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May 1, 2017

Bypassing the restriction system to improve transformation ofStaphylococcus epidermidis.

Staphylococcusepidermidisis the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prostheses. With the exception of two widely-passaged isolates RP62A and 1457, the pathogenesis of infections caused by clinicalS. epidermidisstrains is poorly understood due to the strong genetic barrier that precludes efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinicalS. epidermidisisolates is primarily due to the type I and IV restriction modification systems which act as genetic barriers. Here, we showed…

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May 1, 2017

Efficient genome editing of a facultative thermophile using mesophilic spCas9.

Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates…

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February 9, 2017

DNA target recognition domains in the Type I restriction and modification systems of Staphylococcus aureus.

Staphylococcus aureus displays a clonal population structure in which horizontal gene transfer between different lineages is extremely rare. This is due, in part, to the presence of a Type I DNA restriction–modification (RM) system given the generic name of Sau1, which maintains different patterns of methylation on specific target sequences on the genomes of different lineages. We have determined the target sequences recognized by the Sau1 Type I RM systems present in a wide range of the most prevalent S. aureus lineages and assigned the sequences recognized to particular target recognition domains within the RM enzymes. We used a range…

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February 8, 2017

Habitat generalists or specialists, insights from comparative genomic analyses of Thermosipho lineages

Thermosipho species inhabit various extreme environments such as marine hydrothermal vents, petroleum reservoirs and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents and hotsprings. Comparative genomics and recombination analysis of the genomes of 15 Thermosipho isolates separated them into three species with different habitat distributions, the widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. The three Thermosipho species can also be differentiated on the basis of genome content. For instance the T. africanus…

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November 10, 2016

Complete genome sequence of Brevibacterium linens SMQ-1335.

Brevibacterium linens is one of the main bacteria found in the smear of surface-ripened cheeses. The genome of the industrial strain SMQ-1335 was sequenced using PacBio. It has 4,209,935 bp, a 62.6% G+C content, 3,848 open reading frames, and 61 structural RNAs. A new type I restriction-modification system was identified. Copyright © 2016 de Melo et al.

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November 2, 2016

Novel m4C modification in type I restriction-modification systems.

We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase…

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October 1, 2016

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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September 30, 2016

Novel methyltransferase recognition motif identified in Chania multitudinisentens RB-25(T) gen. nov., sp. nov.

DNA methylation, defined by the addition of a methyl group to adenine or cytosine bases in DNA catalyzed by DNA methyltransferases (MTases), is one of the most studied post-replicative DNA modification mechanism in bacteria (Roberts et al., 2003b). The three forms of nucleotide methylation identified to date are: N6-methyladenine(m6A), N4-methylcytosine (m4C), and 5-methylcytosine (m5C) (Gromova and Khoroshaev, 2003).

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August 20, 2016

Finished genome sequence and methylome of the cyanide-degrading Pseudomonas pseudoalcaligenes strain CECT5344 as resolved by single-molecule real-time sequencing.

Pseudomonas pseudoalcaligenes CECT5344 tolerates cyanide and is also able to utilize cyanide and cyano-derivatives as a nitrogen source under alkaline conditions. The strain is considered as candidate for bioremediation of habitats contaminated with cyanide-containing liquid wastes. Information on the genome sequence of the strain CECT5344 became available previously. The P. pseudoalcaligenes CECT5344 genome was now resequenced by applying the single molecule, real-time (SMRT(®)) sequencing technique developed by Pacific Biosciences. The complete and finished genome sequence of the strain consists of a 4,696,984 bp chromosome featuring a GC-content of 62.34%. Comparative analyses between the new and previous versions of the P.…

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July 21, 2016

Complete genome sequence of highly virulent Haemophilus parasuis serotype 11 strain SC1401.

Haemophilus parasuis, a normal Gram-negative bacterium, may cause Glässer's disease and pneumonia in pigs. This study aims to identify the genes related to natural competence of the serotype 11 strain SC1401, which frequently shows competence and high pathogenicity. SC1401 shows many differences from strains without natural competence within the molecular basis. We performed complete genome sequencing together with restriction modification system analysis to lay the foundation for later study. Copyright © 2016 Dai et al.

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July 1, 2016

Complete nucleotide sequence of pH11, an IncHI2 plasmid conferring multi-antibiotic resistance and multi-heavy metal resistance genes in a clinical Klebsiella pneumoniae isolate.

The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6')-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6')-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium. This plasmid exhibits numerous characteristics, including HipBA and RelBE toxin-antitoxin systems, two major transfer (Tra) regions closely…

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June 6, 2016

Industrial Biotechnology Brochure: Fuel biotech discovery with confident characterization of microbes and their communities

Industrial microbiologists rely on comprehensive genomic information to identify and develop complex biological products. Single Molecule, Real-Time (SMRT) Sequencing delivers a more complete view of individual organisms and microbial communities, fueling research for modern pharmaceutical discovery, environmental remediation, chemical commodity production, and agriculture products.

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June 1, 2016

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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May 19, 2016

Structural insights into DNA sequence recognition by Type ISP restriction-modification enzymes.

Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate…

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