A global survey of small RNA interactors identifies KhpA and KhpB as major RNA-binding proteins in Fusobacterium nucleatum
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ABSTRACT:
ORGANISM(S): Fusobacterium nucleatum subsp. nucleatum ATCC 23726
PROVIDER: GSE246396 | GEO | 2024/02/01
REPOSITORIES: GEO
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Project description:The common oral microbe Fusobacterium nucleatum has recently gained attention when it was found to colonize tumors throughout the human body. Fusobacteria are also interesting in regard to 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. Here, to search for alternate sRNA-associated RBPs in F. nucleatum, we performed a systematic mass spectrometry analysis of proteins that copurified with 19 different sRNAs. Our approach recovered a 6S RNA-RNA polymerase complex in this species and discovered high enrichment of the KH domain proteins KhpA and KhpB with almost any tested sRNA, 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 cellular stability of several of their targets. RNA-seq analysis and cell biological assays suggest that KhpA/B have several physiological functions, including a strong requirement for ethanolamine utilization. Our RBP search and 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.
2024-02-08 | PXD046416 | Pride
Project description: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 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 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’ UTR of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as 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 most 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.
2024-02-08 | PXD047655 | Pride
Project description:The oral commensal Fusobacterium nucleatum can spread to extra-oral sites where it is associated with pathologies as diverse as pre-term birth or cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of RNA regulatory networks that allow this bacterium to adapt to different environmental 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 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’ UTR of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as 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 most 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.
2024-02-08 | GSE249955 | GEO
Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of σ70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.
2010-01-01 | GSE19200 | GEO
Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of σ70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.
2010-01-01 | GSE19196 | GEO
Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of Ï70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media. A microarray was designed for the detection of 101 predicted sRNAs by using OligiWiz version 2.1.3 (Nielsen et al., 2003, Wernersson & Nielsen, 2005). The prokaryotic setting was used to design 50-mers probes located, when possible, at the 3â end of the predicted genes. Probes for 10 negative controls, representing 10 genes of the L. pneumophila Paris plasmid, were also designed. Probes (Illumina) were dissolved in 50% DMSO to a final concentration of 30 uM and printed in triplicate on UltraGAPS coated glass slides (Corning) using a PerkinElmer SpotArray microarray printer. Fifteen micrograms of total RNA was labelled during cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) and amino-allyl dUTP (Sigma). Bacterial genomic DNA was used as the reference channel on each slide to allow comparison of each time point and different samples (Talaat et al., 2002). Five micrograms of genomic DNA (gDNA) was labelled with amino-allyl dUTP by using Klenow fragment and random primers (Invitrogen) at 37 °C for 18 h. DNA was subsequently coupled to the succinimidyl ester fluorescent dye AlexaFluor 546 (for cDNA) or Alexa Fluor 647 (for gDNA) (Invitrogen) following manufacturerâs protocols. Hybridization and data acquisition were performed as previously described (Hovel-Miner et al., 2009).Very low density array, like the sRNA microarray used here, cannot be normalised with common procedure like total intensity or lowess. Local background was removed from spot signal intensity and the noise signal was estimated by recording the average signal intensity of 10 negative controls printed on the chip. Normalisation of signal intensity was carried out by calculating the fold increase over the noise signal value. Correlation of replicates using this normalisation procedure was ⥠0.95.
2010-06-24 | E-GEOD-19196 | biostudies-arrayexpress
Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of Ï70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media. The 70-mer microarray representing all annotated ORFs of L. pneumophila has been previously described (Charpentier et al., 2008). RNA was extracted from ssrS mutant strain and the wild-type strain grown to PE phase. gDNA was used as a reference channel on each slides. Labelling of samples, hybridization strategy and data acquisition were performed as described in the Methods section. Local background was removed from spot signal intensity and normalization was carried out by calculating the fraction over the total signal intensity in both channel as previously described (Faucher et al., 2006). Signal levels that were lower than background in experiments and controls were filtered out. A total of six cDNA to reference ratios were recorded for each time point. Statistical analysis between mutant and wild-type strain was performed using unpaired one-tailed studentâs t-test. Genes were considered as differentially expressed if they demonstrated a ratio to control value of ±2-fold with a p < 0.001.
2010-06-24 | E-GEOD-19200 | biostudies-arrayexpress
Project description:We employed a 14-mer pulldown protocol that was originally developed for eukaryotic miRNA interacting RBPs. In this assay, the sRNA (Dsr2) and a scrambled RNA with the same length were in vitro transcribed with a 5 located, 14 nt long tag and subsequently incubated with D. radiodurans lysates. The resulting co-purified proteins were subjected to LC-MS/MS. Using this alternative approach (hereinafter referred to as 14 mer pull-down), we successfully enriched a total of 66 proteins in the 14 mer Dsr2 samples compared to the control sample. Except for several metabolic and hypothetical proteins, many of these proteins have potential nucleic acid binding functions were enriched, including three ribosomal proteins, two translation initiation factors, two TetR transcriptional regulators, five tRNA modification or processing proteins, DNA polymerase I, and five DNA repair proteins (e.g., RuvB, SbcD, and RecR). DR_2281 is one of the most enriched RBPs which contains a double stranded RNA binding motif (DSBM) but its function is unknown. In contrast, five other proteins (PNPase, RhlB, Ffh, DR_2281, KhpA and KhpB) were known as RBPs previously or contain known RNA binding domains. PNPase, RhlB, and Ffh have been revealed to play crucial roles in oxidative stress response in D. radiodurans by interacting with either oxidized RNA or Signal Recognition Particle RNA (Qpr6). PNPase also plays an indispensable role in paradoxically stabilizing sRNAs bound to other RBPs and promoting their function in gene regulation in E. coli (indicating a similar role of PNPase in D. radiodurans).
2024-05-23 | MSV000094855 | MassIVE
Project description:Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered much attention for its newly discovered prevalence in colorectal and breast cancer tissue. The growing interest in this emerging cancer-associated bacterium sharply contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. Post-transcriptional networks are also unknown, for fusobacteria lack all established small RNA-associated proteins. Here, we present high-resolution global RNA maps for two clinically relevant F. nucleatum subspecies for different growth conditions, and use these to uncover fundamental aspects of fusobacterial gene expression architecture and a previously unknown suite of noncoding RNAs. Developing a new vector for functional analysis of fusobacterial genes, we identify a conserved oxygen-induced small RNA as a post-transcriptional repressor of major porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum to colonize different compartments of the human body.
2021-05-03 | GSE161360 | GEO
Project description:Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum – a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization (eut) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes as well as BMC formation is highly increased in wild type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM1, EutM2, and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN, eutL/eutM1/eutM2, or eutL/eutM1/eutM2/eutN not only affected BMC formation, but also ethanolamine utilization, causing cell growth defects with ethanolamine as nutrient. BMCs also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence.
2024-12-12 | GSE280934 | GEO