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Role of flgA for Flagellar Biosynthesis and Biofilm Formation of Campylobacter jejuni NCTC11168
Role of flgA for Flagellar Biosynthesis and Biofilm Formation of Campylobacter jejuni NCTC11168
Journal of Microbiology and Biotechnology. 2015. Nov, 25(11): 1871-1879
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : April 29, 2015
  • Accepted : July 22, 2015
  • Published : November 28, 2015
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About the Authors
Joo-Sung Kim
Department of Food Biotechnology, Korea University of Science and Technology, Daejeon 305-333, Republic of Korea
jskim@kfri.re.kr
Changwon Park
Korea Food Research Institute, Seongnam 463-746, Republic of Korea
Yun-Ji Kim
Department of Food Biotechnology, Korea University of Science and Technology, Daejeon 305-333, Republic of Korea

Abstract
The complex roles of flagella in the pathogenesis of Campylobacter jejuni , a major cause of worldwide foodborne diarrheal disease, are important. Compared with the wild-type, an insertional mutation of the flgA gene (cj0769c) demonstrated significant decrease in the biofilm formation of C. jejuni NCTC11168 on major food contact surfaces, such as polystyrene, stainless steel, and borosilicate glass. The flgA mutant was completely devoid of flagella and non-motile whereas the wild-type displayed the full-length flagella and motility. In addition, the biofilm formation of the wild-type was inversely dependent on the viscosity of the media. These results support that flagellar-mediated motility plays a significant role in the biofilm formation of C. jejuni NCTC11168. Moreover, our adhesion assay suggests that it plays an important role during biofilm maturation after initial attachment. Furthermore, C. jejuni NCTC11168 wild-type formed biofilm with a net-like structure of extracellular fiber-like material, but such a structure was significantly reduced in the biofilm of the flgA mutant. It supports that the extracellular fiber-like material may play a significant role in the biofilm formation of C. jejuni . This study demonstrated that flgA is essential for flagellar biosynthesis and motility, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.
Keywords
Introduction
Campylobacter jejuni is one of the most common foodborne pathogenic bacteria worldwide. Consumption of poultry, especially chickens, is considered the most common route for human infections of C. jejuni [11 , 34] .
C. jejuni is microaerophilic and susceptible to relatively high oxygen concentrations under normal atmosphere. However, it often confronts and can survive the aerobic conditions that are common in food-associated environments to infect humans. It strongly suggests that C. jejuni should have developed several strategies to overcome the obstacles of those conditions [2 , 20] .
Biofilm formation is considered as one of the important survival strategies bacterial pathogens employ to survive harsh environmental conditions such as acids, metal toxicity, sanitizers, antibiotics, and antimicrobial agents [10 , 16 , 18 , 20 , 29] . It is a biologically active matrix in which bacterial cells are embedded with a self-produced polymeric exopolysaccharide substance [5] . Antimicrobials such as sanitizers may not easily penetrate the three-dimensional matrix and reach the microbial cells embedded in the matrix [16] . Thus, the biofilms formed by foodborne pathogens are more likely to survive the harsh environmental conditions compared with planktonic cells. They can impose a significant threat on public health because microbial cells encased in biofilms can contaminate food products such as in food processing facilities.
C. jejuni has the flagellar apparatus enabling the bacterium to move freely, which is important for its survival in the environment and the colonization of animal host. The flagella have the functions of not only motility but also the secretion of proteins involved in pathogenesis [1 , 15 , 27] . C. jejuni can also form biofilms on various abiotic surfaces such as stainless steel, glass, and plastic [12] . The biofilm is considered to be a potential strategy that C. jejuni , microaerophilic and susceptible to relatively high oxygen concentrations under normal atmosphere, may use to survive hostile environmental conditions such as aerobic conditions [20] . However, further research is still required for clarifying the biofilm formation of C. jejuni . In particular, the molecular mechanism underlying the biofilm formation by C. jejuni is little known and it warrants more detailed studies [2] . In our study, an flgA insertional mutant was generated by a random transposon mutagenesis of the C. jejuni NCTC11168 strain and characterized for the role of flgA in biofilm formation on abiotic surfaces.
Materials and Methods
- Strains, Growth Conditions, and Preparation of Cell Suspension for Biofilm Formation
C. jejuni NCTC11168 purchased from American Type Culture Collection (Manassas, VA, USA) and its derivative mutants were used in this study. They were routinely grown on tryptic soy agar supplemented with 5% (v/v) sheep blood (TSAB) at 37℃ under microaerobic conditions (6-12% O 2 , 5-8% CO 2 ) generated by AnaeroPack-MicroAero (Mitsubishi Gas Chemical Co., Tokyo, Japan). To prepare cells for biofilm formation, C. jejuni strains were streaked on TSAB from -70℃ glycerol stock and grown on TSAB at 37℃ for 44-48 h under microaerobic conditions. Then, the cells were suspended in Mueller-Hinton Broth (MHB) at OD 600 of 0.1-0.2 using sterile cotton swabs, spread at 100 μl on TSAB, and grown at 37℃ for 14-15 h under microaerobic conditions. Finally, the grown cells were suspended in MHB at OD 600 of 0.01 using cell scrapers and vortexing, and used in biofilm formation.
- Preparation of Competent Cells
The previous protocol was followed with modification to prepare competent C. jejuni cells [31] . Briefly, C. jejuni NCTC11168 was grown on TSAB at 37℃ for 40-48 h under microaerobic conditions. The grown cells were suspended in MHB at OD 600 of 0.3-0.4 using sterile cotton swabs. The cell suspension was spread at 100 μl on TSAB and the plates were incubated at 37℃ for about 24 h under microaerobic conditions. The cells were harvested in 2 ml of MHB per plate, pelleted at 9,000 × g for 5 min, and resuspended in 1.2 ml of ice-cold wash buffer (7% sucrose, 15% glycerol). After additional 3 cycles of pelleting at 10,000 × g for 5 min and resuspending, the cells were finally pelleted and then resuspended in 0.25 ml of ice-cold wash buffer. The competent cells prepared were aliquoted at 50 μl in microcentrifuge tubes and stored at -70℃ until use.
- Random Transposon Mutagenesis
The C. jejuni NCTC11168 genome was randomly mutated by electroporation using a non-autonomous transposon containing the Tn903 kanamycin resistance gene and forming a stable complex with the EZ-Tn5 transposase provided in the EZ-Tn5 Tnp Transposome Kit (Epicentre, Madison, WI, USA). A previous protocol was followed with modification for electroporation [31] . The competent cells from -70℃ were slowly thawed on ice. Transposon (20 ng) was added to the competent cells and mixed. Then, the transposon was electroporated into the competent cells in a pre-chilled 0.1 cm electroporation cuvette (Bio-Rad, Hercules, CA, USA) at 2,500 kV and more than 4 ms for time constant. SOC medium was immediately added at 100 μl to the cells, mixed, and spread on TSAB. After 5-6 h incubation at 37℃ under microaerobic conditions, the cells were harvested in 1.5 ml of MHB with cell scrapers, centrifuged at 10,000 × g for 2 min, and the supernatant was removed. The pelleted cells were resuspended in 100 μl of MHB, spread on TSAB supplemented with kanamycin (50 μg/ml), and incubated at 37℃ under microaerobic conditions for 2-4 days. Isolated colonies grown on the plates were individually subcultured onto another kanamycin plates, grown for 1 day, and made into glycerol stocks at -70℃.
- Identification of Transposon-Insertional Site by Direct DNA Sequencing
A previous protocol was followed to identify the transposon-insertional site in C. jejuni genomic DNA [17] . Briefly, the genomic DNA was extracted with an Easy-DNA Kit (Invitrogen, Carlsbad, CA, USA) using the manufacturer’s protocol. For the sequencing reaction, a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used. The sequencing reactions were composed of 16 μl of terminator ready reaction mix, 3 μg of genomic DNA, and 15 pmol of forward primer (KAN-2-FP-1; 5’-ACCTACAACAAAGCTCTCATCAACC-3’) in a 40 μl volume. The sequencing reactions were carried out in 1 cycle of 4 min at 95℃, then 60 cycles of 30 sec at 95℃, 30 sec at 50℃, and 4 min at 60℃. Subsequently, they were purified by using magnetic beads and DNA sequences were obtained using an automated DNA sequencer, ABI 3730xl DNA analyzer (Applied Biosystems).
- RT-PCR Assay
Reverse transcriptase PCR was conducted using a Qiagen Onestep RT-PCR Kit (Qiagen), following the manufacturer’s protocol. The primers used were flgA-F (5’-TATCCAAGCAGCTAGTTTAG-3’) and flgA-R (5’-CCCACTACGATACCTTGC-3’). The reverse transcription was performed at 50℃ for 30 min, followed by the initial PCR activation at 95℃ for 15 min. Then, the PCR amplification was performed at 35 cycles of 94℃ for 45 sec, 52℃ for 45 sec, and 72℃ for 2 min, followed by 1 cycle of 72℃ for 10 min.
- Biofilm Formation on Polystyrene Microtiter Plate
The cell suspension prepared as aforementioned was aliquoted at 100 μl in 96-well polystyrene microtiter plates. After 72 h incubation at 37℃ under microaerobic conditions, the suspension was removed from the plates after four times of pipetting up and down. The inoculated wells were washed two times in 150 μl of sterile deionized water by four times of pipetting up and down each time and completely dried in a 37℃ incubator. Crystal violet solution (1.0%) (Sigma-Aldrich, St. Louis, MO, USA) was added at 100 μl to the wells, incubated at room temperature for 30 min, and then removed. The plates were thoroughly washed in flowing tap water and finally rinsed in deionized water. The plates were dried completely and biofilm-staining crystal violet was dissolved in a 100 μl solution of 30% methanol and 10% acetic acid. The intensity of crystal violet solution was measured at 590 nm with a microplate reader, Infinite M200 Pro NanoQuant (Tecan, Switzerland).
- Biofilm Formation on Stainless Steel Surface
The cell suspension prepared as aforementioned was added at 5 ml to each well of a 12-well tissue culture plate (Corning Inc., Acton, MA, USA) in which autoclaved stainless steel coupons (type 304), 2.0 cm × 1.5 cm × 0.2 cm, had been placed. After 72 h incubation at 37℃ under microaerobic conditions, the coupons were gently washed in 20 ml of sterile deionized water in petri dishes for 10 to 20 sec, and vortexed in 5 ml of sterile 0.85% NaCl solution with ~10 sterile glass beads (4 mm diameter) at high speed for 1 min. Then, the suspension was serially 10-fold diluted in sterile 0.85% NaCl solution up to 10 -6 . Each dilution was spread at 100 μl on TSAB and the spread plates were incubated at 37℃ under microaerobic conditions. After about 48 h incubation, the colonies were enumerated to estimate the number of viable cells attached to the stainless steel surface.
- Biofilm Formation on Borosilicate Glass Surface
The cell suspension prepared as aforementioned was added at 1 ml to sterile borosilicate glass tubes (10 cm × 1.3 cm) and incubated at 37℃ under microaerobic conditions for 72 h. After incubation, the suspension was removed after four times of pipetting up and down. The tubes were washed two times in 1.5 ml of sterile deionized water by four times of pipetting up and down each time. About 10 sterile glass beads (4 mm diameter) and 1 ml of sterile 0.85% NaCl solution were added per tube and the tubes were vortexed at high speed for 1 min. Then, the viable counts were done as in the stainless steel assay.
- Adhesion Assay
The cell suspension prepared as aforementioned was added at 5 ml to each well of 12-well plates in which a sterile borosilicate glass coverslip (18 mm × 18 mm) had been placed in the vertical position. After 20 min incubation at room temperature [21] , the coverslip was briefly rinsed twice in 5 ml of MHB and vortexed in 5 ml of sterile 0.85% NaCl solution with about 10 sterile glass beads. Then, the viable counts were done as in the stainless steel assay. Adhesion frequency (%) was calculated as the number of attached cells divided by the number of total cells studied.
- Scanning Electron Microscopy (SEM) Analysis
The samples were fixed in 2.5% paraformaldehyde-glutaraldehyde buffered w ith 0.1M p hosphate (pH 7 .2) for 2 h, w ashed three times in 0.1 M phosphate buffer for 10 min each time, and postfixed in 1% osmium tetroxide in the same buffer for 2 h. Then, the samples were washed several times in the same buffer and dehydrated in ethanol at gradually increased concentrations. After the ethanol was replaced by isoamyl acetate, the samples were dried using a critical point dryer, sputtered with gold in a sputter coater, and observed using the scanning electron microscope FEI Quanta 250 FEG (FEI, USA) at 10 kV.
- Transmission Electron Microscopy (TEM) Analysis
The cells were fixed, washed, and postfixed as in the SEM preparation. Then, the fixed samples were washed several times in the same buffer, dehydrated in ethanol and propylene oxide, embedded in Epon-812, and then cured for 36 h in a 60℃ oven. Ultra-thin sections were made by an ULTRACUT E (Leica, Austria) ultramicrotome, and stained with uranyl acetate and lead citrate. The prepared samples were observed under a Philips CM 20 transmission electron microscope (Philips, Netherlands) at 100 kV.
- Motility Assay
The motility assay was conducted as previously described [14] . Briefly, the cell suspension was prepared at OD 600 of 0.1 as described above. Pipette tips were dipped in the cell suspension and stabbed into the Mueller-Hinton soft agar (0.4%). Plates wereincubated at 37℃ under microaerobic conditions and the diameter of motility halo was measured at 48 h.
- Statistical Analysis
A t test was used to compare any statistically significant difference between the 11168 wild-type and the flgA mutant ( p < 0.05) .
Results
C. jejuni strain NCTC11168 (hereinafter wild-type) was genetically mutated by random transposon mutagenesis. Each electroporation usually produced 1 to 10 colonies selected by kanamycin resistance, whereas the negative control without transposon produced no colonies under the same conditions (data not shown). About 40 colonies were screened for biofilm formation on polystyrene 96-well microtiter plates. Among them, a transposon mutant, A49(OD 590 of 0.03), displayed a significant (10-fold or more) reduction in biofilm formation compared with that of wild-type (OD 590 of 0.41) after 72 h incubation ( p < 0.05) ( Fig. 1 ). To identify the gene into which the transposon was inserted, a sequencing primer, KAN-2-FP-1, which binds to the end of the transposon, was used to direct sequencing outward. A BLAST search ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) using the identified DNA sequences against the C. jejuni 11168 genome confirmed the flgA gene (cj0769c) flanking the insertional site by the transposon (data not shown). PCR amplification of the flgA gene in the transposon mutant A49 produced a PCR product of about 1.8 kbp, which had increased by about 1.2 kbp corresponding to the size of transposon, compared with the about 650 bp products of wild-type and A16, another transposon mutant in which the transposon was inserted into cj1111c ( Fig. 2 ). It confirms that the insertional mutation indeed occurred in the flgA gene. DNA sequencing analysis on the PCR product identified that the insertional mutation occurred at nucleotide position 526 of the flgA open reading frame (663 bp) (data not shown). The expression of gene flgA was studied in the three strains by RT-PCR analysis ( Fig. 3 ). Whereas the expression of gene flgA was clearly observed in the wild-type and A16 strains, it was almost not detected in the flgA mutant.
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Biofilm formation of C. jejuni NCTC11168 wild-type (WT), flgA mutant (flgA), and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)) in Mueller-Hinton broth on a polystyrene surface in 96-well microtiter plates after 72 or 96 h incubation at 37℃ under microaerobic conditions.

The mutant had significantly reduced biofilm formation at 72 h (* < 0.05) and 96 h (* < 0.05) compared with the NCTC11168 wild-type, respectively. As a control, A16 (WT/tnp) had a similar level of biofilm formation to the wild-type. The measurements are presented after deduction of the MHB negative control for nonspecific binding. The data are presented based on two independent experiments done in triplicate.

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PCR amplification of flgA in C. jejuni NCTC11168 wild-type, flgA mutant, and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)).

The PCR band of mutant (lane 3) was up-shifted by about 1.2 kbp compared with the ones of NCTC11168 wild-type (lane 2) and A16 (lane 4). Lane 1: DNA marker; Lane 2: 11168 wild-type; Lane 3: mutant; Lane 4: A16; Lane 5: negative control, deionized water.

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RT-PCR analysis of the expression of flgA in C. jejuni NCTC11168 wild-type, flgA mutant, and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)).

Lane 1: DNA marker; Lane 2: NCTC11168 wild-type; Lane 3: mutant; Lane 4: A16; Lane 5: negative control, deionized water.

The additional dosage of the genetic elements ( i.e. , transposon) may have negative effects on growth. Therefore, we studied if the decreased biofilm formation by the flgA mutant is simply due to any growth defect. To end this possibility, growth rates for the initial 5 h under similar conditions for biofilm formation were compared among the wild-type, flgA mutant, and A16 strains. The generation time of the flgA mutant was 2.9 ± 0.4 h, while the generation times of wild-type and A16 strains were 2.1 ± 0.8 h and 2.4 ± 0.6 h, respectively. Thus, the flgA mutant showed a slightly reduced growth rate with about 1.3-fold increased generation time compared with that of wild-type. To compensate for the slight growth defect, the biofilm formation of the flgA mutant was studied after an extended incubation time of 96 h instead of 72 h ( Fig. 1 ). The biofilm mass did not change after 96 h compared with that of the 72 h culture in the flgA mutant. As another control, one of the 40 screened colonies, A16 (in which the transposon was inserted into cj1111c, annotated as putative MarC family integral membrane protein), was examined for whether the simple insertion of transposon in the non-flagellar gene can affect the biofilm formation ( Fig. 1 ). On the polystyrene surface, no defect in biofilm formation was found in the A16 strain. These results suggest that neither the slight growth defect nor the simple insertion of transposon in the genome was the reason for the reduced biofilm formation.
Therefore, further studies were conducted to investigate if such a defect in the biofilm formation of the flgA mutant is a common phenomenon irrespective of the types of surfaces, stainless steel and glass ( Fig. 4 A; Fig. 4 B). Compared with the wild-type and A16 strains (7.5-8.0 log CFU/coupon), the flgA mutant formed about 10 times less biofilm on the stainless steel coupon (6.4 log CFU/coupon, p < 0.05) ( Fig. 4 A). The biofilm formation of the flgA mutant strain was also observed on the borosilicate glass surface ( Fig. 4 B). Most biofilms of the wild-type, A16, and the flgA mutant strains formed at the air-liquid interface on the glass surface (data not shown). Whereas both wild-type and A16 strains formed biofilm on borosilicate glass at 6.7 log CFU, respectively, the flgA mutant formed a significantly reduced biofilm at 3.9 log CFU ( p < 0.05) ( Fig. 4 B). Collectively, these results suggest that the full function of flgA is required for the biofilm formation of C. jejuni NCTC11168 on commonly used food contact surfaces. An adhesion assay was conducted on the surface of borosilicate glass to understand the role of flgA in the attachment of food contact surfaces as an initial step in biofilm formation ( Fig. 5 ). The attachment was only slightly reduced (2-fold) in the flgA mutant and A16 strains compared with the wild-type. In contrast to the greatly reduced biofilm formation in the flgA mutant, the similar level of attachment suggests that the full function of flgA is required during maturation of the biofilm after initial attachment on food contact surfaces.
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Biofilm formation of C. jejuni NCTC11168 wild-type (WT), flgA mutant (flgA), and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)) in Mueller-Hinton broth on the surfaces of (A) stainless steel and (B) borosilicate glass after 72 h incubation at 37℃ under microaerobic conditions.

The biofilm formation of mutant was significantly reduced by 1.6-log CFU (* < 0.05) on stainless steel coupon (), and by 2.8-log CFU (* < 0.05) on borosilicate glass tube (), compared with wild-type. The biofilm formation levels of A16 (WT/tnp) were similar to those of wild-type. The data are presented based on three independent experiments done in duplicate.

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Adhesion frequency (%) of C. jejuni NCTC11168 wild-type (WT), flgA mutant (flgA), and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)) on the borosilicate glass surface.

The data presented are based on two independent experiments done in duplicate.

A previous study found extracellular fiber-like material in the biofilm of C. jejuni NCTC11168 on surfaces such as stainless steel [13] . Thus, the scanning electron micrograph was analyzed to study the morphology of biofilm formed at the air-liquid interface on the glass surface ( Fig. 6 ). Consistent with the previous study [9] , both spiral and coccoid cells were found in the biofilm ( Fig. 6 ). Extracellular fiber-like material and its network formation were apparent in the biofilm formed by wild-type on the glass surface, as a previous study demonstrated [13] ( Fig. 6 ). However, such fiber-like material was much reduced in the flgA mutant and no such network formation was found ( Fig. 6 ). Wild-type with transposon inserted in a non-flagellar gene, A16, also yielded extracellular fiber-like material with somewhat reduced level compared with wild-type ( Fig. 6 ).
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SEM images of C. jejuni NCTC11168 wild-type (WT), flgA mutant (flgA), and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)).

The images are the biofilms formed at the air-liquid interface on the borosilicate glass surface. The scale bar represents 5 μm.

Although cj0769c was annotated as flgA , the role of cj0769c in flagellar biosynthesis or function had not been experimentally proven. Therefore, to find out whether flgA actually plays a role in flagellar formation, the TEM image of the flgA mutant was analyzed and compared with those of the wild-type and A16 strains in order to investigate the flagellar morphology ( Fig. 7 ). Whereas the wild-type and A16 strains had the full-length flagella, the flgA mutant was completely devoid of flagella, suggesting that flgA , cj0769c, is involved in flagellar biosynthesis. Because flagella are involved in bacterial motility, the motility was compared on soft agar, MHB supplemented with 0.4% agar ( Fig. 8 ). As expected, the flgA mutant had a significantly reduced motility halo at 0.6 ± 0.1 cm in diameter compared with 4.2 ± 0.4 cm and 3.5 ± 0.5 cm in diameter for the wild-type and A16 strains ( p < 0.01), respectively, based on four independent experiments done in triplicate.
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TEM images of C. jejuni NCTC11168 wild-type (WT), flgA mutant (flgA), and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)).

The mutant lacks flagella on both poles of the cell, whereas wild-type and A16 (WT/tnp) have the full-length flagella. The arrows indicate the flagella. The scale bar represents 500 nm.

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Motility haloes of C. jejuni NCTC11168 wild-type (WT), flgA mutant (flgA), and A16 (the wild-type with transposon inserted in a non-flagellar gene, cj1111c (WT/tnp)) on soft agar, MHB supplemented with 0.4% agar, after inoculation and subsequent incubation for 48 h at 37℃ under microaerobic conditions.
The reduced motility in the flgA mutant suggests that motility may be a major contributor to the biofilm formation of C. jejuni NCTC11168 [12 , 13] . Therefore, glycerol was added to modulate the viscosity of the medium and the biofilm formation was measured at different concentrations of glycerol based on the assumption that the viscosity of the medium will affect the motility of C. jejuni ( Fig. 9 ). As the concentration of glycerol gradually increased from 0.0% to 3.0% in the media, the biofilm formation gradually decreased from OD 590 0.27 to OD 590 0.02 ( Fig. 9 ).
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Effect of viscosity generated by glycerol on the biofilm formation of C. jejuni NCTC11168 wild-type in Mueller-Hinton broth on polystyrene surface of 96-well microtiter plates.

When the glycerol concentration gradually increased from 0.0 to 3.0%, the biofilm formation gradually decreased from 0.27 to 0.02 at OD. The data presented are based on two independent experiments done in triplicate.

In summary, this study demonstrates that flgA is essential for flagellar biosynthesis of C. jejuni . In addition, the data support that it affects the biofilm formation by flagellar-mediated motility and extracellular fiber-like material.
Discussion
Our study clearly shows that an insertional mutation of cj0769c ( flgA ) caused the complete absence of flagella ( Fig. 7 ). This strongly suggests that flgA is essential for flagellar biosynthesis in C. jejuni NCTC11168. It has been reported that the flgA gene is involved in the synthesis of P-ring, a substructure of flagella basal body embedded in peptidoglycan layers in Salmonella [22] . Therefore, C. jejuni flgA is also likely to play its role in P-ring synthesis, which belongs to the earlier stage of flagellar biosynthesis.
In C. jejuni , the expression of flagella basal body genes, flgB and flgD , appears to be dependent on the σ 54 promoter [4 , 33] . In contrast to flgB and flgD , flgA expression is unlikely to be dependent on the σ 54 promoter because we could not identify the proximate upstream region presenting any homology to the putative σ 54 promoter sequence(TTGGAAC…TTGCTT) [4] . It has been known that flagellar biosynthesis in C. jejuni is dependent on the σ 28 , σ 54 , or σ 70 promoter [4 , 33] . Therefore, it will be interesting to confirm which promoter regulates flgA expression in C. jejuni .
Biofilm formation could be one of the strategies that C. jejuni can employ to survive in hostile environments [2] . The motility of C. jejuni may contribute to the biofilm formation; motile cells are able to overcome any repulsive environments and/or to find suitable places [24] to attach onto the food contact abiotic surfaces, including stainless steel, plastic, and glass [23 , 32] , and thereby form a micro-colony, which is considered as an important step [7 , 32] . Moreover, there have been some lines of evidence that motility can affect the biofilm formation of C. jejuni [6 , 12 , 13 , 19 , 25 , 26 , 28] and other bacteria, such as E. coli and Pseudomonas aeruginosa [23 , 24] . Proteins involved in motility exhibited increased expression in the biofilm compared with planktonic cells of C. jejuni 11168 [13] ; flaA flaB double mutants had decreased biofilm formation compared with those of wild-type of C. jejuni M129 on polystyrene surfaces [25] and of C. jejuni NCTC11168 on borosilicate glass surfaces [26] , respectively; and aflagellate mutant maf5 had a defect in pellicle formation [12] .
In this study, we successfully demonstrated that the biofilm formation was significantly reduced by the absence of flagella and/or flagellar-mediated motility, caused by flgA mutation ( Figs. 1 and 4 ). Moreover, the biofilm formation of wild-type cells was also decreased by the increased concentration of glycerol condition ( Fig. 9 ), a viscous environment that can decrease bacterial swimming motility or speed [3 , 30] . Taken together, our results suggest that flagellar-mediated motility seems to be required for biofilm formation in C. jejuni under conditions used in this study. Of interest, our adhesion assay suggests that it is required during the maturation of biofilm in C. jejuni after initial attachment. Such an important role of the flagellar-mediated motility during biofilm maturation is further supported by a previous study demonstrating the enhanced expression among motility-involved proteins of C. jejuni in the biofilm [13] .
The extracellular fiber-like material in the biofilm was previously observed in C. jejuni NCTC11168 [13] . We also confirmed the presence of such extracellular fiber-like material forming a net-like structure in the biofilm of 11168 wild-type ( Fig. 6 ). Interestingly, the flgA mutant devoid of flagella deposited less extracellular fiber-like material into the biofilm compared with wild-type ( Fig. 6 ). This suggests that such fiber-like materials possibly provide integrity to the biofilm matrix by holding the bacterial cells embedded in the matrix tightly. It has been suggested that such fiber-like materials or their components may be secreted by flagella [8] . Therefore, our results suggest that the flagella may play a role in the formation of the extracellular fiber-like material, thereby contributing to the biofilm formation in C. jejuni .
Despite our results, it is premature to generalize that the flagellar-mediated motility and fiber-like material play major roles in the biofilm formation of C. jejuni because different mechanisms may exist for different C. jejuni strains, considering the high genetic diversity among C. jejuni strains. Thus, more studies are necessary to understand the biofilm formation of other C. jejuni strains from food and environmental samples.
Acknowledgements
This research was funded by the Korea Food Research Institute. The assistance in DNA sequencing by Cosmogenetech co. is greatly appreciated and we also thank Dr. Hyun Woo Oh at KRIBB for the assistance in the analysis of TEM and SEM images.
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