Functional characterisation of Hfq and identification of Hfq-dependent sRNAs in Pantoea ananatis

Thesis (PhD)--University of Pretoria, 2020.

The trans-encoded small RNAs (sRNAs) are novel gene expression regulators in bacteria. In response to external stimuli, sRNAs rapidly modulate the expression of genes at the post-transcriptional level to allow bacteria to reprogram its cellular environment for survival and fitness. These regulatory actions of sRNAs are chaperoned by the indispensable RNA-binding protein, Hfq, that stabilizes sRNAs and facilitates the base-pairing between the sRNA and target mRNA. The functional role of this protein in a broad-host-range phytopathogen P. ananatis was determined by constructing an hfq-null mutant strain of P. ananatis LMG 2665T. Overall, deletion of the hfq gene in P. ananatis had a negative effect on motility, biofilm formation, production of quorum sensing autoinducer molecule and pathogenicity of the bacterium, suggesting a collective involvement of Hfq in above-mentioned physiological processes. To identify trans-encoded sRNAs that are dependent on Hfq, strand-specific sRNA sequencing was conducted on total RNA extracted from both the wild-type and hfq mutant strains of P. ananatis at low (OD600 = 0.2) and high (OD600 = 0.6) cell-density conditions. The resulting sRNA transcriptome data were computationally screened for putative sRNAs by applying known sRNA characteristics. The filtering yielded 615 putative sRNAs (302 antisense sRNAs, 249 intergenic sRNAs and 64 gene-overlapping sRNAs). Of these sRNAs, 276 candidate sRNAs were differentially regulated by the loss of hfq (low cell condition = 58, high cell condition = 154 and both cell conditions = 64). Among 276 differentially regulated sRNA candidates, 41 were positive for Rho-independent terminator sequences, which included enterobacteria-conserved Hfq-dependent sRNAs such as ArcZ, FnrS, GlmZ, RprA, RyeB, RyhB, RyhB2, Spot42, SsrA as well as 16 novel P. ananatis sRNAs. Of the 41, the expression profile of nine sRNAs determined by sequencing depth plots and quantitative PCR (qRT-PCR) showed that the abundances of these sRNAs were negatively affected by the absence of the P. ananatis hfq gene, supporting their dependency on Hfq. In addition, computationally predicted target genes of selected sRNAs, whose full-length sequence was determined by 5’ rapid amplification of cDNA ends (5’ RACE) analysis, included those involved in the synthesis of exopolysaccharide, cell wall degrading enzyme and type 6 secretion system. The result suggests possible sRNA-regulation of virulence-related genes. Future works will focus on determining the functional roles of sRNAs and experimentally validating the predicted targets of these sRNAs. Overall, the findings of the current work highlight the importance of Hfq as a global virulence regulator in P. ananatis and identified previously uncharacterized sRNAs that possibly play essential roles in regulating bacterium’s adaptive responses.

Microbiology and Plant Pathology

PhD

Unrestricted