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

Microbiology ◽  
2021 ◽  
Author(s):  
Ian Vandenbussche ◽  
Andrea Sass ◽  
Filip Van Nieuwerburgh ◽  
Marta Pinto-Carbó ◽  
Olga Mannweiler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Type Species ◽  
Cytosine Methylation ◽  
Regulation Of Gene Expression ◽  
Burkholderia Cenocepacia ◽  
Smrt Sequencing ◽  
Content Type ◽  
Link Type ◽  
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 .

PacBio sequencing revealed variation in the microbiota diversity, species richness and composition between milk collected from healthy and mastitis cows

Microbiology ◽  
2021 ◽  
Vol 167 (7) ◽  
Author(s):  
Teng Ma ◽  
Lingling Shen ◽  
Qiannan Wen ◽  
Ruirui Lv ◽  
Qiangchuan Hou ◽  
...  
Keyword(s):  
Single Molecule ◽  
Type Species ◽  
Cow Milk ◽  
Sequencing Technology ◽  
Pacbio Sequencing ◽  
Content Type ◽  
Milk Samples ◽  
Link Type ◽  
Microbiota Diversity ◽  
Important Disease

Mastitis is the economically most important disease of dairy cows. This study used PacBio single-molecule real-time sequencing technology to sequence the full-length 16S rRNAs from 27 milk samples (18 from mastitis and nine from healthy cows; the cows were at different stages of lactation). We observed that healthy or late stage milk microbiota had significantly higher microbial diversity and richness. The community composition of the microbiota of different groups also varied greatly. The healthy cow milk microbiota was predominantly comprised of Lactococcus lactis , Acinetobacter johnsonii , and Bacteroides dorei , while the milk from mastitis cows was predominantly comprised of Bacillus cereus . The prevalence of L. lactis and B. cereus in the milk samples was confirmed by digital droplets PCR. Differences in the milk microbiota diversity and composition could suggest an important role for some these microbes in protecting the host from mastitis while others associated with mastitis. The results of our research serve as useful references for designing strategies to prevent and treat mastitis.

scholarly journals Regulation by the Modulation of Gene Expression Variability

2015 ◽  
Vol 197 (12) ◽  
pp. 1974-1975 ◽  
Author(s):  
David Dubnau
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Content Type ◽  
Expression Variability ◽  
Link Type ◽  
Article Type ◽  
Modulation Of Gene Expression ◽  
The Mean ◽  
Cell Changes ◽  
Expression Variance

Classically, transcription is regulated so that the average expression per cell changes, often with a distribution that extends across the population. Roggiani and Goulian (M. Roggiani and M. Goulian, J. Bacteriol. 197:1976–1987, 2015, doi:http://dx.doi.org/10.1128/JB.00074-15) have shown that this is what happens when thetorCADoperon ofEscherichia coliis induced anaerobically by the addition of trimethylamine-N-oxide (TMAO). However, when the same inducer is added to aerobically growing cells, only a subset of the cells respond, although the mean expression per cell is similar to that obtained anaerobically. Thus, in the presence of oxygen, the variance but not the expression mean is altered. The regulation of gene expression variance appears to be due to noise in the phosphorelay that governstorCADtranscription.

scholarly journals Transcriptome profiling of Variovorax paradoxus EPS under different growth conditions reveals regulatory and structural novelty in biofilm formation

2020 ◽  
Vol 2 (6) ◽  
Author(s):  
Richard J. Fredendall ◽  
Jenny L. Stone ◽  
Michael J. Pehl ◽  
Paul M. Orwin
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Biofilm Formation ◽  
Type Species ◽  
Extracellular Dna ◽  
Differentially Expressed ◽  
Content Type ◽  
Variovorax Paradoxus ◽  
Link Type

We used transcriptome analysis by paired-end strand-specific RNA-seq to evaluate the specific changes in gene expression associated with the transition to static biofilm growth in the rhizosphere plant growth-promoting bacterium Variovorax paradoxus EPS. Triplicate biological samples of exponential growth, stationary phase and static biofilm samples were examined. DESeq2 and Rockhopper were used to identify robust and widespread shifts in gene expression specific to each growth phase. We identified 1711 protein-coding genes (28%) using DESeq2 that had altered expression greater than twofold specifically in biofilms compared to exponential growth. Fewer genes were specifically differentially expressed in stationary-phase culture (757, 12%). A small set of genes (103/6020) were differentially expressed in opposing fashions in biofilm and stationary phase, indicating potentially substantial shifts in phenotype. Gene-ontology analysis showed that the only class of genes specifically upregulated in biofilms was associated with nutrient transport, highlighting the importance of nutrient uptake in the biofilm. The biofilm-specific genes did not overlap substantially with the loci identified by mutagenesis studies, although some were present in both sets. The most highly upregulated biofilm-specific gene is predicted to be a part of the RNA degradosome, which indicates that RNA stability is used to regulate the biofilm phenotype. Two small putative proteins, Varpa_0407 and Varpa_3832, are highly expressed specifically in biofilms and are predicted to be secreted DNA-binding proteins, which may stabilize extracellular DNA as a component of the biofilm matrix. An flp/tad type-IV pilus locus (Varpa_5148–60) is strongly downregulated specifically in biofilms, in contrast with results from other systems for these pili. Mutagenesis confirms that this locus is important in surface motility rather than biofilm formation. These experimental results suggest that V. paradoxus EPS biofilms have substantial regulatory and structural novelty.

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

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Ian Vandenbussche ◽  
Andrea Sass ◽  
Marta Pinto-Carbó ◽  
Olga Mannweiler ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Biofilm Formation ◽  
Regulation Of Gene Expression ◽  
Cell Aggregation ◽  
Burkholderia Cenocepacia ◽  
Model Organisms ◽  
Epigenetic Changes ◽  
Content Type ◽  
Regulatory Systems

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.

scholarly journals Investigation of LuxS-mediated quorum sensing in Klebsiella pneumoniae

2020 ◽  
Vol 69 (3) ◽  
pp. 402-413 ◽  
Author(s):  
Lijiang Chen ◽  
Jonathan J. Wilksch ◽  
Haiyang Liu ◽  
Xiaoxiao Zhang ◽  
Von V. L. Torres ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Type Species ◽  
Microtiter Plate ◽  
Resistant Isolate ◽  
Wild Type ◽  
Content Type ◽  
Link Type

Introduction. Autoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires luxS activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation. Aim. To investigate the role of luxS in biofilm formation and gene expression in the nosocomial pathogen Klebsiella pneumoniae . Methodology. A ΔluxS gene deletion was made in K. pneumoniae KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and ΔluxS strains grown in media supplemented with different carbohydrates. Potential roles of luxS in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide (wzm and wbbM), polysaccharide (pgaA), and type 3 fimbriae (mrkA) synthesis genes in wild-type and ΔluxS mutant biofilm extracts. Results. AI-2 production was dependent on the presence of luxS. AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the ΔluxS mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and ΔluxS mutant using a microtiter plate assay were observed. In ΔluxS mutant biofilm extracts, the expression of wzm was down-regulated, and the expression of pgaA, which encodes a porin for poly-β−1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated. Conclusion. Relationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in K. pneumoniae . These inter-connected processes could be important for bacterial group behaviour and persistence.

scholarly journals DNA methylation epigenetically regulates gene expression in Burkholderia cenocepacia

2020 ◽  
Author(s):  
Ian Vandenbussche ◽  
Andrea Sass ◽  
Marta Pinto-Carbó ◽  
Olga Mannweiler ◽  
Leo Eberl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Single Molecule ◽  
Regulation Of Gene Expression ◽  
Dna Methyltransferases ◽  
Lung Damage ◽  
Burkholderia Cenocepacia ◽  
Model Organisms ◽  
Deletion Mutants ◽  
Tract Infections

AbstractRespiratory 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 has shown to be 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, 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 and motility.ImportanceCF patients diagnosed with B. cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multi-replicon genome and a lot 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 lead to new insights in how to tackle B. cenocepacia infections in CF patients.

scholarly journals Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Esther Blanco-Romero ◽  
David Durán ◽  
Daniel Garrido-Sanz ◽  
Rafael Rivilla ◽  
Marta Martín ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Biofilm Formation ◽  
Type Species ◽  
Global Changes ◽  
Secretion Systems ◽  
Content Type ◽  
Rhizosphere Colonization ◽  
Link Type ◽  
Metabolic Versatility

Rhizosphere colonization by bacteria involves molecular and cellular mechanisms, such as motility and chemotaxis, biofilm formation, metabolic versatility, or biosynthesis of secondary metabolites, among others. Nonetheless, there is limited knowledge concerning the main regulatory factors that drive the rhizosphere colonization process. Here we show the importance of the AmrZ and FleQ transcription factors for adaption in the plant growth-promoting rhizobacterium (PGPR) and rhizosphere colonization model Pseudomonas ogarae F113. RNA-Seq analyses of P. ogarae F113 grown in liquid cultures either in exponential and stationary growth phase, and rhizosphere conditions, revealed that rhizosphere is a key driver of global changes in gene expression in this bacterium. Regarding the genetic background, this work has revealed that a mutation in fleQ causes considerably more alterations in the gene expression profile of this bacterium than a mutation in amrZ under rhizosphere conditions. The functional analysis has revealed that in P. ogarae F113, the transcription factors AmrZ and FleQ regulate genes involved in diverse bacterial functions. Notably, in the rhizosphere, these transcription factors antagonistically regulate genes related to motility, biofilm formation, nitrogen, sulfur, and amino acid metabolism, transport, signalling, and secretion, especially the type VI secretion systems. These results define the regulon of two important bifunctional transcriptional regulators in pseudomonads during the process of rhizosphere colonization.

scholarly journals Eucalyptol inhibits biofilm formation of Streptococcus pyogenes and its mediated virulence factors

2020 ◽  
Vol 69 (11) ◽  
pp. 1308-1318
Author(s):  
Karuppiah Vijayakumar ◽  
Vajravelu Manigandan ◽  
Danaraj Jeyapragash ◽  
Veeraiyan Bharathidasan ◽  
Balaiyan Anandharaj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Streptococcus Pyogenes ◽  
Biofilm Formation ◽  
Type Species ◽  
Inhibitory Concentration ◽  
Antibiofilm Activity ◽  
Dependent Manner ◽  
Biofilm Inhibition ◽  
Content Type ◽  
Link Type

Introduction. Streptococcus pyogenes is a diverse virulent synthesis pathogen responsible for invasive systemic infections. Establishment of antibiotic resistance in the pathogen has produced a need for new antibiofilm agents to control the biofilm formation and reduce biofilm-associated resistance development. Aim. The present study investigates the in vitro antibiofilm activity of eucalyptol against S. pyogenes . Methodology. The antibiofilm potential of eucalyptol was assessed using a microdilution method and their biofilm inhibition efficacy was visualized by microscopic analysis. The biochemical assays were performed to assess the influence of eucalyptol on virulence productions. Real-time PCR analysis was performed to evaluate the expression profile of the virulence genes. Results. Eucalyptol showed significant antibiofilm potential in a dose-dependent manner without affecting bacterial growth. Eucalyptol at 300 µg ml−1 (biofilm inhibitory concentration) significantly inhibited the initial stage of biofilm formation in S. pyogenes . However, eucalyptol failed to diminish the mature biofilms of S. pyogenes at biofilm inhibitory concentration and it effectively reduced the biofilm formation on stainless steel, titanium, and silicone surfaces. The biochemical assay results revealed that eucalyptol greatly affects the cell-surface hydrophobicity, auto-aggregation, extracellular protease, haemolysis and hyaluronic acid synthesis. Further, the gene-expression analysis results showed significant downregulation of virulence gene expression upon eucalyptol treatment. Conclusion. The present study suggests that eucalyptol applies its antibiofilm assets by intruding the initial biofilm formation of S. pyogenes . Supplementary studies are needed to understand the mode of action involved in biofilm inhibition.

scholarly journals Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3877-3884 ◽  
Author(s):  
Celine De Maesschalck ◽  
Filip Van Immerseel ◽  
Venessa Eeckhaut ◽  
Siegrid De Baere ◽  
Margo Cnockaert ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428T and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T could be distinguished from S. pleomorphus ATCC 29734T based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734T. The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428T and LMG 27427. Strain LMG 27428T ( = DSM 26963T) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( = DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756T = ATCC 29734T = DSM 20574T). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA gene sequence similarity to strain LMG 27428T) and Eubacterium biforme DSM 3989T (92.7 % 16S rRNA gene sequence similarity to strain LMG 27428T). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983T = ATCC 27803T = JCM 10261T and that of Holdemanella biformis is DSM 3989T = ATCC 27806T = CCUG 28091T.

scholarly journals Complete Genome Sequence of Pandoraea pnomenusa TF-18, a Multidrug-Resistant Organism Isolated from the Rhizosphere of Rice (Oryza sativa L. subsp. japonica)

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Mihnea R. Mangalea ◽  
Emily K. Luna ◽  
Janet Ziegle ◽  
Christine Chang ◽  
Angela M. Bosco-Lauth ◽  
...  
Keyword(s):  
Single Molecule ◽  
Complete Genome ◽  
Oryza Sativa L ◽  
Gc Content ◽  
Multidrug Resistant ◽  
Selective Medium ◽  
Resistant Organism ◽  
Smrt Sequencing ◽  
Sequencing Technology ◽  
Content Type

Pandoraea pnomenusa strain TF-18 was isolated from the roots of rice seedlings on selective medium containing four classes of antibiotics for isolation of Burkholderia pseudomallei. Using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing technology, we report here a complete genome of 5,499,432 bases, a GC content of 64.8%, and 4,849 coding sequences.

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