Bacterial Small RNAs Research Articles

Differential expression of small RNAs under chemical stress and fed-batch fermentation in E. coli.

BackgroundBacterial small RNAs (sRNAs) are recognized as posttranscriptional regulators involved in the control of bacterial lifestyle and adaptation to stressful conditions. Although chemical stress due to the toxicity of precursor and product compounds is frequently encountered in microbial bioprocessing applications, the involvement of sRNAs in this process is not well understood. We have used RNA sequencing to map sRNA expression in E. coli under chemical stress and high cell density fermentation conditions with the aim of identifying sRNAs involved in the transcriptional response and those with potential roles in stress tolerance.ResultsRNA sequencing libraries were prepared from RNA isolated from E. coli K-12 MG1655 cells grown under high cell density fermentation conditions or subjected to chemical stress with twelve compounds including four organic solvent-like compounds, four organic acids, two amino acids, geraniol and decanoic acid. We have discovered 253 novel intergenic transcripts with this approach, adding to the roughly 200 intergenic sRNAs previously reported in E. coli. There are eighty-four differentially expressed sRNAs during fermentation, of which the majority are novel, supporting possible regulatory roles for these transcripts in adaptation during different fermentation stages. There are a total of 139 differentially expressed sRNAs under chemical stress conditions, where twenty-nine exhibit significant expression changes in multiple tested conditions, suggesting that they may be involved in a more general chemical stress response. Among those with known functions are sRNAs involved in regulation of outer membrane proteins, iron availability, maintaining envelope homeostasis, as well as sRNAs incorporated into complex networks controlling motility and biofilm formation.ConclusionsThis study has used deep sequencing to reveal a wealth of hitherto undescribed sRNAs in E. coli and provides an atlas of sRNA expression during seventeen different growth and stress conditions. Although the number of novel sRNAs with regulatory functions is unknown, several exhibit specific expression patterns during high cell density fermentation and are differentially expressed in the presence of multiple chemicals, suggesting they may play regulatory roles during these stress conditions. These novel sRNAs, together with specific known sRNAs, are candidates for improving stress tolerance and our understanding of the E. coli regulatory network during fed-batch fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2231-8) contains supplementary material, which is available to authorized users.

Bacterial Small Regulatory RNAs and Hfq Protein.

Small regulatory RNA (sRNA) is a unique noncoding RNA involved in regulation of gene expression in both eukaryotic and bacterial cells. This short review discusses examples of positive and negative translation regulation by sRNAs in bacteria and participation of Hfq in these processes. The importance of structure investigation of nucleotide-protein and RNA-protein complexes for designing a model of Hfq interaction with both mRNA and sRNA simultaneously is demonstrated.

Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate.

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins.

Characterization of the salivary microbiome in patients with pancreatic cancer.

Clinical manifestations of pancreatic cancer often do not occur until the cancer has undergone metastasis, resulting in a very low survival rate. In this study, we investigated whether salivary bacterial profiles might provide useful biomarkers for early detection of pancreatic cancer. Using high-throughput sequencing of bacterial small subunit ribosomal RNA (16S rRNA) gene, we characterized the salivary microbiota of patients with pancreatic cancer and compared them to healthy patients and patients with other diseases, including pancreatic disease, non-pancreatic digestive disease/cancer and non-digestive disease/cancer. A total of 146 patients were enrolled at the UCSD Moores Cancer Center where saliva and demographic data were collected from each patient. Of these, we analyzed the salivary microbiome of 108 patients: 8 had been diagnosed with pancreatic cancer, 78 with other diseases and 22 were classified as non-diseased (healthy) controls. Bacterial 16S rRNA sequences were amplified directly from salivary DNA extractions and subjected to high-throughput sequencing (HTS). Several bacterial genera differed in abundance in patients with pancreatic cancer. We found a significantly higher ratio of Leptotrichia to Porphyromonas in the saliva of patients with pancreatic cancer than in the saliva of healthy patients or those with other disease (Kruskal–Wallis Test; P < 0.001). Leptotrichia abundances were confirmed using real-time qPCR with Leptotrichia specific primers. Similar to previous studies, we found lower relative abundances of Neisseria and Aggregatibacter in the saliva of pancreatic cancer patients, though these results were not significant at the P < 0.05 level (K–W Test; P = 0.07 and P = 0.09 respectively). However, the relative abundances of other previously identified bacterial biomarkers, e.g., Streptococcus mitis and Granulicatella adiacens, were not significantly different in the saliva of pancreatic cancer patients. Overall, this study supports the hypothesis that bacteria abundance profiles in saliva are useful biomarkers for pancreatic cancer though much larger patient studies are needed to verify their predictive utility.

Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage.

In E. coli, small RNA GlmZ activates the glmS mRNA by base-pairing in an Hfq dependent manner. When not required, GlmZ is bound by adaptor protein RapZ and recruited to RNase E, which cleaves GlmZ in its base-pairing sequence. Small RNA GlmY counteracts cleavage of GlmZ by sequestration of RapZ. Although both sRNAs are highly homologous, only GlmZ specifically binds Hfq and undergoes cleavage by RNase E. We used domain swapping to identify the responsible modules. Two elements, the 3′ terminal oligo(U) stretch and the base-pairing region enable GlmZ to interact with Hfq. Accordingly, Hfq inhibits cleavage of GlmZ, directing it to base-pairing. Intriguingly, the central stem loop of GlmZ is decisive for cleavage, whereas the sequence comprising the actual cleavage site is dispensable. Assisted by RapZ, RNase E cleaves any RNA fused to the 3′ end of this module. These results suggest a novel mode for RNase E recognition, in which one of the required handholds in the substrate is replaced by an RNA binding protein. This device can generate RNAs of interest in their 5′ monophosphorylated form on demand. As these species are rapidly degraded, this tool allows to regulate gene expression post-transcriptionally by modulation of RapZ levels.

Identification of novel sRNAs involved in biofilm formation, motility, and fimbriae formation in Escherichia coli.

Bacterial small RNAs (sRNAs) are known regulators in many physiological processes. In Escherichia coli, a large number of sRNAs have been predicted, among which only about a hundred are experimentally validated. Despite considerable research, the majority of their functions remain uncovered. Therefore, collective analysis of the roles of sRNAs in specific cellular processes may provide an effective approach to identify their functions. Here, we constructed a collection of plasmids overexpressing 99 individual sRNAs, and analyzed their effects on biofilm formation and related phenotypes. Thirty-three sRNAs significantly affecting these cellular processes were identified. No consistent correlations were observed, except that all five sRNAs suppressing type I fimbriae inhibited biofilm formation. Interestingly, IS118, yet to be characterized, suppressed all the processes. Our data not only reveal potentially critical functions of individual sRNAs in biofilm formation and other phenotypes but also highlight the unexpected complexity of sRNA-mediated metabolic pathways leading to these processes.

Accelerating Discovery and Functional Analysis of Small RNAs with New Technologies.

Over the past decade, bacterial small RNAs (sRNAs) have gone from a biological curiosity to being recognized as a major class of regulatory molecules. High-throughput methods for sampling the transcriptional output of bacterial cells demonstrate that sRNAs are universal features of bacterial transcriptomes, are plentiful, and appear to vary extensively over evolutionary time. With ever more bacteria coming under study, the question becomes how can we accelerate the discovery and functional characterization of sRNAs in diverse organisms. New technologies built on high-throughput sequencing are emerging that can rapidly provide global insight into the numbers and functions of sRNAs in bacteria of interest, providing information that can shape hypotheses and guide research. In this review, we describe recent developments in transcriptomics (RNA-seq) and functional genomics that we expect to help us develop an integrated, systems-level view of sRNA biology in bacteria.

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.

Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia.

Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.

The End Is Not the End: Remnants of tRNA Precursors Live On to Sponge Up Small Regulatory RNAs

Natural RNA sponges sequestering cellular noncoding RNA molecules have been found in diverse organisms. In this issue, Lalaouna et al. (2015) report another type of RNA sponge, showing that stable intermediates of bacterial tRNA processing control endogenous small RNAs.

Target activation by regulatory RNAs in bacteria.

Bacterial small regulatory RNAs (sRNAs) are commonly known to repress gene expression by base pairing to target mRNAs. In many cases, sRNAs base pair with and sequester mRNA ribosome-binding sites, resulting in translational repression and accelerated transcript decay. In contrast, a growing number of examples of translational activation and mRNA stabilization by sRNAs have now been documented. A given sRNA often employs a conserved region to interact with and regulate both repressed and activated targets. However, the mechanisms underlying activation differ substantially from repression. Base pairing resulting in target activation can involve sRNA interactions with the 5(') untranslated region (UTR), the coding sequence or the 3(') UTR of the target mRNAs. Frequently, the activities of protein factors such as cellular ribonucleases and the RNA chaperone Hfq are required for activation. Bacterial sRNAs, including those that function as activators, frequently control stress response pathways or virulence-associated functions required for immediate responses to changing environments. This review aims to summarize recent advances in knowledge regarding target mRNA activation by bacterial sRNAs, highlighting the molecular mechanisms and biological relevance of regulation.

Toxicity of aminoglycoside antibiotics may be due to alternate binding of the human mitochondrial ribosomal RNA

Aminoglycoside antibiotics are a well-known antibiotic family used to treat various bacterial infections in humans and animals, but unfortunately have significant toxicity. These aminoglycoside antibiotics primarily bind to helix 44 of the bacterial small ribosomal subunit RNA at the tRNA receptor aminoacyl site and, thus inhibit protein synthesis by causing misreading or obstructing the translocation process (1). However, recent findings demonstrate that aminoglycosides bind helix 69 of the large subunit RNA as well. Little is known about the interaction of aminoglycosides with the homologous H69 rRNA hairpin of the human host and which may play a role inaminoglycoside toxicity. To gain insight, we compared aminoglycoside binding to the terminal hairpin ofhuman cytoplasmic, human mitochondrial, and Escherichia coli H69, and human mitochondrial and E. coli H44 using UV-monitored thermal stability analysis, circular dichroism (CD), and NMR structural studies. The UV thermal analysis shows that the overallbinding affinities of gentamicin and kanamycin A to the human cytoplasmic, human mitochondrial, and E. coli H69 were similar. CD and 1H 1D and 2D NMR results indicated that aminoglycoside binding enhances the conformational stability by increasing base stacking on human mitochondrial H69. The greater binding induced stability on human mitochondrial H69 and similar binding affinity to that of E. coli suggest that human mitochondrial H69 may be important to understanding the mechanism of aminoglycoside toxicity.

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.

Hfq plays important roles in virulence and stress adaptation in Cronobacter sakazakii ATCC 29544.

Cronobacter spp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated with Cronobacter infection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq in C. sakazakii virulence. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion (3-fold) into animal cells and survival (10(3)-fold) within host cells, and exhibited low resistance to hydrogen peroxide (10(2)-fold). Remarkably, the loss of hfq led to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes.

Multiple approaches for the investigation of bacterial small regulatory RNAs self-assembly.

RNAs are flexible molecules involved in a multitude of roles in the cell. Specifically, noncoding RNAs (i.e., RNAs that do not encode a protein) have important functions in the regulation of biological processes such as RNA decay, translation, or protein translocation. In bacteria, most of those noncoding RNAs have been shown to be critical for posttranscriptional control through their binding to the untranslated regions of target mRNAs. Recent evidence shows that some of these noncoding RNAs have the propensity to self-assemble in prokaryotes. Although the function of this self-assembly is not known and may vary from one RNA to another, it offers new insights into riboregulation pathways. We present here the various approaches that can be used for the detection and analysis of bacterial small noncoding RNA self-assemblies.

Small RNAs in bacteria and archaea: who they are, what they do, and how they do it.

Small RNAs are ubiquitously present regulators in all kingdoms of life. Most bacterial and archaeal small RNAs (sRNAs) act by antisense mechanisms on multiple target mRNAs, thereby globally affecting essentially any conceivable trait-stress responses, adaptive metabolic changes, virulence etc. The sRNAs display many distinct mechanisms of action, most of them through effects on target mRNA translation and/or stability, and helper proteins like Hfq often play key roles. Recent data highlight the interplay between posttranscriptional control by sRNAs and transcription factor-mediated transcriptional control, and cross talk through mutual regulation of regulators. Based on the properties that distinguish sRNA-type from transcription factors-type control, we begin to glimpse why sRNAs have evolved as a second, essential layer of gene regulation. This review will discuss the prevalence of sRNAs, who they are, what biological roles they play, and how they carry out their functions.

Recognition of the small regulatory RNA RydC by the bacterial Hfq protein.

Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

Novosphingobium and its potential role in chronic obstructive pulmonary diseases: insights from microbiome studies.

Bacterial infection of lung airways underlies some of the main complications of COPD, significantly impacting disease progression and outcome. Colonization by bacteria may further synergize, amplify, or trigger pathways of tissue damage started by cigarette smoke, contributing to the characteristic airway inflammation and alveolar destruction of COPD. We sought to elucidate the presence and types of lung bacterial populations in different stages of COPD, aimed at revealing important insights into the pathobiology of the disease. Sequencing of the bacterial small subunit ribosomal RNA gene in 55 well-characterized clinical lung samples, revealed the presence of Novosphingobium spp. (>2% abundance) in lungs of patients with GOLD 3-GOLD 4 COPD, cystic fibrosis and a subset of control individuals. Novosphingobium-specific quantitative PCR was concordant with the sequence data and high levels of Novosphingobium spp. were quantifiable in advanced COPD, but not from other disease stages. Using a mouse model of subacute lung injury due to inhalation of cigarette smoke, bronchoalveolar lavage neutrophil and macrophage counts were significantly higher in mice challenged intratracheally with N. panipatense compared to control mice (p<0.01). Frequencies of neutrophils and macrophages in lung tissue were increased in mice challenged with N. panipatense at room air compared to controls. However, we did not observe an interaction between N. panipatense and subacute cigarette smoke exposure in the mouse. In conclusion, Novosphingobium spp. are present in more severe COPD disease, and increase inflammation in a mouse model of smoke exposure.

Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.