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Use of aminoglycoside 3′ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability

Overview of attention for article published in BMC Research Notes, October 2015
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Title
Use of aminoglycoside 3′ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability
Published in
BMC Research Notes, October 2015
DOI 10.1186/s13104-015-1542-9
Pubmed ID
Authors

Nicole M. Lowden, Laxmi Yeruva, Cayla M. Johnson, Anne K. Bowlin, Derek J. Fisher

Abstract

C hlamydia spp. are obligate, intracellular bacteria that infect humans and animals. Research on these important pathogens has been hindered due to a paucity of genetic tools. We recently adapted a group II intron (GII) mutagenesis platform for creation of ampicillin-selectable gene insertions in C. trachomatis L2. The aims of this study were: (1) to assess the stability of the intron-insertion in an in vivo infection model to gauge the efficacy of this genetic tool for long term animal studies and (2) to expand upon the utility of the method by validating a second selection marker (aadA, conferring spectinomycin resistance) for mutant construction. Intron stability was assessed using a mouse vaginal tract infection model with a C. trachomatis L2 434/Bu incA::GII(bla) mutant. Infections were performed in the absence of selection and isolates shed into the vaginal tract were isolated and expanded in cell culture (also without selection). PCR and inclusion phenotype analysis indicated that the intron was stable for at least 27 days post-infection (at which point bacteria were no longer recovered from the mouse). The aminoglycoside 3' adenyltransferase (aadA) gene was used to create a spectinomycin-selectable GII intron, facilitating the construction of an incA::GII[aadA] C. trachomatis L2 insertion mutant. Both the GII(aadA) intron and our previously reported GII(bla) intron were then used to create an incA::GII(aadA), rsbV1::GII(bla) double mutant. Mutants were confirmed via PCR, sequencing, inclusion morphology (incA only), and western blot. The stability of the intron-insertion during in vivo growth indicates that the GII-insertion mutants can be used to study pathogenesis using the well-established mouse infection model. In addition, the validation of an additional marker for mutagenesis in Chlamydia allows for gene complementation approaches and construction of targeted, double mutants in Chlamydia. The aadA marker also could be useful for other genetic methods. Collectively, our results expand upon the rapidly growing chlamydial genetic toolkit and will aid in the implementation of studies dissecting the contribution of individual genes to infection.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 17 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Germany 1 6%
Unknown 16 94%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 7 41%
Unspecified 2 12%
Student > Master 2 12%
Researcher 1 6%
Other 1 6%
Other 4 24%
Readers by discipline Count As %
Immunology and Microbiology 5 29%
Agricultural and Biological Sciences 3 18%
Unspecified 2 12%
Medicine and Dentistry 2 12%
Biochemistry, Genetics and Molecular Biology 2 12%
Other 3 18%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 01 July 2016.
All research outputs
#4,186,609
of 7,974,292 outputs
Outputs from BMC Research Notes
#987
of 1,956 outputs
Outputs of similar age
#126,699
of 243,103 outputs
Outputs of similar age from BMC Research Notes
#89
of 180 outputs
Altmetric has tracked 7,974,292 research outputs across all sources so far. This one is in the 27th percentile – i.e., 27% of other outputs scored the same or lower than it.
So far Altmetric has tracked 1,956 research outputs from this source. They receive a mean Attention Score of 4.6. This one is in the 36th percentile – i.e., 36% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 243,103 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 37th percentile – i.e., 37% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 180 others from the same source and published within six weeks on either side of this one. This one is in the 41st percentile – i.e., 41% of its contemporaries scored the same or lower than it.