17 research outputs found
Establishment of a Replicating Plasmid in Rickettsia prowazekii
Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen
The Science Performance of JWST as Characterized in Commissioning
This paper characterizes the actual science performance of the James Webb
Space Telescope (JWST), as determined from the six month commissioning period.
We summarize the performance of the spacecraft, telescope, science instruments,
and ground system, with an emphasis on differences from pre-launch
expectations. Commissioning has made clear that JWST is fully capable of
achieving the discoveries for which it was built. Moreover, almost across the
board, the science performance of JWST is better than expected; in most cases,
JWST will go deeper faster than expected. The telescope and instrument suite
have demonstrated the sensitivity, stability, image quality, and spectral range
that are necessary to transform our understanding of the cosmos through
observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures;
https://iopscience.iop.org/article/10.1088/1538-3873/acb29
The James Webb Space Telescope Mission
Twenty-six years ago a small committee report, building on earlier studies,
expounded a compelling and poetic vision for the future of astronomy, calling
for an infrared-optimized space telescope with an aperture of at least .
With the support of their governments in the US, Europe, and Canada, 20,000
people realized that vision as the James Webb Space Telescope. A
generation of astronomers will celebrate their accomplishments for the life of
the mission, potentially as long as 20 years, and beyond. This report and the
scientific discoveries that follow are extended thank-you notes to the 20,000
team members. The telescope is working perfectly, with much better image
quality than expected. In this and accompanying papers, we give a brief
history, describe the observatory, outline its objectives and current observing
program, and discuss the inventions and people who made it possible. We cite
detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space
Telescope Overview, 29 pages, 4 figure
Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii
Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes
Establishment of a replicating plasmid in Rickettsia prowazekii
Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen
Plasmid copy number determination.
<p>Plasmid copy number/rickettsial genome equivalent was determined by comparing genome equivalents of the rickettsial chromosomal gene, <i>rho</i>, and the plasmid-borne <i>gfp<sub>uv</sub></i> gene. Genome equivalents were determined by comparison to standard curves. Two independent biological samples were analyzed in duplicate.</p
Southern blot analysis of DNA extracted from ME-pRAM18dRGA using a probe specific for <i>gfp<sub>UV</sub></i>.
<p>Lanes are labeled as uncut or by the enzyme used in the digestion. Locations of molecular size markers (in kB) are indicated on the left. Plasmid forms in the uncut lane are L- linear, CCC- closed circular, and OC- open circular.</p