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. 2016 Jun 14;6:65.
doi: 10.3389/fcimb.2016.00065. eCollection 2016.

Synthetic Cystic Fibrosis Sputum Medium Regulates Flagellar Biosynthesis Through the flhF Gene in Burkholderia Cenocepacia

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Free PMC article

Synthetic Cystic Fibrosis Sputum Medium Regulates Flagellar Biosynthesis Through the flhF Gene in Burkholderia Cenocepacia

Brijesh Kumar et al. Front Cell Infect Microbiol. .
Free PMC article

Abstract

Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc), a group of at least 18 distinct species that establish chronic infections in the lung of people with the genetic disease cystic fibrosis (CF). The sputum of CF patients is rich in amino acids and was previously shown to increase flagellar gene expression in B. cenocepacia. We examined flagellin expression and flagellar morphology of B. cenocepacia grown in synthetic cystic fibrosis sputum medium (SCFM) compared to minimal medium. We found that CF nutritional conditions induce increased motility and flagellin expression. Individual amino acids added at the same concentrations as found in SCFM also increased motility but not flagellin expression, suggesting a chemotactic effect of amino acids. Electron microscopy and flagella staining demonstrated that the increase in flagellin corresponds to a change in the number of flagella per cell. In minimal medium, the ratio of multiple: single: aflagellated cells was 2:3.5:4.5; while under SCFM conditions, the ratio was 7:2:1. We created a deletion mutant, ΔflhF, to study whether this putative GTPase regulates the flagellation pattern of B. cenocepacia K56-2 during growth in CF conditions. The ΔflhF mutant exhibited 80% aflagellated, 14% single and 6% multiple flagellated bacterial subpopulations. Moreover, the ratio of multiple to single flagella in WT and ΔflhF was 3.5 and 0.43, respectively in CF conditions. The observed differences suggest that FlhF positively regulates flagellin expression and the flagellation pattern in B. cenocepacia K56-2 during CF nutritional conditions.

Keywords: Burkholderia cenocepacia; SCFM; cystic fibrosis; flagellum; flhF; motility.

Figures

Figure 1
Figure 1
Growth kinetics, motility phenotype and flagellin expression analysis of B. cenocepacia K56-2 WT. (A) Growth kinetics of WT strain in SCFM and MOPS-glucose 20 mM. (B,C) Motility analysis. The motility of WT strain was examined in semi-solid SCFM and MOPS-glucose 20 mM 0.3% agar plates. Motility halos were recorded after 24 h of incubation time. Results correspond to three independent experiments and “*” denotes significant p-value (p < 0.05), which was calculated using Student's t-test. (D) Detection of flagellin by Western blot. Whole cell lysates and pure flagellin were run on 12% SDS –PAGE. The flagellin protein was detected using anti-flagellin primary antibody and secondary antibody cross-linked to alkaline phosphatase. Western blot and growth assays were performed twice independently. One representative experiment is shown.
Figure 2
Figure 2
The effect of individual amino acids effect on motility, growth and flagellin expression of B. cenocepacia K56-2 WT. (A) Growth curves of WT strain in the presence of individual amino acids + MOPS-glucose 5 mM. (B) Motility halos of the WT strain in MOPS-glucose 5 mM + individual amino acid at the same concentration found in SCFM. The motility assay was performed three times independently and “*” denotes significant p-values (p < 0.05), obtained using One-Way ANOVA. (C) Western blot of WT whole cell lysates in the same condition as those used for the growth and motility analysis. Lane 1 shows the detection of purified flagellin protein as a positive control. Western blot and growth assays were performed twice independently. One representative experiment is shown.
Figure 3
Figure 3
Flagellar phenotypic analysis of B. cenocepacia K56-2 WT. Transmission electron microscopy (TEM) of the WT strain in SCFM (A) and MOPS-glucose 20 mM (B). (C) Distribution of the WT strain flagellation pattern in SCFM and MOPS-glucose after 24 h of growth. The arrows show the attachment site of flagella. Cell count was based on TEM images (n = 100).
Figure 4
Figure 4
Motility, flagellar morphology and flagellin expression analysis of flhF deletion and overexpression in strains of B. cenocepacia K56-2 WT. Motility (A), flagellation pattern (B) and flagellin expression levels (C) of WT, ΔflhF mutant, ΔflhF+flhF complement and WT+flhF overexpression strain. The motility assay was performed three times independently. One-Way ANOVA was performed to obtain p-values and “*” denotes significant p-values (p < 0.05). The WT motility was used as control for comparisons.
Figure 5
Figure 5
The flhBAFG operon and its transcription activity. (A) Schematic of the flagellar flhBAFG operon. The angle arrow represents the promoter upstream of the flhB gene. To examine transcriptional activity, a 531 bp fragment containing the flhB promoter was amplified and cloned upstream of the lux operon in the bioreporter system. (B) The flhF promoter sequence was transcriptionally fused upstream to luxCDABE gene cassette (flhF-lux) on a plasmid (pflhFprom) and introduced into the WT strain. Bioluminescence was measured hourly in response to activation of the flhF operon promoter in SCFM and MOPS-glucose 20 mM conditions. The transcriptional activity assay was performed three times showing similar results. One representative graph is shown. (C) The graph shows RLU/OD600 at the mid-exponential phase of OD600 2.2 in SCFM and MOPS-glucose 20 mM. “*” denotes significant p-value (p < 0.05), which was calculated using Student's t-test.

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