Antibiotic recalcitrance refers to a slower rate of death for either a bacterial population or a subpopulation of cells upon antibiotic exposure. It complicates treatment of many bacterial infections by contributing to treatment length, treatment failure, disease recurrence, and the emergence of antimicrobial resistance (AMR). Thus, blocking antibiotic recalcitrance could be a powerful strategy for improving treatment outcomes and reducing AMR rates. Here, using a forward genetic method for the isolation of antibiotic-recalcitrant mutants, we isolated two Mycobacterium smegmatis strains with mutations in the tRNA-modifying enzyme adenine-N(1)-methyltransferase. Both mutants were recalcitrant to proteostasis-perturbing antibiotics. We linked these phenotypes to upregulation of the transcriptional regulator WhiB7, highlighting its role as a point of convergence in the regulation of multiple mechanisms of antibiotic recalcitrance and resistance. Further, we identified a mechanism by which the amino acid alanine couples trans -translation to ribosome regulation-dependent, WhiB7-mediated expression of antibiotic resistance and recalcitrance genes, allowing bacterial cells to engage seemingly mutually exclusive mechanisms of survival upon exposure to stress.

Transfer RNA fragments are proposed to regulate numerous processes in eukaryotes, including translation inhibition, epigenetic inheritance, and cancer. In the bacterium Salmonella enterica serovar Typhimurium, 5' tRNA halves ending in 2',3' cyclic phosphate are proposed to bind the RtcR transcriptional activator, resulting in transcription of an RNA repair operon. However, since 5' and 3' tRNA halves can remain base paired after cleavage, the 5' tRNA halves could potentially bind RtcR as nicked tRNAs. Here we report that nicked tRNAs are ligands for RtcR. By isolating RNA from bacteria under conditions that preserve base pairing, we show that many tRNA halves are in the form of nicked tRNAs. Using a circularly permuted tRNA that mimics a nicked tRNA, we show that nicked tRNA ending in 2',3' cyclic phosphate is a better ligand for RtcR than the corresponding 5' tRNA half. In human cells, we show that some tRNA halves similarly remain base paired as nicked tRNAs following cleavage by anticodon nucleases. Our work supports a role for the RNA repair operon in repairing nicked tRNAs and has implications for the functions proposed for tRNA fragments in eukaryotes.

Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.

Ro60 ribonucleoproteins (RNPs), composed of the ring-shaped Ro 60-kDa (Ro60) protein and noncoding RNAs called Y RNAs, are present in all three domains of life. Ro60 was first described as an autoantigen in patients with rheumatic disease, and Ro60 orthologs have been identified in 3% to 5% of bacterial genomes, spanning the majority of phyla. Their functions have been characterized primarily in Deinococcus radiodurans, the first sequenced bacterium with a recognizable ortholog. InD. radiodurans, the Ro60 ortholog enhances the ability of 3'-to-5' exoribonucleases to degrade structured RNA during several forms of environmental stress. Y RNAs are regulators that inhibit or allow the interactions of Ro60 with other proteins and RNAs. Studies of Ro60 RNPs in other bacteria hint at additional functions, since the most conserved Y RNA contains a domain that is a close tRNA mimic and Ro60 RNPs are often encoded adjacent to components of RNA repair systems.

Noncoding Y RNAs are present in both animal cells and many bacteria. In all species examined, Y RNAs tether the Ro60 protein to an effector protein to perform various cellular functions. For example, in the bacterium Deinococcus radiodurans , Y RNA tethers Ro60 to the exoribonuclease polynucleotide phosphorylase, specializing this nuclease for structured RNA degradation. Recently, a new Y RNA subfamily was identified in bacteria. Bioinformatic analyses of these YrlA (Y RNA-like A) RNAs predict that the effector-binding domain resembles tRNA. We present the structure of this domain, the overall folding of which is strikingly similar to canonical tRNAs. The tertiary interactions that are responsible for stabilizing tRNA are present in YrlA, making it a close tRNA mimic. However, YrlA lacks a free CCA end and contains a kink in the stem corresponding to the anticodon stem. Since nucleotides in the D and T stems are conserved among YrlAs, they may be an interaction site for an unknown factor. Our experiments identify YrlA RNAs as a new class of tRNA mimics.

The earliest autoantibodies in lupus are directed against the RNA binding autoantigen Ro60, but the triggers against this evolutionarily conserved antigen remain elusive. We identified Ro60 orthologs in a subset of human skin, oral, and gut commensal bacterial species and confirmed the presence of these orthologs in patients with lupus and healthy controls. Thus, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via cross-reactivity in genetically susceptible individuals. Sera from human anti-Ro60-positive lupus patients immunoprecipitated commensal Ro60 ribonucleoproteins. Human Ro60 autoantigen-specific CD4 memory T cell clones from lupus patients were activated by skin and mucosal Ro60-containing bacteria, supporting T cell cross-reactivity in humans. Further, germ-free mice spontaneously initiated anti-human Ro60 T and B cell responses and developed glomerular immune complex deposits after monocolonization with a Ro60 ortholog-containing gut commensal, linking anti-Ro60 commensal responses in vivo with the production of human Ro60 autoantibodies and signs of autoimmunity. Together, these data support that colonization with autoantigen ortholog-producing commensal species may initiate and sustain chronic autoimmunity in genetically predisposed individuals. The concept of commensal ortholog cross-reactivity may apply more broadly to autoimmune diseases and lead to novel treatment approaches aimed at defined commensal species.

Anti-Ro60 autoantibodies are some of the earliest found in lupus patients and initiate epitope spreading. We identified a subset of commensal microbes with Ro60 orthologs, and hypothesized that Ro60 cross-reactivity may initiate or flare lupus in genetically susceptible individuals. Subjects with systemic and subacute cutaneous lupus erythematosus and healthy controls were recruited and 16S V4 sequencing of the skin, oral, and fecal microbiomes was performed. The presence of commensals with Ro60 orthologs was common among healthy and lupus subjects. Ro60+ lupus subjects had higher mean levels of P. propionicum on the skin than healthy subjects but there was no overall dysbiosis of the microbiota. Lupus memory T cell clones specific for P. propionicum proliferated in response to human Ro60. Similarly, T cell clones specific for B. thetaiotaomicron Ro60 proliferated in response to human Ro60, demonstrating T cell cross-reactivity. Human Ro60+ lupus serum immunoprecipitated P. propionicum Ro60 and its Y RNA binding partner, suggesting B cell cross-reactivity. Mice monocolonized with B. thetaiotaomicron produced serum anti-Ro60 antibodies, and cells from spleen and mesenteric lymph node proliferated in response to both B. thetaiotaomicron Ro60 and human Ro60, demonstrating cross-reactivity and the potential for causality. In summary, Ro60 autoimmune T and B cells from human lupus patients reacted with commensal Ro60 in vitro, and commensal Ro60 triggered anti-human Ro60 responses in vivo. Taken together, these data support that colonization with Ro60 ortholog-producing bacteria may induce and sustain chronic autoimmunity in lupus. This concept may apply more broadly to human autoimmune diseases and could lead to development of novel microbiota-targeted approaches to treat autoimmunity.

BackgroundThe earliest autoantibodies in systemic lupus erythematosus (SLE), a prototypical autoimmune disease, are directed against the RNA binding protein Ro60 but initiating triggers remain unknown. We identified commensal Ro60 orthologs in human skin, oral, and gut microbiomes in a subset of human species. Thus we hypothesize that Ro60‐ortholog‐carrying commensals induce autoimmunity via cross‐reactivity with human Ro60 in genetically susceptible individuals.MethodsV4 16S rDNA samples from SLE and control subjects were sequenced using the MiSeq platform. In addition, species‐specific enrichment by real‐time PCR for Ro60 bacteria (Propionibacterium propionicum, Corynebacterium amycolatum and Bacteroides thetaiotaomicron) was performed. Co‐immunoprecipitation was performed using human SLE serum and Ro60 ortholog‐containing bacterial lysates. Ro60‐specific memory CD4 T cells from SLE patients were cloned using a T cell library assay and stimulated with heat‐killed Ro60 bacteria. Supernatants were screened for cytokine proliferation using Legendplex. Ro60−/− mice were crossed to the TLR7.1 C57BL/6 transgenic lupus model and screened for Ro60 antibodies. Germ‐free mice were monocolonized with B. thetaiotaomicron and tested for anti‐Ro60 antibodies by ELISA. Mesenteric lymph node (MLN) and spleen cells were stimulated with bacterial and human Ro60 to assess for proliferation in vitro.ResultsRo60‐producing gut commensals were prevalent in controls and lupus patients. However, when human serum was used to co‐immunoprecipitate Ro60 and its bound Y RNA from the Ro60+ skin commensal P. propionicum, only antibodies from human Ro60‐positive lupus patients reacted with commensal Ro60. Lack of binding in Ro60‐negative patients or healthy controls supported antibody cross‐reactivity between human and commensal Ro60. Further, Human Ro60‐specific CD4 memory T cell clones proliferated in response to P. propionicum and commensal‐specific peptides, demonstrating T cell cross‐reactivity with commensal Ro60. Cytokine analysis proposes a heterogenous phenotype that is consistent with pathogen‐specific T cell clones. Serum anti‐Ro60 IgG autoantibodies were persistently induced in Ro60−/− TLR7 tg mice, suggesting a microbial trigger in the absence of host Ro60. Finally, MLN and splenic lymphocytes from germ‐free C57BL/6 mice that were monocolonized with B. thetaiotaomicron proliferated in response to bacterial and human Ro60 and sera contained anti‐human Ro60 IgG antibodies.ConclusionsAnti‐human Ro60 IgG in Ro60−/− TLR7 tg mice suggests a microbial trigger of these autoantibodies. Monocolonization with Ro60+ bacteria induced cross‐reactive anti‐human Ro60 responses in vivo. Ortholog cross‐reactivity is underscored by Ro60‐specific T cell clones and sera from lupus patients that reacted with commensal Ro60 in vitro. Our data support a model in which colonization with Ro60 ortholog‐carrying skin and gut bacteria sustain chronic autoreactivity in lupus. Quantifying and targeting Ro60+ bacteria in SLE patients may lead to novel biomarkers and treatment approaches.Ortholog cross‐reactivity is a novel concept that could contribute to the pathogenesis of human autoimmune diseases more broadly.Support or Funding InformationArthritis Foundation

Deinococcus radiodurans RNA ligase (DraRnl) seals 3΄-OH/5΄-PO4 nicks in duplex nucleic acids in which the 3΄-OH nick terminus consists of two or more ribonucleotides. DraRnl exemplifies a widely distributed Rnl5 family of nick-sealing RNA ligases, the physiological functions of which are uncharted. Here we show via gene knockout that whereas DraRnl is inessential for growth of D. radiodurans, its absence sensitizes the bacterium to killing by ionizing radiation (IR). DraRnl protein is present in exponentially growing and stationary phase cells, but is depleted during the early stages of recovery from 10 kGy of IR and subsequently replenished during the late phase of post-IR genome reassembly. Absence of DraRnl elicts a delay in reconstitution of the 10 kGy IR-shattered D. radiodurans replicons that correlates with the timing of DraRnl replenishment in wild-type cells. Complementation with a catalytically dead mutant highlights that nick sealing activity is important for the radioprotective function of DraRnl. Our findings suggest a scenario in which DraRnl acts at genomic nicks resulting from gap-filling by a ribonucleotide-incorporating repair polymerase.

The earliest autoantibodies in lupus are directed against the human autoantigen Ro60, and are common in SLE and SCLE. Certain commensal species found in the human skin, mouth, and gut contain Ro60 orthologs. We hypothesized that these bacteria sustain anti-human Ro60 responses in genetically predisposed hosts and therefore aimed to characterize T and B cell cross-reactivity with commensal Ro60. Skin, mouth, and fecal microbiomes were collected from a pilot cohort (n=10 SLE/SCLE) and tested for Ro60 bacteria by PCR and MiSeq sequencing. Co-immunoprecipitations and proliferation assays of cloned T cells from peripheral blood were used to assess cross-reactivity. Gnotobiotic mice were monocolonized for proof-of-principle in vivo. Ro60-producing gut commensal bacteria were common among lupus patients and healthy controls independent of HLA genotype. However, antibodies from 4/6 Ro60-positive lupus patients co-immunoprecipitated Ro60 and its Y RNA binding partner from the Ro60-containing skin commensal bacterium, P. propionicum, demonstrating antibody cross-reactivity between human and skin commensal-derived Ro60. This was not seen in healthy controls or Ro60-negative lupus patients. Lupus patient memory CD4 T cell clones specific for human Ro60 proliferated in response to P. propionicum, supporting T cell cross-reactivity. Finally, we detected anti-Ro60 IgA in fecal samples from germ-free mice monocolonized with a Ro60-positive gut commensal, B. thetaiotaomicron (n=4, p=0.04), suggesting a causal link between Ro60-producing commensals and anti-human Ro60 autoantibodies. In summary, autoimmune Ro60-specific T and B cells respond to commensal Ro60 orthologs, suggesting that otherwise “harmless” bacteria may have the potential to trigger lupus in genetically predisposed patients. The paradigm of commensal ortholog cross-reactivity may fundamentally change the way we view and treat autoimmune diseases.

Human Ro60 is an RNA binding protein that is commonly targeted in systemic autoimmunity. We identified Ro60 orthologs that are conserved in a subset of skin, oral, and gut commensal species. Since anti-Ro60 antibodies are the earliest autoantibodies detected in lupus patients, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via autoepitope cross-reactivity in genetically susceptible individuals. While Ro60-producing gut commensals were common among both healthy controls and lupus patients, only antibodies from 4 Ro60-positive lupus patients, but not Ro60-negative subjects, co-immunoprecipitated Ro60 and its bound Y RNA from the Ro60-containing skin commensal P. propionicum. This suggests antibody cross-reactivity between human Ro60 antibodies and commensal Ro60. Next, Ro60-specific CCR6+ and CCR6- CD4 memory T cells clones from lupus patients were generated using a T cell library assay. Ro60 CCR6+ T cell clones proliferated in response to P. propionicum, demonstrating T cell cross-reactivity with commensal Ro60. Finally, germ-free mice monocolonized with B. thetaiotaomicron, a Ro60 ortholog-containing gut commensal, produced fecal anti-human Ro60 IgA antibodies (n=4, p=0.04), linking anti-Ro60 commensal responses in vivo with the generation of human Ro60 autoantibodies. In summary, Ro60 autoimmune T and B cells from human lupus patients cross-reacted with commensal Ro60 in vitro and commensal Ro60 triggered anti-Ro60 antibodies in vivo. Taken together, these data suggest that colonization with autoantigen ortholog-carrying species may sustain chronic autoimmunity in patients. This concept could lead to development of novel therapeutic approaches targeted at the human microbiota.

Many bacteria encode an ortholog of the Ro60 autoantigen, a ring-shaped protein that is bound in animal cells to noncoding RNAs (ncRNAs) called Y RNAs. Studies in Deinococcus radiodurans revealed that Y RNA tethers Ro60 to polynucleotide phosphorylase, specializing this exoribonuclease for structured RNA degradation. Although Ro60 orthologs are present in a wide range of bacteria, Y RNAs have been detected in only two species, making it unclear whether these ncRNAs are common Ro60 partners in bacteria. In this study, we report that likely Y RNAs are encoded near Ro60 in >250 bacterial and phage species. By comparing conserved features, we discovered that at least one Y RNA in each species contains a domain resembling tRNA. We show that these RNAs contain nucleotide modifications characteristic of tRNA and are substrates for several enzymes that recognize tRNAs. Our studies confirm the importance of Y RNAs in bacterial physiology and identify a new class of ncRNAs that mimic tRNA.

Many bacteria contain an ortholog of the Ro autoantigen, a ring-shaped protein that binds noncoding RNAs (ncRNAs) called Y RNAs. In the only studied bacterium, Deinococcus radiodurans, the Ro ortholog Rsr functions in heat-stress-induced ribosomal RNA (rRNA) maturation and starvation-induced rRNA decay. However, the mechanism by which this conserved protein and its associated ncRNAs act has been obscure. We report that Rsr and the exoribonuclease polynucleotide phosphorylase (PNPase) form an RNA degradation machine that is scaffolded by Y RNA. Single-particle electron microscopy, followed by docking of atomic models into the reconstruction, suggests that Rsr channels single-stranded RNA into the PNPase cavity. Biochemical assays reveal that Rsr and Y RNA adapt PNPase for effective degradation of structured RNAs. A Ro ortholog and ncRNA also associate with PNPase in Salmonella Typhimurium. Our studies identify another ribonucleoprotein machine and demonstrate that ncRNA, by tethering a protein cofactor, can alter the substrate specificity of an enzyme.

Non-coding RNAs (ncRNAs) called Y RNAs are abundant components of both animal cells and a variety of bacteria. In all species examined, these ~100 nt RNAs are bound to the Ro 60 kDa (Ro60) autoantigen, a ring-shaped protein that also binds misfolded ncRNAs in some vertebrate nuclei. Although the function of Ro60 RNPs has been mysterious, we recently reported that a bacterial Y RNA tethers Ro60 to the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase) to form RYPER (Ro60/Y RNA/PNPase Exoribonuclease RNP), a new RNA degradation machine. PNPase is a homotrimeric ring that degrades single-stranded RNA, and Y RNA-mediated tethering of Ro60 increases the effectiveness of PNPase in degrading structured RNAs. Single particle electron microscopy of RYPER suggests that RNA threads through the Ro60 ring into the PNPase cavity. Further studies indicate that Y RNAs may also act as gates to regulate entry of RNA substrates into the Ro60 channel. These findings reveal novel functions for Y RNAs and raise questions about how the bacterial findings relate to the roles of these ncRNAs in animal cells. Here we review the literature on Y RNAs, highlighting their close relationship with Ro60 proteins and the hypothesis that these ncRNAs function generally to tether Ro60 rings to diverse RNA-binding proteins.

Non-coding RNAs (ncRNAs) called Y RNAs are abundant components of both animal cells and a variety of bacteria. In all species examined, these ~100 nt RNAs are bound to the Ro 60 kDa (Ro60) autoantigen, a ring-shaped protein that also binds misfolded ncRNAs in some vertebrate nuclei. Although the function of Ro60 RNPs has been mysterious, we recently reported that a bacterial Y RNA tethers Ro60 to the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNPase) to form RYPER (Ro60/Y RNA/PNPase Exoribonuclease RNP), a new RNA degradation machine. PNPase is a homotrimeric ring that degrades single-stranded RNA, and Y RNA-mediated tethering of Ro60 increases the effectiveness of PNPase in degrading structured RNAs. Single particle electron microscopy of RYPER suggests that RNA threads through the Ro60 ring into the PNPase cavity. Further studies indicate that Y RNAs may also act as gates to regulate entry of RNA substrates into the Ro60 channel. These findings reveal novel functions for Y RNAs and raise questions about how the bacterial findings relate to the roles of these ncRNAs in animal cells. Here we review the literature on Y RNAs, highlighting their close relationship with Ro60 proteins and the hypothesis that these ncRNAs function generally to tether Ro60 rings to diverse RNA-binding proteins.

研究了Duffing振子对策动力参数与初始值敏感的一致性,基于Duffing振子在不同初值时的仿真发现,不同初值情况下,混沌振子从混沌态过渡到大尺度周期态的过渡过程存在"周混渐变"和"混周渐变"两种,并且在过渡过程中,策动力增大的方向为周期态延时增长,混沌态延时缩短的方向,提出了利用过渡态检测微弱正弦信号的方法,基于"混周渐变"过渡态的检测,能检测的最低信噪比可达-60.7 dB,实验结果表明,这种方法非常适合检测地磁扰动信号Pc2小类中的确定频率信号.更多还原

A improved K-mean cluster method was proposed to detect the randomly occurring small-signal with uncertain frequency,amplitude and phase in broad frequency band.The sophisticate cluster method was based on a feature parameter,which cannot embody the similarity of two or multiple feature parameters between the sample points in the cluster.The improved K-mean cluster method divided the sample points into a number of meshes on the basis of the spectral density,and drew the histogram of every mesh.By the parameters' histogram peak values,the number of cluster centers can be estimated,and these initial cluster centers were selected.According to the minimum Euclidean distance principle,the sample points in every mesh were clustered on the basis of the frequency.The sample points in the subclass have the similarity of frequency and spectral density.A comparative analysis of subclasses between the sample information and the to-be-detected information is conducted.The new subclasses are obtained which correspond to the feature values of randomly occurring small-signal.Experiment results show that randomly occurring small-signal with uncertain frequency can be recognized in a complicated environment.

The consistency of the sensitive dependence on initial conditions and driving force parameter is researched. Under the difference initial conditions, Duffing oscillator is simulated. It is found that the transition process of Duffing oscillator from chaos state to the large scale period state can be divided into "period-to-chaos transition" and "chaos-to-period transition", and the greater the driving force is, the longer the delay time of the period state, the shorter the delay time of the chaos state is. A new method is proposed to detect weak sinusoidal signal by using the transition state. The lowest signal-to-noise ratio based on the transition state of "chaos-to-period transition" which can be detected is down to -60.7 dB. Experimental result shows that the proposed method especially adapted to detect the assigned signal in subclass Pc2 of geomagnetic ultra-low-frequency (ULF) signal.