Iron starvation increases the production of the Pseudomonas aeruginosa RsmY and RsmZ sRNAs in static conditions.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that induces virulence gene expression in response to host-mediated iron starvation. Recently, our laboratory showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions. One of these is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate the expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces the expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. Disruption of either the pqsR gene also eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. Taken together, our results show novel targets of iron regulation that are specific to static growth, highlighting the importance of studying regulatory mechanisms in static communities that may be more representative of growth during chronic infection.IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host-acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primarily relied on shaking bacterial cultures; while these ensure cultural homogeneity, they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

A novel ionizing radiation-induced small RNA, DrsS, promotes the detoxification of reactive oxygen species in Deinococcus radiodurans.

A plethora of gene regulatory mechanisms with eccentric attributes in Deinoccocus radiodurans confer it to possess a distinctive ability to survive under ionizing radiation. Among the many regulatory processes, small RNA (sRNA)-mediated regulation of gene expression is prevalent in bacteria but barely investigated in D. radiodurans. In the current study, we identified a novel sRNA, DrsS, through RNA-seq analysis in D. radiodurans cells while exposed to ionizing radiation. Initial sequence analysis for promoter identification revealed that drsS is potentially co-transcribed with sodA and dr_1280 from a single operon. Elimination of the drsS allele in D. radiodurans chromosome resulted in an impaired growth phenotype under γ-radiation. DrsS has also been found to be upregulated under oxidative and genotoxic stresses. Deletion of the drsS gene resulted in the depletion of intracellular concentration of both Mn2+ and Fe2+ by ~70% and 40%, respectively, with a concomitant increase in carbonylation of intracellular protein. Complementation of drsS gene in ΔdrsS cells helped revert its intracellular Mn2+ and Fe2+ concentration and alleviated carbonylation of intracellular proteins. Cells with deleted drsS gene exhibited higher sensitivity to oxidative stress than wild-type cells. Extrachromosomally expressed drsS in ΔdrsS cells retrieved its oxidative stress resistance properties by catalase-mediated detoxification of reactive oxygen species (ROS). In vitro binding assays indicated that DsrS directly interacts with the coding region of the katA transcript, thus possibly protecting it from cellular endonucleases in vivo. This study identified a novel small RNA DrsS and investigated its function under oxidative stress in D. radiodurans. IMPORTANCE: Deinococcus radiodurans possesses an idiosyncratic quality to survive under extreme ionizing radiation and, thus, has evolved with diverse mechanisms which promote the mending of intracellular damages caused by ionizing radiation. As sRNAs play a pivotal role in modulating gene expression to adapt to altered conditions and have been delineated to participate in almost all physiological processes, understanding the regulatory mechanism of sRNAs will unearth many pathways that lead to radioresistance in D. radiodurans. In that direction, DrsS has been identified to be a γ-radiation-induced sRNA, which is also induced by oxidative and genotoxic stresses. DrsS appeared to activate catalase under oxidative stress and detoxify intracellular ROS. This sRNA has also been shown to balance intracellular Mn(II) and Fe concentrations protecting intracellular proteins from carbonylation. This novel mechanism of DrsS identified in D. radiodurans adds substantially to our knowledge of how this bacterium exploits sRNA for its survival under stresses.

Circulating tumor cell-derived exosome-transmitted long non-coding RNA TTN-AS1 can promote the proliferation and migration of cholangiocarcinoma cells.

BACKGROUND: Exosomes assume a pivotal role as essential mediators of intercellular communication within tumor microenvironments. Within this context, long noncoding RNAs (LncRNAs) have been observed to be preferentially sorted into exosomes, thus exerting regulatory control over the initiation and progression of cancer through diverse mechanisms. RESULTS: Exosomes were successfully isolated from cholangiocarcinoma (CCA) CTCs organoid and healthy human serum. Notably, the LncRNA titin-antisense RNA1 (TTN-AS1) exhibited a conspicuous up-regulation within CCA CTCs organoid derived exosomes. Furthermore, a significant elevation of TTN-AS1 expression was observed in tumor tissues, as well as in blood and serum exosomes from patients afflicted with CCA. Importantly, this hightened TTN-AS1 expression in serum exosomes of CCA patients manifested a strong correlation with both lymph node metastasis and TNM staging. Remarkably, both CCA CTCs organoid-derived exosomes and CCA cells-derived exosomes featuring pronounced TTN-AS1 expression demonstrated the capability to the proliferation and migratory potential of CCA cells. Validation of these outcomes was conducted in vivo experiments. CONCLUSIONS: In conclusion, our study elucidating that CCA CTCs-derived exosomes possess the capacity to bolster the metastasis tendencies of CCA cells by transporting TTN-AS1. These observations underscore the potential of TTN-AS1 within CTCs-derived exosomes to serve as a promising biomarker for the diagnosis and therapeutic management of CCA.

Small regulatory RNA RSaX28 promotes virulence by reinforcing the stability of RNAIII in community-associated ST398 clonotype Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a notorious pathogen that cause metastatic or complicated infections. Hypervirulent ST398 clonotype strains, remarkably increased in recent years, dominated Community-associated S. aureus (CA-SA) infections in the past decade in China. Small RNAs like RNAIII have been demonstrated to play important roles in regulating the virulence of S. aureus, however, the regulatory roles played by many of these sRNAs in the ST398 clonotype strains are still unclear. Through transcriptome screening and combined with knockout phenotype analysis, we have identified a highly transcribed sRNA, RSaX28, in the ST398 clonotype strains. Sequence analysis revealed that RSaX28 is highly conserved in the most epidemic clonotypes of S. aureus, but its high transcription level is particularly prominent in the ST398 clonotype strains. Characterization of RSaX28 through RACE and Northern blot revealed its length to be 533nt. RSaX28 is capable of promoting the hemolytic ability, reducing biofilm formation capacity, and enhancing virulence of S. aureus in the in vivo murine infection model. Through IntaRNA prediction and EMSA validation, we found that RSaX28 can specifically interact with RNAIII, promoting its stability and positively regulating the translation of downstream alpha-toxin while inhibiting the translation of Sbi, thereby regulating the virulence and biofilm formation capacity of the ST398 clonotype strains. RSaX28 is an important virulence regulatory factor in the ST398 clonotype S. aureus and represents a potential important target for future treatment and immune intervention against S. aureus infections.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

Transcriptome fine-mapping in Fusobacterium nucleatum reveals FoxJ, a new σE-dependent small RNA with unusual mRNA activation activity.

The oral commensal Fusobacterium nucleatum can spread to extra-oral sites, where it is associated with diverse pathologies, including pre-term birth and cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of the RNA regulatory networks that allow this bacterium to adapt to its various niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor, σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped the 5' and 3' ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156-nucleotide sRNA previously misannotated as the 5' untranslated region (UTR) of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as is FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5' and 3' UTRs in F. nucleatum and expands our understanding of the σE response in this evolutionarily distant bacterium.IMPORTANCEThe oral microbe Fusobacterium nucleatum can colonize secondary sites, including cancer tissue, and likely deploys complex regulatory systems to adapt to these new environments. These systems are largely unknown, partly due to the phylogenetic distance of F. nucleatum to other model organisms. Previously, we identified a global stress response mediated by σE that displays functional conservation with the envelope stress response in Proteobacteria, comprising a coding and noncoding regulatory arm. Through global identification of transcriptional start and stop sites, we uncovered the small RNA (sRNA) FoxJ as a novel component of the noncoding arm of the σE response in F. nucleatum. Together with its companion sRNA FoxI, FoxJ post-transcriptionally modulates the synthesis of envelope proteins, revealing a conserved function for σE-dependent sRNAs between Fusobacteriota and Proteobacteria. Moreover, FoxJ activates the gene expression for several targets, which is a mode of regulation previously unseen in the noncoding arm of the σE response.

A global survey of small RNA interactors identifies KhpA and KhpB as major RNA-binding proteins in Fusobacterium nucleatum.

The common oral microbe Fusobacterium nucleatum has recently drawn attention after it was found to colonize tumors throughout the human body. Fusobacteria are also interesting study systems for bacterial RNA biology as these early-branching species encode many small noncoding RNAs (sRNAs) but lack homologs of the common RNA-binding proteins (RBPs) CsrA, Hfq and ProQ. To search for alternate sRNA-associated RBPs in F. nucleatum, we performed a systematic mass spectrometry analysis of proteins that co-purified with 19 different sRNAs. This approach revealed strong enrichment of the KH domain proteins KhpA and KhpB with nearly all tested sRNAs, including the σE-dependent sRNA FoxI, a regulator of several envelope proteins. KhpA/B act as a dimer to bind sRNAs with low micromolar affinity and influence the stability of several of their target transcripts. Transcriptome studies combined with biochemical and genetic analyses suggest that KhpA/B have several physiological functions, including being required for ethanolamine utilization. Our RBP search and the discovery of KhpA/B as major RBPs in F. nucleatum are important first steps in identifying key players of post-transcriptional control at the root of the bacterial phylogenetic tree.

Balanced cell division is secured by two different regulatory sites in OxyS RNA.

The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.

Elevated levels of IL-6 in IgA nephropathy patients are induced by an epigenetically driven mechanism modulated by viral and bacterial RNA.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis and the role of IL-6 in pathogenesis is becoming increasingly important. A recent whole genome DNA methylation screening in IgAN patients identified a hypermethylated region comprising the non-coding RNA Vault RNA 2-1 (VTRNA2-1) that could explain the high IL-6 levels. METHODS: The pathway leading to IL-6 secretion controlled by VTRNA2-1, PKR, and CREB was analyzed in peripheral blood mononuclear cells (PBMCs) isolated from healthy subjects (HS), IgAN patients, transplanted patients with or without IgAN. The role of double and single-strand RNA in controlling the pathway was investigated. RESULTS: VTRNA2-1 was downregulated in IgAN compared to HS and in transplanted IgAN patients (TP-IgAN) compared to non-IgAN transplanted (TP). The loss of the VTRNA2-1 natural restrain in IgAN patients caused PKR hyperphosphorylation, and consequently the activation of CREB by PKR, which, in turn, led to high IL-6 production, both in IgAN and in TP-IgAN patients. IL-6 levels could be decreased by the PKR inhibitor imoxin. In addition, PKR is normally activated by bacterial and viral RNA, and we found that both the RNA poly(I:C), and the COVID-19 RNA-vaccine stimulation significantly increased the IL-6 levels in PBMCs from HS but had an opposite effect in those from IgAN patients. CONCLUSION: The discovery of the upregulated VTRNA2-1/PKR/CREB/IL-6 pathway in IgAN patients may provide a novel approach to treating the disease and may be useful for the development of precision nephrology and personalized therapy by checking the VTRNA2-1 methylation level in IgAN patients.

Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA.

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Thermus thermophilus Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection.

Recent advances in prokaryotic Argonaute proteins (pAgos) as potential genome-editing tools have provided new insights into the development of pAgos-based nucleic acid detection platforms. However, pAgos-based isothermal detection remains challenging. Here, we report a true isothermal amplification strategy, termed Thermus thermophilus Argonaute-based thermostable exponential amplification reaction (TtAgoEAR), to detect RNA with ultrasensitivity and single-nucleotide resolution at a constant temperature of 66 °C. We demonstrate the reliable detection of lncRNA, mRNA, and virus RNA with attomolar sensitivity and that TtAgoEAR can be applied to detect RNA targets in in cell lines, saliva, and tissues. We utilize this assay to distinguish pancreatic cancer cells carrying the mutation from wild-type cells with as little as 2 ng of RNA material. We also show that TtAgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that TtAgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.

A Pseudomonas aeruginosa small RNA regulates chronic and acute infection.

The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.

Comparison of plasmid stabilization systems during heterologous isopropanol production in fed-batch bioreactor.

Strain robustness during production of recombinant molecules is of major interest to ensure bioprocess profitability. The heterogeneity of populations has been shown in the literature as a source of instability in bioprocesses. Thus, the heterogeneity of the population was studied by evaluating the robustness of the strains (stability of plasmid expression, cultivability, membrane integrity and macroscopic cell behavior) during well-controlled fedbatch cultures. On the context of microbial production of chemical molecules, isopropanol (IPA) has been produced by recombinant strains of Cupriavidus necator. Plasmid stability was monitored by the plate count method to assess the impact of isopropanol production on plasmid stability, depending on implanted plasmid stabilization systems for strain engineering designs. With the reference strain Re2133/pEG7c, an isopropanol titer of 15.1 g·L-1 could be achieved. When the isopropanol concentration has reached about 8 g. L-1, cell permeability increased (up to 25 %) and plasmid stability decreased significantly (up to 1.5 decimal reduction rate) resulting in decreased isopropanol production rates. Bioprocess robustness under isopropanol producing conditions was then investigated with two plasmid construction strategies (1) Post Segregational Killing hok/sok (in Re2133/pEG20) and (2) expression of GroESL chaperon proteins (in Re2133/pEG23). Plasmid stability for strain Re2133/pEG20 (PSK hok/sok) appears to be improved up to 11 g. L-1 of IPA compared to the reference strain (8 g. L-1 IPA). Nevertheless, cell permeability followed the same dynamic as the reference strain with a drastic increase around 8 g. L-1 IPA. On the contrary, the Re2133/pEG23 strain made it possible to minimize the cell permeability (with a constant value at 5 % IP permeability) and to increase the growth capacities in response to increased isopropanol concentrations but plasmid stability was the weakest. The metabolic burden, linked to either the overexpression of GroESL chaperones or the PSK hok/sok system, seems to be deleterious for the overall isopropanol production compared to the reference strain (RE2133/pEG7c) even if we have shown that the overexpression chaperones GroESL improve membrane integrity and PSK system hok/sok improve plasmid stability as long as isopropanol concentration does not exceed 11 g L- 1.

sRNA21, a novel small RNA, protects Mycobacterium abscessus against oxidative stress.

BACKGROUND: During infection, Mycobacterium abscessus encounters numerous environmental changes and adapts to them using a variety of complex mechanisms. Non-coding small RNAs (sRNAs) have been shown in other bacteria to be involved in post-transcriptional regulatory pathways, including environmental stress adaptation. However, the potential role of sRNAs in the resistance to oxidative stress in M. abscessus was not clearly described. METHODS: In the present study, we analyzed putative sRNAs identified by RNA-sequencing (RNA-seq) experiments in M. abscessus ATCC_19977 under oxidative stress, and the transcription profiles of sRNAs with differential expression were verified by quantitative reverse transcription-PCR (qRT-PCR). Six sRNA overexpression strains were constructed, and the differences in growth curves between these strains and the control strain were verified. An upregulated sRNA under oxidative stress was selected and named sRNA21. The survival ability of the sRNA21 overexpression strain was assessed, and computer-based approaches were used to predict the targets and pathways regulated by sRNA21. The total ATP production and NAD+ /NADH ratio of the sRNA21 overexpression strain were measured. The expression level of antioxidase-related genes and the activity of antioxidase were tested to confirm the interaction of sRNA21 with the predicted target genes in silico. RESULTS: In total, 14 putative sRNAs were identified under oxidative stress, and the qRT-PCR analysis of six sRNAs showed comparable results to RNA-seq assays. Overexpression of sRNA21 in M. abscessus increased cell growth rate and intracellular ATP level before and after peroxide exposure. The expression of genes encoding alkyl hydroperoxidase and superoxide dismutase was significantly increased, and superoxide dismutase activity was enhanced in the sRNA21 overexpression strain. Meanwhile, after sRNA21 overexpression, the intracellular NAD+ /NADH ratio decreased, indicating changes in redox homeostasis. CONCLUSIONS: Our findings show that sRNA21 is an oxidative stress-induced sRNA that increases M. abscessus survival and promotes the expression of antioxidant enzymes under oxidative stress. These findings may provide new insights into the adaptive transcriptional response of M. abscessus to oxidative stress.

The Small RNA NcS25 Regulates Biological Amine-Transporting Outer Membrane Porin BCAL3473 in Burkholderia cenocepacia.

Regulation of porin expression in bacteria is complex and often involves small-RNA regulators. Several small-RNA regulators have been described for Burkholderia cenocepacia, and this study aimed to characterize the biological role of the conserved small RNA NcS25 and its cognate target, outer membrane protein BCAL3473. The B. cenocepacia genome carries a large number of genes encoding porins with yet-uncharacterized functions. Expression of the porin BCAL3473 is strongly repressed by NcS25 and activated by other factors, such as a LysR-type regulator and nitrogen-depleted growth conditions. The porin is involved in transport of arginine, tyrosine, tyramine, and putrescine across the outer membrane. Porin BCAL3473, with NcS25 as a major regulator, plays an important role in the nitrogen metabolism of B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is a Gram-negative bacterium which causes infections in immunocompromised individuals and in people with cystic fibrosis. A low outer membrane permeability is one of the factors giving it a high level of innate resistance to antibiotics. Porins provide selective permeability for nutrients, and antibiotics can also traverse the outer membrane by this means. Knowing the properties and specificities of porin channels is therefore important for understanding resistance mechanisms and for developing new antibiotics and could help in overcoming permeability issues in antibiotic treatment.

A New Bacterial Adenosine-Derived Nucleoside as an Example of RNA Modification Damage.

The fields of RNA modification and RNA damage both exhibit a plethora of non-canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2-methylthio-1,N6-ethenoadenosine (ms2 ϵA), which is related to 1,N6-ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms2 ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2-methylthio-N6-isopentenyladenosine (ms2 i6 A). The relative abundance of ms2 ϵA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage.

Multifaceted Interplay between Hfq and the Small RNA GssA in Pseudomonas aeruginosa.

Behind the pathogenic lifestyle of Pseudomonas aeruginosa exists a complex regulatory network of intertwined switches at both the transcriptional and posttranscriptional levels. Major players that mediate translation regulation of several genes involved in host-P. aeruginosa interaction are small RNAs (sRNAs) and the Hfq protein. The canonical role of Hfq in sRNA-driven regulation is to act as a matchmaker between sRNAs and target mRNAs. Besides, the sRNA CrcZ is known to sequester Hfq and abrogate its function of translation repression of target mRNAs. In this study, we describe the novel sRNA GssA in the strain PA14 and its multifaceted interplay with Hfq. We show that GssA is multiresponsive to environmental and physiological signals and acts as an apical repressor of key bacterial functions in the human host such as the production of pyocyanin, utilization of glucose, and secretion of exotoxin A. We suggest that the main role of Hfq is not to directly assist GssA in its regulatory role but to repress GssA expression. In the case of pyocyanin production, we suggest that Hfq interplays with GssA also by converging a positive effect on this pathway. Furthermore, our results indicate that both Hfq and GssA play a positive role in anaerobic growth, possibly by regulating the respiratory chain. On the other hand, we show that GssA can modulate not only Hfq expression at both transcriptional and posttranscriptional levels but also that of CrcZ, thus potentially influencing the pleiotropic role of Hfq. IMPORTANCE The pathogenic lifestyle of the bacterium Pseudomonas aeruginosa, a leading cause of life-threatening infections in the airways of cystic fibrosis patients, is based on the fine regulation of virulence-associated factors. Regulatory small RNAs (sRNAs) and the RNA-binding protein Hfq are recognized key components within the P. aeruginosa regulatory networks involved in host-pathogen interaction. In this study, we characterized in the highly virulent P. aeruginosa strain PA14 the novel sRNA GssA. We found that it can establish a many-sided reciprocal interplay with Hfq which goes beyond the canonical mechanism of direct physical interaction that had previously been characterized for other sRNAs. Given that the Hfq-driven regulatory network of virulence factors is very broad and important for the progression of infection, we consider GssA as a new RNA target that can potentially be used to develop new antibacterial drugs.

E. coli 6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides.

The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S RNA 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for noncanonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the conserved 6S RNA template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions.

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.