Date of Award

12-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Microbiology, Molecular Biology and Biochemistry

Research Advisor

Robert Belland, Ph.D.

Committee

James E. Bina, Ph.D. Yan Cui, Ph.D. Thomas P. Hatch, Ph.D. Richard E. Lee, Ph.D.

Keywords

Chlamydia trachomatis, Developmental cycle, non-coding RNAs, Carbenicillin persistence, IFN-gamma persistence, RNA Circularization

Abstract

Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-g and b-lactams, giving rise to an abnormal growth state termed persistence. Relatively little is known about the regulatory mechanisms that control temporal gene expression during the developmental cycle or the control of persistence and reactivation. Here we have examined the expression of a newly defined family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation (Using IFNγ and Carbenicillin). Non-coding RNAs were initially identified using an intergenic tiling microarray and were confirmed by Northern blotting. A group of 10 ncRNAs were mapped and characterized and compared to the previously described chlamydial ncRNAs (IhtA, pCHL antisense transcripts). The 5’ and 3’ ends of the ncRNAs were determined using an RNA circularization procedure. Promoter predictions indicated that all ncRNAs were expressed from s66 promoters and 9 ncRNAs contained non-templated 3’ poly-A or poly-AG additions.

Expression of ncRNAs was studied by Northern blotting during i) the normal developmental cycle, ii) IFN-g-induced persistence, and iii) carbenicillin-induced persistence. Differential temporal expression during the developmental cycle was seen for all ncRNAs and distinct differences in expression were seen during IFN-g and carbenicillin-induced persistence and reactivation.

Two of the studied ncRNAs were cis acting antisense molecules (CTIG270 and CTIG153). Expression of CTIG270 in a surrogate E. coli system along with its target gene ftsI proved that it was an antisense RNA, and it effectively controlled ftsI availability. A screening system was developed to determine the targets of potential trans-acting ncRNAs. The screen was composed of two compatible plasmids, a high copy effector plasmid expressing the ncRNA and a low/medium copy target plasmid expressing chlamydial library. Target plasmid contained translational fusion between chlamydial library and a positive/negative selection fusion system (ccdB/CAT fusion/TEV protease). The selection process to identify potential targets for ncRNAs was carried out over two stages. Stage one was to eliminate self ligated plasmids and non translational fusions, in this phase, only CAT+ ccdB resistant cells survived chloramphenicol selection i.e. cells representing true translational fusions survived antibiotic selection. Plasmids enriched for by stage one selection were subjected to stage two selection. In stage two ccdB sensitive cells were double transformed with both target plasmid and effector plasmid. Only in instances when ncRNA inhibits translation of ccdB would cells survive this selection. Stage two selection enriched for target(s) of ncRNA under test. We show in details the construction of this screening system and its functional aspect.

DOI

10.21007/etd.cghs.2009.0001

Comments

One year embargo expired December 2010.

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