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

2020 ◽  
Author(s):  
Olga Mannweiler ◽  
Marta Pinto-Carbó ◽  
Martina Lardi ◽  
Kirsty Agnoli ◽  
Leo Eberl
Keyword(s):  
Burkholderia Cepacia ◽  
Essential Gene ◽  
Epigenetic Modification ◽  
Burkholderia Cepacia Complex ◽  
Phenotypic Traits ◽  
Opportunistic Pathogens ◽  
Smrt Sequencing ◽  
Wide Range ◽  
Methylation Patterns ◽  
The Impact

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.

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

2021 ◽  
Author(s):  
Olga Mannweiler ◽  
Marta Pinto-Carbó ◽  
Martina Lardi ◽  
Kirsty Agnoli ◽  
Leo Eberl
Keyword(s):  
Dna Methylation ◽  
Burkholderia Cepacia ◽  
Membrane Vesicle ◽  
Restriction Enzymes ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Phenotypic Traits ◽  
Opportunistic Pathogens ◽  
Smrt Sequencing ◽  
Foreign Dna

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.

scholarly journals The Impact of Natural Dietary Compounds and Food-Borne Mycotoxins on DNA Methylation and Cancer

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2004 ◽  
Author(s):  
Terisha Ghazi ◽  
Thilona Arumugam ◽  
Ashmika Foolchand ◽  
Anil A. Chuturgoon
Keyword(s):  
Dna Methylation ◽  
Bioactive Compounds ◽  
Epigenetic Modification ◽  
Bioactive Molecules ◽  
Methyl Donors ◽  
Food Borne ◽  
One Carbon Metabolism ◽  
Aberrant Dna Methylation ◽  
Methylation Patterns ◽  
The Impact

Cancer initiation and progression is an accumulation of genetic and epigenetic modifications. DNA methylation is a common epigenetic modification that regulates gene expression, and aberrant DNA methylation patterns are considered a hallmark of cancer. The human diet is a source of micronutrients, bioactive molecules, and mycotoxins that have the ability to alter DNA methylation patterns and are thus a contributing factor for both the prevention and onset of cancer. Micronutrients such as betaine, choline, folate, and methionine serve as cofactors or methyl donors for one-carbon metabolism and other DNA methylation reactions. Dietary bioactive compounds such as curcumin, epigallocatechin-3-gallate, genistein, quercetin, resveratrol, and sulforaphane reactivate essential tumor suppressor genes by reversing aberrant DNA methylation patterns, and therefore, they have shown potential against various cancers. In contrast, fungi-contaminated agricultural foods are a source of potent mycotoxins that induce carcinogenesis. In this review, we summarize the existing literature on dietary micronutrients, bioactive compounds, and food-borne mycotoxins that affect DNA methylation patterns and identify their potential in the onset and treatment of cancer.

scholarly journals Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

2016 ◽  
Vol 60 (10) ◽  
pp. 6003-6012 ◽  
Author(s):  
Saira Ahmad ◽  
Jean Tyrrell ◽  
William G. Walton ◽  
Ashutosh Tripathy ◽  
Matthew R. Redinbo ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Lung Disease ◽  
Biofilm Formation ◽  
Burkholderia Cepacia ◽  
Burkholderia Cepacia Complex ◽  
Upper Airways ◽  
Content Type ◽  
Innate Defense ◽  
Opportunistic Bacteria ◽  
The Impact

ABSTRACTThe opportunistic bacteria of theBurkholderia cepaciacomplex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, includingB. cenocepaciastrain J2315 (50% inhibitory concentration [IC50] = 0.28 μM), and reduced biofilm formation and attachment (IC50= 0.11 μM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease.

scholarly journals Distinguishing Species of the Burkholderia cepacia Complex and Burkholderia gladioli by Automated Ribotyping

2000 ◽  
Vol 38 (5) ◽  
pp. 1876-1884 ◽  
Author(s):  
Sylvain Brisse ◽  
Cees M. Verduin ◽  
Dana Milatovic ◽  
Ad Fluit ◽  
Jan Verhoef ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Burkholderia Cepacia Complex ◽  
Opportunistic Pathogens ◽  
Burkholderia Gladioli ◽  
Microbial Characterization ◽  
Automated Ribotyping ◽  
Level Cluster ◽  
Identification Analysis ◽  
Epidemiological Characterization

Several species belonging to the genus Burkholderia are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs. Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory. In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species from diverse sources, including several publicly accessible collections. The main outcome of this analysis was that all strains were typeable and that strains of Burkholderia gladioli and of each species of the B. cepacia complex, includingB. multivorans, B. stabilis, and B. vietnamiensis, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that withinB. gladioli, B. multivorans, and B. cepacia genomovar VI, the different riboprints identified always clustered together. Riboprints of B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for genomovar identification. Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments. The automated approach allows very rapid and reliable identification and epidemiological characterization of strains and generates an easily manageable database suited for expansion with information on additional bacterial isolates.

Microbiology of CF lung disease

2015 ◽  
Author(s):  
John Govan ◽  
Andrew Jones
Keyword(s):  
Lung Disease ◽  
Chronic Infection ◽  
Burkholderia Cepacia ◽  
Bacterial Pathogens ◽  
Control Measures ◽  
Burkholderia Cepacia Complex ◽  
Opportunistic Pathogens ◽  
Haemophilus Influenza ◽  
Infection Control Measures ◽  
Regular Screening

This chapter presents the microbiology of CF and describes the classical bacterial pathogens including Staphylococcus aureus, Haemophilus influenza, Pseudomonas aeruginosa and organisms of the Burkholderia cepacia complex. The dominant of these is P. aeruginosa. Infections with other opportunistic pathogens including non-tuberculous mycobacteria, Stenotrophomonas maltophila, and Achromobacter (Alcaligenes) xylosoxidans are also encountered. This chapter details measures to prevent the onset of chronic infection with these organisms include regular screening of respiratory tract samples for bacterial pathogens and the use of aggressive antibiotic therapy to eradicate initial infection before the pathogen can adapt to the environment of the CF lung. Patient-to-patient spread of transmissible strains of bacterial pathogens has led to the implementation of strict infection control measures at CF centres, including patient segregation. In addition to bacterial pathogens, the contribution of fungal infection in CF lung disease is increasingly recognized.

scholarly journals Redox-active secondary metabolites act as interspecies modulators of antibiotic resilience

2021 ◽  
Author(s):  
Lucas A. Meirelles ◽  
Dianne K. Newman
Keyword(s):  
Secondary Metabolites ◽  
Burkholderia Cepacia ◽  
Defense Responses ◽  
Microbial Interactions ◽  
Opportunistic Pathogens ◽  
Burkholderia Gladioli ◽  
Redox Active ◽  
Wide Range ◽  
Genes Encoding

ABSTRACTBacterial opportunistic pathogens make a wide range of secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of two redox-active secondary metabolites, pyocyanin and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin is made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. Both molecules alter the susceptibility profile of pathogenic species within the “Burkholderia cepacia complex” to different antibiotics, either antagonizing or potentiating their effects, depending on the drug’s class. Defense responses regulated by the redox-sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

scholarly journals Current Advances in Burkholderia Vaccines Development

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2671
Author(s):  
Guanbo Wang ◽  
Paulina Zarodkiewicz ◽  
Miguel A. Valvano
Keyword(s):  
Burkholderia Cepacia ◽  
Burkholderia Pseudomallei ◽  
Vaccine Development ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Antimicrobial Treatment ◽  
Burkholderia Cepacia Complex ◽  
Attractive Alternative ◽  
Wide Range ◽  
Substantial Progress

The genus Burkholderia includes a wide range of Gram-negative bacterial species some of which are pathogenic to humans and other vertebrates. The most pathogenic species are Burkholderia mallei, Burkholderia pseudomallei, and the members of the Burkholderia cepacia complex (Bcc). B. mallei and B. pseudomallei, the cause of glanders and melioidosis, respectively, are considered potential bioweapons. The Bcc comprises a subset of Burkholderia species associated with respiratory infections in people with chronic granulomatous disease and cystic fibrosis. Antimicrobial treatment of Burkholderia infections is difficult due to the intrinsic multidrug antibiotic resistance of these bacteria; prophylactic vaccines provide an attractive alternative to counteract these infections. Although commercial vaccines against Burkholderia infections are still unavailable, substantial progress has been made over recent years in the development of vaccines against B. pseudomallei and B. mallei. This review critically discusses the current advances in vaccine development against B. mallei, B. pseudomallei, and the Bcc.

scholarly journals Draft Genome Sequence of Pseudomonas sp. Strain Ep R1 Isolated from Echinacea purpurea Roots and Effective in the Growth Inhibition of Human Opportunistic Pathogens Belonging to the Burkholderia cepacia Complex

2017 ◽  
Vol 5 (20) ◽  
Author(s):  
Valentina Maggini ◽  
Luana Presta ◽  
Elisangela Miceli ◽  
Marco Fondi ◽  
Emanuele Bosi ◽  
...  
Keyword(s):  
Medicinal Plant ◽  
Genome Sequence ◽  
Burkholderia Cepacia ◽  
Draft Genome ◽  
Echinacea Purpurea ◽  
Burkholderia Cepacia Complex ◽  
Draft Genome Sequence ◽  
Antimicrobial Compounds ◽  
Pseudomonas Sp ◽  
Opportunistic Pathogens

ABSTRACT In this announcement, we detail the draft genome sequence of the Pseudomonas sp. strain Ep R1, isolated from the roots of the medicinal plant Echinacea purpurea. The elucidation of this genome sequence may allow the identification of genes associated with the production of antimicrobial compounds.

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