Bacterial Small Non-coding RNAs Research Articles

Multi-omics characterization and identification of small non-coding RNAs in Spiroplasma eriocheiris

Spiroplasma eriocheiris is the major pathogen in the aquaculture shrimp and crab tremor disease; its infection causes heavy losses in aquaculture. Previous investigations concentrated on how the host responds to S. eriocheiris infection, with little known on the regulation of virulence and pathogenesis by the bacteria themselves. A body of increasing evidence shows that bacterial small noncoding RNAs (sRNAs) play a crucial part in regulating bacterial virulence and pathogenesis; however, whether there are some sRNAs in S. eriocheiris and how those sRNAs regulate virulence and pathogenesis are still unclear. This study analyzed multi-omics data integration to identify the potential sRNAs and their mediated regulatory network in S. eriocheiris. First, through an integration analysis of the public genomic and transcriptomic data, 54 potential sRNAs were identified in S. eriocheiris with a 50–250 nt length distribution and the base composition mainly AT. Next, sRNA profiling of the in vitro logarithmic and decline phase of S. eriocheiris, as well as the early and onset stage of infection in Eriocheir sinensis, was performed, showing that 4 sRNAs (SR01, SR05, SR08, and S04) were both down-regulated in the logarithmic and the onset stage of infection. Subsequently, integrated with sRNAs target gene prediction and target gene expression analysis, a regulatory network composed of four sRNAs and 96 target genes was constructed. Last, an RNA pull-down experiment was performed on the SR01, which exhibited the highest abundance and the highest number of target genes, resulting in 37 highly credible target genes with multiple genes related to bacterial virulence, such as GTPases and virulence factor coding genes. Collectively, this study identified the sRNAs in S. eriocheiris and revealed their mediated regulatory network for regulating virulence and pathogenesis, thus paving a new avenue for disease prevention and control in aquaculture.

Small RNAs in the Antarctic bacterium Pseudomonas extremaustralis responsive to oxygen availability and oxidative stress.

Bacterial small non-coding RNAs (sRNAs) play key roles as genetic regulators, mediating in the adaptability to changing environmental conditions and stress responses. In this work, we analysed putative sRNAs identified by RNA-seq experiments in different aeration conditions in the extremophile bacterium P. extremaustralis. These analyses allowed the identification of 177 putative sRNAs under aerobiosis (A), microaerobiosis (M) and microaerobiosis after H2 O2 exposure (m-OS). The size and transcription profile of eight sRNAs with differential expression were verified by Northern blot. sRNA40, with unknown function but conserved in other Pseudomonas species, was selected to perform overexpression experiments followed by RNA-seq analysis. The overexpression of sRNA40 in P. extremaustralis resulted in significant expression changes of 19 genes with 14 differentially upregulated and five downregulated. Among the upregulated genes, eight transcripts corresponded to components of secretion systems, such as gspH, gspK, and gspM, belonging to the Type II secretion system, and rspO and rspP from Type III secretion system. Our results showed a novel sRNA which expression was triggered by low oxygen levels, and whose overexpression was associated with upregulation of selected components of protein secretion systems.

Genome-wide screen and functional analysis in Xanthomonas reveal a large number of mRNA-derived sRNAs, including the novel RsmA-sequester RsmU.

Although bacterial small noncoding RNAs (sRNAs) are known to play a critical role in various cellular processes, including pathogenesis, the identity and action of such sRNAs are still poorly understood in many organisms. Here we have performed a genome‐wide screen and functional analysis of the sRNAs in Xanthomonas campestris pv. campestris (Xcc), an important phytopathogen. The 50–500‐nt RNA fragments isolated from the wild‐type strain grown in a virulence gene‐inducing condition were sequenced and a total of 612 sRNA candidates (SRCs) were identified. The majority (82%) of the SRCs were derived from mRNA, rather than specific sRNA genes. A representative panel of 121 SRCs were analysed by northern blotting; 117 SRCs were detected, supporting the contention that the overwhelming majority of the 612 SRCs identified are indeed sRNAs. Phenotypic analysis of strains overexpressing different candidates showed that a particular sRNA, RsmU, acts as a negative regulator of virulence, the hypersensitive response, and cell motility in Xcc. In vitro electrophoretic mobility shift assay and in vivo coimmunoprecipitation analyses indicated that RsmU interacted with the global posttranscriptional regulator RsmA, although sequence analysis displayed that RsmU is not a member of the sRNAs families known to antagonize RsmA. Northern blotting analyses demonstrated that RsmU has two isoforms that are processed from the 3′‐untranslated region of the mRNA of XC1332 predicted to encode ComEA, a periplasmic protein required for DNA uptake in bacteria. This work uncovers an unexpected major sRNA biogenesis strategy in bacteria and a hidden layer of sRNA‐mediated virulence regulation in Xcc.

SRNA OsiA Stabilizes Catalase mRNA during Oxidative Stress Response of Deincoccus radiodurans R1.

Deinococcus radiodurans adapts to challenging environments by modulating gene expression in response to oxidative stress. Recently, bacterial small noncoding RNAs (sRNAs) have been presumed to participate in the transcriptional or translational regulation of stress-responsive genes. We found 24 sRNAs that may be involved in the oxidative stress response of D. radiodurans by deep RNA sequencing. Moreover, a typical stress-inducible sRNA, IGR_3053, named OsiA, was predicted to bind to the mRNA of katA, katE, and sodC by the bioinformatics method. An osiA knockout of D. radiodurans displayed increased sensitivity to H2O2 and the decreased catalase activity and total antioxidant activity, suggesting that OsiA probably serves as a regulator in the adaptation to oxidative environments. Further microscale thermophoresis results demonstrated that OsiA can directly bind to the mRNA of katA, sodC, and katE. The stability test result of katA mRNA showed that its half-life was 2 min in the osiA mutant compared with 5 min in the wildtype(wt) strain. Our results indicated that OsiA can enhance the stability of katA mRNA and the activity of KatA and consequently the oxidation resistance of D.radiodurans. We are the first one to explore the super-strong oxidative stress resistance of D.radiodurans at the level of post-transcriptional regulation, and found a new pathway that provides a new explanation for the long-term adaptability of D.radiodurans in extreme environments.

EsR240, a non-coding sRNA, is required for the resistance of Edwardsiella tarda to stresses in macrophages and for virulence

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators that respond to environmental changes and pathogenic conditions. In this study, 13 novel sRNAs were identified in the intracellular pathogen, Edwardsiella tarda (E. tarda) ET13 strain, based on RNA sequencing and bioinformatic analyses. Eight of the 13 putative sRNAs from the ET13 strain were transcribed (as indicated by RT-PCR) following exposure to different stresses. The transcription levels of three sRNAs (EsR128, EsR139 and EsR240) were all highly induced under these stress conditions. Northern blot hybridization was employed to verify that EsR240 was expressed in the ET13 strain under both logarithmic and stationary growth phases, and that it formed a single copy transcript in the chromosomes of the ET13 strain. The precise start and end points of EsR240 were determined using 5’and 3’ RACE. The conservation of EsR240 was in agreement with the characteristics of sRNA, as indicated by a BLAST analysis. Furthermore, the survival rates of EsR240 mutant were lower than the rates of the wild type ET13 under stress conditions. When the infection time was extended 4 or 6 h, the CFUs of the wild type bacteria increased more significantly within macrophages compared to the mutant. When the intra-peritoneal (i.p.) route of infection was used in mice, the bacterial loads of the tissues in the mice infected with the wild type bacteria were significantly higher than in the mice infected with the mutants. The virulence of the EsR240 mutant was 6.79-fold lower than the wild type bacterium based on the LD50. In addition, the IntaRNA program was used to predict the target genes of EsR240. Out of the top 10 predicted target genes, 9 genes were regulated by EsR240. These target genes may encode FtsH protease modulator YccA, Na+ and H+ antiporters, FtsX-like permease family protein, glycoside hydrolases or various other proteins. Therefore, EsR240 may positively regulate its target genes in E. tarda to maintain intracellular survival within host macrophages and to increase its virulence.

Transcriptional noise and exaptation as sources for bacterial sRNAs.

Understanding how new genes originate and integrate into cellular networks is key to understanding evolution. Bacteria present unique opportunities for both the natural history and experimental study of gene origins, due to their large effective population sizes, rapid generation times, and ease of genetic manipulation. Bacterial small non-coding RNAs (sRNAs), in particular, many of which operate through a simple antisense regulatory logic, may serve as tractable models for exploring processes of gene origin and adaptation. Understanding how and on what timescales these regulatory molecules arise has important implications for understanding the evolution of bacterial regulatory networks, in particular, for the design of comparative studies of sRNA function. Here, we introduce relevant concepts from evolutionary biology and review recent work that has begun to shed light on the timescales and processes through which non-functional transcriptional noise is co-opted to provide regulatory functions. We explore possible scenarios for sRNA origin, focusing on the co-option, or exaptation, of existing genomic structures which may provide protected spaces for sRNA evolution.

An improved method for identification of small non-coding RNAs in bacteria using support vector machine

Bacterial small non-coding RNAs (sRNAs) are not translated into proteins, but act as functional RNAs. They are involved in diverse biological processes like virulence, stress response and quorum sensing. Several high-throughput techniques have enabled identification of sRNAs in bacteria, but experimental detection remains a challenge and grossly incomplete for most species. Thus, there is a need to develop computational tools to predict bacterial sRNAs. Here, we propose a computational method to identify sRNAs in bacteria using support vector machine (SVM) classifier. The primary sequence and secondary structure features of experimentally-validated sRNAs of Salmonella Typhimurium LT2 (SLT2) was used to build the optimal SVM model. We found that a tri-nucleotide composition feature of sRNAs achieved an accuracy of 88.35% for SLT2. We validated the SVM model also on the experimentally-detected sRNAs of E. coli and Salmonella Typhi. The proposed model had robustly attained an accuracy of 81.25% and 88.82% for E. coli K-12 and S. Typhi Ty2, respectively. We confirmed that this method significantly improved the identification of sRNAs in bacteria. Furthermore, we used a sliding window-based method and identified sRNAs from complete genomes of SLT2, S. Typhi Ty2 and E. coli K-12 with sensitivities of 89.09%, 83.33% and 67.39%, respectively.

Identification and validation of sRNAs in Edwardsiella tarda S08.

Bacterial small non-coding RNAs (sRNAs) are known as novel regulators involved in virulence, stress responsibility, and so on. Recently, a lot of new researches have highlighted the critical roles of sRNAs in fine-tune gene regulation in both prokaryotes and eukaryotes. Edwardsiella tarda (E. tarda) is a gram-negative, intracellular pathogen that causes edwardsiellosis in fish. Thus far, no sRNA has been reported in E. tarda. The present study represents the first attempt to identify sRNAs in E. tarda S08. Ten sRNAs were validated by RNA sequencing and quantitative PCR (qPCR). ET_sRNA_1 and ET_sRNA_2 were homolous to tmRNA and GcvB, respectively. However, the other candidate sRNAs have not been reported till now. The cellular abundance of 10 validated sRNA was detected by qPCR at different growth phases to monitor their biosynthesis. Nine candidate sRNAs were expressed in the late-stage of exponential growth and stationary stages of growth (36~60 h). And the expression of the nine sRNAs was growth phase-dependent. But ET_sRNA_10 was almost expressed all the time and reached the highest peak at 48 h. Their targets were predicted by TargetRNA2 and each sRNA target contains some genes that directly or indirectly relate to virulence. These results preliminary showed that sRNAs probably play a regulatory role of virulence in E. tarda.

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation.

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

In silico prediction and qPCR validation of novel sRNAs in Propionibacterium acnes KPA171202.

Propionibacterium acnes is an anaerobic, Gram-positive, opportunistic pathogen known to be involved in a wide variety of diseases ranging from mild acne to prostate cancer. Bacterial small non-coding RNAs are novel regulators of gene expression and are known to be involved in, virulence, pathogenesis, stress tolerance and adaptation to environmental changes in bacteria. The present study was undertaken keeping in view the lack of predicted sRNAs of P. acnes KPA171202 in databases. This report represents the first attempt to identify sRNAs in P. acnes KPA171202. A total of eight potential candidate sRNAs were predicted using SIPHT, one was found to have a Rfam homolog and seven were novel. Out of these seven predicted sRNAs, five were validated by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. The expression of these sRNAs was quantified in different growth phases by qPCR (quantitative PCR). They were found to be expressed in both exponential and stationary stages of growth but with maximum expression in stationary phase which points to a regulatory role for them. Further investigation of their targets and regulatory functions is in progress.

Genome-wide analyses of small non-coding RNAs in streptococci.

Streptococci represent a diverse group of Gram-positive bacteria, which colonize a wide range of hosts among animals and humans. Streptococcal species occur as commensal as well as pathogenic organisms. Many of the pathogenic species can cause severe, invasive infections in their hosts leading to a high morbidity and mortality. The consequence is a tremendous suffering on the part of men and livestock besides the significant financial burden in the agricultural and healthcare sectors. An environmentally stimulated and tightly controlled expression of virulence factor genes is of fundamental importance for streptococcal pathogenicity. Bacterial small non-coding RNAs (sRNAs) modulate the expression of genes involved in stress response, sugar metabolism, surface composition, and other properties that are related to bacterial virulence. Even though the regulatory character is shared by this class of RNAs, variation on the molecular level results in a high diversity of functional mechanisms. The knowledge about the role of sRNAs in streptococci is still limited, but in recent years, genome-wide screens for sRNAs have been conducted in an increasing number of species. Bioinformatics prediction approaches have been employed as well as expression analyses by classical array techniques or next generation sequencing. This review will give an overview of whole genome screens for sRNAs in streptococci with a focus on describing the different methods and comparing their outcome considering sRNA conservation among species, functional similarities, and relevance for streptococcal infection.

Identification of a Novel Small Non-Coding RNA Modulating the Intracellular Survival of Brucella melitensis.

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators respond to environmental changes, stressful conditions, and pathogenesis. In this study, by using a combined bioinformatic and experimental approach, eight novel sRNA genes were identified in intracellular pathogen Brucella melitensis. BSR0602, one sRNA that was highly induced in stationary phase, was further examined and found to modulate the intracellular survival of B. melitensis. BSR0602 was present at very high levels in vitro under stresses similar to those encountered during infection in host macrophages. Furthermore, BSR0602 was found to be highly expressed in the spleens of infected mice, suggesting its potential role in the control of pathogenesis. BSR0602 targets the mRNAs coding for gntR, a global transcriptional regulator, which is required for B. melitensis virulence. Overexpression of BSR0602 results in distinct reduction in the gntR mRNA level. B. melitensis with high level of BSR0602 is defective in bacteria intracellular survival in macrophages and defective in growth in the spleens of infected mice. Therefore, BSR0602 may directly inhibit the expression of gntR, which then impairs Brucellae intracellular survival and contributes to Brucella infection. Our findings suggest that BSR0602 is responsible for bacterial adaptation to stress conditions and thus modulate B. melitensis intracellular survival.

3′-UTRs as a source of regulatory RNAs in bacteria

Bacterial small non-coding RNAs, sRNAs, have up to now been identified primarily in intergenic regions. Chao et al reveal that the 3'-region of mRNAs is another rich reservoir of sRNAs.

Computational prediction and transcriptional analysis of sRNAs in Nitrosomonas europaea

Bacterial small noncoding RNAs (sRNAs) have been discovered in many genetically well-studied microorganisms and have been shown to regulate critical cellular processes at the post-transcriptional level. In this study, we used comparative genomics and microarray data to analyze the genome of the ammonia-oxidizing bacterium Nitrosomonas europaea for the presence and expression of sRNAs. Fifteen genes encoding putative sRNAs (psRNAs) were identified. Most of these genes showed altered expression in a variety of experimental conditions. The transcripts of two psRNAs were further characterized by mapping their 5'- and 3'-ends and by real-time PCR. The results of these analyses suggested that one of them, psRNA11, is involved in iron homeostasis in N. europaea.

Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation

Bacterial small noncoding RNAs (sRNAs) generally recognize target mRNAs in the 5' region to prevent 30S ribosomes from initiating translation. It was thought that the mRNA coding sequence (CDS) was refractory to sRNA-mediated repression, because elongating 70S ribosomes have an efficient RNA helicase activity that prevents stable target pairing. We report that the Hfq-associated MicC sRNA silences Salmonella typhimurium ompD mRNA via a <or=12-bp RNA duplex within the CDS (codons 23-26) that is essential and sufficient for repression. MicC does not inhibit translational initiation at this downstream position but instead acts by accelerating RNase E-dependent ompD mRNA decay. We propose an alternative gene-silencing pathway within bacterial CDS wherein sRNAs repress targets by endonucleolytic mRNA destabilization rather than by the prototypical inhibition of translational initiation. The discovery of CDS targeting markedly expands the sequence space for sRNA target predictions in bacteria.

Identification of differentially expressed small non‐coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics

Bacterial small non-coding RNAs (sRNAs) are being recognized as novel widespread regulators of gene expression in response to environmental signals. Here, we present the first search for sRNA-encoding genes in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, performed by a genome-wide computational analysis of its intergenic regions. Comparative sequence data from eight related α-proteobacteria were obtained, and the interspecies pairwise alignments were scored with the programs eQRNA and RNAz as complementary predictive tools to identify conserved and stable secondary structures corresponding to putative non-coding RNAs. Northern experiments confirmed that eight of the predicted loci, selected among the original 32 candidates as most probable sRNA genes, expressed small transcripts. This result supports the combined use of eQRNA and RNAz as a robust strategy to identify novel sRNAs in bacteria. Furthermore, seven of the transcripts accumulated differentially in free-living and symbiotic conditions. Experimental mapping of the 5′-ends of the detected transcripts revealed that their encoding genes are organized in autonomous transcription units with recognizable promoter and, in most cases, termination signatures. These findings suggest novel regulatory functions for sRNAs related to the interactions of α-proteobacteria with their eukaryotic hosts.

Translational repression is sufficient for gene silencing by bacterial small noncoding RNAs in the absence of mRNA destruction

Stress-induced Hfq-binding small RNAs of Escherichia coli, SgrS and RyhB, down-regulate the expression of target mRNAs through base-pairing. These small RNAs form ribonucleoprotein complexes with Hfq and RNase E. The regulatory outcomes of the RNase E/Hfq/small RNA-containing ribonucleoprotein complex (sRNP) are rapid degradation of target mRNAs and translational inhibition. Here, we ask to what extent the sRNP-mediated mRNA destabilization contributes to the overall silencing of target genes by using strains in which the rapid degradation of mRNA no longer occurs. We demonstrate that translational repression occurs in the absence of sRNP-mediated mRNA destabilization. We conclude that translational repression is sufficient for gene silencing by sRNP. One possible physiological role of mRNA degradation mediated by sRNP is to rid the cell of translationally inactive mRNAs, making gene silencing irreversible.