scholarly journals Bifidobacterium -Escherichia coli Shuttle Vector Series pKO403, with Temperature-Sensitive Replication Origin for Gene Knockout in Bifidobacterium

2022 ◽  
Author(s):  
Hend Altaib ◽  
Yuka Ozaki ◽  
Tomoya Kozakai ◽  
Kouta Sakaguchi ◽  
Izumi Nomura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cloning ◽  
Replication Origin ◽  
Gene Knockout ◽  
Shuttle Vector ◽  
Temperature Sensitive ◽  
Content Type ◽  
Shuttle Vectors ◽  
Restriction Sites

A series of Bifidobacterium - Escherichia coli shuttle vectors (pKO403- lacZ′ -Cm, pKO403- lacZ′ -Sp, pKO403- lacZ′ -p15A) were constructed based on the pKO403 backbone, which carries a temperature-sensitive replication origin. These vectors carry the lacZ′ α fragment, overhung by two facing type IIS restriction sites, for blue-white selection and seamless gene cloning.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

scholarly journals Establishment of a Tractable Genetic Transformation System in Veillonella spp.

2012 ◽  
Vol 78 (9) ◽  
pp. 3488-3491 ◽  
Author(s):  
Jinman Liu ◽  
Zhoujie Xie ◽  
Justin Merritt ◽  
Fengxia Qi
Keyword(s):  
Escherichia Coli ◽  
Genetic Transformation ◽  
Shuttle Vector ◽  
Transformation System ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Transformation System

ABSTRACTWe have constructed the firstEscherichia coli-Veillonellashuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinicalVeillonellastrain. A highly transformableVeillonellastrain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for futureVeillonellagenetic studies.

scholarly journals Disruption of a Type II Endonuclease (TDE0911) Enables Treponema denticola ATCC 35405 To Accept an Unmethylated Shuttle Vector

2011 ◽  
Vol 77 (13) ◽  
pp. 4573-4578 ◽  
Author(s):  
Jiang Bian ◽  
Chunhao Li
Keyword(s):  
Southern Blot ◽  
Shuttle Vector ◽  
Cell Extract ◽  
Treponema Denticola ◽  
Type Ii ◽  
Restriction Digestion ◽  
Content Type ◽  
Shuttle Vectors ◽  
Genetic Manipulations ◽  
Dna Restriction

ABSTRACTThe oral spirocheteTreponema denticolais associated with human periodontal disease.T. denticolaATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared toT. denticolaATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence ofT. denticolaATCC 35405. In this report, we hypothesize thatT. denticolaATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome ofT. denticolaATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant ofTDE0911failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulateT. denticola, in particular ATCC 35405, and other strains that may carry similar endonucleases.

scholarly journals PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

2013 ◽  
Vol 80 (4) ◽  
pp. 1477-1481 ◽  
Author(s):  
Karina Klevanskaa ◽  
Nadja Bier ◽  
Kerstin Stingl ◽  
Eckhard Strauch ◽  
Stefan Hertwig
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

scholarly journals TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

2015 ◽  
Vol 83 (5) ◽  
pp. 1893-1903 ◽  
Author(s):  
Daniela Gutiérrez ◽  
Mirka Pardo ◽  
David Montero ◽  
Angel Oñate ◽  
Mauricio J. Farfán ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Coli Strain ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Temperature Sensitive ◽  
Content Type ◽  
E Coli ◽  
Colonization Factor ◽  
Adhesion Level

EnterotoxigenicEscherichia coli(ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avianE. colistrains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with atleAmutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression oftleAconferred the capacity for adherence to nonadherentE. coliHB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.

scholarly journals Rapid Accumulation of Motility-Activating Mutations in Resting Liquid Culture ofEscherichia coli

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Darren J. Parker ◽  
Pınar Demetci ◽  
Gene-Wei Li
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Liquid Medium ◽  
Regulatory Networks ◽  
Gene Knockout ◽  
Single Gene ◽  
Point Mutations ◽  
Content Type ◽  
E Coli ◽  
Keio Collection

ABSTRACTExpression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains ofEscherichia colipossess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in theE. colisingle-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motileE. colistrains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of theflhDCoperon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring.IMPORTANCEThe ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotileEscherichia colibacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains ofE. coli.

scholarly journals The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

2012 ◽  
Vol 56 (6) ◽  
pp. 3216-3223 ◽  
Author(s):  
Tina Manzhu Kang ◽  
Jessica Yuan ◽  
Angelyn Nguyen ◽  
Elinne Becket ◽  
Hanjing Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Gene Knockout ◽  
Aminoglycoside Antibiotic ◽  
Double Strand Breaks ◽  
Repair Capacity ◽  
Content Type ◽  
Strand Breaks ◽  
Knockout Mutants ◽  
Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

scholarly journals Functions of a GyrBA Fusion Protein and Its Interaction with QnrB and Quinolones

2015 ◽  
Vol 59 (11) ◽  
pp. 7124-7127 ◽  
Author(s):  
Chunhui Chen ◽  
Regis Villet ◽  
George A. Jacoby ◽  
David C. Hooper
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Dna Gyrase ◽  
Temperature Sensitive ◽  
Type Ii ◽  
Wild Type ◽  
Interacting Protein ◽  
Content Type ◽  
Negative Supercoiling

ABSTRACTIn order to study the interactions betweenEscherichia coliDNA gyrase and the gyrase interacting protein QnrBin vivo, we constructed agyrB-gyrAfusion and validated its ability to correct the temperature-sensitive growth ofgyrAandgyrBmutants. Like wild-typegyrA, thegyrB-gyrAfusion complemented a quinolone-resistantgyrAmutant to increase susceptibility. It functioned as an active type II topoisomerase, catalyzed negative supercoiling of DNA, was inhibited by quinolone, and was protected by QnrB.

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