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Sunday, September 22, 2019

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene…

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Sunday, September 22, 2019

Characterizing the DNA methyltransferases of Haloferax volcanii via bioinformatics, gene deletion, and SMRT Sequencing.

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeonHaloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase…

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Sunday, September 22, 2019

The structure of a conserved telomeric region associated with variant antigen loci in the blood parasite Trypanosoma congolense

African trypanosomiasis is a vector-borne disease of humans and livestock caused by African trypanosomes (Trypanosoma spp.). Survival in the vertebrate bloodstream depends on antigenic variation of Variant Surface Glycoproteins (VSGs) coating the parasite surface. In T. brucei, a model for antigenic variation, monoallelic VSG expression originates from dedicated VSG expression sites (VES). Trypanosoma brucei VES have a conserved structure consisting of a telomeric VSG locus downstream of unique, repeat sequences, and an independent promoter. Additional protein-coding sequences, known as “Expression Site Associated Genes (ESAGs)”, are also often present and are implicated in diverse, bloodstream-stage functions. Trypanosoma congolense is a related…

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Sunday, September 22, 2019

The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools.

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The…

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Sunday, September 22, 2019

Improved nucleic acid extraction protocols for Ganoderma boninense, G. miniatocinctum and G. tornatum.

The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm…

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Friday, July 19, 2019

Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.

Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an…

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Friday, July 19, 2019

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically…

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Sunday, July 7, 2019

Complete genome sequence of the Clostridium difficile laboratory strain 630¿ erm reveals differences from strain 630, including translocation of the mobile element CTn 5.

Background Clostridium difficile strain 630¿erm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns.ResultsIn addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than…

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Sunday, July 7, 2019

Complete genome sequencing of Pandoraea pnomenusa RB38 and molecular characterization of its N-acyl homoserine lactone synthase gene ppnI.

In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar…

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Sunday, July 7, 2019

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors.

The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis…

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Sunday, July 7, 2019

Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme.

Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum.Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By…

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Sunday, July 7, 2019

Identification of a Pseudomonas aeruginosa PAO1 DNA methyltransferase, its targets, and physiological roles.

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N(6)-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes…

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Sunday, July 7, 2019

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation.

Butanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of…

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Sunday, July 7, 2019

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n?=?14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species.…

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Sunday, July 7, 2019

A roadmap for gene system development in Clostridium.

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron…

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