scholarly journals CRISPRbuilder-TB: “CRISPR-builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in mycobacterium tuberculosis complex using SRA

2021 ◽  
Vol 17 (3) ◽  
pp. e1008500
Author(s):  
Christophe Guyeux ◽  
Christophe Sola ◽  
Camille Noûs ◽  
Guislaine Refrégier
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Species ◽  
Direct Repeats ◽  
Crispr Locus ◽  
The Public ◽  
Whole Process ◽  
General Rules ◽  
Genetic Mechanisms ◽  
Set Up

Mycobacterium tuberculosis complex (MTC) CRISPR locus diversity has long been studied solely investigating the presence/absence of a known set of spacers. Unveiling the genetic mechanisms of its evolution requires a more exhaustive reconstruction in a large amount of representative strains. In this article, we point out and resolve, with a new pipeline, the problem of CRISPR reconstruction based directly on short read sequences in M. tuberculosis. We first show that the process we set up, that we coin as “CRISPRbuilder-TB” (https://github.com/cguyeux/CRISPRbuilder-TB), allows an efficient reconstruction of simulated or real CRISPRs, even when including complex evolutionary steps like the insertions of mobile elements. Compared to more generalist tools, the whole process is much more precise and robust, and requires only minimal manual investigation. Second, we show that more than 1/3 of the currently complete genomes available for this complex in the public databases contain largely erroneous CRISPR loci. Third, we highlight how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that describes MTC-CRISPR diversity and suggests general rules for its evolution. This work opens perspectives for an in-depth exploration of M. tuberculosis CRISPR loci diversity and of mechanisms involved in its evolution and its functionality, as well as its adaptation to other CRISPR locus-harboring bacterial species.

scholarly journals Exhaustive reconstruction of the CRISPR locus in Mycobacterium tuberculosis complex using short reads

2019 ◽  
Author(s):  
Christophe Guyeux ◽  
Christophe Sola ◽  
Guislaine Refrégier
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Silico ◽  
Structural Characteristics ◽  
Computational Method ◽  
Direct Repeats ◽  
Crispr Locus ◽  
Short Read ◽  
General Rules ◽  
Genetic Mechanisms

AbstractSpoligotyping, a graphical partial display of the CRISPR locus that can be produced in vitro or in silico, is an important tool for analyzing the diversity of given Mycobacterium tuberculosis complex (MTC) isolates. As other CRISPR loci, this locus is made up of an alternation between direct repeats and spacers, and flanked by cas genes. Unveiling the genetic mechanisms of its evolution requires to have a fairly large amount of fully reconstructed loci among all MTC lineages.In this article, we point out and resolve the problem of CRISPR reconstruction based on short read sequences. We first show that more than 1/3 of the currently assembled genomes available for this complex contain a CRISPR locus erroneously reconstructed, and errors can be very significant. Second, we present a new computational method allowing this locus to be reconstructed extensively and reliably in silico using short read sequencing runs. Third, using this method, we describe new structural characteristics of CRISPR locus by lineages. We show how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that presents general rules for the evolution of the CRISPR locus in MTC.This work opens new perspectives for a larger exploration of CRISPR loci diversity and of mechanisms involved in its evolution and its functionality.

scholarly journals Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Guislaine Refrégier ◽  
Christophe Sola ◽  
Christophe Guyeux
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Insertion Sequence ◽  
Recent Common Ancestor ◽  
Direct Repeats ◽  
Evolutionary Mechanisms ◽  
Evolutionary Patterns ◽  
Crispr Locus ◽  
Most Recent Common Ancestor ◽  
Low Diversity ◽  
Set Up

Abstract Background Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes. Results We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats. Conclusion This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.

scholarly journals Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Guislaine Refrégier ◽  
Christophe Sola ◽  
Christophe Guyeux
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Insertion Sequence ◽  
Recent Common Ancestor ◽  
Direct Repeats ◽  
Evolutionary Mechanisms ◽  
Evolutionary Patterns ◽  
Crispr Locus ◽  
Most Recent Common Ancestor ◽  
Low Diversity ◽  
Set Up

AbstractDiversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes.We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs.We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats.This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.

scholarly journals Labour sharing promotes coexistence in atrazine degrading bacterial communities

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Loren Billet ◽  
Marion Devers ◽  
Nadine Rouard ◽  
Fabrice Martin-Laurent ◽  
Aymé Spor
Keyword(s):  
Bacterial Communities ◽  
Gene Loss ◽  
Bacterial Species ◽  
Single Species ◽  
Microbial Consortia ◽  
The Public ◽  
Herbicide Atrazine ◽  
Set Up ◽  
Multiple Species ◽  
Isolated Species

AbstractMicrobial communities are pivotal in the biodegradation of xenobiotics including pesticides. In the case of atrazine, multiple studies have shown that its degradation involved a consortia rather than a single species, but little is known about how interdependency between the species composing the consortium is set up. The Black Queen Hypothesis (BQH) formalized theoretically the conditions leading to the evolution of dependency between species: members of the community called ‘helpers’ provide publicly common goods obtained from the costly degradation of a compound, while others called ‘beneficiaries’ take advantage of the public goods, but lose access to the primary resource through adaptive degrading gene loss. Here, we test whether liquid media supplemented with the herbicide atrazine could support coexistence of bacterial species through BQH mechanisms. We observed the establishment of dependencies between species through atrazine degrading gene loss. Labour sharing between members of the consortium led to coexistence of multiple species on a single resource and improved atrazine degradation potential. Until now, pesticide degradation has not been approached from an evolutionary perspective under the BQH framework. We provide here an evolutionary explanation that might invite researchers to consider microbial consortia, rather than single isolated species, as an optimal strategy for isolation of xenobiotics degraders.

scholarly journals Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Kuan Hu ◽  
Ashley T. Jordan ◽  
Susan Zhang ◽  
Avantika Dhabaria ◽  
Amanda Kovach ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Structure Prediction ◽  
Bacterial Species ◽  
Normal Growth ◽  
Anion Transport ◽  
Content Type ◽  
Reading Frame ◽  
Anion Transporters ◽  
Anion Transporter

ABSTRACT We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

scholarly journals Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

2016 ◽  
Vol 60 (9) ◽  
pp. 5232-5237 ◽  
Author(s):  
Xia Yu ◽  
Guirong Wang ◽  
Suting Chen ◽  
Guomei Wei ◽  
Yuanyuan Shang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Bacterial Species ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type ◽  
Lowenstein Jensen

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

scholarly journals Internet Thinking for Omnimedia Reconstruction of News Production and Dissemination Mechanism of Chinese Traditional Mass Media

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Shaoshuo Cai ◽  
Fen Xie
Keyword(s):  
Mass Communication ◽  
News Production ◽  
The Public ◽  
Whole Process ◽  
Social Events ◽  
Audience Interaction ◽  
Media Platform ◽  
Set Up ◽  
Media Integration ◽  
Innovation Mechanism

<p>This paper briefly combs the practical innovation of Chinese traditional media in news production and dissemination mechanism, focuses on the on the analysis of subjective and objective factors that restrict the innovation and reform of its original mechanism, and puts forward the reconstruction and innovation mechanism of news production and dissemination in the omnimedia era with the thinking of the Internet. Set up service thinking, set up an all-round agenda, report major social events in the whole process to build a dynamic media platform; Set up the user's thinking, carry out all-round mass communication, meet the audience's personalized news consumption needs in all aspects to build a multi-dimensional platform; Set up open thinking, conduct audience interaction in all directions, strengthen the public participation experience in the whole process to build a multi-terminal communication platform; Set up platform thinking, expand the communication effect in all directions, give full play to the efficiency of media integration in all aspects to provide the multi-form experience.</p>

scholarly journals Mycobacterium tuberculosis β-Carbonic Anhydrases: Novel Targets for Developing Antituberculosis Drugs

2019 ◽  
Vol 20 (20) ◽  
pp. 5153 ◽  
Author(s):  
Aspatwar ◽  
Kairys ◽  
Rala ◽  
Parikka ◽  
Bozdag ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
In Vivo Studies ◽  
Close Relative ◽  
Carbonic Anhydrases ◽  
In Vitro Inhibition ◽  
Inhibition Studies

The genome of Mycobacterium tuberculosis (Mtb) encodes three β-carbonic anhydrases (CAs, EC 4.2.1.1) that are crucial for the life cycle of the bacterium. The Mtb β-CAs have been cloned and characterized, and the catalytic activities of the enzymes have been studied. The crystal structures of two of the enzymes have been resolved. In vitro inhibition studies have been conducted using different classes of carbonic anhydrase inhibitors (CAIs). In vivo inhibition studies of pathogenic bacteria containing β-CAs showed that β-CA inhibitors effectively inhibited the growth of pathogenic bacteria. The in vitro and in vivo studies clearly demonstrated that β-CAs of not only mycobacterial species, but also other pathogenic bacteria, can be targeted for developing novel antimycobacterial agents for treating tuberculosis and other microbial infections that are resistant to existing drugs. In this review, we present the molecular and structural data on three β-CAs of Mtb that will give us better insights into the roles of these enzymes in pathogenic bacterial species. We also present data from both in vitro inhibition studies using different classes of chemical compounds and in vivo inhibition studies focusing on M. marinum, a model organism and close relative of Mtb.

scholarly journals Urease Activity Represents an Alternative Pathway for Mycobacterium tuberculosis Nitrogen Metabolism

2012 ◽  
Vol 80 (8) ◽  
pp. 2771-2779 ◽  
Author(s):  
Wenwei Lin ◽  
Vanessa Mathys ◽  
Emily Lei Yin Ang ◽  
Vanessa Hui Qi Koh ◽  
Julia María Martínez Gómez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Source ◽  
Nitrogen Metabolism ◽  
Urease Activity ◽  
Alternative Pathway ◽  
Bacterial Species ◽  
Deficient Mutant ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTUrease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficientMycobacterium tuberculosisstrain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage onM. tuberculosisin macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impairedin vitrogrowth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity inM. tuberculosisnitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion thatM. tuberculosisvirulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment.

scholarly journals Labour sharing promotes coexistence in atrazine degrading bacterial communities

2019 ◽  
Author(s):  
Loren Billet ◽  
Marion Devers ◽  
Nadine Rouard ◽  
Fabrice Martin-Laurent ◽  
Aymé Spor
Keyword(s):  
Gene Loss ◽  
Bacterial Species ◽  
Single Species ◽  
Microbial Consortia ◽  
Future Research ◽  
The Public ◽  
Herbicide Atrazine ◽  
Set Up ◽  
Multiple Species

SUMMARYMicrobial communities exert a pivotal role in the biodegradation of xenobiotics including pesticides1. In the case of atrazine, multiple studies have shown that its degradation involved a consortia rather than a single species2,3,4,5, but little is known about how interdependency between the species composing the consortium is set up. The Black Queen Hypothesis (BQH) formalized theoretically the conditions leading to the evolution of dependency between species6: members of the community called ‘helpers’ provide publicly common goods obtained from the costly degradation of a compound, while others called ‘beneficiaries’ take advantage of the public goods, but lose access to the primary resource through adaptive degrading gene loss. Here, we test whether liquid media supplemented with the herbicide atrazine could support coexistence of bacterial species through BQH mechanisms. We observed the establishment of dependencies between species through atrazine degrading gene loss. Labour sharing between members of the consortium led to coexistence of multiple species on a single resource and improved atrazine degradation potential. Until now, pesticide degradation has not been approached from an evolutionary perspective under the BQH framework. We provide here an evolutionary explanation that might invite researchers to consider microbial consortia, rather than single isolated species, as an optimal strategy for isolation of xenobiotics degraders. Also, we anticipate that future research should focus on the bioaugmentation with stabilized and tightly structured microbial degrading consortia as an effective solution forin situbioremediation of sites polluted with recalcitrant compounds.

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