Characterization of an Anaplasma Mutant Generated by Transposon Mutagenesis

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Material Information

Title:
Characterization of an Anaplasma Mutant Generated by Transposon Mutagenesis
Physical Description:
1 online resource (124 p.)
Language:
english
Creator:
Crosby, Francy L
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
BARBET,ANTHONY F
Committee Co-Chair:
DARK,MICHAEL JAMES
Committee Members:
DAME,JOHN B
ABBOTT,JEFFREY R
GULIG,PAUL A

Subjects

Subjects / Keywords:
anaplasmosis -- himar1 -- transposition -- transposon
Veterinary Medicine -- Dissertations, Academic -- UF
Genre:
Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The large amounts of data generated by genomics, transcriptomics and proteomics have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classic genetic mutation. One example is the definition of genes associated with virulence. This dissertation describes the characterization of a red fluorescent and spectinomycin and streptomycin resistant A. marginale mutant generated by Himar1 transposon mutagenesis. Characterization at the molecular level as shown here using high throughput genome sequencing to determine the Himar1-A.marginale genome junctions established that the transposon sequences were integrated within the coding region of the omp10 gene. This gene is arranged within an operon with AM1225 at the 5 end and with omp9, omp8, omp7 and omp6 arranged in tandem at the 3 end. RNA analysis to determine the effects of the transposon insertion on the expression of omp10 and downstream genes revealed that the Himar1 insertion not only reduced the expression of omp10 but also that of downstream genes. Transcript expression from omp9, and omp8 dropped by more than 90% in comparison with their counterparts in wild-type A. marginale. Immunoblot analysis showed a reduction in the production of Omp9 protein in these mutants compared to wild-type A.marginale. Transposon insertion within the omp10 and alteration of its expression and that of downstream genes did not affected the growth of these omp10 mutants in vitro in tick and endothelial cell culture. However these organisms were unable to infect immunocompetent cattle red blood cells in vivo. This work reports for the first time the method to promote transposon mutagenesis in Anaplasma marginale and a detailed characterization of the first Anaplasma marginale transformants obtained by this method.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Francy L Crosby.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: BARBET,ANTHONY F.
Local:
Co-adviser: DARK,MICHAEL JAMES.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-11-30

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2014
System ID:
UFE0046612:00001