Epigenetic Switch Driven by DNA Inversions Dictates Phase Variation in Streptococcus pneumoniae.

DNA methylation is an important epigenetic mechanism for phenotypic diversification in all forms of life. We previously described remarkable cell-to-cell heterogeneity in epigenetic pattern within a clonal population of Streptococcus pneumoniae, a leading human pathogen. We here report that the epigenetic diversity is caused by extensive DNA inversions among hsdSA, hsdSB, and hsdSC, three methyltransferase hsdS genes in the Spn556II type-I restriction modification (R-M) locus. Because hsdSA encodes the sequence recognition subunit of this type-I R-M DNA methyltransferase, these site-specific recombinations generate pneumococcal cells with variable HsdSA alleles and thereby diverse genome methylation patterns. Most importantly, the DNA methylation pattern specified by the HsdSA1 allele leads to the formation of opaque colonies, whereas the pneumococci lacking HsdSA1 produce transparent colonies. Furthermore, this HsdSA1-dependent phase variation requires intact DNA methylase activity encoded by hsdM in the Spn556II (renamed colony opacity determinant or cod) locus. Thus, the DNA inversion-driven ON/OFF switch of the hsdSA1 allele in the cod locus and resulting epigenetic switch dictate the phase variation between the opaque and transparent phenotypes. Phase variation has been well documented for its importance in pneumococcal carriage and invasive infection, but its molecular basis remains unclear. Our work has discovered a novel epigenetic cause for this significant pathobiology phenomenon in S. pneumoniae. Lastly, our findings broadly represents a significant advancement in our understanding of bacterial R-M systems and their potential in shaping epigenetic and phenotypic diversity of the prokaryotic organisms because similar site-specific recombination systems widely exist in many archaeal and bacterial species.

Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae.

Reversible or phenotypic tolerance to antibiotics within microbial populations has been implicated in treatment failure of chronic infections and development of persister cells. However, the molecular mechanisms regulating phenotypic drug tolerance are largely unknown. In this study, we identified a four-gene operon in Streptococcus pneumoniae that contributes to phenotypic tolerance to vancomycin (ptv). RNA sequencing, quantiative reverse transcriptase PCR, and transcriptional luciferase reporter experiments revealed that transcription of the ptv operon (consisting of ptvR, ptvA, ptvB, and ptvC) is induced by exposure to vancomycin. Further investigation showed that transcription of the ptv operon is repressed by PtvR, a PadR family repressor. Transcriptional induction of the ptv operon by vancomycin was achieved by transcriptional derepression of this locus, which was mediated by PtvR. Importantly, fully derepressing ptvABC by deleting ptvR or overexpressing the ptv operon with an exogenous promoter significantly enhanced vancomycin tolerance. Gene deletion analysis revealed that PtvA, PtvB, and PtvC are all required for the PtvR-regulated phenotypic tolerance to vancomycin. Finally, the results of an electrophoretic mobility shift assay with recombinant PtvR showed that PtvR represses the transcription of the ptv operon by binding to two palindromic sequences within the ptv promoter. Together, the ptv locus represents an inducible system in S. pneumoniae in response to stressful conditions, including those caused by antibiotics.IMPORTANCE Reversible or phenotypic tolerance to antibiotics within microbial populations is associated with treatment failure of bacterial diseases, but the underlying mechanisms regulating phenotypic drug tolerance remain obscure. This study reports our finding of a multigene locus that contributes to inducible tolerance to vancomycin in Streptococcus pneumoniae, an important opportunistic human pathogen. The vancomycin tolerance phenotype depends on the PtvR transcriptional repressor and three predicted membrane-associated proteins encoded by the ptv locus. This represents the first example of a gene locus in S. pneumoniae that is responsible for antibiotic tolerance and has important implications for further understanding bacterial responses and phenotypic tolerance to antibiotic treatment in this and other pathogens.

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases.

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types.

qDNAmod: a statistical model-based tool to reveal intercellular heterogeneity of DNA modification from SMRT sequencing data.

In an isogenic cell population, phenotypic heterogeneity among individual cells is common and critical for survival of the population under different environment conditions. DNA modification is an important epigenetic factor that can regulate phenotypic heterogeneity. The single molecule real-time (SMRT) sequencing technology provides a unique platform for detecting a wide range of DNA modifications, including N6-methyladenine (6-mA), N4-methylcytosine (4-mC) and 5-methylcytosine (5-mC). Here we present qDNAmod, a novel bioinformatic tool for genome-wide quantitative profiling of intercellular heterogeneity of DNA modification from SMRT sequencing data. It is capable of estimating proportion of isogenic haploid cells, in which the same loci of the genome are differentially modified. We tested the reliability of qDNAmod with the SMRT sequencing data of Streptococcus pneumoniae strain ST556. qDNAmod detected extensive intercellular heterogeneity of DNA methylation (6-mA) in a clonal population of ST556. Subsequent biochemical analyses revealed that the recognition sequences of two type I restriction-modification (R-M) systems are responsible for the intercellular heterogeneity of DNA methylation initially identified by qDNAmod. qDNAmod thus represents a valuable tool for studying intercellular phenotypic heterogeneity from genome-wide DNA modification.

Detecting DNA modifications from SMRT sequencing data by modeling sequence context dependence of polymerase kinetic.

DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering the same local sequence contexts of interest. We develop an Empirical Bayesian hierarchical model for incorporating historical data. Our results show that the model could greatly increase DNA modification detection accuracy, and reduce requirement of control data coverage. For some DNA modifications that have a strong signal, a control sample is not even needed by using historical data as alternative to control. Thus, sequencing costs can be greatly reduced by using the model. We implemented the model in a R package named seqPatch, which is available at https://github.com/zhixingfeng/seqPatch.

DEGseq: an R package for identifying differentially expressed genes from RNA-seq data.

SUMMARY: High-throughput RNA sequencing (RNA-seq) is rapidly emerging as a major quantitative transcriptome profiling platform. Here, we present DEGseq, an R package to identify differentially expressed genes or isoforms for RNA-seq data from different samples. In this package, we integrated three existing methods, and introduced two novel methods based on MA-plot to detect and visualize gene expression difference. AVAILABILITY: The R package and a quick-start vignette is available at http://bioinfo.au.tsinghua.edu.cn/software/degseq

Detecting and phasing minor single-nucleotide variants from long-read sequencing data.

Cellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, and co-infection of multiple pathogens. Detecting and phasing minor variants play an instrumental role in deciphering cellular genetic heterogeneity, but they are still difficult tasks because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, provide an opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrate that iGDA can accurately reconstruct haplotypes in closely related strains of the same species (divergence ≥0.011%) from long-read metagenomic data.

Molecular detection of colistin resistance genes (mcr-1, mcr-2 and mcr-3) in nasal/oropharyngeal and anal/cloacal swabs from pigs and poultry.

Antimicrobial resistance against colistin has emerged worldwide and is threatening the efficacy of colistin treatment of multi-resistant Gram-negative bacteria. In this study, PCRs were used to detect mcr genes (mcr-1, mcr-2, mcr-3) in 213 anal and 1,339 nasal swabs from pigs (n = 1,454) in nine provinces of China, and 1,696 cloacal and 1,647 oropharyngeal samples from poultry (n = 1,836) at live-bird markets in 24 provinces. The mcr-1 prevalences in pigs (79.2%) and geese (71.7%) were significantly higher than in chickens (31.8%), ducks (34.6%) and pigeons (13.1%). The mcr-2 prevalence in pigs was 56.3%, significantly higher than in chickens (5.5%), ducks (2.3%), geese (5.5%) and pigeons (0%). The mcr-3 prevalences in pigs (18.7%), ducks (13.8%) and geese (11.9%) were significantly higher than in chickens (5.2%) and pigeons (5.1%). In total, 173 pigs and three chickens were positive for all three mcr genes. The prevalences of the mcr were significantly higher in nasal/oropharyngeal swabs than in the anal /cloacal swabs. Phylogenetic studies identified 33 new mcr-2 variants and 12 new mcr-3 variants. This study demonstrates high prevalences of mcr in pigs and poultry in China, and indicates there is need for more thorough surveillance and control programs to prevent further selection of colistin resistance.

[Free jejunum reconstruction and laryngeal preservation for squamous cell carcinoma in the pharyngoesophageal junction].

OBJECTIVE: Try to use free jejunum flaps reconstruction and laryngeal preservation for squamous cell carcinoma (SCC) in the pharyngoesophageal junction. METHODS: Thirteen patients who underwent resections of SCC in the pharyngoesophageal junction with free jejunal interposition from August 2007 to December 2012 were reviewed. Of them, 8 had T3 lesions, 4 had T4 lesions, and one had radiation failure with rT2 lesion. Ten patients were treated with postoperative radiotherapy with a average dosage of 56 Gy. RESULTS: The 3 year over all survival rate was 47.9% and disease-specific survival rate was 34.2%. The surgical complications occurred in 9 patients (9/13), including one death and one flap failure. Five patients (5/13) had permanent tracheal canulation, 10 patients (10/13) resumed oral feeding and all patients achieved reasonable speech. CONCLUSION: Free jejunum interposition can be used to reconstruct surgical defect of SCC in the pharyngoesophageal junction, thus preserving the larynx and ensuring a better quality of life for the patients.

[Expression and significance of survivin and PCNA in sinonasal inverted papilloma].

OBJECTIVE: To explore the expression and significance of survivin and proliferating cell nuclear antigen (PCNA) on the occurrence, proliferation, recurrence and carcinogenesis of the sinonasal inverted papilloma (SNIP). METHOD: Immunohistochemical method was used to detect the expression of survivin and PCNA in 10 cases of nasal cavity mucosal (NM), 45 cases of SNIP and 9 cases of canceration SNIP. RESULT: The positive expression of survivin and PCNA increased gradually in NM,SNIP and canceration PCNA group, and there were significant difference between the three groups. But there was no correlation between survivin and PCNA in the tissue of SNIP (r = 0.135, P > 0.05). CONCLUSION: Survivin and PCNA are involved in the growth and carcinogenesis of SNIP.

Identifying differentially spliced genes from two groups of RNA-seq samples.

Recent study revealed that most human genes have alternative splicing and can produce multiple isoforms of transcripts. Differences in the relative abundance of the isoforms of a gene can have significant biological consequences. Identifying genes that are differentially spliced between two groups of RNA-sequencing samples is an important basic task in the study of transcriptomes with next-generation sequencing technology. We use the negative binomial (NB) distribution to model sequencing reads on exons, and propose a NB-statistic to detect differentially spliced genes between two groups of samples by comparing read counts on all exons. The method opens a new exon-based approach instead of isoform-based approach for the task. It does not require information about isoform composition, nor need the estimation of isoform expression. Experiments on simulated data and real RNA-seq data of human kidney and liver samples illustrated the method's good performance and applicability. It can also detect previously unknown alternative splicing events, and highlight exons that are most likely differentially spliced between the compared samples. We developed an NB-statistic method that can detect differentially spliced genes between two groups of samples without using a prior knowledge on the annotation of alternative splicing. It does not need to infer isoform structure or to estimate isoform expression. It is a useful method designed for comparing two groups of RNA-seq samples. Besides identifying differentially spliced genes, the method can highlight on the exons that contribute the most to the differential splicing. We developed a software tool called DSGseq for the presented method available at http://bioinfo.au.tsinghua.edu.cn/software/DSGseq.

Compound heterozygous mutations of SLC26A4 in 4 Chinese families with enlarged vestibular aqueduct.

OBJECTIVE: Enlarged vestibular aqueduct is the most common inner ear malformation in individuals with sensorineural hearing loss. Mutations in SLC26A4 can cause non-syndromic EVA. To date, more than 170 SLC26A4 mutations have been described. The aim of the present study was to detect and report genetic causes of four unrelated Chinese families with hearing loss. METHODS: We evaluated 4 families presenting bilateral enlarged vestibular aqueducts and describe the clinical and molecular characteristics of 5 patients. RESULTS: The SLC26A4 gene was sequenced in 23 members of these 4 Chinese families with EVA, and the patients were found to carry 4 compound heterozygous mutations, p.G197R and p.S391R, IVS7-2A>G, p.I188T and c.1746 del G, p.V659L and p.T410M, and p.T94I and p.G197R, none of which have been reported previously. CONCLUSIONS: These results emphasize the necessity of considering the complete DNA sequencing of the SLC26A4 gene in molecular diagnosis of deafness, especially when phenotypes such as congenital, invariable, and progressive hearing loss with EVA are present.

Novel mutations of SLC26A4 in Chinese patients with nonsyndromic hearing loss.

CONCLUSIONS: This study demonstrated high prevalence of GJB2, SLC26A4, and mtDNA A1555G mutations in Chinese patients with nonsyndromic hearing loss and discovered eight novel mutations in SLC26A4. Most of these novel mutations were predicted pathogenic variants. OBJECTIVES: Nonsyndromic hearing loss is the most common neurosensory deafness where the majority of patients have highly diversified genetic defects. This study aimed to define the genetic profile of deafness in a Chinese population with potential to discover novel mutations. METHODS: A total of 227 segregating deaf students and 200 individuals with normal hearing were enrolled. With the Sanger sequencing chemistry, direct sequencing was performed on entire coding regions of GJB2, GJB3, SLC26A4, and mtDNA m.C1494T and m.A1555G. RESULTS: Direct sequencing analysis revealed that 53 (23.35%) of 227 patients carried at least 1 mutant allele in GJB2, 40 (17.62%) patients in SLC26A4, 5 (2.20%) patients in mtDNA A1555G, and 1 (0.44%) patient in mtDNA C1494T mutations. Four patients carried three unclassified mutations in GJB3 genes. Overall 38 mutant variants were detected in this cohort of patients, including 8 novel mutations in SLC26A4. The eight novel variants were six missense substitutions (p.V163L, p.G222S, p.A456D, p.N457I, p.C466Y, p.F667L), one nonsense mutation (p.W472X), and one frameshift (p.Asn612Ilefs×23).

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.