Exosialidases (also termed neuraminidases; E.C. 188.8.131.52) are glycoside hydrolases that catalyze removal of a single terminal sialic acid from a subterminal sugar in an oligosaccharide. They are widely distributed in biology, having been found in prokaryotes, eukaryotes and certain viruses. Most characterized prokaryotic sialidases derive from organisms that are pathogenic or commensal with mammals. Less is known about sialidases from noncommensal microorganisms, including those that thrive in an extreme environmental niche like hypersaline ponds, evaporation salterns or thermal springs. In this study, we sought to explore if active sialidases could be identified from organisms that populate a thermal spring.
To address this question, we constructed a fosmid library in Escherichia coli from metagenomic DNA that
had been isolated from green microbial mats from the Dixie Hot Spring in Nevada. A total of 616 E. coli clones, each
having a fosmid with an insert of ~40 kb of environmental DNA, were created and arrayed in microtiter plates for
screening. The library was screened for sialidases with two substrates: 2′-(4-methylumbelliferyl)-α-D-Nacetylneuraminic
acid (4MU-Neu5Ac) and 5-bromo-4-chloro-3-indolyl α-D-N-acetylneuraminic acid (X-Neu5Ac).
A single E. coli clone having sialidase activity was identified using both substrates. The fosmid was isolated from this strain, sequenced using the Pacific Biosciences DNA sequencing platform, and encoded ORFs were predicted with MetaGeneMark. The DNA sequence did not match any reported sequences from known microorganisms. Additionally, none of the predicted ORFs showed homology to existing sialidase families. Tn5 mutagenesis was conducted to identify the 505 amino acid ORF responsible for the enzymatic activity. BLASTP using the ORF’s deduced protein sequence analysis indicated that it was a member of a small family of bacterial “hypothetical” proteins with no known function. The protein was recombinantly over-expressed in E. coli and was shown to hydrolyze a variety of sialic acid containing substrates. Additionally, protein NMR showed that the enzyme functions via an inverting catalytic mechanism, a biochemical property distinct from known exosialidases that each function via a retaining mechanism. This unique inverting exosialidase defines a novel CAZy glycoside hydrolase family that has been designated GH154.
The periplasmic protease/chaperone DegP (HtrA) plays a key role in the quality control of many proteins in the periplasm of E. coli. Proteins that fail to fold in the periplasm can be proteolysed, while others are chaperoned to their native folded state by DegP. In a ΔdegP strain, E. coli is unable to survive the protein folding stress at elevated temperatures. Utilizing this phenotype, we developed a plasmid-based selection of suppression of heat-induced lethality in a ΔdegP strain. Plasmid libraries of various prokaryotic genomes were screened for proteins that overcame heat-induced lethality. Initial hits indicate novel mechanisms of overcoming periplasmic stress, such as the periplasmic expression of a cytoplasmic GrpE homolog and the cytoplasmic expression of an unknown protein.
Open chromatin profiling integrates information across diverse regulatory elements to reveal the transcriptionally active genome. Tn5 transposase and DNase I sequencing-based methods prefer native or high cell numbers. Here, we describe NicE-seq (nicking enzyme assisted sequencing) for high-resolution open chromatin profiling on both native and formaldehyde-fixed cells using 25 to 250K cells. Lower cell numbers (25 and 250 cells) require lower amounts of enzyme mix. NicE-seq captures and reveals open chromatin sites (OCSs) and transcription factor occupancy at single nucleotide resolution, coincident with DNase hypersensitive and ATAC-seq (Tn5 transposase based) sites at a low sequencing burden. OCSs correlate with RNA polymerase II occupancy and active chromatin marks, while displaying a contrasting pattern to CpG methylation. Decitabine-mediated hypomethylation of HCT116 displays higher numbers of OCSs.
Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their geneLc material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the DelPia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α- putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2ʹ-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635–7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy) methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.
Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share about 17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics we directly compare SpyCas9 and SauCas9 activities. In contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.
The Polymerase Chain Reaction (PCR) is an integral part of many NGS sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. We utilized a single-molecule sequencing assay to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage caused by thermocycling.
Much of modern biology and medicine depends on the ability to accurately determine the identity of a target, whether virus, tumor, mystery infection, or even an uncertain type of food. Fortunately, all living things and viruses carry unique identifiers in their DNA or RNA genomes. Once a sequence identifier is determined, we still need to be able to find it, using a nucleic acid amplification method for precisely identifying a particular sequence. This amplification can be analyzed to see what is made and how much of a particular DNA was present in the sample to begin with. And only the specific target of interest will be amplified, enabling accurate detection of the sequences that we are looking for. With demands and applications for nucleic acid identification growing all the time, new methods have been developed to allow detection more easily, rapidly, and in more settings. A popular example is loop-mediated isothermal amplification (LAMP) which works at a single temperature (isothermal) using DNA polymerases with a special ability to go through double-stranded DNA without heating (“strand displacement”) activity. NEB developed a novel colorimetric format that reacts to DNA synthesis by changing color from pink to yellow as a direct visual response to DNA polymerase adding bases to the growing DNA products. This simple readout of amplification, paired with the speed and robustness of LAMP make for a useful diagnostic tool, with LAMP being used for easy detection of targets everywhere from farms to doctors’ offices, and recently, the International Space Station! If it has DNA or RNA, we can find it, and LAMP will let it be done easily, rapidly and right where you need the answer. While there are plenty of those places on Earth, we’ll soon have them in space too, and with methods like LAMP our astronauts, their food supplies, and their homes can be kept safe.
Molecular diagnostic methods to detect DNA and RNA targets represent a significant and growing focus for point-of-care and rapid testing. Though varied in application, design, and even mechanism, these methods all rely on enzymes to carry out the reactions. NEB has long provided reagents and enzymes to enable molecular biology research, including providing reliable and novel materials for molecular diagnostic applications. Isothermal amplification methods have emerged as a promising option for reliable point-of-care diagnostics and our research and development on these techniques have produced novel and more versatile isothermal DNA polymerases for faster and robust amplification; WarmStart® technology to permit room-temperature reaction set-up and consistent performance; and colorimetric LAMP detection for field and point-of-care applications. For customization of methods and use, reagents are tested for stability in glycerol-free formats and compatibility with lyophilization. And to enable a wide range of intended applications, we have developed reagents that support the next generation of tests in isothermal (SDA, NASBA, LAMP) and PCR or qPCR methods. Developing better reagents for molecular diagnostics is essential to broader adoption and reach of these powerful tools.
Next Generation Sequencing (NGS) is expanding its applications. Laboratories are increasingly implementing NGS and incrementing the number of samples to process. The ability to construct high quality sequencing libraries in a fast turnaround time has become critical. Automating sequencing library preparation reduces bottlenecks, enables higher throughput and minimizes human errors. This work describes the flexible, automated NEBNext® Ultra™ II DNA library preparation protocols on the TECAN® Freedom EVO® NGS workstation. This simple workflow allows for highly reproducible library preparation from a wide range of DNA input (picograms to a microgram), and with variable quality (from intact to heavily degraded FFPE samples). Automation of this protocol on the Freedom EVO NGS workstation enables flexible sample numbers from 1-96, and minimizes hands-on time with minimal user intervention. High input (200ng) and low input (500pg) human and yeast genomic DNA libraries generated on the Freedom EVO NGS workstation have comparable library performance (high yield, absence of adaptor dimer) to those obtained from manual libraries. NEXTSeq® sequencing data shows high quality libraries. The high percentage of aligned reads (>97.7% mapped reads and >99.03% mapped in pairs) and the low percentage of chimeras (<1%) and adaptor-mapping reads (<0.001%) observed indicate that the Tecan automation of the NEBNext Ultra II DNA Library Prep workflow enables high quality sequence data, even with very low input amounts. GC coverage information obtained indicates that automated Ultra II DNA libraries have very uniform coverage across the range of GC content. This automated method provides a much-needed resource for the reliable preparation of DNASeq libraries from a broad range of sample types and input amounts.
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