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.
The Burkholderia cepacia complex (Bcc) displays a wealth of metabolic diversity with great biotechnological potential, but the utilization of these bacteria is limited by their opportunistic pathogenicity to humans. The third replicon of the Bcc, megaplasmid pC3 (0.5 to 1.4 Mb, previously chromosome 3), is important for various phenotypes, including virulence, antifungal, and proteolytic activities and the utilization of certain substrates. Approximately half of plasmid pC3 is well conserved throughout sequenced Bcc members, while the other half is not. To better locate the regions responsible for the key phenotypes, pC3 mutant derivatives of Burkholderia cenocepacia H111 carrying large deletions (up to 0.58 Mb) were constructed with the aid of the FLP-FRT (FRT, flippase recognition target) recombination system from Saccharomyces cerevisiae. The conserved region was shown to confer near-full virulence in both Caenorhabditis elegans and Galleria mellonella infection models. Antifungal activity was unexpectedly independent of the part of pC3 bearing a previously identified antifungal gene cluster, while proteolytic activity was dependent on the nonconserved part of pC3, which encodes the ZmpA protease. To investigate to what degree pC3-encoded functions are dependent on chromosomally encoded functions, we transferred pC3 from Burkholderia cenocepacia K56-2 and Burkholderia lata 383 into other pC3-cured Bcc members. We found that although pC3 is highly important for virulence, it was the genetic background of the recipient that determined the pathogenicity level of the hybrid strain. Furthermore, we found that important phenotypes, such as antifungal activity, proteolytic activity, and some substrate utilization capabilities, can be transferred between Bcc members using pC3. IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of closely related bacteria with great biotechnological potential. Some strains produce potent antifungal compounds and can promote plant growth or degrade environmental pollutants. However, their agricultural potential is limited by their opportunistic pathogenicity, particularly for cystic fibrosis patients. Despite much study, their virulence remains poorly understood. The third replicon, pC3, which is present in all Bcc isolates and is important for pathogenicity, stress resistance, and the production of antifungal compounds, has recently been reclassified from a chromosome to a megaplasmid. In this study, we identified regions on pC3 important for virulence and antifungal activity and investigated the role of the chromosomal background for the function of pC3 by exchanging the megaplasmid between different Bcc members. Our results may open a new avenue for the construction of antifungal but nonpathogenic Burkholderia hybrids. Such strains may have great potential as biocontrol strains for protecting fungus-borne diseases of plant crops.
23Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia 24 often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to 25 tackle these infections might emerge from the field of epigenetics, as DNA methylation 26 has shown to be an important regulator of gene expression. The present study focused 27 on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2, 28 and their role in regulating gene expression. In silico predicted DNA MTase genes 29 BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the 30 resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes 31 in biofilm structure and cell aggregation, ΔBCAM0992 was less motile. B. cenocepacia 32 wild type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the 33 same phenotype as DNA MTase deletion mutants. Single-Molecule Real-Time 34 sequencing was used to characterize the methylome of B. cenocepacia, including 35 methylation at the origin of replication, and motifs CACAG and GTWWAC were 36 identified as targets of BCAL3494 and BCAM0992, respectively. All genes with 37 methylated motifs in their putative promoter region were identified and qPCR 38 experiments showed an upregulation of several genes, including biofilm and motility 39 related genes, in MTase deletion mutants with unmethylated motifs, explaining the 40 observed phenotypes in these mutants. In summary, our data confirm that DNA 41 methylation plays an important role in regulating the expression of B. cenocepacia 42 genes involved in biofilm formation and motility. 43 44 3 Importance 45 CF patients diagnosed with B. cenocepacia infections often experience rapid 46 deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large 47 multi-replicon genome and a lot remains to be learned about regulation of gene 48 expression in this organism. From studies in other (model) organisms, it is known that 49 epigenetic changes through DNA methylation play an important role in this regulation. 50 The identification of B. cenocepacia genes of which the expression is regulated by DNA 51 methylation and identification of the regulatory systems involved in this methylation are 52 likely to lead to new insights in how to tackle B. cenocepacia infections in CF patients. 53 54
Bacterial genomes can be methylated at particular motifs by methyltransferases (M). This DNA modification allows restriction endonucleases (R) to discriminate between self and foreign DNA. While the accepted primary function of such restriction modification (RM) systems is to degrade incoming foreign DNA, other roles of RM systems and lone R or M components have been found in genome protection, stability and the regulation of various phenotypes. The Burkholderia cepacia complex (Bcc) is a group of closely related opportunistic pathogens with biotechnological potential. Here, we constructed and analysed mutants lacking various RM components in the clinical Bcc isolate Burkholderia cenocepacia H111 and used SMRT sequencing of single mutants to assign the B. cenocepacia H111 Ms to their cognate motifs. DNA methylation is shown to affect biofilm formation, cell shape, motility, siderophore production and membrane vesicle production. Moreover, DNA methylation had a large effect on the maintenance of the Bcc virulence megaplasmid pC3. Our data also suggest that the gp51 M-encoding gene, which is essential in H111 and is located within a prophage, is required for maintaining the bacteriophage in a lysogenic state, thereby ensuring a constant, low level of phage production within the bacterial population. Importance While genome sequence determines an organism’s proteins, methylation of the nucleotides themselves can confer additional properties. In bacteria, Ms modify specific nucleotide motifs to allow discrimination of ‘self’ from ‘non-self’ DNA, e.g. from bacteriophages. Restriction enzymes detect ‘non-self’ methylation patterns and cut foreign DNA. Furthermore, methylation of promoter regions can influence gene expression and hence affect various phenotypes. In this study, we determined the methylated motifs of four strains from the Burkholderia cepacia complex of opportunistic pathogens. We deleted all genes encoding the restriction and modification components in one of these strains, Burkholderia cenocepacia H111. It is shown that DNA methylation affects various phenotypic traits, the most noteworthy being lysogenicity of a bacteriophage and maintenance of a virulence megaplasmid.
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