Investigation of Burkholderia cepacia Complex Methylomes via Single-Molecule, Real-Time Sequencing and Mutant Analysis.

Bacterial genomes can be methylated at particular motifs by methyltransferases (MTs). This DNA modification allows restriction endonucleases (REs) 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 RE or MT 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 analyzed mutants lacking various RM components in the clinical Bcc isolate Burkholderia cenocepacia H111 and used single-molecule, real-time (SMRT) sequencing of single mutants to assign the B. cenocepacia H111 MTs 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 MT-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 the genome sequence determines an organism's proteins, methylation of the nucleotides themselves can confer additional properties. In bacteria, MTs modify specific nucleotide motifs to allow discrimination of "self" from "nonself" DNA, e.g., from bacteriophages. Restriction enzymes detect "nonself" 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.

Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia, an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia.

Use of Synthetic Hybrid Strains To Determine the Role of Replicon 3 in Virulence of the Burkholderia cepacia Complex.

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.IMPORTANCE The 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.

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures.

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.

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

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.