DNA Methylation Epigenetically Regulates Gene Expression in Burkholderia cenocepacia and Controls Biofilm Formation, Cell Aggregation, and Motility

Abstract

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2 and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. B. cenocepacia wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation, cell aggregation, and motility.IMPORTANCE CF patients diagnosed with Burkholderia cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of B. cenocepacia cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle B. cenocepacia infections in CF patients.

Keywords: Burkholderia; DNA methylation; epigenetics.

FIG 1

Effect of DNA MTase deletion on biofilm structure, cell aggregation, and pellicle formation in B. cenocepacia J2315 and K56-2. (A) Microscopic images of LIVE/DEAD-stained biofilms, grown in microtiter plate wells for 24 h. White bar (200 μm) for scale. (B) Clustering of cells in planktonic cultures, quantified with flow cytometry. (C) Pellicle formation inside glass tubes after 24 h of static incubation. Left pictures represent unstained samples, and right pictures display pellicles stained with crystal violet. n = 3; *, P < 0.05 compared to wild type; error bars represent the standard error of the mean (SEM). WT, wild type; ΔL, deletion mutant ΔBCAL3494; ΔM, deletion mutant ΔBCAM0992.

FIG 2

Swimming motility of DNA MTase deletion mutants. Diameters were measured after 24 h (K56-2) or 32 h (J2315). n = 3; *, P < 0.05 compared to wild type, error bars represent the SEM. WT, wild type; ΔL, deletion mutant ΔBCAL3494; ΔM, deletion mutant ΔBCAM0992; ΔL pJH2 and ΔM pJH2, mutant strains with empty vector pJH2 (vector control); cΔL pJH2 and cΔM pJH2, deletion mutants complemented with genes BCAL3494 and BCAM0992.

FIG 3

Effect of DNA MTase inhibitor sinefungin on biofilm and pellicle formation, cell aggregation, and motility. (A) Pellicle formation inside glass tubes after 24 h of static incubation. (B) Clustering of planktonic cultures analyzed with flow cytometry. (C) Microscopic images of LIVE/DEAD-stained biofilms, grown on plastic surfaces in microtiter plates for 24 h. (D) Swimming motility of treated and untreated samples. n = 3; *, P < 0.05 compared to wild type; error bars represent the SEM. WT, wild type; +S, medium supplemented with 50 μg/ml sinefungin.

FIG 4

Genomic position of all methylated CACAG motifs. Black circles represent the four replicons of B. cenocepacia; black ticks mark the motif locations. The total number of methylated CACAG motifs and methylated CACAG motifs in promoter regions, per replicon (red, replicon 1; green, replicon 2; yellow, replicon 3; gray, plasmid), is shown on the large and small inner circle, respectively. The positions and names of genes with methylated promoter regions are indicated with colored labels (same color code).

FIG 5

Genomic position of all methylated GTWWAC motifs. Black circles represent the four replicons of B. cenocepacia; black ticks mark the motif locations. The total number of methylated GTWWAC motifs and methylated GTWWAC motifs in promoter regions, per replicon (red, replicon 1; green, replicon 2; yellow, replicon 3; gray, plasmid), is shown on the large and small inner circle, respectively. The positions and names of genes with methylated promoter regions are indicated with colored labels (same color code).

FIG 6

Position of methylated motifs relative to gene start for genes of which the expression is upregulated in DNA MTase deletion mutants. J2315 + K56-2, upregulation in both strains; K56-2, upregulation in strain K56-2 only. (A) Genes with methylated CACAG motifs in their corresponding promoter region. (B) Genes with methylated GTWWAC motifs in their corresponding promoter region. The motifs are marked in bold; the positions of −10 and −30/35 elements in bacterial promoters are framed.

FIG 7

eGFP production in B. cenocepacia J2315 strains harboring a pJH2 plasmid that contains a BCAL1515 promoter-eGFP construct (A), a BCAM0820 promoter-eGFP construct (B), or a BCAL0079 promoter-eGFP construct (C). BCAL1515 and BCAM0820 are associated with methylation of the CACAG motif by DNA MTase BCAL3494, and BCAL0079 is associated with methylation of the GTWWAC motif by DNA MTase BCAL0992. n = 3; *, P < 0.05 compared to wild type; error bars represent the SEM. WT, wild type; ΔL, deletion mutant ΔBCAL3494; ΔM, deletion mutant ΔBCAM0992.

FIG 8

Methylation in the origin of replication of the different replicons in B. cenocepacia J2315. SMRT sequencing was used to detect methylated CACAG (red triangles) and GTWWAC (green triangles) motifs within these regions. DnaA boxes (TTATCCACA, consensus sequence of DnaA boxes in E. coli) are indicated in the figure. CACAG motifs were frequently found to be part of a previously discovered 7-mer (sense, CTGTGCA; antisense, TGCACAG) (34). The positions of these 7-mers are indicated with an asterisk. nt1, nucleotide 1; parABS genes, responsible for chromosome segregation in B. cenocepacia.

FIG 9

(A) DAPI staining of B. cenocepacia wild type and MTase mutants at 4 h, 8 h, 16 h, and 24 h. (B) The nucleoid size relative to the cell size (ratio, R) was calculated by dividing the surface area occupied by the nucleoid (section B) by the total surface area of the cell (section A). (C) Comparison of ratio R of wild type and deletion mutants (average over all time points). WT, wild type; ΔL, ΔBCAL3494; ΔM, ΔBCAM0992. n = 100 for each strain; error bars represent the SEM; *, P < 0.001 compared to wild type.

FIG 10

Proposed mechanism of regulation of gene expression in B. cenocepacia. (1) Methylated motifs in the promoter region of the gene are bound by a TF, acting as repressor (OFF state). (2) In the absence of methylation in the promoter region, the TF dissociates from the motif and vacates the promoter region. (3) The sigma factor is no longer sterically hindered by a repressor and is able to bind to the promoter region. (4) RNA polymerase can access the promoter region and start transcription of the gene (ON state).