AbstractThe Burkholderia cepacia complex (Bcc) is a group of 22 closely related opportunistic pathogens which produce a wide range of bioactive secondary metabolites with great biotechnological potential, for example in biocontrol and bioremediation.This study aimed to investigate methylation in the Bcc by SMRT sequencing, and to determine the impact of restriction-methylation (RM) systems on genome protection and stability and on phenotypic traits. We constructed and analysed a mutant lacking all RM components in the clinical isolate B. cenocepacia H111. We show that a previously identified essential gene of strain H111, gp51, encoding a methylase within a prophage region, is required for maintaining the bacteriophage in a lysogenic state. We speculate that epigenetic modification of a phage promoter provides a mechanism for a constant, low level of phage production within the bacterial population. We also found that, in addition to bacteriophage induction, methylation was important in biofilm formation, cell shape, motility, siderophore production and membrane vesicle production. Moreover, we found that DNA methylation had a massive effect on the maintenance of the smallest replicon present in this bacterium, which is essential for its virulence.In silico investigation revealed the presence of two core RM systems, present throughout the Bcc and beyond, suggesting that the acquisition of these RM systems occurred prior to the phylogenetic separation of the Bcc. We used SMRT sequencing of single mutants to experimentally assign the B. cenocepacia H111 methylases to their cognate motifs. Analysis of the distribution of methylation patterns suggested roles for m6A methylation in replication, since motifs recognised by the core Type III RM system were more abundant at the replication origins of the three H111 replicons, and in regions encoding functions related to cell motility and iron uptake.Author summaryWhile nucleotide sequence determines an organism’s proteins, methylation of the nucleotides themselves can confer additional properties. In bacteria, methyltransferases methylate specific 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 phenotype. In this study, we determined the methylated motifs of four strains from the Burkholderia cepacia complex of opportunistic pathogens. Three novel motifs were found, and two that were previously identified in a related species. We deleted the genes encoding the restriction and modification components in a representative strain from among the four sequenced. In this study, methylation is shown to affect various phenotypes, among which maintenance of the lysogenic state of a phage and segregational stability of the smallest megareplicon are most remarkable.
Possibilities in Identification of Genomic Species of Burkholderia cepacia Complex by PCR and RFLP