An investigation of Burkholderia cepacia complex methylomes via SMRT sequencing and mutant analysis

The 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 summary While 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.

cenocepacia H111. We show that a previously identified essential gene of strain H111, gp51, encoding 23 a methylase within a prophage region, is required for maintaining the bacteriophage in a lysogenic 24 state. We speculate that epigenetic modification of a phage promoter provides a mechanism for a 25 constant, low level of phage production within the bacterial population. We also found that, in addition 26 to bacteriophage induction, methylation was important in biofilm formation, cell shape, motility, 27 siderophore production and membrane vesicle production. Moreover, we found that DNA methylation 28 had a massive effect on the maintenance of the smallest replicon present in this bacterium, which is 29 essential for its virulence.

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In silico investigation revealed the presence of two core RM systems, present throughout the Bcc and 31 beyond, suggesting that the acquisition of these RM systems occurred prior to the phylogenetic 32 separation of the Bcc. We used SMRT sequencing of single mutants to experimentally assign the B.

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cenocepacia H111 methylases to their cognate motifs. Analysis of the distribution of methylation 34 patterns suggested roles for m6A methylation in replication, since motifs recognised by the core Type 35 III RM system were more abundant at the replication origins of the three H111 replicons, and in regions 36 encoding functions related to cell motility and iron uptake.

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Author summary 38 While nucleotide sequence determines an organism's proteins, methylation of the nucleotides 39 themselves can confer additional properties. In bacteria, methyltransferases methylate specific motifs 40 to allow discrimination of 'self' from 'non-self' DNA, e.g. from bacteriophages. Restriction enzymes 41 detect 'non-self' methylation patterns and cut foreign DNA. Furthermore, methylation of promoter

Introduction Verification of loss of methylation in an RM null mutant, and of the methylase cognate to each
246 recognition motif 247 We used SMRT sequencing to assign the methylated motifs present in the H111 genome to their 248 cognate RM systems. Three methylated motifs were detected in the H111 genome. The genomes of 249 single mutants in the Type I RM on C1 (I35_3254), the Type II methylase on C2 (I35_2582), and the 250 Type III RM system on C1 (I35_1825) were subjected to SMRT sequencing, allowing the predicted 251 motifs 5'-CAG-NNNNNN-TTYG-3', GTWWAC and CACAG to be confirmed for these methylases, 252 respectively.

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The genome of the RM null mutant was also subjected to SMRT sequencing, to verify loss of all 254 methylated motifs by mapping the data from the RM null mutant against the obtained methylome 255 data of the H111 wildtype. No m6A or m4C modifications were detected in the RM null mutant.

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Transmission electron microscopy (TEM) was used to investigate the presence of phages and phage-370 like structures in the supernatants of H111 wild type and the clean RM system null mutant, which our 371 RNAseq analysis suggested was increased in the RM null mutant. In the electron micrographs, we 372 observed the presence of phages and phage tails, either from partially assembled phages or tailocins 373 (bacteriocins) (Fig 6). Furthermore, we discovered long fibres indicating flagella, and other tube-like 374 structures of unknown function. We also observed membrane vesicles of varying sizes. To compare vesicle production between the wild type and the null strain, membrane vesicles were collected and 376 quantified by staining with FM 1-43 fluorescent dye, which binds the cell membrane. We observed a 377 2.2-fold increase in MV production in the RM null mutant compared to the H111 wild type (Fig 6 panel 378 C), probably as a result of phage-triggered cell lysis (Turnbull et al., 2016).

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Chemical and phenotypic assays for pyochelin production support methylation pattern observations

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Genes involved in the biosynthesis and utilization of the siderophore pyochelin showed a 383 transcriptional increase in the RM system null mutant compared to H111 (

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This study aimed to shed light on the involvement of DNA methyltransferases in the regulation of 514 important cellular processes, as well as to unravel the impact of RM systems on bacterial phenotypes.

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Our work has confirmed the role of methylases and RM systems in genome protection and stability 516 and has suggested involvement in phenotypes such as biofilm formation, siderophore production, 517 motility, and prophage induction.