scholarly journals Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

2016 ◽  
Author(s):  
Aaron Smargon ◽  
David B.T. Cox ◽  
Neena Pyzocha ◽  
Kaijie Zheng ◽  
Ian M. Slaymaker ◽  
...  
Keyword(s):  
Rna Interference ◽  
Essential Gene ◽  
Sequence Database ◽  
Flanking Sequence ◽  
E Coli ◽  
Adaptive Immune ◽  
Associated Proteins ◽  
Adaptive Immune Systems ◽  
Target Rna

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

scholarly journals Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

2020 ◽  
Vol 48 (10) ◽  
pp. 5624-5638
Author(s):  
Thomas Jacobsen ◽  
Fani Ttofali ◽  
Chunyu Liao ◽  
Srinivas Manchalu ◽  
Benjamin N Gray ◽  
...  
Keyword(s):  
Selective Pressure ◽  
Amino Acid Identity ◽  
Human Cells ◽  
High Sequence Identity ◽  
Guide Rnas ◽  
Adaptive Immune ◽  
Translation Systems ◽  
Adaptive Immune Systems ◽  
Single Effector

Abstract CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G’s in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

scholarly journals Optimization of Methods for the Accurate Characterization of Whole Blood Neutrophils

2021 ◽  
Author(s):  
Ashley N. Connelly ◽  
Richard P.H. Huijbregts ◽  
Harish C. Pal ◽  
Valeriya Kuznetsova ◽  
Marcus D. Davis ◽  
...  
Keyword(s):  
Whole Blood ◽  
Neutrophil Activation ◽  
Immune Defense ◽  
Experimental Conditions ◽  
Adaptive Immune ◽  
Blood Neutrophils ◽  
One Step ◽  
And Function ◽  
Adaptive Immune Systems

Abstract Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4ºC and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.

scholarly journals Characterization of 67 Confirmed Clustered Regularly Interspaced Short Palindromic Repeats Loci in 52 Strains of Staphylococci

2021 ◽  
Vol 12 ◽  
Author(s):  
Ying Wang ◽  
Tingting Mao ◽  
Yinxia Li ◽  
Wenwei Xiao ◽  
Xuan Liang ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Direct Repeat ◽  
Minimum Free Energy ◽  
Important Species ◽  
Crispr Locus ◽  
Adaptive Immune ◽  
Associated Proteins ◽  
The Relationship ◽  
Cas Proteins

Staphylococcus aureus (S. aureus), which is one of the most important species of Staphylococci, poses a great threat to public health. Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune platform to combat foreign mobile genetic elements (MGEs) such as plasmids and phages. The aim of this study is to describe the distribution and structure of CRISPR-Cas system in S. aureus, and to explore the relationship between CRISPR and horizontal gene transfer (HGT). Here, we analyzed 67 confirmed CRISPR loci and 15 companion Cas proteins in 52 strains of Staphylococci with bioinformatics methods. Comparing with the orphan CRISPR loci in Staphylococci, the strains harboring complete CRISPR-Cas systems contained multiple CRISPR loci, direct repeat sequences (DR) forming stable RNA secondary structures with lower minimum free energy (MFE), and variable spacers with detectable protospacers. In S. aureus, unlike the orphan CRISPRs away from Staphylococcal cassette chromosome mec (SCCmec), the complete CRISPR-Cas systems were in J1 region of SCCmec. In addition, we found a conserved motif 5′-TTCTCGT-3′ that may protect their downstream sequences from DNA interference. In general, orphan CRISPR locus in S. aureus differed greatly from the structural characteristics of the CRISPR-Cas system. Collectively, our results provided new insight into the diversity and characterization of the CRISPR-Cas system in S. aureus.

scholarly journals Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

2021 ◽  
Author(s):  
Alexander J Meeske ◽  
Matthew C Johnson ◽  
Logan T Hille ◽  
Benjamin P Kleinstiver ◽  
Joseph Bondy-Denomy
Keyword(s):  
Nucleic Acid ◽  
Rna Cleavage ◽  
Nucleic Acid Binding ◽  
Bacterial Strains ◽  
Bioinformatics Analyses ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Bona Fide ◽  
Adaptive Immune Systems

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.

scholarly journals Characterization of the glnK-amtB Operon of Azotobacter vinelandii

1998 ◽  
Vol 180 (12) ◽  
pp. 3260-3264 ◽  
Author(s):  
Dietmar Meletzus ◽  
Paul Rudnick ◽  
Natalie Doetsch ◽  
Andrew Green ◽  
Christina Kennedy
Keyword(s):  
Azotobacter Vinelandii ◽  
Essential Gene ◽  
Transport Proteins ◽  
Ammonium Transport ◽  
Translation Product ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Type Strains

ABSTRACT To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nifgene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized. Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was cotranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote.glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.

scholarly journals Proteome dynamics in persisting and resuscitating bacterial cells

2020 ◽  
Author(s):  
Maja Semanjski ◽  
Fabio Gratani ◽  
Till Englert ◽  
Viktor Beke ◽  
Nicolas Nalpas ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Treatment Time ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Time Resolved ◽  
E Coli ◽  
Associated Proteins ◽  
Label Incorporation ◽  
Insight Into

AbstractBacterial persister cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of bacterial persistence is hindered by low count of persisting cells and the need for their isolation. Here we used sustained addition of stable isotope-labeled lysine to selectively label the proteome of hipA-induced persisting and hipB-induced resuscitating E. coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-hour measurement of label incorporation allowed detection of over 500 newly synthetized proteins in persister cells, demonstrating low but widespread protein synthesis. Many essential proteins were newly synthesized and several ribosome-associated proteins showed unusually high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized ABC transporters, restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. This dataset provides an unprecedented insight into the processes governing persistence and resuscitation of bacterial cells.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Antimicrobial Resistance and Virulence Characterization of E. Coli Isolated from Subclinical Mastitic Sheep and Goats

2018 ◽  
Vol 34 (3) ◽  
pp. 267-278
Author(s):  
Ashraf A. Abd El-Tawab ◽  
Mohamed G. Aggour ◽  
Fatma I. El- Hofy ◽  
Marwa M. Y. El- Mesalami
Keyword(s):  
Antimicrobial Resistance ◽  
Sheep And Goats ◽  
E Coli

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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