April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Characterization of a Small, Noncoding RNA in Pseudomonas aeruginosa
Author Affiliations & Notes
  • Victoria Addis
    Department of Ophthalmology, Georgetown University/Washington Hospital Center, Washington, DC
  • Diana Pastrana
    Laboratory of Cellular Oncology, National Cancer Institute, Rockville, MD
  • David FitzGerald
    Biotherapy Section, National Cancer Insitute, Rockville, MD
  • Footnotes
    Commercial Relationships Victoria Addis, None; Diana Pastrana, None; David FitzGerald, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5715. doi:
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      Victoria Addis, Diana Pastrana, David FitzGerald; Characterization of a Small, Noncoding RNA in Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5715.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

Pseudomonas aeruginosa is a gram-negative, opportunistic pathogen that causes serious ophthalmic infection including corneal ulceration. Pseudomonas utilizes virulence factors and processes such as quorum sensing (QS) to cause morbidity. QS is regulated by chemical signaling molecules, termed autoinducers, that enable regulation of gene expression in response to changes in cell density. Pseudomonas has two interdependent QS systems (Las/Rhl) which function to control the expression of numerous genes and virulence factors. In this study, we characterize a small, noncoding RNA (sRNA) from the Pseudomonas genome and show that it is intricately involved in QS.

 
Methods
 

Bioinformatics was used to identify a candidate sRNA sequence from the Pseudomonas genome and 5'/3'Race and primer extension were used to map the ends of this sRNA. Using Northern blot analysis, we determined expression levels of the transcript over time in both wild-type as well as autoinducer-mutant Pseudomonas strains. We electroporated the candidate sRNA into the autoinducer-mutant Pseudomonas and performed experiments to study autoinducer production upon up-regulation of vector, and, as a result, sRNA transcription.

 
Results
 

In wild-type Pseudomonas, the sRNA is initially transcribed in mid-exponential growth phase and increases in amount through early stationary phase (Figure 1). An autoinducer-mutant strain makes no detectable amount of the sRNA under normal growth conditions, however transcription is restored upon addition of autoinducers to the growth medium. When a vector containing the sRNA sequence is electroporated into the autoinducer-mutant strain and turned-on, transcription of the sRNA is induced both from the vector and possibly from the chromosome as well.

 
Conclusions
 

In this study, we identified a putative sRNA in the intergenic region of the Pseudomonas genome. We demonstrate that Pseudomonas expresses this sRNA in response to increased cell density and that expression is dependent on autoinducers. We hope that by increasing our understanding of the pathogenesis of Pseudomonas infection, we will be better equipped to develop new strategies for treatment of Pseudomonas-related ophthalmic infections.

 
 
Northern blot analysis of the expression level of the sRNA over time in wild-type Pseudomonas. The sRNA is initially transcribed in mid-exponential phase (OD600 = 0.4A) and made in increasing amounts through early stationary phase.
 
Northern blot analysis of the expression level of the sRNA over time in wild-type Pseudomonas. The sRNA is initially transcribed in mid-exponential phase (OD600 = 0.4A) and made in increasing amounts through early stationary phase.
 
Keywords: 594 microbial pathogenesis: experimental studies • 664 pseudomonas • 533 gene/expression  
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