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Project 3

Discovery and functional analysis of small regulatory RNAs (sRNAs)

Principal Investigator: Caroline Harwood

Goals and Objectives

The goal of this project is to identify and characterize the functions of small regulatory RNAs (sRNAs) whose expression is associated with seven clinically relevant resistance traits of A. baumannii. We will also seek to identify sRNAs that contribute to traits of starvation resistance and desiccation resistance that enable A. baumannii to persist outside the human host, since this is its natural reservoir. sRNAs are expressed in response to specific environmental signals, they are synthesized rapidly and they act quickly to control gene expression at the translational level. We hypothesize that sRNAs help A. baumannii to cope with cell envelope and metabolic stresses associated with starvation, antibiotic, biocide and desiccation exposure.

Our objectives are as follows:

  1. Identification–discovery of sRNAs from A. baumannii transcriptome data. The transcriptomes of A. baumannii cells that have been subjected to a variety of different stresses will be determined.
  2. Verification of candidate sRNA by Northern blot and identification of ‘5 start sites of the sRNAs
  3. Identification of sRNA function. This will include phenotypic array analysis of sRNA mutants and analysis of effects of transient expression of the sRNA on global gene expression.

Background

In all bacteria examined, small regulatory RNAs (sRNAs) participate in gene regulation, most often at the level of translation. sRNAs are synthesized rapidly at reduced metabolic cost relative to proteins and facilitate rapid adaptation to a variety of stresses including cell envelope stress, oxidative stress and iron limitation. The connection between antibiotic stress and sRNAs has been explored in only a limited way and in very few bacteria. We are unaware of any studies connecting sRNAs to stresses associated with biocides or desiccation. sRNAs have not yet been described from A. baumannii, thus maximizing opportunities for new discoveries. Identification of sRNAs that are important for antibiotic or biocide resistance or traits associated with desiccation tolerance increases opportunities for interfering with these properties in a targeted way. Many sRNAs remodel metabolic flux and it is possible that direct interference with their activities may make cells more sensitive to antibiotics. sRNAs tend to be controlled by very sensitive promoters and thus they may be excellent reporters for stresses encountered by cells.