scholarly journals ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Kenan C. Murphy ◽  
Samantha J. Nelson ◽  
Subhalaxmi Nambi ◽  
Kadamba Papavinasasundaram ◽  
Christina E. Baer ◽  
...  
Keyword(s):  
Drug Targets ◽  
Single Step ◽  
Selection Marker ◽  
Recombination Site ◽  
New Paradigm ◽  
Content Type ◽  
Simultaneous Integration ◽  
Bacterial Chromosomes ◽  
Recombinant Formation ◽  
A Site

ABSTRACTTwo efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for “oligonucleotide-mediatedrecombineering followed byBxb1integrasetargeting”) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this “drag and drop” strategy by the construction of insertions or deletions in over 100 genes inMycobacteriumtuberculosisandM. smegmatis.IMPORTANCEWe sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use inM. smegmatisandM. tuberculosis. By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1attPsite into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries anattBsite and a drug selection marker, we show both formation of a chromosomalattPsite and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

scholarly journals ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes

2018 ◽  
Author(s):  
Kenan C. Murphy ◽  
Samantha J. Nelson ◽  
Subhalaxmi Nambi ◽  
Kadamba Papavinasasundaram ◽  
Christina E. Baer ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Genome Engineering ◽  
Target Genes ◽  
Dna Recombination ◽  
Single Step ◽  
Recombination Site ◽  
New Paradigm ◽  
Double Stranded Dna ◽  
Simultaneous Integration ◽  
A Site

ABSTRACTCurrent methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT‐ and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes inM. tuberculosisandM. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target‐ or modification-specific double-stranded DNA recombination substrates.

scholarly journals Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Paras Jain ◽  
Tsungda Hsu ◽  
Masayoshi Arai ◽  
Karolin Biermann ◽  
David S. Thaler ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Single Step ◽  
Open Reading Frame ◽  
Model Systems ◽  
Effective Vaccine ◽  
Temperature Sensitive ◽  
Content Type ◽  
Reading Frame ◽  
New Methods

ABSTRACTSpecialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulentMycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library ofM. tuberculosisnull strains, where each strain is deleted for a single defined open reading frame inM. tuberculosis.IMPORTANCEThis work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulentM. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.

scholarly journals In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1649-1663
Author(s):  
Oliver Z Nanassy ◽  
Kelly T Hughes
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Dna Inversion ◽  
Hin Recombinase ◽  
One Step ◽  
A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

“The cut and thrust of industrial relations” – bullying by another name?

2018 ◽  
Vol 33 (1) ◽  
pp. 2-13 ◽  
Author(s):  
Pat Drake
Keyword(s):  
Women Leaders ◽  
Industrial Relations ◽  
Illustrative Case ◽  
University Leadership ◽  
Public Identity ◽  
Content Type ◽  
Female Self ◽  
Emotional Work ◽  
Differences Of Opinion ◽  
A Site

Purpose The purpose of this paper is to offer a feminography, that is a “narration of a female self in a feminist age” (Abrams, 2017) by presenting a conceptual analysis, derived from experience, of email providing a form of discourse – that the author calls finger-speak – through which unexamined gender positioning caricatures a person’s identity. In so doing, the paper provides an illustrative case of a female manager being positioned through email to “know her place, perform it and feel it” (Hey, 2011). Design/methodology/approach An analysis of email foregrounds “finger-speak” as a form of digital conversation and through which people in universities may be positioned publicly but without their consent in relation to unexamined norms and assumptions. For women, it is argued, these norms are ageist and sexist. In this paper, fragments of finger-speak are collated to provide a reading of how mixing gendered norms with apparent differences of opinion constructs, via unexamined sexism, a public identity and then undermines it. Findings Through the case presented, the author argues that, because of a shared but unarticulated shadow over women as leaders, email lays the ground for subsequent scapegoating in such a manner that the woman takes responsibility for structural challenges that rightly belong to the organisation. Originality/value The contribution that email makes to constructing female identity in public is new, complementing other work that publicly characterises women leaders, through film (Ezzedeen, 2015), and through published writing such as autobiography (Kapasi et al., 2016). Emotional work undertaken by women in university leadership is so far under-represented in public, and email is a site through which this work becomes visible.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

Diagnosis of spontaneous intracranial hypotension by using magnetic resonance myelography

2000 ◽  
Vol 92 (5) ◽  
pp. 873-876 ◽  
Author(s):  
Akira Matsumura ◽  
Izumi Anno ◽  
Hiroshi Kimura ◽  
Eiichi Ishikawa ◽  
Tadao Nose
Keyword(s):  
Magnetic Resonance ◽  
Intracranial Hypotension ◽  
Direct Evidence ◽  
Spontaneous Intracranial Hypotension ◽  
Content Type ◽  
Highly Sensitive ◽  
Csf Leakage ◽  
Leakage Site ◽  
A Site ◽  
Mr Myelography

✓ The authors describe a case of spontaneous intracranial hypotension in which the leakage site was determined by using magnetic resonance (MR) myelography. This technique demonstrated the route of cerebrospinal fluid (CSF) leakage, whereas other methods failed to show direct evidence of leakage. Magnetic resonance myelography is a noninvasive method that is highly sensitive in detecting CSF leakage. This is the first report in which a site of CSF leakage was detected using MR myelography.

The auditing profession and the key audit matter reporting requirement

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Dessalegn Getie Mihret ◽  
Monika Kansal ◽  
Mohammad Badrul Muttakin ◽  
Tarek Rana
Keyword(s):  
Standard Setting ◽  
International Standards ◽  
Financial Reports ◽  
Content Type ◽  
Reporting Requirement ◽  
Regulatory Rule ◽  
Rule Setting ◽  
A Site ◽  
Relationship Of

Purpose This study aims to examine the setting of International Standards on Auditing (ISA) 701 on disclosing key audit matters (KAMs) to explore the role of standard setting in maintaining or reconstituting the relationship of the auditing profession with preparers and users of financial reports. Design/methodology/approach This study draws on concepts from the sociology of the professions literature and the regulatory space metaphor. Data comprises comment letters and other documents pertaining to the setting of ISA 701. Findings The study shows that the KAM reporting requirement is part of the ongoing re-calibration of the regulatory arrangements governing auditing, which started in the early 2000s. This study interprets standard setting as a site for negotiating the relationships between linked ecologies in the audit regulatory space, namely, the auditing profession, preparers of financial statements and users of audited reports. This study identifies three processes involved in setting ISA 701, namely, reconstitution of the rules governing auditors’ reports as a link between the three ecologies, preserving boundaries between the auditing profession and preparers and negotiation aimed at balancing competing interests of the interrelated ecologies. Originality/value The study offers insights into the role of regulatory rule setting as a central medium through which the adaptive relationship of the profession with its environment is negotiated.

The role of site manager transformational leadership in the construction project success

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Masoud Zavari ◽  
Mohammad Reza Afshar
Keyword(s):  
Construction Projects ◽  
Project Success ◽  
Construction Project ◽  
Content Type ◽  
Team Innovation ◽  
Mediating Role ◽  
A Site ◽  
Site Manager ◽  
Site Managers

PurposeIn the construction industry, site managers are responsible for the day-to-day on-site running of a project. Site managers are required not only to ensure that work is done safely, on time, within budget and to the right quality standards, but also to manage any delays or problems encountered on-site during a construction project. Thus, a site manager has a significant position in the success of a construction project. However, there is a lack of studies considering the role of the leadership style of a site manager in construction project success. In this study, not only the effect of the transformational leadership (TL) style of site managers on the success of construction projects is assessed, but also the mediating role of team-building (TB) and team innovation between TL and project success is studied.Design/methodology/approachThe proposed hypotheses are tested by comparing the performance criteria of construction projects with the TL style of site managers. The success criteria of the projects are examined using project documents and client opinions, and the TL of site managers, TB aspects and team innovation in projects are assessed by asking from their subordinates. The Pearson correlation coefficient is employed to investigate the relation between every two variables.FindingsThe results illustrate that the TL of a site manager is directly related to project success; thus, the findings would confirm the importance of selecting appropriate site managers for construction projects. Moreover, while there is a mediating role of TB between TL and construction project success, team innovation does not have a mediating impact in design-bid-build (DBB) construction projects.Originality/valueAs there is a lack of studies assessing the TL effects on the success of construction projects and the significant role of site managers, this study is one of the first researches that tested these impacts and evaluated the mediating role of TB and team innovation between TL and project performance.

scholarly journals A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency inCampylobacter jejuni

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Ximin Zeng ◽  
Zuowei Wu ◽  
Qijing Zhang ◽  
Jun Lin
Keyword(s):  
Comparative Genomics ◽  
Heat Shock ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Campylobacter Jejuni ◽  
Selection Marker ◽  
Sequencing Analysis ◽  
Content Type ◽  
Restriction Modification ◽  
Modification Enzyme

ABSTRACTConjugation is an important mechanism for horizontal gene transfer inCampylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency inCampylobacterspp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC)C. jejunistrain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFCC. jejunistrain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFCC. jejunistrain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (akaCjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFCC. jejunistrains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest theEscherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency inC. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency inC. jejuni.IMPORTANCEConjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance.Campylobacter jejuni, the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency inC. jejuniare still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency inC. jejuni. The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.

Tribology study of hydroxyapatite (HAp) scaffold blended single based binder via rheology and mechanical properties

2017 ◽  
Vol 69 (3) ◽  
pp. 414-419
Author(s):  
Mimi Azlina Abu Bakar ◽  
Siti Norazlini Abd Aziz ◽  
Muhammad Hussain Ismail
Keyword(s):  
Mechanical Properties ◽  
Injection Molding ◽  
Palm Stearin ◽  
Polymeric Materials ◽  
High Energy ◽  
Single Step ◽  
Injection Molding Process ◽  
Plastic Behavior ◽  
Molding Process ◽  
Content Type

Purpose This paper aims to investigate the vital characteristic of an innovative ceramic injection molding (CIM) process for orthopedic application with controlled porosity and improved tribological and mechanical properties which were affected by complex tribological interactions, whether lubricated like hip implants and other artificial prostheses. The main objective is to maximize the usage of palm stearin as a single based binder as the function of flow properties during injection molding process. Design/methodology/approach The binder used in this present study consists of 100 per cent palm stearin manufactured by Kempas Oil Sdn Bhd and supplied by Vistec Technology Sdn Bhd. The feedstock was prepared by using a Z-blade mixer (Thermo Haake Rheomix OS) and Brabender mixer model R2400. The feedstock prepared was injection molded using a manually operated vertical benchtop machine with an average pressure of about 5-7 bars. The firing step included the temporary holds at intermediate temperatures to burn out organic binders. At this stage, the green molded specimen was de-bound using a single-step wick-debinding method. Findings The maximum content of ceramic material is applied to investigate the efficiencies of net formulation that can be achieved by ceramic materials. The longer the viscosity will change with shear rate, the higher the value of n obtained instead. From the slope of the curves obtained in Figure 3, the value of n for the feedstock was determined to be less than 1, which indicates a pseudoplastic behavior and suitability for the molding process. Moreover, high shear sensitivity is important in producing complex and intrinsic specimens which are leading products in the CIM industry. Originality/value The feedstock containing HAp powder and palm stearin binder was successfully prepared at very low temperature of 70°C, which promoting a required pseudo-plastic behavior during rheological test. The single binder palm stearin should be optimized in other research works carried out, as palm stearin is most preferred compared to other polymeric materials that provided high energy consumption when subjected to the sintering process. Besides the binder is widely available in Malaysia, low cost and harmless effect during debinding process.

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