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July 7, 2019

Genomics of Helicobacter pylori

As Helicobacter pylori infects half the world’s population and displays an extensive intraspecies diversity, genomics is a powerful tool to understand evolution and disease, to identify factors that confer higher risk of severe sequelae, and to find new approaches for therapy both among bacterial and host targets. In line with these objectives, this review article summarizes the major findings in Helicobacter genomics in papers published between April 2016 and March 2017.

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July 7, 2019

Simultaneous production of Anabaenopeptins and Namalides by the cyanobacterium Nostoc sp. CENA543.

Anabaenopeptins are a diverse group of cyclic peptides, which contain an unusual ureido linkage. Namalides are shorter structural homologues of anabaenopeptins, which also contain an ureido linkage. The biosynthetic origins of namalides are unknown despite a strong resemblance to anabaenopeptins. Here, we show the cyanobacterium Nostoc sp. CENA543 strain producing new (nostamide B-E (2, 4, 5, and 6)) and known variants of anabaenopeptins (schizopeptin 791 (1) and anabaenopeptin 807 (3)). Surprisingly, Nostoc sp. CENA543 also produced namalide B (8) and the new namalides D (7), E (9), and F (10) in similar amounts to anabaenopeptins. Analysis of the complete Nostoc sp. CENA543 genome sequence indicates that both anabaenopeptins and namalides are produced by the same biosynthetic pathway through module skipping during biosynthesis. This unique process involves the skipping of two modules present in different nonribosomal peptide synthetases during the namalide biosynthesis. This skipping is an efficient mechanism since both anabaenopeptins and namalides are synthesized in similar amounts by Nostoc sp. CENA543. Consequently, gene skipping may be used to increase and possibly broaden the chemical diversity of related peptides produced by a single biosynthetic gene cluster. Genome mining demonstrated that the anabaenopeptin gene clusters are widespread in cyanobacteria and can also be found in tectomicrobia bacteria.

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July 7, 2019

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni-derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase (ctsM), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM(+) (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni, whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria.

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July 7, 2019

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena.

DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

DNA methylation profiling using long-read Single Molecule Real-Time bisulfite sequencing (SMRT-BS).

For the past two decades, bisulfite sequencing has been a widely used method for quantitative CpG methylation detection of genomic DNA. Coupled with PCR amplicon cloning, bisulfite Sanger sequencing allows for allele-specific CpG methylation assessment; however, its time-consuming protocol and inability to multiplex has recently been overcome by next-generation bisulfite sequencing techniques. Although high-throughput sequencing platforms have enabled greater accuracy in CpG methylation quantitation as a result of increased bisulfite sequencing depth, most common sequencing platforms generate reads that are similar in length to the typical bisulfite PCR size range (~300-500 bp). Using the Pacific Biosciences (PacBio) sequencing platform, we developed single molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplexing and long read lengths. SMRT-BS is reproducible and was found to be concordant with other lower throughput quantitative CpG methylation methods. Moreover, the ability to sequence up to ~1.5-2.0 kb amplicons, when coupled with an optimized bisulfite-conversion protocol, allows for more thorough assessment of CpG islands and increases the capacity for studying the relationship between single nucleotide variants and allele-specific CpG methylation.

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July 7, 2019

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori.

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ?modH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

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July 7, 2019

Genome-wide epigenetic studies in chicken: A review

Over the years, farmed birds have been selected on various performance traits mainly through genetic selection. However, many studies have shown that genetics may not be the sole contributor to phenotypic plasticity. Gene expression programs can be influenced by environmentally induced epigenetic changes that may alter the phenotypes of the developing animals. Recently, high-throughput sequencing techniques became sufficiently affordable thanks to technological advances to study whole epigenetic landscapes in model plants and animals. In birds, a growing number of studies recently took advantage of these techniques to gain insights into the epigenetic mechanisms of gene regulation in processes such as immunity or environmental adaptation. Here, we review the current gain of knowledge on the chicken epigenome made possible by recent advances in high-throughput sequencing techniques by focusing on the two most studied epigenetic modifications, DNA methylation and histone post-translational modifications. We discuss and provide insights about designing and performing analyses to further explore avian epigenomes. A better understanding of the molecular mechanisms underlying the epigenetic regulation of gene expression in relation to bird phenotypes may provide new knowledge and markers that should undoubtedly contribute to a sustainable poultry production.

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July 7, 2019

Unlocking the biological potential of Euglena gracilis: evolution, cell biology and significance to parasitism

Photosynthetic euglenids are major components of aquatic ecosystems and relatives of trypanosomes. Euglena gracilis has considerable biotechnological potential and great adaptability, but exploitation remains hampered by the absence of a comprehensive gene catalogue. We address this by genome, RNA and protein sequencing: the E. gracilis genome is >2Gb, with 36,526 predicted proteins. Large lineage-specific paralog families are present, with evidence for flexibility in environmental monitoring, divergent mechanisms for metabolic control, and novel solutions for adaptation to extreme environments. Contributions from photosynthetic eukaryotes to the nuclear genome, consistent with the shopping bag model are found, together with transitions between kinetoplastid and canonical systems. Control of protein expression is almost exclusively post-transcriptional. These data are a major advance in understanding the nuclear genomes of euglenids and provide a platform for investigating the contributions of E. gracilis and its relatives to the biosphere.

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July 7, 2019

Effects of genome structure variation, homeologous genes and repetitive DNA on polyploid crop research in the age of genomics.

Compared to diploid species, allopolyploid crop species possess more complex genomes, higher productivity, and greater adaptability to changing environments. Next generation sequencing techniques have produced high-density genetic maps, whole genome sequences, transcriptomes and epigenomes for important polyploid crops. However, several problems interfere with the full application of next generation sequencing techniques to these crops. Firstly, different types of genomic variation affect sequence assembly and QTL mapping. Secondly, duplicated or homoeologous genes can diverge in function and then lead to emergence of many minor QTL, which increases difficulties in fine mapping, cloning and marker assisted selection. Thirdly, repetitive DNA sequences arising in polyploid crop genomes also impact sequence assembly, and are increasingly being shown to produce small RNAs to regulate gene expression and hence phenotypic traits. We propose that these three key features should be considered together when analyzing polyploid crop genomes. It is apparent that dissection of genomic structural variation, elucidation of the function and mechanism of interaction of homoeologous genes, and investigation of the de novo roles of repeat sequences in agronomic traits are necessary for genomics-based crop breeding in polyploids. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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July 7, 2019

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the a-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future. © 2015 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019

Complete genome sequence of Celeribacter marinus IMCC12053(T), the host strain of marine bacteriophage P12053L.

Isolated from coastal seawater from Yellow Sea of Korea, Celeribacter marinus IMCC12053 was used as the host bacterium for bacteriophage P12053L. Here we report the complete genome sequence of strain IMCC12053 for further study of the marine bacteriophage P12053L functional genes. Single molecule real-time technology (PacBio RSII) was used for the single circular chromosome that is 3,096,705 base pairs in length and the GC content is 56.24%. It contains 3155 ORFs with 45 tRNAs and 6 rRNAs genes. N(6)-methyladenosine patterns were also investigated for 32 unmethylated genes and intergenic regions that covered many regulators and phage genes as well as ribosomal RNA genes and tRNA genes. Cryptic N(4)-methylcytosine pattern was investigated to speculate GpC methylase activity throughout the genome. Comparative genomics with other Celeribacter genomes were carried out for polyaromatic hydrocarbon degradation, but there were no aromatic ring oxygenases in IMCC12053 when compared to Celeribacter indicus P73. Copyright © 2015 Elsevier B.V. All rights reserved.

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July 7, 2019

CauloBrowser: A systems biology resource for Caulobacter crescentus.

Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of high-throughput spatiotemporal databases including data from gene expression profiling experiments (microarrays, RNA-seq, ChIP-seq, ribosome profiling, LC-ms proteomics), gene essentiality studies (Tn-seq), genome wide protein localization studies, and global chromosome methylation analyses (SMRT sequencing). A major challenge involves the integration of these diverse data sets into one comprehensive community resource. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica.

Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvAB(OFF)) and a lineage with shorter O-antigen chains (OpvAB(ON)). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1-4). OpvAB(OFF) and OpvAB(ON) cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvAB(OFF) state, GATC1 and GATC3 are non-methylated, whereas GATC2 and GATC4 are methylated; (ii) in the OpvAB(ON) state, GATC2 and GATC4 are non-methylated, whereas GATC1 and GATC3 are methylated. We provide evidence that such DNA methylation patterns are generated by OxyR binding. The higher stability of the OpvAB(OFF) lineage may be caused by binding of OxyR to sites that are identical to the consensus (OBSA and OBSc), while the sites bound by OxyR in OpvAB(ON) cells (OBSB and OBSD) are not. In support of this view, amelioration of either OBSB or OBSD locks the system in the ON state. We also show that the GATC-binding protein SeqA and the nucleoid protein HU are ancillary factors in opvAB control.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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