ClpP has also been shown to impact biofilm formation in Pseudomonas aeruginosa (38), Staphylococcus aureus (17), and Staphylococcus mutans (33).

While there are several reasons for the enhanced survivability of cells within a biofilm to environmental stresses, including lower growth rates and limited diffusion through the matrix (14, 19), a stress response under optimal growth conditions in biofilm-grown cells does not seem to be a common feature in the proteomes of other bacteria examined to date (with the exception of L. monocytogenes). This finding raises interesting questions regarding stress and how this might impact the survival capability of campylobacter. It is also of interest to note that both C. jejuni and L. monocytogenes are invasive; perhaps this unusual response is common in the biofilms of other invasive bacteria.

The requirement for flagella in the initial stages of biofilm formation was confirmed through the examination of an aflagellate flhA mutant (8), which was found to be unable to attach to glass fibers and form a biofilm. FlhA influences both virulence and {sigma}28/{sigma}54-regulated genes, including the class II and III flagellar genes (8). This finding is consistent with other studies of various bacterial species on the role of flagella in the initial cell attachment phase of biofilm formation (38, 62). An additional motility gene that impacts biofilm formation in Aeromonas hydrophila (28), and for which we observed enhanced protein expression in C. jejuni biofilm-grown cells, is CheA. Inactivation of flhA also resulted in a loss in the ability to form pellicle at the air-liquid interface of cultures. Although most biofilm research has focused on cell interactions on solid surfaces immersed in liquid, it has become clear that the air-liquid interface also represents a suitable site for cell-to-cell interactions leading to the formation of pellicles or interfacial biofilms, and many bacteria share this property (5, 9, 46, 47). Furthermore, mutant analysis has indicated that this phenotype is related to biofilm formation on solid surfaces in different species of bacteria, including Vibrio parahaemolyticus (15), Salmonella enterica serovar Typhimurium (50), and P. aeruginosa (18, 61). An examination of wild-type campylobacter cells taken from the pellicle demonstrated retention of the normal spiral morphology, and like the solid-surface biofilm cells, they appeared to remain in a viable state. Thus, we believe that the pellicle assay represents a rapid screening method for the selection of campylobacter mutants with altered biofilm-forming capabilities.

Insertional inactivation of genes that impact the composition or morphology of the flagellar filament was found to produce either a delay (flaA, flaB, fliA, flhA) or loss (flaG) in pellicle-forming ability. Several of these mutations produced altered flagellar morphologies, ranging from aflagellate (flhA) to stubby (flaA, fliA) to cells expressing extremely long filaments (flaG). The delay in pellicle formation in these mutants underlines the important role of the intact flagellar filament in the cell-to-cell interactions required for pellicle formation. The flaG mutant was unusual in that it lacked the ability to form a pellicle. However, it may be that the very long flagellar filaments hinder the intercellular associations that would lead to the development of these interfacial biofilms. Alternatively, the protein composition or glycan arrangement of the filament may have changed in this mutant. We are currently examining the effect of flaG mutation in C. jejuni further. Biofilm-grown cells also exhibited enhanced expression of two proteins previously shown to play a role in adherence to various cultured cell lines, Peb1 (40) and FlaC (51). Peb1 is a putative aspartate/glutamate binding component of an ABC transporter, and inactivation of the gene significantly reduces both C. jejuni invasiveness and attachment capability (40; M. Leon-Kempis and D. Kelly, Abstr. 13th Int. Worksh. Campylobacter, Helicobacter Rel. Organisms, abstr. B28, p. 42, 2005). Similarly, FlaC binds to the surface of HEp-2 cells with the corresponding null mutant demonstrating reduced invasiveness (51). The finding that both proteins have enhanced expression in biofilm-grown cells suggests an involvement for these proteins in the phenotype, a suggestion supported by our finding that inactivation of flaC delayed pellicle formation despite the presence of wild-type flagellar morphology and motility in this mutant.

The continued enhanced expression of motility-related proteins in mature biofilms formed on glass fiber filters remains the most unusual aspect of this study. There are two possible explanations for this observation. First, it may be that flagellar filaments are important in the formation of the three-dimensional matrix, either through aiding cell-to-cell contact or perhaps as a component of the extracellular matrix. These possibilities are supported by previous work on the autoagglutination of campylobacters, where it was also found that an aflagellate mutant no longer autoagglutinated (36), and by the fact that additional surface appendages such as fimbriae (2) or other surface-associated proteins are important in the formation of mature biofilms in other bacteria (30). A second possibility that may explain the enhanced expression of motility proteins is the requirement for the flagellar export pathway for secretion of FlaC (51), which was also expressed at an enhanced level in the biofilm-grown cells and appears to be involved in early pellicle formation.

In conclusion, we have demonstrated that C. jejuni 11168 can attach to various surfaces and form a biofilm. The proteome of biofilm-grown cells was different from planktonic cells grown at 37°C, demonstrating enhanced expression of proteins involved in the flagellar motility complex, stress response, and adhesion. Not only are flagella required for both biofilm and pellicle formation in C. jejuni, but the continued enhanced expression of the motility apparatus in mature biofilms indicates a role for flagella in the initial maintenance of the phenotype. Future studies should be aimed at understanding the relevance of biofilms in the C. jejuni life cycle by comparing other strains, temperatures, surfaces, and atmospheric conditions. Detailed analyses of the flagellar biosynthetic pathway, stress response, adhesion mechanisms, and extracellular matrix are warranted in order that a better understanding of this novel C. jejuni phenotype may be gained.


   ACKNOWLEDGMENTS
 
We thank Luc Tessier for mass spectrometry, Brendan Wren for kindly providing the flaA and flaB mutants, Elham Kheradpir for technical support, and Tom Devecseri for assistance with figures.

Funding to J.K. and C.M.S. was provided through the NRC Genomics and Health Initiative.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada. Phone: (613) 990-1569. Fax: (613) 952-9092. E-mail: christine.szymanski{at}nrc-cnrc.gc.ca. Back

{dagger} Present address: Agriculture and Agri-Food Canada, Kentville, Nova Scotia B4N 1J5, Canada. Back

{ddagger} Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, June 2006, p. 4312-4320, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.01975-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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