Translational T-box riboswitches bind tRNA by modulating conformational flexibility.

T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.

Transcriptome analysis and molecular characterization of novel small RNAs in Mycobacterium tuberculosis Lineage 1.

Mycobacterium tuberculosis (Mtb), the tuberculosis-causing agent, exhibits diverse genetic lineages, with known links to virulence. While genomic and transcriptomic variations between modern and ancient Mtb lineages have been explored, the role of small non-coding RNA (sRNA) in post-translational gene regulation remains largely uncharted. In this study, Mtb Lineage 1 (L1) Sabahan strains (n = 3) underwent sRNA sequencing, revealing 351 sRNAs, including 23 known sRNAs and 328 novel ones identified using ANNOgesic. Thirteen sRNAs were selected based on the best average cut-off value of 300, with RT-qPCR revealing significant expression differences for sRNA 1 (p = 0.0132) and sRNA 29 (p = 0.0012) between Mtb L1 and other lineages (L2 and L4, n = 3) (p > 0.05). Further characterization using RACE (rapid amplification of cDNA ends), followed by target prediction with TargetRNA3 unveils that sRNA 1 (55 base pairs) targets Rv0506, Rv0697, and Rv3590c, and sRNA 29 (86 base pairs) targets Rv33859c, Rv3345c, Rv0755c, Rv0107c, Rv1817, Rv2950c, Rv1181, Rv3610c, and Rv3296. Functional characterization with Mycobrowser reveals these targets involved in regulating intermediary metabolism and respiration, cell wall and cell processes, lipid metabolism, information pathways, and PE/PPE. In summary, two novel sRNAs, sRNA 1 and sRNA 29, exhibited differential expression between L1 and other lineages, with predicted roles in essential Mtb functions. These findings offer insights into Mtb regulatory mechanisms, holding promise for the development of improved tuberculosis treatment strategies in the future.

A novel small non-coding RNA 562 mediates the virulence of Pseudomonas plecoglossicida by regulating the expression of fliP, a key component of flagella T3SS.

Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.

Beyond its preferential niche: Brucella abortus RNA down-modulates the IFN-γ-induced MHC-I expression in epithelial and endothelial cells.

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

A novel small RNA PhaS contributes to polymyxin B-heteroresistance in carbapenem-resistant Klebsiella pneumoniae.

In recent years, polymyxin has been used as a last-resort therapy for carbapenem-resistant bacterial infections. The emergence of heteroresistance (HR) to polymyxin hampers the efficacy of polymyxin treatment by amplifying resistant subpopulation. However, the mechanisms behind polymyxin HR remain unclear. Small noncoding RNAs (sRNAs) play an important role in regulating drug resistance. The purpose of this study was to investigate the effects and mechanisms of sRNA on polymyxin B (PB)-HR in carbapenem-resistant Klebsiella pneumoniae. In this study, a novel sRNA PhaS was identified by transcriptome sequencing. PhaS expression was elevated in the PB heteroresistant subpopulation. Overexpression and deletion of PhaS were constructed in three carbapenem-resistant K. pneumoniae strains. Population analysis profiling, growth curve, and time-killing curve analysis showed that PhaS enhanced PB-HR. In addition, we verified that PhaS directly targeted phoP through the green fluorescent protein reporter system. PhaS promoted the expression of phoP, thereby encouraging the expression of downstream genes pmrD and arnT. This upregulation of arnT promoted the 4-amino-4-deoxyL-arabinosaccharide (L-Ara4N) modification of lipid A in PhaS overexpressing strains, thus enhancing PB-HR. Further, within the promoter region of PhaS, specific PhoP recognition sites were identified. ONPG assays and RT-qPCR analysis confirmed that PhaS expression was positively modulated by PhoP and thus up-regulated by PB stimulation. To sum up, a novel sRNA enhancing PB-HR was identified and a positive feedback regulatory pathway of sRNA-PhoP/Q was demonstrated in the study. This helps to provide a more comprehensive and clear understanding of the underlying mechanisms behind polymyxin HR in carbapenem-resistant K. pneumoniae.

Identification of intracellular bacteria from multiple single-cell RNA-seq platforms using CSI-Microbes.

The study of the tumor microbiome has been garnering increased attention. We developed a computational pipeline (CSI-Microbes) for identifying microbial reads from single-cell RNA sequencing (scRNA-seq) data and for analyzing differential abundance of taxa. Using a series of controlled experiments and analyses, we performed the first systematic evaluation of the efficacy of recovering microbial unique molecular identifiers by multiple scRNA-seq technologies, which identified the newer 10x chemistries (3' v3 and 5') as the best suited approach. We analyzed patient esophageal and colorectal carcinomas and found that reads from distinct genera tend to co-occur in the same host cells, testifying to possible intracellular polymicrobial interactions. Microbial reads are disproportionately abundant within myeloid cells that up-regulate proinflammatory cytokines like IL1Β and CXCL8, while infected tumor cells up-regulate antigen processing and presentation pathways. These results show that myeloid cells with bacteria engulfed are a major source of bacterial RNA within the tumor microenvironment (TME) and may inflame the TME and influence immunotherapy response.

Variations in seminal microbiota and their functional implications in chickens adapted to high-altitude environments.

Seminal fluid, once believed to be sterile, is now recognized as constituting a complex and dynamic environment inhabited by a diverse community of micro-organisms. However, research on the seminal microbiota in chickens is limited, and microbiota variations among different chicken breeds remain largely unexplored. In this study, we collected semen samples from Beijing You Chicken (BYC) and Tibetan Chicken (TC) and explored the characteristics of the microbiota using 16S rRNA gene sequencing. Additionally, we collected cloacal samples from the TC to control for environmental contamination. The results revealed that the microbial communities in the semen were significantly different from those in the cloaca. Firmicutes and Actinobacteriota were the predominant phyla in BYC and TC semen, respectively, with Lactobacillus and Phyllobacterium being the dominant genera in each group. Additionally, the seminal microbiota of BYC exhibited greater richness and evenness than that of TC. Principal coordinate analysis (PCoA) indicated significant intergroup differences between the seminal microbiotas of BYC and TC. Subsequently, by combining linear discriminant analysis effect size and random forest analyses, we identified Lactobacillus as the predominant microorganism in BYC semen, whereas Phyllobacterium dominated in TC semen. Furthermore, co-occurrence network analysis revealed a more intricate network in the BYC group than in the TC group. Additionally, unique microbial functional characteristics were observed in each breed, with TC exhibiting metabolic features potentially associated with their ability to adapt to high-altitude environments. The results of this study emphasized the unique microbiota present in chicken semen, which may be influenced by genetics and evolutionary history. Significant variations were observed between low-altitude and high-altitude breeds, highlighting the breed-specific implications of the seminal microbiota for reproduction and high-altitude adaptation.

Bifidobacterium bifidum SAM-VI Riboswitch Conformation Change Requires Peripheral Helix Formation.

The Bifidobacterium bifidum SAM-VI riboswitch undergoes dynamic conformational changes that modulate downstream gene expression. Traditional structural methods such as crystallography capture the bound conformation at high resolution, and additional efforts would reveal details from the dynamic transition. Here, we revealed a transcription-dependent conformation model for Bifidobacterium bifidum SAM-VI riboswitch. In this study, we combine small-angle X-ray scattering, chemical probing, and isothermal titration calorimetry to unveil the ligand-binding properties and conformational changes of the Bifidobacterium bifidum SAM-VI riboswitch and its variants. Our results suggest that the SAM-VI riboswitch contains a pre-organized ligand-binding pocket and stabilizes into the bound conformation upon binding to SAM. Whether the P1 stem formed and variations in length critically influence the conformational dynamics of the SAM-VI riboswitch. Our study provides the basis for artificially engineering the riboswitch by manipulating its peripheral sequences without modifying the SAM-binding core.

Multiplexing Imaging of Closely Located Single-Nucleotide Mutations in Single Cells via Encoded in situ PCR.

Mutation accumulation in RNAs results in closely located single-nucleotide mutations (SNMs), which is highly associated with the drug resistance of pathogens. Imaging of SNMs in single cells has significance for understanding the heterogeneity of RNAs that are related to drug resistance, but the direct "see" closely located SNMs remains challenging. Herein, we designed an encoded ligation-mediated in situ polymerase chain reaction method (termed enPCR), which enabled the visualization of multiple closely located SNMs in bacterial RNAs. Unlike conventional ligation-based probes that can only discriminate a single SNM, this method can simultaneously image different SNMs at closely located sites with single-cell resolution using modular anchoring probes and encoded PCR primers. We tested the capacity of the method to detect closely located SNMs related to quinolone resistance in the gyrA gene of Salmonella enterica (S. enterica), and found that the simultaneous detection of the closely located SNMs can more precisely indicate the resistance of the S. enterica to quinolone compared to the detection of one SNM. The multiplexing imaging assay for SNMs can serve to reveal the relationship between complex cellular genotypes and phenotypes.

Exploring a unique class of flavoenzymes: Identification and biochemical characterization of ribosomal RNA dihydrouridine synthase.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

Utilizing RNA-seq Data to Infer Bacterial Transcription Termination Sites and Validate Predictions.

The transcription termination process is an important part of the gene expression process in the cell. It has been studied extensively, but many aspects of the mechanism are not well understood. The widespread availability of experimental RNA-seq data from high-throughput experiments provides a unique opportunity to infer the end of the transcription units genome wide. This data is available for both Rho-dependent and Rho-independent termination pathways that drive transcription termination in bacteria. Our book chapter gives an overview of the current knowledge of Rho-independent transcription termination mechanisms and the prediction approaches currently deployed to infer the termination sites. Thereafter, we describe our method that uses cluster hairpins to detect Rho-independent transcription termination sites. These clusters are a group of hairpins that lies at <15 bp from each other and are together capable of enforcing the termination process. The idea of a group of hairpins being extensively used for transcription termination is new, and results show that at least 52% of the total cases are of this type, while in the remaining cases, a single strong hairpin is capable of driving transcription termination. The reads derived from the RNA-seq data for corresponding bacteria have been used to validate the predicted sites. The predictions that match these RNA-seq derived sites have higher confidence, and we find almost 98% of the predicted sites, including alternate termination sites, to match the RNA-seq data. We discuss the features of predicted hairpins in detail for a better understanding of the Rho-independent transcription termination mechanism in bacteria. We also explain how users can use the tools developed by us to do transcription terminator predictions and design their experiments through genome-level visualization of the transcription termination sites from the precomputed INTERPIN database.

Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni.

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.

GUT MICROBIOME DIVERSITY OF THREE RHINOCEROS SPECIES IN EUROPEAN ZOOS.

The wild rhinoceros populations have declined drastically in the past decades because the rhinoceros are heavily hunted for their horns. Zoological institutions aim to conserve rhinoceros populations in captivity, but one of the challenges of ex situ conservation is to provide food sources that resemble those available in the wild. Considering that the mammalian gut microbiota is a pivotal player in their host's health, the gut microbiota of rhinoceros may also play a role in the bioavailability of nutrients. Therefore, this study aims to characterize the fecal microbiome composition of grazing white rhinoceros (WR; Ceratotherium simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) as well as the browsing black rhinoceros (BR; Diceros bicornis) kept in European zoos. Over the course of 1 yr, 166 fecal samples in total were collected from 9 BR (n = 39), 10 GOHR (n = 56), and 14 WR (n = 71) from 23 zoological institutions. The bacterial composition in the samples was determined using 16S rRNA gene Illumina sequencing. The fecal microbiomes of rhinoceros clustered by species, with BR clustering more distantly from GOHR and WR. Furthermore, the data report clustering of rhinoceros microbiota according to individual rhinoceros and institutional origin, showing that zoological institutions play a significant role in shaping the gut microbiome of rhinoceros species. In addition, BR exhibit a relatively higher microbial diversity than GOHR and WR. BR seem more susceptible to microbial gut changes and appear to have a more diverse microbiome composition among individuals than GOHR and WR. These data expand on the role of gut microbes and can provide baseline data for continued efforts in rhinoceros conservation and health status.

Caulobacter crescentus RNase E condensation contributes to autoregulation and fitness.

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA's 5' untranslated region (UTR). While the 5' UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacterium Caulobacter crescentus RNase E contains a similar 5' UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal intrinsically disordered region (IDR) of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDR's ability to promote phase separation correlates with enhanced 5' UTR cleavage, suggesting that phase separation of RNase E with the 5' UTR enhances autoregulation. Finally, using growth competition experiments, we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5' UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing thiC Riboswitch in Escherichia coli.

Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.

Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue.

BACKGROUND: The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. METHODS: We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). FINDINGS: We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. INTERPRETATION: RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. FUNDING: Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

An RNA pseudoknot mediates toxin translation and antitoxin inhibition.

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion.

sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.

Bradyrhizobium japonicum HmuP is an RNA-binding protein that positively controls hmuR operon expression by suppression of a negative regulatory RNA element in the 5' untranslated region.

The hmuR operon encodes proteins for the uptake and utilization of heme as a nutritional iron source in Bradyrhizobium japonicum. The hmuR operon is transcriptionally activated by the Irr protein and is also positively controlled by HmuP by an unknown mechanism. An hmuP mutant does not express the hmuR operon genes nor does it grow on heme. Here, we show that hmuR expression from a heterologous promoter still requires hmuP, suggesting that HmuP does not regulate at the transcriptional level. Replacement of the 5' untranslated region (5'UTR) of an HmuP-independent gene with the hmuR 5'UTR conferred HmuP-dependent expression on that gene. Recombinant HmuP bound an HmuP-responsive RNA element (HPRE) within the hmuR 5'UTR. A 2 nt substitution predicted to destabilize the secondary structure of the HPRE abolished both HmuP binding activity in vitro and hmuR expression in cells. However, deletion of the HPRE partially restored hmuR expression in an hmuP mutant, and it rescued growth of the hmuP mutant on heme. These findings suggest that the HPRE is a negative regulatory RNA element that is suppressed when bound by HmuP to express the hmuR operon.