Towards the complete small RNome of Acinetobacter baumannii
ABSTRACT: In recent years, the Gram-negative bacterium Acinetobacter baumannii has garnered considerable attention for its unprecedented capacity to rapidly develop resistance to antibacterial therapeutics. This is coupled with the seemingly epidemic emergence of new hyper-virulent strains. Although strain-specific differences for A. baumannii isolates have been well described, these studies have primarily focused on proteinaceous factors. At present, only limited publications have investigated the presence and role of small regulatory RNA (sRNA) transcripts. Herein, we perform such an analysis, describing the RNA-seq-based identification of 78 A. baumannii sRNAs in the AB5075 background. Together with six previously identified elements, we include each of these in a new genome annotation file, which will serve as a tool to investigate regulatory events in this organism. Our work reveals that the sRNAs display high expression, accounting for >50 % of the 20 most strongly expressed genes. Through conservation analysis we identified six classes of similar sRNAs, with one found to be particularly abundant and homologous to regulatory, C4 antisense RNAs found in bacteriophages. These elements appear to be processed from larger transcripts in an analogous manner to the phage C4 molecule and are putatively controlled by two further sRNAs that are strongly antisense to them. Collectively, this study offers a detailed view of the sRNA content of A. baumannii, exposing sequence and structural conservation amongst these elements, and provides novel insight into the potential evolution, and role, of these understudied regulatory molecules. This study is based on the annotation of novel sRNAs on basis of an Acinetobacter baumannii RNA sequencing dataset. Each sample was generated by pooling three independent biological replicate RNA preps
Project description:In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome anno- tation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions. Four RNAseq data sets in total. Each sample was generated by pooling three independent biological replicate RNA preps
Project description:Staphylococcus aureus is an important human pathogen that causes life-threatening infections, and is resistant to the majority of our antibiotic arsenal. This resistance is complicated by the observation that most antibacterial agents target actively growing cells, thus, proving ineffective against slow growing populations, such as cells within a biofilm or in stationary phase. Recently, our group generated updated genome annotation files for S. aureus that not only include protein-coding genes but also regulatory and small RNAs. As such, these annotation files were used to perform a transcriptomic analysis in order to understand the metabolic and physiological changes that occur during transition from active growth to stationary phase; with a focus on sRNAs. We observed ∼24% of protein-coding and 34% of sRNA genes displaying changes in expression by ≥3-fold. Collectively, this study adds to our understanding of S. aureus adaptation to nutrient-limiting conditions, and sheds new light onto the contribution of sRNAs to this process. Bacterial cells were grown in TSB medium at 37°C with shaking for 3h (exponential growth phase) or 16h (stationary growth phase).
Project description:This study is the first to show transfer of regulatory bacterial sRNAs from bacterial OMVs to host cells. Demonstration of transfer of bacterial sRNA from Pseudomonas aeruginosa OMVs to host cells in two cell types. RNA isolated from PA14 OMV exposed primary human bronchial epithelial cells or CFBE41o- bronchial epithelial cells as well as unexposed control cells was analyzed by small RNA-Seq. Primary cell exposures were performed on two donors and exposures of CFBE41o- cells were done in triplicate.
Project description:Characterization of the sRNA content of P. aeruginosa OMVs compared to whole cells. Result: OMVs contain differentially packaged sRNAs. Whole cell PA14 and OMVs from 3 separate preparations.
Project description:Total RNA isolated from mid-log-grown cultures of A. baumannii and mutant strain in three independent times. Expression profile of A. baumannii and its protein kinase muatant was compared. Overall design: Agilent one-color experiment, Organism: Acinetobacter baumannii, Agilent Custom Acinetobacter baumannii 8x15k Microarray designed by Genotypic Technology Private Limited (AMADID: 079361).
Project description:Total RNA isolated from mid-log-grown cultures of A. baumannii and mutant strain in three independent times. Expression profile of A. baumannii and its byk mutant was compared. Overall design: Agilent one-color experiment, Organism: Acinetobacter baumannii, Agilent Custom Acinetobacter baumannii 8x15k Microarray designed by Genotypic Technology Private Limited (AMADID: 079361).
Project description:Background miRNAs and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep sRNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique tasiRNA biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that ten of the 19 miR828-targeted MYBs undergo siRNA biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusion Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. Identification of apple miRNAs and their targets from four different tissues
Project description:Transcriptional profiling of A. oleivorans DR1 cells harboring pAST2. Plasmid pAST2 is a tetracycline-resistance plasmid which was isolated from activated sludge (Hong et al., 2014). The complete plasmid sequence was deposited in the National Center for Biotechnology Information (NCBI) GenBank under accession number KC734561 [PMID: 24337108]. Acquisition of TC resistance through plasmid uptake is related to loss of biological fitness and affected host gene expression in oil-degrading soil bacterium, Acinetobacter oleivorans DR1. To identify genes in A. oleivorans DR1 harboring pAST2, the cells were grown to exponential phase (OD600 ~0.4) and total RNA was extracted using an RNeasy Mini kit (Qiagen, USA) following the manufacturer's instructions.
Project description: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. Overall design: sRNA profiles from P. atrosepticum SCRI1043 cells existing under growth-promoting and starvation conditions were generated using Strand-specific RNA sequencing