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Discovery and profiling of small RNAs responsive to stress conditions in the plant pathogen Pectobacterium atrosepticum

ABSTRACT: Small RNAs (sRNAs) have emerged as important regulatory molecules and have been studied in several bacteria. However, to date, there have been no whole-transcriptome studies on sRNAs in any of the Soft Rot Enterobacteriaceae (SRE) group of pathogens. Although the main ecological niches for these pathogens are plants, a significant part of their life cycle is undertaken outside their host within the adverse soil environment. However, the mechanisms of SRE adaptation to this harsh nutrient-deficient environment are poorly understood. In the study reported herein, by using strand-specific RNA-seq analysis and in silico sRNA predictions, we describe the sRNA pool of Pectobacterium atrosepticum and reveal numerous sRNA candidates, including those that are induced during starvation-activated stress response. Consequently, strand-specific RNA-seq enabled detection of 137 sRNAs and sRNA candidates under starvation conditions; 25 of these sRNAs were predicted for this bacterium in silico. Functional annotations were computationally assigned to 68 sRNAs. The expression of sRNAs in P. atrosepticum was compared under growth-promoting and starvation conditions: 63 sRNAs were differentially expressed with 57 sRNAs up-regulated under nutrient-deficient conditions. Conservation analysis using BLAST (e-value < 0.001) showed that most of the identified sRNAs are conserved within the SRE. Subsequently, we identified 9 novel sRNAs within the P. atrosepticum genome. A large number of these sRNAs is starvation-induced, suggesting their role in bacterial adaptive response. Finally, this work provides a basis for future experimental characterization and validation of sRNAs in plant pathogens.

ORGANISM(S): Pectobacterium atrosepticum SCRI1043

PROVIDER: GSE68547 | GEO | 2016/01/26

SECONDARY ACCESSION(S): PRJNA283104

REPOSITORIES: GEO

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Project description:Regulatory RNAs (sRNAs) are now considered as major players in many physiological and adaptive responses in pathogenic bacteria. sRNAs have been extensively studied in Gram-negative bacteria, but less information is available in Gram-positive pathogens. There is a spread of multidrug-resistant (MDR) opportunistic organisms, grouped as “ESKAPE” pathogens, which comprise enterococci, a leading cause of hospital-acquired infections and outbreaks with emergence of MDR isolates, especially vancomycin-resistant Enterococcus faecium (VREF). Note that no information about sRNA expression is known in this major opportunistic pathogen. By transcriptomic and genomic analyses using E. faecium Aus0004 reference strain, 249 transcribed IGRs, including sRNA candidates, were detected and, using a series of cut-offs, this set was lowered down to 54 sRNAs while 7 that were predicted based on comparative sequence analysis. RNA-seq was performed with and without subinhibitory concentrations (SIC) of daptomycin, a cyclic lipopeptide antibiotic used for VREF infections. Under daptomycin SIC exposure, 260 genes (9.1% of the genome) had a significant alteration of expression including 80 upregulated genes and 180 downregulated genes. Among the repressed genes, a large proportion (55%) coded for proteins involved in carbohydrate and transport metabolism. Also, we focused on the 9 sRNAs exhibiting the highest expression, and all of them were confirmed as expressed along bacterial growth by Northern blots and qPCR. Out of these 9 sRNAs, four had significantly lower or higher expression in the presence of daptomycin SIC, and therefore responded to antibiotic exposure. Finally, we also tested the expression of these 9 sRNAs in a collection of isogenic Aus0004 mutants with increasing levels of daptomycin resistance, and we observed by qPCR that some sRNAs had a significantly modified expression in daptomycin resistance mutants. It highlights the significant implication of some of the E. faecium sRNAs in the early steps of the development of daptomycin resistance. This is the first experimental genome-wide sRNA identification in Gram-positive E. faecium, a leading cause of hospital acquired infections.

Project description:As integral parts of gene regulatory networks, small regulatory RNAs (sRNAs) play important roles in the adaptation of bacteria to environmental conditions. To adapt to iron limitation, many bacteria exploit an "iron-sparing" response mediated by Fur-regulated sRNAs such as RyhB of E. coli, allowing them to reduce the synthesis of iron-utilizing proteins not essential for growth. In this study, we aimed to confirm the regulatory role of the sRNA IsrR (Iron-sparing response Regulator) of S. aureus and to characterize the IsrR targetome in order to better understand the pathogens adaptation to iron-limited conditions often encountered in the host. To identify potential IsrR targets, two different experimental approaches were used. In the first, proteome profiles of S. aureus HG001 were compared to those of an isogenic ΔisrR mutant under iron-limited conditions. In the second approach, the effects of IsrR on the proteome under iron-rich conditions were analyzed using a strain in which IsrR was constitutively expressed from a plasmid. In silico target prediction was performed using the CopraRNA2 tool. Presence of IsrR caused a large number of consistent changes in protein levels both under iron-limited or iron-rich conditions. Among the proteins exhibiting significantly different abundance, 63 were in common between both experiments. Since sRNAs can have direct and indirect effects on global gene expression, the experimental approach was combined with in silico target prediction. In this way, very likely direct IsrR targets were identified, which include catalase (KatA) and three tricarboxylic acid (TCA) cycle enzymes (CitB, SdhA, SucA). Follow-up analyses confirmed that IsrR negatively affects KatA and CitB enzymatic activities. In contrast to most identified IsrR targets, the predicted interaction region of IsrR with the katA mRNA does not overlap the ribosome binding site, suggesting that IsrR cannot only inhibit translation, but also directly affect mRNA stability. A putative IsrR targetome was identified. In agreement with Fur-regulated sRNAs of other organisms, IsrR negatively affects the expression of TCA cycle and heme biosynthesis enzymes.

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Discovery and profiling of small RNAs responsive to stress conditions in the plant pathogen Pectobacterium atrosepticum

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