scholarly journals Development of a Modified Gentamicin Resistance Cassette for Genetic Manipulation of the Oral Spirochete Treponema denticola

2012 ◽  
Vol 78 (6) ◽  
pp. 2059-2062 ◽  
Author(s):  
Jiang Bian ◽  
J. Christopher Fenno ◽  
Chunhao Li
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Manipulation ◽  
Targeted Mutagenesis ◽  
Treponema Denticola ◽  
Content Type ◽  
Reliable Tool ◽  
Gentamicin Resistance

ABSTRACTHerein, we report that a modified gentamicin cassette and a PCR-based method can be used for targeted mutagenesis of the oral spirocheteTreponema denticola. This approach minimizes polar effects and spontaneous antibiotic resistance. Therefore, it can serve as a reliable tool for genetic manipulation ofT. denticola.

scholarly journals Natural Transformation of Gallibacterium anatis

2012 ◽  
Vol 78 (14) ◽  
pp. 4914-4922 ◽  
Author(s):  
Bodil M. Kristensen ◽  
Sunita Sinha ◽  
John D. Boyce ◽  
Anders M. Bojesen ◽  
Joshua C. Mell ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Natural Transformation ◽  
Genetic Manipulation ◽  
Targeted Mutagenesis ◽  
Chromosomal Dna ◽  
Natural Competence ◽  
Content Type ◽  
Transformation Procedure ◽  
High Transformation ◽  
Dna Types

ABSTRACTGallibacterium anatisis a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function inG. anatis, we have established methods for transformation and targeted mutagenesis. The genusGallibacteriumbelongs to thePasteurellaceae, a group with several naturally transformable members, includingHaemophilus influenzae. Bioinformatics analysis identifiedG. anatishomologs of theH. influenzaecompetence genes, and natural competence was induced inG. anatisby the procedure established forH. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies withG. anatischromosomal DNA and with linearized plasmid DNA carryingG. anatissequences. Both DNA types integrated into theG. anatischromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10−4transformants CFU−1. Transformation was also efficient with circular plasmid containing noG. anatisDNA; this resulted in the establishment of a self-replicating plasmid. Nine diverseG. anatisstrains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. TheG. anatisgenome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, butG. anatisdid preferentially take up its own DNA over that ofEscherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation ofG. anatis.

scholarly journals Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola

2015 ◽  
Vol 81 (13) ◽  
pp. 4329-4338 ◽  
Author(s):  
Yuebin Li ◽  
John Ruby ◽  
Hui Wu
Keyword(s):  
Genetic Manipulation ◽  
Periodontal Diseases ◽  
Genetic System ◽  
Kanamycin Resistance ◽  
Treponema Denticola ◽  
Oral Pathogen ◽  
Content Type ◽  
Kanamycin Resistance Cassette ◽  
Allelic Replacement ◽  
Kanamycin Cassette

ABSTRACTTreponema denticolahas been recognized as an important oral pathogen of the “red complex” bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence ofT. denticoladue to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation ofT. denticolaATCC 35405. Compared to the widely usedermF-ermAMcassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistantT. denticolacolonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames ofT. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional intrans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had theflgEgene coding for PF hook protein inactivated. The integration of the full-lengthflgEgene into the genome of theflgEmutant rescued all of the defects associated with theflgEmutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation ofT. denticolathat will facilitate the characterization of virulence factors attributed to this important oral pathogen.

scholarly journals Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
MaFeng Liu ◽  
Li Zhang ◽  
Li Huang ◽  
Francis Biville ◽  
DeKang Zhu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Natural Transformation ◽  
Genetic Manipulation ◽  
Gene Knockout ◽  
Targeted Mutagenesis ◽  
Chromosomal Dna ◽  
Content Type ◽  
Riemerella Anatipestifer ◽  
The Family ◽  
First Time

ABSTRACT Riemerella anatipestifer is a member of the family Flavobacteriaceae and a major causative agent of duck serositis. Little is known about its genetics and pathogenesis. Several bacteria are competent for natural transformation; however, whether R. anatipestifer is also competent for natural transformation has not been investigated. Here, we showed that R. anatipestifer strain ATCC 11845 can uptake the chromosomal DNA of R. anatipestifer strain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established for R. anatipestifer ATCC 11845. Targeted mutagenesis gave transformation frequencies of ∼10−5 transformants. Competition assay experiments showed that R. anatipestifer ATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such as Escherichia coli DNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ∼10−9 transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containing R. anatipestifer DNA fragments (transformation frequencies of ∼10−7 transformants). Finally, we found that the R. anatipestifer RA-CH-1 strain was also naturally transformable, suggesting that natural competence is widely applicable for this species. The findings described here provide important tools for the genetic manipulation of R. anatipestifer. IMPORTANCE Riemerella anatipestifer is an important duck pathogen that belongs to the family Flavobacteriaceae. At least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms of R. anatipestifer remain largely unknown because no genetic tools are available for this bacterium. At present, natural transformation has been found in some bacteria but not in R. anatipestifer. For the first time, we showed that natural transformation occurred in R. anatipestifer ATCC 11845 and R. anatipestifer RA-CH-1. Then, we established an easy gene knockout method in R. anatipestifer based on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis in R. anatipestifer.

scholarly journals Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

2015 ◽  
Vol 81 (22) ◽  
pp. 7888-7892 ◽  
Author(s):  
Christopher J. Pappas ◽  
Mathieu Picardeau
Keyword(s):  
Genetic Manipulation ◽  
Immunoblot Analysis ◽  
Targeted Mutagenesis ◽  
Pcr Analysis ◽  
Leptospira Interrogans ◽  
Transcription Activator ◽  
Promoter Regions ◽  
Control Of Gene Expression ◽  
Content Type

ABSTRACTLeptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs withinLeptospiraspp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for thelacO-like region upstream ofbgaLwastransinserted in the saprophyteLeptospira biflexa(the TALEβgalstrain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgalstrain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions ofligAandligBwascisinserted into the pathogenLeptospira interrogans(TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALEligclones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1and TALElig2, which were avirulentin vivoand nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains ofLeptospiraspp., providing an additional genetic manipulation tool for this genus.

scholarly journals Screening of a Leptospira biflexa Mutant Library To Identify Genes Involved in Ethidium Bromide Tolerance

2014 ◽  
Vol 80 (19) ◽  
pp. 6091-6103 ◽  
Author(s):  
Helena Pětrošová ◽  
Mathieu Picardeau
Keyword(s):  
Antibiotic Resistance ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Genetic Manipulation ◽  
Content Type ◽  
Transposon Insertion ◽  
Damage Repair ◽  
Transposon Mutants ◽  
Living Species ◽  
Increased Sensitivity

ABSTRACTLeptospiraspp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyteL. biflexais a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants inL. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduceL. biflexasusceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n= 11), transport (n= 6), membrane structure (n= 5), stress response (n= 2), DNA damage repair (n= 1), and other processes (n= 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth ofL. biflexain the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenicL. interrogans.

scholarly journals Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

2014 ◽  
Vol 80 (12) ◽  
pp. 3597-3603 ◽  
Author(s):  
Vicki Adams ◽  
Radhika Bantwal ◽  
Lauren Stevenson ◽  
Jackie K. Cheung ◽  
Milena M. Awad ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Genes ◽  
Dna Fragments ◽  
Site Specific ◽  
Content Type ◽  
E Coli ◽  
Marker Recycling ◽  
A Site

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

scholarly journals pyrFas a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

2015 ◽  
Vol 82 (4) ◽  
pp. 1346-1352 ◽  
Author(s):  
Kurni Kurniyati ◽  
Chunhao Li
Keyword(s):  
Resistant Mutant ◽  
Selection Marker ◽  
Treponema Denticola ◽  
Gene Encoding ◽  
Content Type ◽  
Genetic Manipulations ◽  
Solid Agar ◽  
Monophosphate Decarboxylase ◽  
Marker Free ◽  
Gentamicin Resistance

ABSTRACTThe pathophysiology ofTreponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate thatpyrF, a gene encoding orotidine-5′-monophosphate decarboxylase, can be used as a counterselectable marker inT. denticolato construct marker-free mutants.T. denticolais susceptible to 5-fluoroorotic acid (5-FOA). To establish apyrF-based counterselectable knockout system inT. denticola, thepyrFgene was deleted. The deletion conferred resistance to 5-FOA inT. denticola. Next, a single-crossover mutant was constructed by reintroducingpyrFalong with a gentamicin resistance gene (aacC1) back into the chromosome of thepyrFmutant at the locus of choice. In this study, we choseflgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgEin) regained the susceptibility to 5-FOA. Finally, FlgEinwas plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgEout) were obtained and characterized by PCR and Southern blotting analyses. The results showed that theflgEgene was deleted and FlgEoutwas free of selection markers (i.e.,pyrFandaacC1). Compared to previously constructedflgEmutants that contain an antibiotic selection marker, the deletion offlgEin FlgEouthas no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity ofT. denticola.

scholarly journals Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Ellen N. Kersh ◽  
Cau D. Pham ◽  
John R. Papp ◽  
Robert Myers ◽  
Richard Steece ◽  
...  
Keyword(s):  
Public Health ◽  
Antibiotic Resistance ◽  
Neisseria Gonorrhoeae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Whole Genome ◽  
Content Type ◽  
Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

scholarly journals Draft Genome Sequence of the Shrimp Pathogen Vibrio parahaemolyticus ST17.P5-S1, Isolated in Peninsular Malaysia

2018 ◽  
Vol 7 (11) ◽  
Author(s):  
Sridevi Devadas ◽  
Subha Bhassu ◽  
Tze Chiew Christie Soo ◽  
Fatimah M. Yusoff ◽  
Mohamed Shariff
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Vibrio Parahaemolyticus ◽  
East Coast ◽  
Draft Genome ◽  
Antibiotic Resistance Genes ◽  
Peninsular Malaysia ◽  
Penaeus Vannamei ◽  
Content Type ◽  
Acute Hepatopancreatic Necrosis Disease

We sequenced the genome of Vibrio parahaemolyticus strain ST17.P5-S1, isolated from Penaeus vannamei cultured in the east coast of Peninsular Malaysia. The strain contains several antibiotic resistance genes and a plasmid encoding the Photorhabdus insect-related (Pir) toxin-like genes, pirAvp and pirBvp, associated with acute hepatopancreatic necrosis disease (AHPND).

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