The role of cydB gene in the biofilm formation by Campylobacter jejuni

Abstract

Campylobacter jejuni is a major cause of food- and water-borne bacterial infections in humans. A key factor helping bacteria to survive adverse environmental conditions is biofilm formation ability. Nonetheless, the molecular basis underlying biofilm formation by C. jejuni remains poorly understood. Around thirty genes involved in the regulation and dynamics of C. jejuni biofilm formation have been described so far. We applied random transposon mutagenesis to identify new biofilm-associated genes in C. jejuni strain 81-176. Of 1350 mutants, twenty-four had a decreased ability to produce biofilm compared to the wild-type strain. Some mutants contained insertions in genes previously reported to affect the biofilm formation process. The majority of identified genes encoded hypothetical proteins. In the library of EZ-Tn5 insertion mutants, we found the cydB gene associated with respiration that was not previously linked with biofilm formation in Campylobacter. To study the involvement of the cydB gene in biofilm formation, we constructed a non-marked deletion cydB mutant together with a complemented mutant. We found that the cydB deletion-mutant formed a weaker biofilm of loosely organized structure and lower volume than the parent strain. In the present study, we demonstrated the role of the cydB gene in biofilm formation by C. jejuni.

Keywords: C. jejuni; cydB; Biofilm; Transposon mutagenesis.

Fig. 1

Biofilm formation of C. jejuni 81–176 Tn5 mutants based on CV staining of cells after 72 h of growth. The data show mean absorbance relative to the wild type strain. Error bars show the standard deviations from at least three independent experiments. Statistically significant results were considered when p-value < 0.05. Black bars show the cydB gene mutant and wild type strain.

Fig. 2

A. Biofilm formation of C. jejuni strains. Bacterial suspensions (OD₆₀₀ = 0.2) of 81–176 WT, ΔcydB, and comp-cydB strains were incubated statically for 72 h at 42 °C, and stained with CV. Then OD₅₇₀ was measured to quantify biofilm formation. An asterisk represents statistical significance (P < 0.05). B Biofilms on plate after 72-h incubation based on CV staining.

Fig. 3

Biofilm microstructure observed by scanning electron microscopy at 72 h. SEM results show that the ∆cydB mutant strains had a lower biofilm volume and irregular structure (magnification × 2500 and × 10,000).

Fig. 4

Representative CLSM image of live (SYTO 9), dead (PI) and merged bacterial cells in C. jejuni biofilms (A). Percentage of live (green) and dead cells (red) calculated from three randomly selected images. Data are presented as mean ± SD (B).

Fig. 5

Dynamics of biofilm formation in the microfluidic channel (BioFlux 1000z) for Campylobacter jejuni strains during 48 h of incubation under flow conditions (0.2 dynes/cm2); mean ± SD, n = 3; * statistically different from control p < 0.05.

Fig. 6

Growth curves of C. jejuni strains. C. jejuni 81–176 WT, ΔcydB, and comp-cydB strains were initially adjusted to OD₆₀₀ of 0.05, and then cultivated in MH broth under microaerobic conditions at 42 °C for 39 h.

Fig. 7

Motility of C. jejuni strains after 24-h incubation. Bacterial suspension (OD₆₀₀ = 0.2) was stabbed using 10 µl pipette into the middle of 0.4% MH agar plate and incubated overnight at 42º C in a microaerobic atmosphere.